bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP45: The Roles Of Telomeric Repeat Binding Factor 2-interacting Protein (TERF2IP) K240 Sumoylation In Endothelial Cells On Atherogenesis
Authors: Imanishi Masaki , Velatooru Loka , Ko Kyung ae , Heo Kyung-Sun , Thomas Tamlyn , GI YOUNG JIN , Fujiwara Keigi , Le Nhat T , Kotla Sivareddy , Abe Junichi
Published: May 2022

Background and Objectives:
It is not yet clear how the pro-atherogenic signaling events in endothelial cells (ECs) such as those that lead to EC senescence, apoptosis and activation are interconnected and promote atherosclerotic plaque formation in the area exposed to disturbed blood flow (d-flow). TERF2IP, a member of the shelterin complex of the telomere, regulates all three pathological events. We investigated the role of TERF2IP K240 SUMOylation in the process of d-flow-induced atherosclerotic plaque formation.

Methods and Results:
We found that d-flow increased TERF2IP K240 SUMOylation in ECs and that it was suppressed by a p90RSK specific inhibitor, FMK-MEA. This SUMOylation was independent of TERF2IP S205 phosphorylation. The d-flow-induced senescence, DNA damage, and apoptosis were inhibited in ECs with TERF2IP depletion or point-mutated phosphorylation (S205A) and SUMOylation (K240R) sites. NF-κB activation induced by d-flow or overexpression of p90RSK was also significantly inhibited in ECs overexpressing the TERF2IP S205A phosphorylation mutant. However, cells overexpressing the TERF2IP K240R SUMOylation mutant showed no effect on the d-flow or p90RSK-medaited NF-κB activation. To determine the biological function of TERF2IP K240 SUMOylation, we generated TERF2IP K240R knock-in (KI) mice and examined d-flow-induced atherosclerotic plaque formation using partial left carotid ligation model mice fed a high-fat diet after AAV8-PCSK9 injection. We found no differences in body weights and cholesterol levels between TERF2IP K240R KI and wild type control (WT) mice, but plaque formation was significantly inhibited in the KI mice compared to WT animals (Oil Red O positive area (%): 19.0 +/- 12.5 (KI mice, n=8) vs 61.2 +/- 24.3 (WT mice, n=7), p = 0.0008). Bone marrow from WT mice were transplanted into KI and WT mice, which were then injected with AAV8-PCSK9 virus and fed a high-fat diet for 16 weeks, but we still found that plaque formation was inhibited in the KI mice.

Conclusion:
TERF2IP SUMOylation plays a role in in EC senescence but not in activation. The significant inhibition of plaque formation in the TERF2IP K240R KI mice is due to downregulation of TERF2IP SUMOylation in ECs not in myeloid cells.
Abstract MP49: Macrophage-mediated Extracellular Digestive Exophagy Of Aggregated LDL Is Responsible For The Formation Of Cholesterol Crystals In Atherosclerotic Plaques
Authors: Ma Cheng-I Jonathan J , Barbosa Lorenzi Valeria , Maxfield Frederick R
Published: May 2022

Atherosclerosis is a disease that develops overtime through dyslipidemia and chronic inflammation. Formation of cholesterol crystals in the atherosclerotic plaques is a major contributor to the inflammatory response. However, how these crystals form in the atherosclerotic plaques remained poorly understood. We have observed that macrophage-mediated extracellular digestive exophagy of aggregated LDL releases a large amount of free cholesterol in the extracellular space. Therefore, we hypothesized that this release of cholesterol may play a major role in formation of cholesterol crystals in plaques. We have shown previously that TLR4 knockout in macrophages compromises digestive exophagy of aggregated LDL. Thus, to evaluate the role of digestive exophagy in cholesterol crystal formation, we examined cryosections of aortic plaques from

Ldlr
-/-

mice that were gamma-irradiated and reconstituted with WT or

Tlr4
-/-

bone marrows. Interestingly, TLR4 KO in macrophages reduce both free cholesterol and cholesterol crystal in plaques compared to WT macrophages, suggesting a role of digestive exophagy in cholesterol crystal formation. To investigate whether digestive exophagy of aggregated LDL results in inflammatory response, we cultured bone-marrow derived macrophages expressing the inflammasome marker ASC-citrine with aggregated LDL over time. We observed a gradual increase in number of inflammasomes over a 24 h time period. We also observed extracellular cholesterol crystal formation as early as 2 h after the addition of aggregated LDL. Moreover, treating bone marrow-derived macrophages with acetylated LDL did not result in the formation of extracellular cholesterol crystals, suggesting this process is specific to a subset of modified LDL in the vessel wall. Overall, our data provide evidence that macrophage-mediated digestive exophagy of aggregated LDL is a major contributor to the formation of extracellular cholesterol crystal found in atherosclerotic lesions.
Abstract P102: False Lumen Volume Is A Predictor Of Successful Thoracic Endovascular Aortic Repair
Authors: Khabaz Kameel , Sankary Seth , Milner Ross , Pocivavsek Luka
Published: May 2022

Objectives:
Thoracic endovascular aortic repair (TEVAR) is becoming the treatment modality of choice for type B aortic dissection (TBAD). Using CTA imaging, clinicians track dissection evolution; however, clinical imaging analysis is limited to linearized measurements of maximum diameter that poorly predict post-operative outcomes. Achieving post-operative thrombosis of the false lumen is a desired characteristic that is thought to prevent aneurysmal degeneration, complication, and reintervention. We hypothesize that decrease in false lumen volume can be used to accurately predict successful TEVAR, as defined by lack of reintervention.

Methods:
We studied 20 TBAD patients that underwent TEVAR, 10 with desired outcomes, defined by the absence of post-operative reintervention, and 10 with failed outcomes, defined by the presence of reintervention. Utilizing CTA imaging, we performed pre- and post-operative segmentations of the false lumen for each patient. We calculated the volume of the false lumen of each scan. We performed two-sample t-tests on the null hypothesis that false lumen volume does not change post-TEVAR. We also tested the null hypothesis that patients with desired outcomes and poor outcomes have equal false lumen volumes.

