bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapeutic potential of wild type TMEM43 overexpression in a mouse model of arrhythmogenic cardiomyopathy type 5
Authors: Lalaguna L , Lopez-Olaneta M , Villalba-Orero M , Rey-Martin A , Garcia-Pavia P , Lara-Pezzi E
Published: October 2022

Background and purpose

Arrhythmogenic cardiomyopathy type 5 (ACM5) is the most severe subtype of arrhythmogenic cardiomyopathy, caused by a point mutation in TMEM43 (p.S358L). Pathologically, the disease is characterised by dilation of the cardiac chambers and fibro-fatty replacement of the myocardium, which results in heart failure and sudden cardiac death. The function of TMEM43 and the molecular mechanism underlying ACM5 are very poorly understood, precluding the development of effective therapeutic options.

We have previously observed the interaction between the wild-type (WT) and pS358L forms of TMEM43, suggesting that WT-TMEM43 could quench the detrimental effects of the mutant protein by forming a heterodimer. Therefore, we propose that the overexpression of WT-TMEM43 could be beneficial for ACM5 progression.

Methods and results

Using our transgenic mouse models overexpressing either WT- or pS358L-TMEM43, we have generated a double transgenic mouse overexpressing both forms of the protein. The functional effects were analysed by echocardiography and electrocardiography at 5, 16 and 24 weeks of age. The lifespan of double transgenic (DT) mice increased by 10 weeks compared to mice overexpressing the mutant TMEM43 (p<0.0001). Echocardiography results show an improved systolic function of both ventricles, as observed by an increased left ventricular ejection fraction (LVEF, p<0.001) and tricuspid annular plane systolic excursion (TAPSE, p<0.01), which is already evident at 16 weeks of age. This is accompanied by an improvement in cardiac conduction, with a reduced p wave duration (p<0.001) and QRS duration (p<0.001) and increased QRS amplitude (p<0.0001) in DT mice compared to the mutant mice. In addition, histological analysis of heart samples shows a reduced fibrotic content in the myocardium of DT mice at 24 weeks of age (p<0.0001). Furthermore, the delayed raise in serum cardiac troponin I levels suggests cardiomyocyte necrosis is also delayed in the therapeutic mouse model.

Conclusion

Overall, this data provides evidence that an overexpression of WT-TMEM43 delays the onset of ACM5 phenotype and could potentially be a novel therapeutic approach for ACM5.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation
Association between inflammation and left ventricular thrombus formation following ST-elevation myocardial infarction
Authors: Holzknecht M , Lechner I , Reindl M , Tiller C , Fink P , Mayr A , Klug G , Bauer A , Reinstadler S J , Metzler B
Published: October 2022

Background

Current evidence suggests a link between the inflammatory state and left ventricular thrombus (LVT) formation following ST-elevation myocardial infarction (STEMI). However, a comprehensive study investigating the association between inflammatory biomarkers and LVT diagnosed by cardiac magnetic resonance (CMR) is lacking.

Purpose

The present study aimed to investigate the association of biochemical markers of inflammation with LVT as assessed by CMR imaging among patients with STEMI.

Methods

We studied 309 patients with acute STEMI treated with primary percutaneous coronary intervention (pPCI) from the prospective MARINA-STEMI cohort study. Concentrations of high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), white blood cell count (WBCc), fibrinogen and D-dimer were measured two days after STEMI. Infarct characteristics and presence of LVT were assessed with the use of contrast-enhanced CMR at a median of 4 (interquartile range [IQR] 3–5) days after pPCI.

Results

In total, 309 STEMI patients (18% female) with a median age of 57 (IQR 52–65) years were included. An LVT was observed in 8% (n=24) of the overall cohort and in 15% of patients with an anterior STEMI. Hs-CRP (OR: 2.16, 95% CI: 1.54–3.02, p<0.001), IL-6 (OR: 2.38, 95% CI: 1.48–3.81, p<0.001) and fibrinogen levels (OR: 2.05, 95% CI: 1.40–3.00, p<0.001) were significantly associated with presence of LVT. Among all assessed inflammatory biomarkers, only hs-CRP was independently associated with LVT after adjustment for markers of inflammation and CMR parameters (OR: 1.77, 95% CI: 1.21–2.59, p=0.004).

Conclusion

In patients with STEMI treated with pPCI, inflammatory markers (hs-CRP, IL-6 and fibrinogen) are associated with the presence of LVT. However, only hs-CRP was independently associated with the occurrence of LVT, highlighting the key role of CRP as clinical risk marker for LVT formation in STEMI patients treated with pPCI.

Funding Acknowledgement

Type of funding sources: Public Institution(s). Main funding source(s): Austrian Science Fund (FWF)Austrian Society of Cardiology
Pathogenic variants damage cell compositions and single cell transcription in cardiomyopathies
Authors: Reichart D , Lindberg E L , Maatz H , Miranda A , Viveiros A , Shvetsov N , Lee M , Kanemaru K , Milting H , Noseda M , Oudit G , Heinig M , Seidman J G , Huebner N , Seidman C E
Published: October 2022

Pathogenic variants in genes that cause dilated (DCM) and arrhythmogenic cardiomyopathies (ACM) convey high risks for the development of heart failure (HF) through unknown mechanisms. Using single nucleus RNA sequencing (snRNAseq), we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, non-ischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single cell resolution. Together these data illuminate both shared and distinct cellular and molecular architectures of human HF and suggest novel candidate therapeutic targets.

