bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 392: C-C Chemokine Receptor Type 5 Deficiency Exacerbates The Severity Of Vasculitis In A Mouse Model Of Kawasaki Disease
Authors: Singh Shubhnita , Galley Joseph C , Bruder Nascimento Ariane , Bruder-Nascimento Thiago B
Published: November 2022

Kawasaki disease (KD) is an acute vasculitis and a common cause of acquired heart disease in children. C-C chemokine receptor type 5 (CCR5) is a receptor involved in the resolution of inflammation by eliminating chemical mediators and recruiting anti-inflammatory cells into the inflammatory site. Whether CCR5 plays a vascular protective effect in KD, it is still to be clarified. We tested the hypothesis that CCR5 reduces the vasculitis by decreasing the chemokine and cytokine vascular signaling. KD was induced by the
Candida albicans
water-soluble fraction model (CAWS, 4mg per injection/day for 5 days) in male control (CCR5+/+) and CCR5 deficient (CCR5-/-) mice. Mice were divided in 3 CAWS treatment groups: 1 single CAWS injection(G1) to identify the chemokines and cytokines levels, 2-days post CAWS treatment (G7) to understand the acute effects of KD, and 28-days post CAWS treatment (G28) to study the chronic effects of KD. Heart and abdominal aorta (AA) were harvested for vascular characterization. We found 4 to 10-times fold increase in C-C Motif Chemokine Ligand (CCL) 1, 3, and 5 (CCR5 ligands) and CCR5 gene expression in hearts and AA from CCR5+/+ in G1. Circulating IL1β and CCL5 levels were elevated in CCR5+/+ G1, which was worsened in CCR5-/-, this pattern persisted in G7 and G28, but in less intensity. No sign of vasculitis in coronary arteries and aortic root (scored by immune cells infiltration) or abdominal aorta aneurysm (AAA) were detected in G7 in CCR5+/+ and CCR5-/-, but a vascular dysfunction, reduced relaxation to acetylcholine and increased contractility to thromboxane in AA, was found, such changes were pronounced in CCR5-/-[Maximal response (mN): CCR5+/+: 4.65 ± 0.2; CCR5+/+ G7: 6.795 ± 0.3*; CCR5-/- G7: 9.750 ± 0.7
#
, *P<0.05 vs CCR5+/+;
#
P<0.05 vs CCR5+/+ G1]. Vasculitis in coronary arteries, aortic root, AAA formation, and a drastic loss of vascular contractility in AA were observed in CCR5+/+ and CCR5-/- G28 but was more exacerbated in later.Initial data show that CCR5 plays a protective role in the genesis of vasculitis in a mouse model of KD by regulating the chemokines and cytokines availability and signaling. For future directions, we hope to identify the vascular signaling activated by this intensified availability of chemokines and cytokines.
Abstract 401: Epigenetic Regulation At
LRP1
Cardiovascular Disease Risk Locus And Assessment Of Cellular Function In Human Ipscs-derived Smooth Muscle Cells
Authors: LIU LU , JOUVE Charlène , Hulot Jean-Sébastien , Georges Adrien , Bouatia-Naji Nabila
Published: November 2022

Background/Introduction:
Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of myocardial infarction in young and middle-aged women. A common genetic variant, rs11172113, located in
LRP1
(
low density lipoprotein receptor-related protein 1)
first intron, was associated with several vascular diseases, including SCAD, coronary artery disease and migraine. However, the biological mechanisms through which rs11172113 influence
LRP1
function in the context of arterial lesions is not fully understood.

Methods:
We applied luciferase reporter assay to measure the different enhancer activities between 2 alleles of rs11172113 in rat SMCs and human iPSC-derived SMCs. We edited iPSCs prior to differentiation using CRISPR-Cas9 to generate 100 bp deletion of the enhancer region containing rs11172113. We also created frame-shift indels in exons 2 or 5 of
LRP1
in iPSCs to generate SMCs knockouts and differentiated into SMCs. We then performed a proteomic and transcriptomic characterization of
LRP1
knockout effect in these iPSC-derived SMCs.

Results:
Reporter assay confirmed that rs11172113 belongs to a genomic region showing enhancer activity of 2 alleles
in vitro
. The transcription factor MECP2 increased the enhancer capacity in both alleles. iPSCs with homozygous deletion of rs11172113 enhancer region presented the same pluripotency compared with wild type. We found that the deletion of rs11172113 enhancer region decreased the expression of
LRP1
. LRP1 knockout decreased capacity of cell proliferation and migration in iPSC derived SMCs. Transcriptomic analysis in iPSC derived SMCs suggests
LRP1
knockout decreased the expression of genes involved in vascular smooth muscle cells microenvironment, such as
decorin
(
DCN
), and contraction, such as
ryanodine receptor 2
(
RYR2
). We are currently evaluating the effect of
LRP1
on SMC contraction and extracellular matrix synthesis.

Conclusions:
We confirmed rs11172113 to regulate
LRP1
expression in iPSCs derived synthetic and contractile SMCs. Our results support
LRP1
effect on SMCs cellular function alteration as a potential mechanism in genetic susceptibility for vascular disease.
Abstract 404: Basement Membrane Of Small Diameter Vascular Extracellular Matrix Scaffolds Modulate Human Endothelial Cell Quiescence
Authors: Shortreed Nicholas A , Lopera-Higuita Manuela , Dasari Surendra , Griffiths Leigh G
Published: November 2022