Results:
We found a statistically significant difference in the pre-operative false lumen volume of patients with desired outcomes post-TEVAR versus patients with failed outcomes (p=0.014). However, we found no statistically significant change in false lumen volume of patients with successful or failed TEVAR.

Conclusions:
Pre-operative false lumen volume distinguishes desired versus failed post-TEVAR outcomes. While our initial hypothesis was not proven, our results identify that pre-operative false lumen volume is a viable predictor of TEVAR success. The clinical implication of our finding is that patients with a large false lumen volume may not be ideal candidates for an endovascular approach to TBAD repair.
Abstract P104: The Transcription Factor Erg Governs Chromatin Accessibility And Gene Expression Programs Underlying Vascular Dysfunction In Aortic Endothelial Cells
Authors: Botts Steven R , Khyzha Nadiya , Kumaragurubaran Rathnakumar , Wu Ruilin , Raju Sneha , Prajapati Kamalben , Howe Kathryn L , Fish Jason E
Published: May 2022

Aims:
The transcription factor ERG has emerged as an important regulator of vascular function through its ability to repress inflammation in endothelial cells (ECs) and ERG dysregulation is associated with chronic inflammation in atherosclerosis and aortic aneurysms. To characterize the cellular mechanisms that underlie this regulation, we deleted
ERG
in human aortic ECs and assessed genome-wide chromatin accessibility and gene expression to identify dysregulated pathways. Furthermore, we have begun to investigate ERG expression in mouse atherosclerotic plaque.

Methods:
CRISPR/Cas9 was used to delete
ERG
exon 6 in immortalized human aortic ECs. Wildtype or Δ
ERG
ECs were profiled with ATAC-seq and RNA-seq (n=2-4). GREAT and clusterProfiler were used to identify enriched pathways (log
2
FC>|1|; FDR<0.05). Regulatory regions were defined as 5kb upstream and 1kb downstream of transcription start sites with ≤1Mb nearest gene extension. To determine if ERG expression is altered during atherosclerosis, 8-week-old
Ldlr
−/−
mice were fed a 12-week high-cholesterol diet (1.25%) before sacrifice. Ascending aortic sections were stained with anti-CD68 and anti-ERG (n=2). Images were obtained using a Leica SP8 confocal microscope.

Results:
Pathway analysis of >21,000 differentially accessible chromatin regions identified 67 enriched processes in Δ
ERG
ECs. Of these, the top 20% included disrupted wound healing, abnormal aorta morphology, and aneurysm, which were driven by association with proatherogenic and proaneurysmal genes including
PDGFRB
and
TGFBR1
. Gene expression analysis revealed that loss of ERG contributes to endothelial to mesenchymal transition (increased
KLF4
and
SNAI2
), inflammation (increased
IL18
), and dysregulated extracellular matrix and cell adhesion (increased
FBLN2
and
VCAN
). In the mouse aorta, preliminary data suggest that ERG expression may be elevated in EC nuclei overlying plaque compared to non-atherosclerotic regions.

Conclusions:
The loss of ERG has a broad impact on chromatin accessibility and gene expression in aortic ECs, with enrichment of vascular dysfunction pathways. Further investigation will establish how altered ERG expression contributes to
in vivo
models of atherosclerosis and aortic aneurysms.
Abstract P107: Celastrol Supplementation Profoundly Activates Aortic Mmp-9 And Abolishes Sexual Dimorphism Of Abdominal Aortic Aneurysm In Mice.
Authors: Javidan Aida , Jiang Weihua , Yang Lihua , Subramanian Venkateswaran
Published: May 2022

Background and Objective:
Abdominal Aortic Aneurysms (AAAs) are permanent dilations of the abdominal aorta with greater than 80% mortality after rupture. AAA prevalence is 4-5 times greater in males than females. AAA formation involves a complex process of destruction of aortic media through degradation of extracellular matrix proteins, elastin and collagen. Angiotensin II (AngII) infusion model of AAA in mice recapitulates major features of human AAA mainly male gender specificity. Celastrol, a pentacyclic triterpene from the root extracts of Thunder God Vine (
Tripterygium wilfordii
), strongly suppressed AngII-induced cardiovascular complications in mice. The purpose of this study is to test the effect of Celastrol supplementation on AngII-induced AAAs in mice.

Methods and Results:
Male and female LDL receptor -/- mice (8 weeks old; n= 12 per group) were fed a fat-enriched diet (21% wt/wt fat; 0.15% wt/wt cholesterol) supplemented with or without Celastrol (10mg/kg/day) for 5 weeks. After 1 week of diet feeding, mice were infused with AngII (500 or 1000 ng/kg/min) for 28 days by osmotic minipumps. Dietary supplementation of celastrol significantly promoted AngII-induced abdominal aortic luminal dilation (Con = 1.25 ± 0.05 versus Celas = 1.55 ± 0.09 mm, P<0.05) and external aortic width (Con= 1.01 ± 0.09 versus Celas = 1.62 ± 0.14 mm, P<0.05) in male mice as measured by ultra-sonography and ex vivo measurement, with 90% incidence (10/11) compared to 36% (4/11) in control group. Interestingly, celastrol supplementation to AAA female mice, resulted in a significant increase in AngII-induced aortic luminal dilation (Con = 1.17 ± 0.30 versus Celas = 1.57 ± 0.21 mm, P<0.001) and AAA formation (Con= 0.94 ± 0.03 versus Celas = 1.36 ± 0.25 mm, P<0.05) mice with 80% incidence (12/15) compared to 6% (1/15) in control group. Celastrol supplementation dramatically increased AngII-induced aortic leukocyte accumulation, MMP-9 activity and medial elastin degradation in both male and female mice compared to saline and AngII controls.