Funding Acknowledgement

Type of funding sources: Other. Main funding source(s): Chan Zuckerberg FoundationLeducq Foundation
DNA damage induced by radiation exposure from cardiac catheterization – an analysis in patients and operators
Authors: Ishida T , Jin Y , Yaegashi D , Ishida M , Sakai C , Yamaki T , Nakazato K , Tashiro S , Takeishi Y
Published: October 2022

Background

The biological effects of low-dose radiation from cardiac imaging or intervention remain largely unknown. This study aimed to evaluate the effects of ionized radiation from cardiac catheterization on genomic DNA integrity and inflammatory cytokines in patients and operators.

Methods

Peripheral mononuclear cells (MNCs) were isolated from patients (n=52) and operators (n=35) before and after coronary angiography and/or percutaneous coronary intervention. Expression of gammaH2AX, a marker for DNA double-strand breaks, was measured by immunofluorescence. Dicentric chromosomes (DICs), a form of chromosome aberrations, were assayed using a fluorescent in situ hybridization technique.

Results

In the patient MNCs, the numbers of gammaH2AX foci and DICs increased after cardiac catheterization by 101±75% and 28±99%, respectively (P<0.05 for both). The mRNA expressions of interleukin (IL)-1α, IL-1β, leukemia inhibitory factor (LIF) and caspase-1 were significantly increased by radiation exposure from cardiac catheterization. The increase in IL-1β was significantly correlated with that of gammaH2AX, but not with dose area product. In the operators, neither gammaH2AX foci nor DICs level was changed, but IL-1β mRNA was significantly increased. Protein expression of IkappaBα was significantly decreased in both groups.

Conclusions

DNA damage was increased in the MNCs of patients, but not of operators, who underwent cardiac catheterization. Inflammatory cytokines were increased both in the patients and operators, presumably through activation of NF-kappaB. Further efforts to reduce radiation exposure from cardiac catheterization are necessary both for patients and operators.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grants-in-Aid for Scientific Research (KAKENHI)
Long non-coding RNA TERRA regulates DNA damage and survival of endothelial cells and cardiomyocytes: implications for aging
Authors: Bink D I , Pham T P , Van Bergen A , Stanicek L , Van Der Ven D , Groenen E M J , Kaagman L , Jonk M , Hofmann P , Dimmeler S , Boon R A
Published: October 2022

Introduction

Ageing is the major risk factor for cardiovascular disease. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. One of the hallmarks of aging is telomere attrition. Non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are molecules of 0.2–10kb in length which are transcribed from the subtelomeres and telomeres of chromosomes and might play a role in cardiovascular ageing.

Purpose

This study aims to characterize the role of TERRA in aging of the cardiovascular system.

Methods and results

TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). Increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donors (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found with increasing passage in human umbilical vein endothelial cells (HUVECs) (p=0.014) and IPSC-derived cardiomyocytes (p=0.011). After h20q-TERRA knockdown with LNA GapmeRs, HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed an increase in apoptosis (p=0.015) and telomeric DNA damage (p=0.011) and a decrease in telomere length (p<0.001), while lentiviral TERRA-repeat overexpression had the opposite effect (p=0.016, p=0.031, p<0.001, resp.). Apoptosis (p=0.012) and telomeric DNA damage (p=0.007) were also increased after the knockdown of h20q-TERRA in human cardiomyocytes. An apoptosis pathway profiler array in HUVECs showed that the expression of the antioxidant PON2 was decreased after knockdown of h20q-TERRA (p=0.040). PON2 expression was increased after TERRA overexpression (p=0.003). RNA immunoprecipitation revealed that TERRA can bind to PON2. Silencing PON2 in TERRA overexpressing cells diminished the TERRA-mediated decrease in caspase activation, suggesting a detrimental role for PON2 in caspase activation and endothelial cell survival.

Conclusion

Our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival.

Funding Acknowledgement

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020
Genome-wide association study on cerebral white-matter hyperintensities in 36,577 individuals
Authors: Jespersen J , Ahlberg G , Andreasen L , Ghouse J , Frederiksen K S , Haunsoe S , Svendsen J H , Frikke-Schmidt R , Olesen M S , Bundgaard H
Published: October 2022

Background/Introduction

A quarter of all strokes are caused by cerebral small vessel disease (CSVD), which is also the most common pathology underlying vascular dementia. [1] White matter hyperintensities (WMH), a radiological marker detectable on magnetic resonance imaging (MRI), can serve as a proxy for CSVD. WMH is associated with stroke, increased risk of dementia and functional decline in older age. [2,3]

Purpose

We aimed at investigating the genetic architecture of WMH using cerebral MRI data.

Methods

We used imaging data ∼40,000 individuals from the UK Biobank, a large population-based, prospective cohort study. We selected individuals with available total volume of WMH from T1 and T2_FLAIR images.

We performed a genome-wide association study (GWAS) on autosomal genetic variants assuming an additive model based on genotype dosages with BOLT-LMM treating WMH as outcome.

We applied LD score regression (LDSC software) to estimate the genetic correlation between WMH traits and traits selected based on availability and relevance for cardiovascular disease.

Results

We included a total of 36,577 individuals with available quality controlled cerebral MRI data.

Genome-wide analysis identified 20 loci of statistical significance, six of which are not previously reported (Fig. 1). Within these novel loci, the following genes are located in proximity to lead variants: EHBP1, OTX1, WDPCP, VCAN, WNT16, FAM3C, ERI1, PRAG1, CACNB2, MTHFSD, FOXL1, FOXC2. Using BOLT, we found the heritability of WMH to be 37%.

We investigated genetic correlation between WMH and multiple phenotypes (Fig. 2). We observed the highest correlation with small vessel stroke (rg=0.56, P=3.9x10–4) Interestingly, WMH was genetically correlated with left atrial volume (rg=0.24, P=2.8x10–3).

Conclusion

In a GWAS on WMH acquired from cerebral MRI, we identified 20 significant loci, of which six are novel. This genetic study provides insights on the biological understanding and epidemiology of CSVD.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): John and Birthe Meyer FoundationThe Hallas-Møller Emerging Investigator Novo Nordisk
Adverse epigenetic signatures driven by histone methyltransferase EZH2 contribute to hyperglycaemia-induced endothelial dysfunction
Authors: Sanchez-Ceinos J , Hussain S , Waheed-Khan A , Zhang L , Pernow J , Cosentino F
Published: October 2022

Introduction

Posttranslational modifications of histones play a critical role in regulating gene expression in health and disease. Activation of histone methyltransferase enhancer of zeste homolog 2 (EZH2) catalyses repressive trimethylation of histone 3 at lysine residue 27 (H3K27me3) and favours reactive oxygen species (ROS) generation as well as pro-inflammatory changes. Oxidative stress and inflammation are linked to hyperglycaemia-induced endothelial dysfunction.

Purpose

The present study was designed to investigate whether EZH2 may represent a novel therapeutic target against hyperglycaemia-induced abnormal phenotypes in the endothelium.

Methods

The effect of EZH2 was tested in aortas from male mice (3–5 months old), human aortic endothelial cells (HAECs) and aortic endothelial cells isolated from diabetic individuals (D-HAECs) in the presence and in the absence of pharmacological inhibitor GSK126 (5 μM) or gene silencing of EZH2. HAECs and aortic rings were exposed either to normal glucose (5 mM) or high glucose (25 mM) for 20 hrs. Expression levels of H3K27me3, EZH2, oxidant and pro-inflammatory genes were determined by RT-PCR, western blotting, and protein activation assay kits. Superoxide anion production was measured by electron spin resonance spectroscopy. Binding of H3K27me3 to the promoter region of candidate genes was evaluated by chromatin immunoprecipitation (ChIP). Endothelium-dependent and independent relaxations were assessed in aortic rings by organ chamber experiments.

Results

Immunoblotting showed a significant increase in EZH2 activity measured as H3K27me3 in HAECs exposed to high glucose and in D-HAEC. ChIP assays showed that binding of H3K27me3 to the promoter of anti-oxidant and transcription factor JunD genes triggers ROS generation and activation of inflammatory pathways. Interestingly enough, histone methyltransferase EZH2 inhibitor GSK126 or specific siRNA blunting H3K27me3 abolished hyperglycaemia-induced oxidative stress and inflammation. Moreover, targeting EZH2 rescued endothelial function.

Conclusions

These results uncover adverse epigenetic signatures underlying endothelial dysfunction in diabetes. Targeting EZH2 may attenuate oxidative and inflammatory transcriptional programmes and thus prevent vascular disease in this setting.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): Swedish Research Council, the Swedish Heart & Lung Foundation, and King Gustav V and Queen Victoria Foundation.
Fast and more affordable CYP2C19 LOF testing to personalized of clopidogrel therapy
Authors: Akkaif M A , Sha'aban A , Daud N A A , Noor D A M , Musa N F , Sk Abdul Kader M A , Ibrahim B
Published: October 2022

Background

Clopidogrel is an antiplatelet drug used to treat coronary artery disease (CAD) to reduce blood clotting by inhibiting platelet activity. However, many patients experience ischemic events due to changes in clopidogrel metabolism attributed to various genetic and non-genetic factors. CYP2C19 is one of the important genes responsible for the metabolism of clopidogrel, but this gene has polymorphisms in cytochrome P450 (CYP), and this CYP2C19 Loss-of-function (LoF) polymorphisms impair clopidogrel metabolism between individuals and races. Therefore, genetic testing is an effective strategy to personalized clopidogrel and reduce the incidence of ischemic events in CAD patients.

Purpose

We sought to develop a nested allele-specific multiplex polymerase chain reaction (PCR) method for the detection of CYP2C9 LOF alleles.

Method

Genomic DNA was extracted from blood taken from 7 healthy volunteers using a commercial DNA extraction kit. A two-step PCR method was developed. First, DNA was subjected to a first PCR used to amplify exons 4 and 5 simultaneously in a single reaction tube. Second, The products of 1st PCR were then used as a template in the second PCR to detect CYP2C9 polymorphisms alleles (CYP2C19*2 and *3) using allele-specific primers. The test results were validated via sequencing.

Result

We successfully detected clinically important CYP2C19 LOF polymorphisms alleles by the nested allele-specific multiplex PCR method (Figure 1). This method is fast and doesn't require high-quality and expensive laboratory equipment. It was reproducible and specific and can reliably detect CYP2C19 variants. Direct sequencing of target variants has been used to validate this strategy further, and the amplified sequences were 100% identical to the CYP2C9 sequence.

Conclusion

The developed methods were specific to identify polymorphisms in CYP2C19 (rs4244285 and rs4986893). However, to understand ethnic differences and their potential implications for clopidogrel therapy, we highly recommend identifying relevant genetic polymorphisms before clinical manifestations arise.

Funding Acknowledgement

Type of funding sources: None.
Circadian nuclear receptor Rev-erbalpha is expressed by platelets and potentiates platelet activation and thrombus formation
Authors: Shi J , Tong R , Zhou M , Gao Y , Zhao Y , Chen Y , Liu W , Li G , Lu D , Meng G , Hu L , Yuan A , Lu X , Pu J
Published: October 2022

Background

Adverse cardiovascular events have day/night patterns with peaks in the morning, potentially related to endogenous circadian clock control of platelet activation. Circadian nuclear receptor Rev-erbα is an essential and negative component of the circadian clock.