Conclusion:
These findings demonstrate that celastrol supplementation to LDLr -/- mice ablates sexual dimorphism and promotes AngII-induced AAA formation, which is associated with increased leukocytic infiltration and MMP-9 activation.
Abstract P108: Inflammatory Factors And Metalloproteinases Levels Are Elevated In Aorta Of Patients With Abdominal Aortic Aneurysm
Authors: Michelon Barbosa Jessyca , Corsi Carlos A , Mestriner Fabiola L , Mesquita Carolina D , Couto Ariel E , Campos Lígia C , Dugaich Vinícius F , Jordani Maria Cecília , Joviliano Edwaldo E , Évora Paulo Roberto B , Polonis Katarzyna , Ribeiro Maurício S , Becari Ribeiro Christiane
Published: May 2022

Introduction:
The pathophysiology of abdominal aortic aneurysm (AAA) remains poorly understood. The identification of the mechanisms involved in growth, progression, and the risk of aortic rupture could bring a novel treatment perspective and improve patient outcomes. Inflammatory mediators and metalloproteinases (MMPs) 2 and 9 have been shown to contribute to the pathophysiology of AAA in humans. It is widely recognized that these mediators produce both local (tissue) and systemic (circulation) responses. Therefore, in this study, we sought to characterize the inflammatory and MMP-2 and MMP-9 profiles in the aorta and plasma in patients with AAA.

Methodology:
Plasma and aorta tissue from AAA patients(n=31)were obtained during conventional AAA correction surgeries at Ribeirão Preto Medical School Hospital-University of São Paulo. The control samples were obtained from organ donors without AAA (n=15). The levels of inflammatory markers (IL-6,IL-8,TGFbeta) and MMPs were measured by radio immunoassay and zymography, respectively, in plasma and aorta.

Results:
Levels of IL-6 (p=0.001), IL-8 (p=0.01) and TGF beta (p=0.05) were significantly elevated in the aorta of AAA patients, but not in plasma, when compared to control group. Interestingly, levels of IL-6 (p=0.05) and IL-8 (p=0.02) were lower both in the aorta and plasma of controls. MMP-2 and MMP-9 levels were upregulated in plasma (p=0.02 and p=0.0007, respectively) and in aorta (p=0.017 and p=0.03, respectively) of AAA patients when compared to controls. The tissular levels of MMP-2 and MMP-9 were upregulated in the aorta in both AAA patients (p=0.001 and p=0.0001, respectively) and controls (p=0.008 and p=0.001, respectively) when compared to plasmatic levels of respective groups.

Conclusion:
Our data show that a profile of AAA patients is characterized by locally elevated inflammation factors and MMPs, and systemically elevated MMPs levels. This might suggest that the local response in aortais an important pathway in the pathophysiology of AAA development.
Abstract P109: Angiotensin 1-7 Attenuates Angiotensin II-induced Aortic Aneurysm In A Preclinical Murine Model
Authors: Jadli Anshul , Gomes Karina , Ballasy Noura , Patel Vaibhav B
Published: May 2022

Background:
Aortic aneurysm (AA) is a vascular disease that involves extracellular matrix degeneration of the aorta wall leading to dilatation and eventually aorta wall rupture. Aortic aneurysms contribute significantly to global morbidity and mortality due to vascular diseases. Elusive pathophysiology of initiation and progression of aortic aneurysm led to the absence of clinically relevant therapeutic interventions against aortic aneurysm. Though the role of AngII in the development and progression of AA is widely studied, the effect of Ang 1-7 on hallmarks of AA i.e. SMCs apoptosis, aorta matrix remodeling, and inflammation are poorly understood.

Methods and Results:
In the present study, we have investigated Ang 1-7-mediated protective effect on the AngII-infused murine model. AngII-infused ApoEKO murine model showed aortic dilatation in the thoracic and abdominal region with matrix remodeling. Infusion of Ang 1-7 rescued the phenotype evident by echocardiography. While histology staining showed excessive matrix remodeling i.e. collagen deposition in thoracic aorta and immunofluorescence showed VSMCs cell death in the abdominal aorta, suggesting different mechanisms underlying the development of TAA and AAA. The inflammatory markers, MMP2, MMP9, and TNF-α were significantly reduced in the thoracic aorta post-infusion of Ang 1-7. The Ang 1-7 treatment led to a reduced phenotypic switch in abdominal VSMCs evident by increased expression of ACTA2, MyH11, Calponin and reduced expressions of MMP2, MMP9, Collagen I, and collagen III. The apoptosis assay using flow cytometry showed attenuation of cell death of abdominal VSMCs after infusion of Ang 1-7 in a murine model of AA. The analysis of apoptosis-associated proteins, Caspase 3 and 8, showed reduced expression in the abdominal aorta.

Conclusion:
The study showed mitigation of AngII-mediated AA by Ang 1-7 infusion. Ang 1-7 treatment attenuated AngII-induced TAA by suppressing aortic matrix remodeling and AAA by inhibiting VSMCs apoptosis and vascular inflammation. Ang 1-7 can be a potential therapeutic alternative for the treatment of AA.
Abstract P113: Multi Omics Analysis Identified Fndc1 And Mxra5 As Novel Extracellular Matrix Proteins In Aortic Stenosis
Authors: Bouchareb Rihab , Guaque-Orlate Sandra , Snider Justin , Zaminski Devyn , Anyanwu Anelechi , Stelzer Paul , Lebeche Djamel
Published: May 2022

Calcified aortic valve disease (CAVD) affects over six million Americans and is associated with changes in valve leaflets' mechanical properties, resulting in impaired valvular blood flow. Currently, there is no viable pharmacological treatment to stop the disease's progression or activate mineral regression. The only effective therapy to treat CAVD is aortic valve replacement (AVR) or transcatherization (TAVR). Therefore, it is imperative to understand the molecular mechanisms leading to aortic valve mineralization to identify new pharmacological targets. In this work, we examined the expression of extracellular matrix proteins (ECM) during valve calcification.