Purpose

We aim to investigate the expression profile and biological function of Rev-erbα in platelets.

Methods and results

Here we report the presence and functions of circadian nuclear receptor Rev-erbα in human and mouse platelets. Both human and mouse platelet Rev-erbα showed a circadian rhythm that positively correlated with platelet aggregation. Global Rev-erbα knockout and platelet-specific Rev-erbα knockout mice exhibited defective in hemostasis as assessed by prolonged tail-bleeding times. Rev-erbα deletion also reduced ferric chloride-induced carotid arterial occlusive thrombosis, prevented collagen/epinephrine-induced pulmonary thromboembolism, and protected against microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. In vitro thrombus formation assessed by CD41-labeled platelet fluorescence intensity was significantly reduced in Rev-erbα knockout mouse blood. Platelets from Rev-erbα knockout mice exhibited impaired agonist-induced aggregation responses, integrin αIIbβ3 activation and α-granule release. Consistently, pharmacological inhibition of Rev-erbα by specific antagonists decreased platelet activation markers in both mouse and human platelets. Mechanistically, mass spectrometry and co-immunoprecipitation analyses revealed that Rev-erbα potentiated platelet activation via oligophrenin-1-mediated RhoA/ERM (ezrin/radixin/moesin) pathway.

Conclusion

We provide the first evidence that circadian protein Rev-erbα is functionally expressed in platelets and potentiates platelet activation and thrombus formation. Rev-erbα may serve as a novel therapeutic target for managing thrombosis-based cardiovascular disease.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): This work was supported by grants from the National Science Fund for Distinguished Young Scholars (81625002), the National Natural Science Foundation of China (81930007).
Thrombin receptor protease-activated receptor 1 (PAR1) related cytotoxic CD8+ T cell activity is associated with atrial myopathy and pro-inflammatory immune response in early atrial fibrillation
Authors: Friebel J , Wegner M , Lammel S , Bloebaum L , Jakobs K , Puccini M , Landmesser U , Rauch U
Published: October 2022

Background and aims

Cytotoxic CD8+ T cells mediate myocardial damage in patients with virus induced myocarditis, plaque erosion during acute coronary syndrome, and promote adverse post-ischemic cardiac remodeling. There is emerging evidence to support a link between inflammation and atrial fibrillation (AF). Protease-activated receptor 1 (PAR1) is the thrombin receptor expressed on platelets, but it is also expressed in endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, and CD8+ T cells. The role of CD8+ T cells and PAR1 in atrial myopathy and early AF have not been studied.

Methods

In 80 non-anticoagulated patients with a first documented episode of AF, and 20 control subjects without AF but with a comparable cardiovascular risk profile (e.g. SCORE2, CHA2DS2-VASc, p>0.05), we studied PAR1-related cytotoxic T cell activity in the first hours (up to 24 h) of AF clinical manifestation. The Apolipoprotein E–knock out (ApoEko) mouse is a commonly used model of metabolic syndrome that develops atrial myopathy (structural correlate of AF). ApoEko male mice (Apoetm1Unc with a C57BL/6 genetic background) were fed for six weeks with a standard chew, western-type diet (high fat and high sucrose = WD) ± the specific PAR1 inhibitor vorapaxar.

Results

High levels of circulating, activated CD8+ T cells (CD8+CD57+ T cells with pro-inflammatory and high cytotoxic properties) were present in patients with early AF (flow cytometry). Stimulation of cytotoxic T cells, that expressed the thrombin receptor PAR1, that were isolated from patients with new onset of AF, released pro-inflammatory cytokines (e.g. IFN-γ, TNF-α). Enhanced cytotoxic activity of CD8+ T cells was suggested by elevated plasma levels of effector molecules (e.g. granulysin and granzyme).

In ApoEko mice fed a WD, atrial myopathy as the structural correlate for AF, was paralleled by increased expression of effector molecules that were associated with CD8+ T cell activity (like perforin, granzyme A, granzyme B, IFN-γ). Cytotoxic T cell activity disrupted the release of the potective atrial natriuretic peptide, ANP, and increased signaling pathways associated with atrial fibrosis (e.g. TGF-β, cTGF, TGF-β receptor). The specific PAR1 inhibitor, vorapaxar, reduced inflammatory T cell activity, transcription of fibrotic mediators, and reconstituted ANP secretion in atria of ApoEko mice.

Conclusions

Targeting the FXa/FIIa-PAR1-CD8+ axis might be a promising approach to reduce atrial fibrosis and inflammation.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BIH Charité Clinician Scientist Program
SWATH proteomic analysis from the standard care versus celecoxib outcome trial identifies renal biomarkers are associated with early cardiovascular events amongst NSAID users
Authors: Vaja R , Ferreira P M , Want L , Ahmetaj-Shala B , Gashaw H , Kirkby N S , Mitchell J A
Published: October 2022

Background

Despite being one of the most commonly used and readily available drugs worldwide, we still do not understand why non-steroidal anti-inflammatory drugs (NSAIDs) increase cardiovascular risk. A proteomics approach may provide mechanistic insight into how COX-2 protects the cardiovascular system.

Purpose

To use SWATH proteomics to identify cardiovascular biomarkers in NSAID users who go onto have a cardiovascular event.

Methods

A case control study was designed using the Standard Care Versus Celecoxib Outcome Trial (SCOT) biobank (1). Serum samples from 228 NSAID users with osteoarthritis were analysed. From these, an exploratory analysis of 15 individuals who had a cardiovascular event within 6 months were compared to 213 individuals who did not. SWATH proteomics was used to identify biomarkers and findings were validated by immunoassay.

Results

Seventy-eight proteins were detected at significantly different levels in serum from participants who did or did not have an event within 6 months of enrolment. Of these the greatest increase in the event group was in levels of the renal dysfunction marker, cystatin C (Log Fold difference 1.4, p<0.001). Validation using a renal biomarker immunoassay panel confirmed a higher level of cystatin C (Z-score 0.6 vs −0.04, p: 0.02) as well as a lower level of uromodulin (Z-score −0.5 vs 0.03, p: 0.036) in the event group. In agreement, mass spectrometry analysis identified a higher level of the cardio-renal biomarker asymmetric dimethylarginine (ADMA) in the event group (Z-score 0.4 vs −0.03, p: 0.07) (Figure 1).

Conclusion

Renal biomarkers are associated with early cardiovascular events amongst NSAID users in the SCOT cohort. Thus, changes in renal function should be investigated as a mechanistic or predictive factor in this population at elevated cardiovascular risk.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation
Protective effects of COVID-19 vaccination on endothelial function of patients with COVID-19
Authors: Lampsas S , Oikonomou E , Souvaliotis N , Papamikroulis G A , Lytra T , Sarantos S , Mystakidi V C , Pantelidis P , Kalogeras K , Theofilis P , Tsatsaragkou A , Siasos G , Tsioufis C , Tousoulis D , Vavuranakis M
Published: October 2022

Background

Endotheliilitis COVID-19 related endothelial dysfunction plays a key role in the cardiovascular complications of the disease. Vaccine against SARS-CoV-2 protects against severe COVID-19 and from adverse effects. We evaluated the impact of vaccination on COVID-19 induced endothelial dysfunction.

Methods

We enrolled 45 patients hospitalized for COVID-19 (either vaccinated or not against SARS-CoV-2). Clinical information and laboratory findings were collected, and brachial artery flow-mediated dilation (FMD) was evaluated as a measure of endothelial function. Subjects without COVID-19 were used as the control group. All patients were hospitalized in a medical ward classified according to the World Health Organization (WHO) scale.

Results

There was no difference in age (62±10 years vs. 65±8 years, p=0.12) and male sex prevalence (56% vs. 49%, p=0.53) between patients with COVID-19 and control subjects. Of the patients with COVID-19, 44% (20) were vaccinated against SARS-CoV-2. FMD was impaired in patients with COVID-19 compared to controls (4.35±3.56% vs. 7.36±2.91%, p<0.001). In patients with COVID-19, FMD was impaired in non-vaccinated subjects compared to vaccinated (2.05±2.41% vs. 7.24±2.52%, p<0.001). There was no difference in FMD between controls and vaccinated against COVID-19 patients (7.36±2.91% vs. 7.24±2.52%, p=0.86). There was no difference in the WHO scale clinical status for vaccinated and not vaccinated COVID-19 subjects (For Vaccinated WHO scale 3: 35%; scale 4: 35%; scale 5: 30% vs. For Non-vaccinated WHO scale 3: 20; scale 4: 60%; scale 5: 20%, p=0.24).

Conclusion

Hospitalized patients with COVID-19 present endothelial dysfunction in the acute phase of the disease. Endothelial function in unvaccinated patients with COVID-19 is impaired compared to control subjects as well as compared to vaccinated patients with COVID-19. This data provides insights on the protective role of vaccination against COVID-19 related endotheliitis.

Funding Acknowledgement

Type of funding sources: None.
GPR55 deficiency in B-cells promotes atherosclerosis and regulates plasma cell maturation
Authors: Guillamat-Prats R , Hering D , Rami M , Haerdtner C , Santovito D , Rinne P , Pagano S , Nicolas Vuilleumier N , Schmid S , Janjic A , Enard W , Weber C , Maegdefessel L , Hilgendorf I , Steffens S
Published: October 2022

Atherosclerosis is a chronic and multifactorial disease accompanied by an imbalance between resolving and pro-inflammatory lipid mediators. Targeting lipid signaling might offer new therapeutical targets for improving the clinical outcome in cardiovascular disease patients. We considered lysophosphatidylinositol (LPI) and its receptor G protein-coupled receptor (GPR)55 as a potential modulator of atherosclerosis. Its role in regulating atherosclerosis and B cell function is unknown.

We hypothesize that GPR55 signaling affects atherosclerosis by regulating B cell function.

Atherosclerotic plaques were compared between apolipoprotein-E-deficient (ApoE−/−) and ApoE−/−Gpr55−/− mice after 4 to 16 weeks Western Diet (WD; 0.15% cholesterol; n=12–15 per group). To test the role of B cell GPR55, we generated mixed chimeras by irradiating low density lipoprotein receptor deficient (Ldlr−/−) mice and reconstituting with a mixture of μMT and wildtype or μMT and Gpr55−/− bone marrow cells. Circulating B cells were sorted and bulk RNA sequencing analysis was performed. We performed atheroma plaque characterization, qPCR and ELISA of tissue lysates and measure plasma immunoglobulins. Circulating and tissue leukocyte counts were determined.