Method:
We used both RNAseq (n=18) and Proteomics (n=15) on explanted human valves of two different human cohorts to study the modulation of ECM proteins. Proteins were isolated from the fibrotic and the calcific side of each valve tissue. We next confirm our data in isolated human valve cells. Cells were treated with calcifying medium for one week and secreted ECM proteins were analyzed at different time points: Baseline, 3 days, and 7 days.

Results:
our network analysis revealed an interaction between ECM, metabolic, complement, and lipids transporters proteins. We identified SPP1, VTN, THSB2, MATN2, FNDC1, and MXRA5 as the common modulated ECM protein in both RNAseq and proteomics data set.

Conclusion:
This study yields new insight into ECM proteins expression during the aortic valve calcification process, and identified two new ECM proteins, MXRA5 and FNDC1, in aortic stenosis. The disruption of ECM proteins was more significant in the calcified stage and it was accompanied by a down-regulation of many metabolic enzymes.
Abstract P114: Statins, High-density Lipoprotein, And Acute Kidney Injury After Endovascular Aortic Repair
Authors: Patrick Jordan T , Smith Derek K , Smith Loren E
Published: May 2022

Acute kidney injury (AKI) after endovascular aortic repair (EVAR) occurs in 20% of patients and is an independent predictor of death. Higher preoperative high-density lipoprotein (HDL) cholesterol concentration before revascularization of chronic limb ischemia is associated with a lower risk of AKI. Further, preoperative statin use has been shown to be associated with a decreased risk of AKI after EVAR. We hypothesized that both preoperative statin use and higher preoperative HDL are associated with a lower risk of AKI in patients undergoing EVAR.

Charts from adult patients that underwent EVAR were selected using the ICD codes (n=251). Each chart was reviewed for demographic and medical information, preoperative and postoperative serum creatinine concentrations, and preoperative HDL concentration. One hundred and three patients had available statin medication, serum creatinine, and HDL concentrations. The associations between statin dose, HDL concentration and maximum serum creatinine change from baseline in the first 48 postoperative hours was assessed using multivariable linear regression models, adjusted for AKI risk factors. Preoperative HDL concentration was not correlated with preoperative statin dose (Pearson’s R=0.0515, p=0.57). Higher preoperative statin doses (atorvastatin equivalents) and higher preoperative HDL concentrations were associated with lower serum creatinine rise (p=0.042 and 0.002 respectively, Figure). When both preoperative statin dose and preoperative HDL concentration were included in the model, both variables lost significance, suggesting HDL may mediate the relationship between preoperative statin dose and postoperative AKI.

Higher preoperative HDL concentrations and statin doses were associated with less postoperative AKI. Future work involves identifying the biological mechanism underlying these associations as a first step towards developing new therapies to prevent AKI following EVAR.
Abstract P115: Building A Structurally Sound System To Understand SR-BI Function
Authors: Powers Hayley R , Jenjak Shawn E , Volkman Brian F , Sahoo Daisy
Published: May 2022

The interaction of scavenger receptor BI (SR-BI) and high density lipoproteins (HDL) is the key step for the bodily removal of cholesterol. The HDL/SR-BI interaction is one of the concluding steps of the reverse cholesterol transport (RCT) pathway. In RCT, cholesterol within atherosclerotic plaques is taken up by HDL particles, which then dock on SR-BI at the surface of the liver, where cholesterol can be delivered converted into bile for excretion. Humans with mutations in the gene encoding SR-BI display elevated HDL-cholesterol levels and an increased risk of cardiovascular disease (CVD). Therefore, understanding the interaction between HDL and SR-BI is crucial to discovering ways to lower plasma cholesterol and modulate CVD risk. Due to the importance of SR-BI, the goal of these studies is to resolve a structure of functional full-length human SR-BI. We have expressed and purified full-length SR-BI using an insect cell Sf9 system. In order to assess SR-BI’s functions within this system, Sf9 cells were infected with baculovirus encoding empty vector or SR-BI, or left uninfected and plated into culture dishes. Expression of SR-BI significantly increased HDL cellular association and uptake of HDL-cholesteryl esters compared to empty vector and uninfected cells. Additionally, free cholesterol efflux was increased upon SR-BI expression. Lastly, SR-BI in Sf9 cells maintained its ability to form higher order oligomers in cells, which is crucial, as SR-BI oligomerization is important in cholesterol transport. Additionally, size exclusion chromatography shows that purified SR-BI is able to form oligomers in micelles. Lastly, using microscale thermophoresis, we demonstrated that purified SR-BI binds to its ligands (apolipoprotein A-I, holoparticle HDL, and oxidized LDL) with high affinity, regardless of the glycosylation state of SR-BI. Together, these assays represent the first steps in resolving a high-resolution structure of human full-length SR-BI and provide promise for delineating the ways in which the HDL/SR-BI relationship allows for efficient cholesterol clearance.
Abstract P120: Heparin-warfarin Therapy Could Be An Alternative Treatment For Diabetes Mellitus In Addition To Being A Standard Therapy For Pulmonary Vein Thrombi.
Authors: Takeuchi Hidekazu
Published: May 2022

Background:
Our previous study showed that warfarin or direct oral anticoagulants (DOACs) dissolved pulmonary vein thrombi (PVTs). However, in some instances, warfarin or DOACs could not dissolve PVT. It is unclear whether heparin-warfarin therapy affects PVT and diabetes mellitus (DM).

Methods and Results:
Our patient was a 74 year-old male. The patient had cerebral infarction one year prior; at that time, he was not treated with any medications. The patient was examined, and it was found that he had diabetes mellitus, hypertension and dyslipidemia. His HbA1c was 9.5%. The patient was treated with medications including ipraglifilozin L-proline (50 mg), sitagliptin (50 mg), repaglinide (0.25 mg) and clopidogrel (75 mg). One year after discharge, we detected PVTs in the patient’s left lower pulmonary vein (LLPV) via cardiac CT and transesophageal echocardiography (TEE). He was treated with standard doses of heparin and warfarin after stopping clopidogrel. The patient’s HbA1c was 6.2% at admission. After one month of treatment with heparin-warfarin, the PVTs were almost dissolved. The patient showed a tendency of hypoglycemia the day after admission, so the three glucose-lowering medications were discontinued within nine days. One month later, HbA1c was 6.1% without DM medications. After discharge, warfarin was continued, and blood glucose levels gradually increased. Three months later, HbA1c was 7.3%, and 4 months later, HbA1c was 7.8%, so DM medications were restarted. PVTs can release microclots, which can occlude the microvessels of all organs, leading to cell dysfunction. Heparin-warfarin can decrease microclots and reopen microvessels. Thus, blood glucose could be taken into cells, decreasing blood glucose levels. There is a possibility that DM could be prevented if we treat PVTs in their early stage. Compared to other similar cases, this patient showed hypoglycemia rather early, which might be caused by the interaction between repaglinide and clopidogrel.

Conclusion:
One month of heparin-warfarin therapy almost completely dissolved PVTs. During that course, DM medications were stopped in order to prevent hypoglycemia. During the next four months of warfarin therapy, the blood glucose levels gradually increased without DM medications.
Abstract P123: Effects Of Lipid Control On Coronary Calcification
Authors: Alam Mustafa , Savinova Olga V
Published: May 2022

Introduction:
Lipid lowering drugs are some of the most prescribed medications to control coronary artery disease (CAD). The long-term lipid lowering effects on coronary artery calcium (CAC) progression remain a subject of controversy with inconclusive results from multiple literature reviews. The purpose of this meta-analysis is to elucidate the relationship between lipid control and CAC progression.

Methods:
A computerized search of MEDLINE was conducted using the search terms “coronary”, “calcification”, “statin”, and “lipid lowering.” Search filters included studies in English performed on human subjects, clinical trials, and journal articles. Studies were included if they measured mean value changes from baseline in coronary calcium using cardiac computed tomography. Data were extracted from the articles according to the Cochrane manual. RevMan 5.4 was used to aggregate the studies and report statistics.

Results:
A total of 13 studies were included with a total of 6671 patients. LDL reduction amongst the studies was significant as compared to placebo or lower dose lipid lowering therapy (standardized mean difference [SMD]: -0.71; 95% CI: -0.79, -0.64; p<0.00001). Study heterogeneity was not significant amongst the CAC progression studies (p=0.19). An increase in CAC was statistically significant (standardized mean difference [SMD]: 0.10; 95% CI: 0.02, 0.17; p=0.009).

Conclusion:
Lipid lowering therapy shows efficacy in LDL reduction however, it does not lead to coronary calcium reduction. Further studies are needed to elucidate the long-term effects of lipid powering on CAC and the prognostic value of CAC in CAD parents receiving optimal therapy.
Abstract P124: Post Acute Infection Of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads To Systemic Inflammation And Podocyte Injury.
Authors: Nandula Seshagiri , Sen Sabyasachi , Brichacek Beda , Janapala Rajesh Naidu
Published: May 2022

Introduction:
COVID-19 attributed to SARS-CoV2 infection is a world-wide pandemic. SARS-CoV2 has been associated with cardiovascular disease and diabetes. At a cellular level, the infection causes endothelial cell dysfunction (ECD), which is manifested by entities such as microvascular thrombosis. ECD is quite common in type 2 diabetes mellitus (T2DM) where the renal podocyte dysfunction is often an early manifestation of microvascular complication. In this study we explored whether presence of hyperglycemia predisposes to increased SARS-CoV2 infection and whether co-morbid presence of COVID-19 and hyperglycemia predisposes to cardio-metabolic complications such as diabetic kidney disease (DKD). To estimate kidney damage we evaluated albuminuria and podocyte markers in urine exosomes from SARS-CoV2 patients at 10 days, 6 months and 12 months post COVID-19 infection.

Methods:
Blood and Urine samples from SARS-CoV2 patients post acute phase of infection were procured from the core facility. Peripheral blood mononuclear cells (PBMNCs) and urine exosomes were isolated and podocyte protein markers such as Nephrin (Nep), Podocalyxin (PODXL) and Wilm’s Tumour-1 (WT-1) were identified by western blot analysis.

Results:
Our results showed that human kidney cells on exposure to hyperglycemia (5 vs 25mM) upregulates expression of TMPRSS2 gene, a key receptor for SARS-CoV2, by 5 fold. Next we examined blood and urine samples from COVID-19, subjects (n=16). As all examined subjects had blood glucose levels above 400mg%, we used samples from T2DM subjects with no COVID-19 infection, as a comparator. COVID-MNCs showed persistent over-expression of IL-6 and TNFa (4-fold) even at 12 months post infection. Urine-exosomal-Nephrin protein band expression was 10-fold higher than T2DM-No-COVID urine-exosomal-Nephrin protein band Similar trend was noted on estimating PODXL and WT-1.

Conclusions:
A persistent inflammatory marker over-expression at 12 months in COVID samples indicate possible long standing renal damage. Increased podocyte specific protein loss at 12 months compared to samples from T2DM subjects with similar GFR also indicates persistent kidney damage which may lead to renal failure and hypertension.
Abstract P126: Pro-Inflammatory Macrophage Secretome Promotes Cardiac Fibroblast Activation
Authors: Kremastiotis Georgios , Ascione Raimondo , Johnson Jason L , George Sarah J
Published: May 2022

Introduction:
Following myocardial infarction, an acute immune response occurs, and infiltrating monocytes and monocyte-derived macrophages migrate into the infarct. Under the influence of intricate pro- and anti-inflammatory stimuli, macrophages drive cardiac fibroblast (CF) activation. The interaction between macrophages and CFs regulates fibrosis-driven wound healing.