We confirmed Gpr55 expression on circulating B cells, which was higher compared to T and myeloid cells. ApoE−/−Gpr55−/− mice had significantly larger plaques after 4 & 16 weeks WD compared to ApoE−/−, with increased body weight & cholesterol levels. In addition, global Gpr55 deficiency resulted in enhanced aortic pro-inflammatory cytokine mRNA expression, a massively upregulated IgG levels and increased counts of splenic germinal center and plasma cells. ApoE−/−Gpr55−/− B-cell RNA-seq analysis showed 460 differential expressed genes compared to ApoE−/−. The main pathways affected were calcium ion transport, immunoglobulin production, T & B cell activation, and cellular response to stress. B cell specific Gpr55 deficiency blunted the metabolic effects but still translated in larger atherosclerotic plaques and elevated plasma IgG levels.

Both global and B cell-restricted Gpr55 deficiency promotes atherosclerosis and is associated with a pro-inflammatory phenotype.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The authors received funds from the Deutsche Forschungsgemeinschaft (STE1053/6-1, STE1053/8-1 to S.S. and SFB1123 to S.S., C.W. and L.M.), the German Ministry of Research and Education (DZHK FKZ 81Z0600205 to S.S.) and the LMU Medical Faculty FöFoLe program (1061 to R.G.P.). I.H. is supported by the DFG (HI1573/2 and CRC1425 #422681845).
Diagnostic ability of miR-19a and Let-7f along with platelet and leukocyte extracellular vesicles in acute ischemic stroke patients
Authors: Eyileten C , Nowak A , Shahzadi A , Jarosz-Popek J , Jakubik D , Gasecka A , Van Der Pol E , Wolska M , Mirowska-Guzel M , Czlonkowska A , Postula M
Published: October 2022

Background

Ischemic stroke (IS) is one of the most frequent causes of death. Since miRNAs have been illustrated to play an important role in various processes through regulation of multiple genes, and platelet function, their utility as novel biomarkers should be determined.

Purpose

We aimed to analyze the circulating platelet-derived miR-19a-3p, miR-186-5p, Let-7f, platelet-extracellular vesicles (EVs), leukocyte-EVs, and endothelial-EVs levels 24-h and 7-days after IS as novel diagnostic and prognostic/predictive biomarkers in 28 acute IS and 35 control patients.

Methods

Blood samples of 28 patients diagnosed with acute IS with hyper platelet reactivity were collected 24-h and 7-days after stroke and 35 age- and gender-matched individuals free of stroke with multiple risk factors for cardiovascular disease. Platelet reactivity was assessed by AA-, TRAP-, ADP-induced platelet aggregometry. PlasmaRNA was extracted; quality RNA was assessed: fluorometric assay; RT-PCR for miRNAs expression measurement and flow-cytometry for EVs determination p<0.05.

Results

Patients with IS on day-1 had significantly higher platelet reactivity assessed by AA-induced platelet aggregometry compared to controls (p=0.001). Patients with normal platelet activation had significantly higher miR-186-5p expression levels compared to patients with HPR at day-1 acute-stroke (p=0.034). Seven days after acute stroke, expression levels of miR-186-5p significantly decreased in the same patients with normal platelet reactivity (p=0.036). Patients with HPR had significantly elevated platelet-EVs (CD62) concentration compared to patients with normal platelet reactivity at the day of 1 acute-stroke (p=0.012). Similarly, patients with HPR had significantly higher leukocyte-EVs (CD45) concentration compared to patients with normal platelet function at day-1 acute-stroke (p=0.002). Diagnostic values of baseline miRNAs and EVs were evaluated with receiver operating characteristic curve analysis. ROC curve showed that pooling the miR-19a-3p expressions, platelet-EVs, and leukocyte-EVs concentration yielded a higher AUC than the value of each individual biomarker as AUC was 0.893 (95% CI, 0.79–0.99). Patients with moderate stroke had significantly elevated miR-19a-3p expression levels compared with patients with minor stroke at the day of acute IS. AUC in ROC curve analysis was 0.867, (95% CI, 0.74–0.10) p=0.001.

Conclusions

Our analysis showed alteration of circulating miRNAs and EVs after IS. Combination of miR-19a-3p, Let-7f, platelet-EVs and leukocyte-EVs might have a diagnostic value for acute stroke and miR-19a-3p can predict the severity of stroke in IS patients.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre - Narodowe Centrum Nauki
Leptin mediates protective effects on the vasculature
Authors: Filipova V , Stuerzebecher P E , Kralisch S , Schubert M R , Hoffmann A , Oliveira F , Sheikh B , Blueher M , Kogel A , Scholz M , Miehle K , Ebert T , Laufs U , Toenjes A , Boeckel J N
Published: October 2022

Introduction and purpose

Lipodystrophy (LD) syndromes are characterized by the loss of adipose tissue resulting in metabolic complications and accelerated atherosclerosis. The systemic concentration of the adipokine leptin is reduced in LD as a result of adipose tissue deficiency. A therapeutical option to treat LD is the substitution of leptin, which improves metabolic complications and reduces mortality. However, the vascular effects of leptin remain largely unknown.

Here we analyze the direct effects of leptin on the vascular system and the development of atherosclerosis.

Methods and results

Treatment of human endothelial cells (ECs) with leptin reduced endothelial inflammation and the process of endothelial-to-mesenchymal transition (EndMT) (CNN1, −41.4%, p<0.05, n=4). In addition, leptin administration prevented the EndMT-induced increase of endothelial permeability. The protective effect of leptin on EndMT was confirmed in vivo in a combined lipodystrophic and atherosclerosis-prone mouse model (LDLR−/−; aP2-nSrebp1c). Treatment of the mice with leptin (3.0 mg/kg body weight daily for 8 weeks) decreased EndMT. Leptin showed no effect on plaques size but reduced the protrusion of plaques in atherosclerotic areas of the aortic roots (−31%, p<0.05, n=4–6).

Cytokine screening revealed an increase of the growth differentiation factor 15 (GDF15) in serum of LD patients (+26.2%, p<0.05, n=53–58) and in ECs after EndMT (+138%, p<0.05, n=6743–10920). This increase was reversed using leptin treatment in ECs undergoing EndMT, in the LD mice model, and in LD patients after 4 weeks of leptin administration. Indeed, treatment of endothelial cells with GDF15 induced EndMT (CNN1, +7.7-fold-control, p<0.05, n=3), and impaired EC barrier function. Neutralizing antibodies targeting GDF15 inhibited EndMT-mediated expression of mesenchymal genes (CNN1, −54%, p<0.05, n=4). The treatment of ECs with serum from LD patients induced EndMT and the increase of mesenchymal marker expression was inhibited with additional administration with neutralizing antibodies targeting GDF15 (CNN1, −28%, p<0.05, n=3).

Conclusion

Our findings indicate that EndMT is part of the cardiovascular disease progression in lipodystrophy syndromes. Leptin treatment has direct protective vascular effects by preventing inflammation, EndMT, and maintaining endothelial integrity.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)
Left ventricular volumes on positron emission tomography: validation against cardiovascular magnetic resonance
Authors: Han Y , Ahmed A I , Saad J M , Malahfji M , Al-Mallah M H
Published: October 2022

Introduction

Cardiovascular magnetic resonance (CMR) is the gold standard for determination of left ventricular volumes (LVV), including end diastolic volume (LVEDV) and end systolic volume (LVESV). With incorporation of ECG gating, the quantitative analysis of ventricular function can be achieved in Positron Emission Tomography (PET) studies. However, the accuracy of LVV quantification using PET has not been shown in previous studies.

Purpose

We aimed to assess the inter-study variability of LVEDV and LVESV between CMR and PET.

Methods

Consecutive patients who underwent both PET and CMR within 1 year were identified from prospective institutional registries. LVV on PET was compared against CMR as a reference standard using several statistical measures of agreement.

Results

The study population consisted of 233 patients (mean age 60.8±13.6 years, 64.8% male). The median (IQR) duration between CMR and PET was 41 (11–96) days. The median (IQR) LVEDV values were 144 (107–196) on PET and 178 (138–236) on CMR (mean difference 36.09±2.36, p<0.001). As shown in Figure 1, there was a good correlation (Spearman rho=0.88, p<0.001; Intraclass Correlation Coefficient 0.88, 95% CI 0.84–0.90, P<0.001) with moderate limits of agreement (95% limits of agreement −108.17–35.99). A total of 0% and 3.9% of the cohort had LVEDV >150 ml/m2 by CMR and PET respectively. Overall results were similar with LVESV values and similar in subgroup analysis of patients with a low EF (<40%), a coronary artery disease scar, LV hypertrophy and of patients with defibrillators.

Conclusion

Our analysis has shown a good correlation of LVV between PET and CMR, indicating PET derived LVV can accurately be used in the diagnosis and management of patients.

Funding Acknowledgement

Type of funding sources: None.
Early stage atherosclerosis regression using combined sonotherapy and mitoxantrone-mediated sonoporation therapy
Authors: Mehrad H
Published: October 2022

Background

In atherosclerosis, local inflammation and associated macrophage activity can lead to foam cell-rich plaque formation, which results to plaque rupture and thrombosis, making inflammation an important therapeutic target in cardiovascular disease. The early stage of atherosclerosis is characterized by the accumulation of low-density lipoprotein (LDL) droplets, leading to the creation of foam cells. Foam cells are generated by uncontrolled uptake of modified LDL, especially oxidized LDL (oxLDL).

Purpose

In this study, we developed an experimental focused ultrasound-based combined sonotherapy and sonoporation therapy system, and investigated its effectiveness on macrophage-derived foam cells reduction, wherein a high resolution diagnostic ultrasound is adjuncted with combination therapy system, with a goal of increased safety.

Methods

Briefly, New Zealand white rabbits underwent primary balloon dilatation injury at the right common carotid artery followed by a 1.5% cholesterol-rich diet injury for three weeks. Histopathology results showed the early stage atherosclerosis formation in all of the rabbits' arteries. Then common carotid arteries of the treatment group at the lesion region, treated using extracorporeal low-level pulsed-focused ultrasound (P=11 W, F=1.1 MHz, DF=50%) sonotherapy accompanied by mitoxantrone-loaded PESDA (Perfluorocarbon-Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2–5x105 bubbles/ml)-mediated sonoporation therapy. Foam cells density were evaluated in the treatment group compared with the other groups using B-mode ultrasonography and histopathology.

Results

Results from B-mode ultrasonography and histopathology showed a significant reduction in the mean value for immune cells and foam cells density within the early stage atherosclerotic lesion in the treatment group compared with the other groups (p<0.05).

Conclusion

Enhanced sonoporation effect of ultrasound, induced by collapsed PESDA microbubbles accompanied by anti-inflammatory effect of sonotherapy and antineoplastic and cytotoxic effect of mitoxantrone can cause to reduce the immune cells and foam cells density within the lesion. These finding provide the basis for developing low-level pulsed-focused ultrasound-based combined sonotherapy and sonoporation therapy for a successful clinical application in the treatment of the early stage atherosclerosis.

Funding Acknowledgement

Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab
Coronary microvascular disease assessed by 82Rb-PET-CT is an independent prognostic marker of all-cause mortality
Authors: Hojstrup S , Hansen K , Talleruphuus U , Marner L , Galatius S , Rauf M , Bjerking L , Jakobsen L , Christiansen E , Bouchelouche K , Prescott E
Published: October 2022

Background

Coronary microvascular disease (CMD) is a major contributor to e.g. heart failure and angina pectoris, as well as being associated with an increased risk of adverse events. CMD is diagnosed by reduced myocardial blood flow reserve (MBFR), preferably by Positron emission tomography myocardial perfusion CT (PET-CT).