Hypothesis:
We hypothesised that pro- and anti-inflammatory macrophages divergently effect CF activation. We aimed to identify macrophage proteins that regulate CFs as potential intervention targets.

Methods:
Monocytes from healthy volunteers were differentiated towards pro- or anti-inflammatory macrophages using GM-CSF (20 ng/mL) and M-CSF (20 ng/mL) or M-CSF (40 ng/mL) alone, respectively. CF activation was characterised by α-SMA expression, contraction, and migration. Proteomics were performed on macrophage secretome and subjected to bioinformatics (IPA, Qiagen). CFs were cultured with macrophage-conditioned medium (CM) in the presence of recombinant proteins or neutralising antibodies, as indicated.

Results:
Pro-inflammatory macrophage CM enhanced CF activation, as evidenced by elevated α-SMA expression (1.6-fold change, p<0.05, n=7), contraction (1.3-fold change, p<0.05, n=4), and migration (1.2-fold change, p<0.05, n=4) in comparison to anti-inflammatory macrophage CM. Bioinformatics and validative Western blotting revealed that CTSZ and CXCL10 were significantly differentially expressed, and they were upregulated in the pro- and anti-inflammatory macrophage secretome, respectively. Neutralisation of CTSZ or addition of recombinant CXCL10 to pro-inflammatory macrophage CM reduced CF α-SMA expression, supporting a causal role for CTSZ and CXCL10 in CF activation. Finally, CTSZ cleaved CXCL10
in vitro
(33% reduction in 2 hours, 37
o
C), which, subsequently, abrogated the suppression of α-SMA expression by CXCL10 in CFs.

Conclusions:
Our findings indicate that macrophage phenotype exerts divergent effects on CF behaviour, with pro-inflammatory macrophages promoting CF activation, through enhanced CTSZ secretion and associated CXCL10 cleavage, revealing both proteins as potential targets for modulating fibrosis-driven wound healing.
Abstract P127: Myeloid-specific PKM2 Deletion Reduces Atherosclerosis By Limiting Inflammation
Authors: Doddapattar Prakash , Dev Rishabh , Ghatge Madankumar , Jain Manish , Dhanesha Nirav , Lentz Steven R , Chauhan Anil K
Published: May 2022

Introduction:
The underlying cause of coronary artery disease (CAD) is atherosclerosis, which is a pathological response to chronic inflammation and hyperlipidemia. The onset of atherogenesis is characterized by infiltration of myeloid cells, including monocytes followed by fatty streak formation and progressive accumulation of smooth muscle cells (SMCs). These microenvironmental changes dictate the balance between inflammatory and anti-inflammatory macrophages. Pyruvate kinase M2 (PKM2), a glycolytic enzyme, is highly expressed in activated proinflammatory macrophages. The mechanistic role of PKM2 in atherosclerosis remains unknown.

Hypothesis:
We hypothesize that PKM2 promotes macrophage migration in response to MCP-1 and mediates atherosclerosis by increasing inflammation.

Methods and Results:
PKM2 was upregulated in macrophages of Ldlr
-/-
mice fed a high-fat "Western" diet compared with a control chow diet. We generated the novel myeloid cell-specific PKM2
fl/fl
LysMCre
+/-
on a Ldlr-deficient background (PKM2
fl/fl
LysMCre
+/-
Ldlr
-/-
) and evaluated atherosclerosis after 14 weeks high-fat “Western” diet feeding. Controls were littermate PKM2
fl/fl
LysMCre
-/-
Ldlr
-/-
mice. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. (P<0.05, n=10-12 mice/group). Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2
-/-
mice compared with control mice that was associated with decreased plasma levels of pro-inflammatory cytokines, including MCP-1, and reduced transmigration of macrophages in response to MCP-1. Macrophages isolated from myeloid-specific PKM2
-/-
mice fed a high-fat "Western" diet exhibited reduced expression of pro-inflammatory genes, including MCP-1, IL-1β, IL-12, and increased expression of the anti-inflammatory genes Arg1 and IL-10. Inhibiting PKM2 nuclear translocation in bone marrow-derived macrophages led to a significant reduction in MCP-1 and IL-1β levels and reduced transmigration of macrophages.

Conclusion:
Genetic deletion of PKM2 in myeloid cells reduces atherosclerosis by limiting inflammation.
Abstract P128: Reducing Inflammation In Coronary Atherosclerosis: A Renewed Look At Colchicine
Authors: Alam Mustafa , Savinova Olga V , Jhamnani Sunny
Published: May 2022

Introduction:
Atherosclerosis is an inflammatory disease. Randomized controlled trials (RCT) have demonstrated that targeting inflammation is important for the secondary prevention of major adverse cardiovascular events (MACE) in patients with acute coronary syndrome (ACS) and chronic coronary artery disease (CAD). Out of these trials, low-dose colchicine has emerged as an inexpensive therapy for coronary atherosclerosis. This meta-analysis focuses to understand colchicine’s anti-inflammatory efficacy via reduction in high sensitivity C-Reactive Protein (hs-CRP) in patients with ACS and CAD.

Methods:
A computerized search of MEDLINE was conducted to retrieve journal articles reporting the results in human studies performed from January 1, 2005 to January 1, 2021 using the keywords: “Colchicine” AND “Coronary” OR “CRP” OR “Coronary Artery Disease.” Studies were included if they measured hs-CRP changes from baseline and colchicine or placebo were given to patients with ACS or CAD. Studies were graded on quality via the Jadad scoring criteria.

Results:
A total of nine studies with a total population of 1502 patients and mean Jadad score of 4 were included in the meta-analysis. Five studies were RCT. The mean/median age ranged from 57 to 67 years while the duration of therapy ranged from one day to one year. Overall, colchicine treatment reduced hs-CRP levels compared with placebo (standardized mean difference [SMD]: -0.45; 95% CI: -0.68, -0.22; p=0.0001).