Purpose

We aim to determine whether reduced MBFR is associated with an increased hazard of all-cause mortality independently of the extent of perfusion defects in patients suspected of obstructive coronary artery disease.

Method

We conducted a multicenter study of all patients referred for 82Rubidium PET-CT imaging between January 2018 and August 2020. Rest and stress examinations were performed using standard imaging protocols. Percentage of perfusion defects were calculated based on summed rest- and difference score. CMD was defined as MBFR ≤2. Patients were followed for all-cause mortality through national registries with no loss to follow-up.

Results

Among the 7156 patients studied, 61.8% were men, median age was 69 [61–76 IQR] years, 14.1% had LVEF ≤40%, 58.4% had a previous diagnosis of ischemic heart disease (IHD), 20.1% had atrial fibrillation and 38.9% had MBFR ≤2.

A total of 571 (7.8) deaths were observed, more frequently in MBFR ≤2 compared to MBFR >2 (4.2% vs 13.2%, p<0.001). MBFR was significantly associated with reversible hypoperfusion (r2=−0.33, p<0.0001). In Kaplan-Meier estimation MBFR ≤2 was significantly associated to all-cause mortality in the overall population as well as in clinically relevant subgroups defined by the extent of reversible and/or irreversible perfusion defects (p<0.05 for all, fig. 1). In multivariate Cox-analysis adjusting for age, sex, Charlson's Co-morbidity index, eGFR, LVEF and LVEF-reserve and stratifying by diabetes, MBFR ≤2 remained a robust predictor of all-cause mortality with a HR 1.73, 95% CI: 1.62–2.19, p<0.0001 (fig. 2). No interaction was found between MBFR and reversible hypoperfusion. In subgroup analysis including only patients with no reversible perfusion defects (n=3095), MBFR ≤2 was still strongly associated with a HR of 2.00, 95% CI: 1.29–3.11, p<0.001 for all-cause mortality.

Conclusion

MBFR ≤2 is a robust predictor of all-cause mortality independently of the extent of reversible- and/or irreversible perfusion defects. Information of MBFR should be incorporated in the clinical risk stratification of patients being investigated for ischemia.

Funding Acknowledgement

Type of funding sources: Public hospital(s). Main funding source(s): Research Committee, Bispebjerg & Frederiksberg University Hospital, scientific scholarship
SGLT-2 inhibition by empagliflozin exerts neutral effects on experimental arterial thrombosis in a murine model of low-grade inflammation
Authors: Liberale L , Kraler S , Puspitasari Y , Bonetti N , Akhmedov A , Ministrini S , Montecucco F , Marx N , Lehrke M , Hartmann N U K , Beer J H , Paneni F , Luescher T F , Camici G G
Published: October 2022

Introduction

Low-grade inflammation couples dysmetabolic states to insulin resistance and atherosclerotic cardiovascular disease (ASCVD). Selective sodium-glucose co-transporter 2 (SGLT-2) inhibition by empagliflozin improves clinical outcomes in patients with ASCVD independently of glucose-lowering. Yet, its mechanism of action remains largely undetermined.

Purpose

We aimed to test whether empagliflozin affects arterial thrombus formation in baseline conditions or low-grade inflammation, a systemic milieu shared among patients with ASCVD.

Methods

Sixteen-week-old C57BL/6 mice were randomly assigned to acute administration of empagliflozin (25 mg/kg BW) or vehicle, of which a subgroup was pre-treated biweekly over 4 weeks with super-low-dose lipopolysaccharide (LPS; 5 ng/kg BW), before carotid thrombosis was induced by photochemical injury. The translational value of these findings was investigated in primary human aortic endothelial cells (HAECs) and plasma samples of patients randomized to empagliflozin therapy.

Results

The between-group difference in doppler-flow probe detected time-to-occlusion (TTO) remained within the predefined equivalence margin (Δ=|10.50|), irrespective of low-grade inflammation (95% confidence interval [CI], −9.82 to 8.85 and −9.20 to 9.69), while glucose dropped by 1.64 and 4.84 mmol/l, respectively (Fig. 1). Ex vivo platelet aggregometry suggests similar platelet activation status, corroborated by unchanged circulating platelet-factor 4 (PF4) plasma levels. In concert, carotid PAI-1 expression and TF activity remained unaltered upon SGLT-2 inhibition, and no difference in plasma D-dimer levels was detected, suggesting comparable coagulation cascade activation and fibrinolytic activity (Fig. 1). In HAECs pre-treated with LPS, empagliflozin neither changed TF activity nor PAI-1 expression (Fig. 2). Accordingly, among patients with established ASCVD or at high cardiovascular (CV) risk randomized to 10 mg empagliflozin daily signatures of thrombotic (i.e., TF) and fibrinolytic activity (i.e., PAI-1) remained unchanged, while plasma glucose declined significantly during 3 months of follow-up (Fig. 2).

Conclusion

SGLT-2 inhibition by empagliflozin does not impact experimental arterial thrombus formation, neither under baseline conditions nor during sustained low-grade inflammation, and has no impact on proxies of thrombotic/fibrinolytic activity in patients with ASCVD. The beneficial pleiotropic effects of empagliflozin are likely independent of pathways mediating thrombosis.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science FoundationSwiss Heart FoundationFoundation for Cardiovascular Research–Zurich Heart House