Conclusion:
Colchicine reduces hs-CRP in patients with ACS and CAD. This finding supports the role of colchicine in the reduction of inflammation associated with ACS or CAD. Further randomized control trials are needed to quantify long-term inflammatory control and subsequent MACE reduction.
Abstract P129: Ablation Of TNAP (tissue-nonspecific Alkaline Phosphatase) In Macrophages Does Not Affect Atherosclerotic Plaque Calcification Or Cardiovascular Physiology
Authors: Shamsian Ethan , Than Sandy , Canellos Maria , Singh Mohnish , Savinova Olga V
Published: May 2022

Introduction:
Increased expression of TNAP (tissue-nonspecific alkaline phosphatase) in macrophages exacerbates atherosclerotic calcification in a mouse model. However, whether or not TNAP is necessary in the progression of calcification, or for proper cardiac function has yet been studied.

Objective:
The purpose of this study was to test the effect of TNAP ablation in macrophages on atherosclerotic plaque calcification and cardiovascular physiology, and compare it to a wild type situation.

Methods:
Macrophage-specific WHC-mTNAP knockout (KO) mice were produced in our colony by intercrossing floxed
alpl
transgenic mice (
alpl
gene encodes TNAP) and macrophage-specific CRE recombinase mice, in which CRE was expressed under the control of the lysozyme gene promoter. The macrophage TNAP KO strain was developed on the background of homozygous WHC (“wicked high cholesterol”) mutation in the low density lipoprotein receptor gene. WHC-TNAP KO (n=10) and their WHC littermates (n=9) were placed on an atherosclerosis inducing diet at 8 weeks of age. The mice were maintained on the diet for 44 weeks, or until they turned 52 weeks. Microcomputed tomography (microCT) analyses of calcification in the aortic roots and arches were performed
ex vivo
. Echocardiographic studies were performed to assess cardiac structure and function at 52 weeks of age. Data were analyzed via a 2-way ANOVA to calculate the effect of TNAP ablation on calcification and cardiovascular physiology while adjusting for sex.

Results:
CT data showed no significant difference in calcification levels between the two genotypes. Cardiac parameters such as left ventricular (LV) mass, LV diameter and wall thickness, ejection fraction, fractional shortening and cardiac output also showed no signifgant difference between the two genotypes.

Conclusion:
Our data suggest that, despite TNAP expression in macrophages being sufficient to induce calcification of atherosclerotic plaques, it is not necessary in the calcification process. To the extent of our study, the ablation of TNAP in macrophages does not appear to have any consequences in a mouse model of atherosclerosis.
Abstract P130: Inflammation-Mediated Modification Of Extracellular Matrix Generates A New Adhesive Substrate For Integrin α
D
β
2
-Dependent Macrophage Retention During Chronic Inflammation
Authors: Forgey Cady , Bailey William , Casteel Jared , Scarbrough Haley , Podrez Eugene A , Byzova Tatiana V , Yakubenko Valentin P
Published: May 2022

The accumulation of pro-inflammatory macrophages in the inflamed vascular wall is a critical step in atherogenesis. The mechanism of macrophage retention within the site of inflammation is not understood yet. High adhesion that prevents macrophage migration is one of the potential mechanisms. We previously showed that integrin α
D
β
2
is upregulated on pro-inflammatory macrophages, promotes macrophage retention, and contributes to atherogenesis. However, we have not identified a key ligand for α
D
β
2
within the tissue, since α
D
β
2
does not interact with major ECM proteins, collagens, and laminins. We recently found that during acute inflammation, the oxidation of docosahexaenoic acid (DHA) leads to the generation of end product carboxyethylpyrrole (CEP), which forms an adduct with fibrinogen and albumin via ε-amino group of lysines. Moreover, we revealed that macrophages adhered to CEP-modified albumin in α
D
β
2
-dependent manner. Now we are testing a hypothesis that DHA oxidation is a universal mechanism during chronic inflammatory diseases that promotes the generation of CEP adducts with different ECM proteins and forms α
D
β
2
-mediated strong anchorage of macrophages, which is critical for macrophage retention during chronic inflammation. We detected CEP-modified proteins in digested atherosclerotic lesions by western blot. In vitro DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin but not with collagen I. Using α
D
β
2
-transfected HEK293 cells, WT and α
D
-/-
mouse macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading to compare with natural macrophage ligands. Using site-directed mutagenesis, we generated mutant α
D
I-domains and α
D
β
2
-transfected cells with single amino acid substitutions. Applying protein-protein binding and adhesion assays we detected one amino acid within integrin α
D
, K
246
, which is critical for α
D
β
2
binding to CEP-modified proteins. In summary, we propose a new mechanism of macrophage retention, which is based on inflammatory modifications of ECM with DHA end-product, CEP. The identification of a binding site for CEP-modified proteins within α
D
β
2
will help to develop a blocking reagent for the treatment of the inflammatory component of atherosclerosis.
Abstract P132: Heterogeneously Expressed Bcl6 Controls Endothelial Inflammatory Response
Authors: Valenzuela Nicole M
Published: May 2022

We recently reported that
BCL6
, a DNA binding protein important in germinal center responses and lymphoncogenesis, was heterogeneously expressed across human endothelial cells (EC). Its expression correlated with more intense inflammatory responses. Here, we asked what role BCL6 might play in endothelial inflammation. We examined public expression and epigenomic data and tested BCL6 function
in vitro
. BCL6 was significantly enriched in cardiac microvascular and endocardial EC, associated with cardiovascular disease in GWAS studies, and was elevated in rejecting cardiac transplant biopsies. The chromatin landscape around BCL6 reflected that of a superenhancer in EC, with high H3K27ac and H3K4me3 signal indicative of active/poised transcription. EC treated with TNFα, IFNγ or IL-1β upregulated BCL6 within 2-3hr, maintained through 24hr. The BCL6 gene had putative NFκB/REL transcription factor binding sequences, and p65/RelA binding to the BCL6 gene increased more than 8-fold under TNFα or IL-1β stimulation. BCL6 DNA binding sites were found in EC adhesion molecule and chemokine genes, including
VCAM1
,
CXCL2
,
CX3CL1
,
SELE
,
CCL5
; as well as NFKB genes themselves. Perturbation of BCL6 complexes with inhibitors (FX1, BI-3812) led to dramatic changes in the TNFα-induced transcriptome and at the protein level in endothelial cells. For example, FX1 and BI-3812 significantly blocked TNFα-induction of
VCAM1
and
CXCL2
/GROβ, but upregulated
CCL5
/RANTES and
CXCL10
/IP-10. Interestingly, TNFα-induced expression of miR155, a pro-inflammatory microRNA important in atherosclerosis, was also suppressed. We hypothesize that BCL6 is a novel NFκB-inducible regulator of vascular inflammation. Surprisingly, BCL6 corepressor antagonism both enhanced and suppressed TNFα mediated EC activation. Therefore BCL6 performs transcriptional modification that is context and cell-type dependent. Its role in vascular inflammation merits further investigation.
Abstract P136: First Steps In Uncovering Mechanisms In Late Onset Preeclampsia
Authors: Griffin Jakara , Lindemer Brian , Krolikowski John G , Lohr Nicole L , Weihrauch Dorothee
Published: May 2022

Preeclampsia is a leading cause of maternal morbidity and mortality worldwide, complicating 2-8% of pregnancies. The etiology of PE is poorly understood, the role of placental extracellular matrix (pECM) in PE is understudied. Extravillous trophoblasts (HTR8/svneo) and pECM are crucial in placental blood vessel formation, including spiral arteries. The objective is to identify changes in signaling mechanisms in late onset PE and how HTR8/svneo respond. Additionally, we explored a novel therapeutic to restore HTR8/svneo phenotype by phototherapy using a 670nm LED light. Western blot analysis shows eNOS, phospho-eNOS and Hsp90 expressions are unchanged in late onset PE placental samples despite previous reports in early onset of PE. The association of Hsp90 to eNOS and eNOS to phospho-eNOS is reduced in the PE sample. Soluble Flt, a diagnostic marker for PE, levels are increased in media from HTR8/svneo trophoblasts cultured on PE pECM. Endothelial function is significantly reduced in an isolated vessel as determined by acetylcholine stimulation. 670nm light reverses this impairment. Migration of HTR8/SVneo on PE pECM is reduced (control 86.29±18.58, PE 30.16±15.49) but proliferation of HTR8/SVneo on PE pECM is unaffected in patients (control: 59.125±6262.9, PE:67.125±7706.5). Proliferation and migration are improved by 670nm light (proliferation control 101.375±2075.4; PE 106.875±4870.7, migration control 125±11.97, PE 144.84±16.01). An increase of apoptotic HTR8/SVneo cultured on PE pECM is observed compared to healthy pECM in patients control 2.4±0.24, PE 3±0.4 samples. 670nm light treatment decreases the number of apoptotic cells, control 1.2 ±0.58, PE 1.4±0.5.

Conclusion:
In late onset preeclampsia, endothelial function is decreased, sFlt release is increased, the eNOS/Hsp90/p-eNOS axis is slightly reduced pointing to O
2
-
synthesis. Migration is decreased likely due to impaired trophoblast invasion, but proliferation is unaffected. 670nm light treatment can reverse some of the effects of the preeclamptic microenvironment. Extracellular matrix is altered in late onset PE leading to an abnormal HTR8/SVneo phenotype. 670nm light restores the phenotype of HTR8/SVneo on PE pECM suggesting 670nm light as a therapeutic treatment.
Abstract P137: Hypercholesterolemia Aggravates In-stent Restenosis In Rabbits By Escalating Vascular Inflammation
Authors: Inamdar Vaishali , Zviman Menekhem , Bratinov George , Fitzpatrick Emmett o , Gardiner Kristin , Alferiev Ivan S , Levy Robert J , Stachelek Stanley J , Fishbein Ilia
Published: May 2022

Background:
Hypercholesterolemia (HC) has previously been shown to augment restenotic response in several animal models. However, the mechanistic aspects of in-stent restenosis (ISR) on an HC background are not fully understood.

Methods:
HC was induced in 5 NZW rabbits by hypercholesterolemic diet (HCD) fed for 4 weeks prior to bilateral implantation of stainless steel stents in the iliac location. The diet was continued until sacrifice. In parallel, stents were deployed in the iliac arteries of 5 normocholesterolemic (NC) rabbits. All animals were euthanized 4 weeks after stenting. Harvested arteries were formalin-fixed. The stent struts were dissolved in a mixture of nitric and hydrofluoric acids. The destented arteries were paraffin-embedded, sectioned, stained according to the Verhoeff-vanGieson method, and the lumen area, neointimal thickness, neointimal area, neointima-to-media ratio, and percent of luminal stenosis were determined morphometrically. A semiquantitative scale was used to assess the intensity and spread of TNFa expression by immunohistochemistry (IHC). The prevalence of peri-strut macrophages (MΦ) was determined by IHC as a percentage of a strut circumference infiltrated with MΦ.

Results:
HC diet drastically increased severity of ISR (Fig). The corresponding values of the lumen area, neointimal thickness, neointimal area, neointima-to-media ratio and percent of luminal stenosis for the groups of NC and HC animals were 1.86±0.44 vs 1.48±0.54 (p<0.05), 0.06±0.02 vs 0.31±0.14 (p<0.0001), 0.66±0.16 vs 2.07±0.56 (p<0.0001), 1.99±0.11 vs 4.57±0.76 (p<0.0001), and 26.35±4.78 vs 57.89±13.71 (p<0.0001). Compared to NC animals, TNFa immunopositivity and MΦ infiltration of peri-strut areas increased in HC group animals 1.81- and 2.58-fold, respectively (p<0.001 for both).

Conclusions:
The inflammatory response to stent deployment is intensified in HC metabolic conditions, leading to the augmented neointimal expansion and ISR.