Chronic vascular diseases affect over 25 million patients in the U.S, alone. While non-invasive therapies are available, approximately 4.5 million individuals are estimated to require a vascular bypass annually, worldwide. Autologous vascular grafts remain the standard of care; yet the absence of a suitable donor vessel results in approximately one third of patients being ineligible for autologous grafting. “Off-the-shelf” alternatives have been proposed, but have had limited pre-clinical success, primarily due to graft failure via thrombosis. Thrombotic failure risk has been shown to be dramatically reduced by the formation of a luminal quiescent endothelial cell monolayer. Recently, our group engineered an unfixed, antigen-removed (AR) extracellular matrix (ECM) scaffold from bovine saphenous vein (SV) which is minimally immunogenic and avoids
in vivo
thrombosis. However, the mechanism by which AR-ECM SV scaffolds prevent thrombosis remains unknown. In this study, we utilized cell culture, immunofluorescence, and RNA-seq to assess the hypothesis that hAECs seeded on the basement membrane (BM) surface of AR-ECM SV scaffolds adopt a quiescent phenotype. Cells seeded on the BM surface proliferated and underwent significantly greater XY migration than those seeded on the non-BM surface. Additionally, unlike non-BM seeded cells, BM seeding resulted in quiescent hAEC phenotype (i.e., apical polarization of podocalyxin and adherence junction protein (i.e., cadherin and β-catenin) colocalization). Finally, the transcriptional phenotype of hAECs seeded on the BM surface was similar to cells treated with simvastatin (i.e. “quiescent”) and significantly different from those treated with TNFα (i.e. “activated”) (n=6/group), as assessed by gene set enrichment and t-SNE analysis. We conclude that seeding hAECs on the BM surface of AR-SV ECM scaffolds induces a favorable “quiescent” phenotype, while an unfavorable “activated” phenotype is avoided. Ultimately, these results show that the BM surface of AR-SV-ECM scaffolds possesses inherent characteristics which promote quiescent hAEC behavior and resultant vascular homeostasis and will help inform future pre-clinical studies of this novel small diameter vascular grafting biomaterial.
Abstract 411: The Development Of New Therapies Targeting Pneumonia-Accelerated Atherosclerosis
Authors: Stotts Cameron , Rayner Katey J
Published: November 2022

Background:
Pneumonia is inflammation of the lungs caused by a bacterial or viral infection. Recent studies show an association between pneumonia and an increased risk of a heart attack. It is proposed that inflammatory pathways are left uncontrolled, which exacerbates atherosclerotic vascular disease. As a result, there is a need to develop therapeutics for attenuating pneumonia-associated inflammation to reduce atherosclerotic plaque build-up and improve concomitant cardiac complications.

A normal course of inflammation begins with an initial pro-inflammatory phase and ends with a resolution phase, which is marked by tissue return to homeostasis. During pneumonia, resolution pathways are impaired. Returning resolution processes to normal function may prove to be an effective strategy to attenuate prolonged pneumonia-associated inflammation. Resolution pathways are mediated by fatty acid derivatives termed specialized pro-resolving lipid mediators (SPMs), the metabolites of poly-unsaturated fatty acids such as eicosapentaenoic acid (EPA). EPA is used locally by cells and readily degraded in the body. To overcome this barrier, EPA can be encapsulated in nanoparticle carriers to increase bioavailability and therapeutic delivery. We hypothesize that delivery of EPA nanoparticles into the lungs may control the persistent inflammatory response following pneumonia and reduce atherosclerotic burden.

Results:
In vitro
in mouse bone-marrow derived macrophages stimulated with gram-negative or gram-positive toxins, EPA nanoparticles reduced the production of pro-inflammatory markers including IL-6 and TNFa measured using western blot, qPCR and ELISA. To test the efficacy of EPA-nanoparticles
in vivo
, we have developed a mouse model of atherosclerosis combined with
S. pneumoniae
lung infection. We are currently evaluating the impact of infection with
S.pneumoniae
on atherosclerosis progression and will determine whether EPA-nanoparticles can be used to prevent atherosclerotic inflammation and plaque progression.

Clinical Impact:
The nanoparticles used are already FDA approved, and regular consumption of EPA has well supported benefits. As such, it is possible that this project may translate into a clinical setting more readily.
Abstract 412: Transcriptomic Analysis Reveals That Lipid Loading Of Distinct Intimal Myeloid Cell Subpopulations Precedes Inflammation In Early Atherogenesis
Authors: Scipione Corey A , Hyduk Sharon J , Polenz Chanele K , Althagafi Marwan G , Ibrahim Hisham M , MacParland Sonya A , Cybulsky Myron I
Published: November 2022

Hypercholesterolemia (HC) is a key risk factor of atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. Dietary HC in

Ldlr
-/-

mice induces intimal myeloid cells to develop into foam cells, constituting nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intima during the earliest stages of atherogenesis, and correlate them to cellular responses. Using
en face
enzymatic digestion and intimal cell microisolation, transcriptomic analysis was performed on the LC. Intimal cells were isolated from the LC of the ascending aortic arch of
Ldlr
-/-
mice at 0, 5, and 14 days of CRD feeding. Bulk RNA-seq and RT-qPCR found that genes associated with lipid-loaded macrophages (e.g.,
Abcg1, Lgals3
) were progressively elevated at 5 and 14 days of CRD. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not detected at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of
Ldlr
-/-
mice revealed multiple sub-populations of myeloid, endothelial, and other cell types. A shift in the
C1qb
+
,
Mmp12
+
myeloid subpopulations was observed as the duration of cholesterol feeding increased - with expansion of a macrophage population rich in lipid-responsive transcripts. This finding is consistent with an increase in macrophage number and the presence of Nile Red
+
CD68
+
foam cells observed by immunoconfocal microscopy at 5 days of CRD. However, a robust inflammatory signature was not detected in this macrophage cluster. Interestingly, an inflammatory signature similar to that reported in early atherogenesis was identified in a distinct
Ccr2
+
myeloid population with no signature of lipid loading. This suggests that the inflammation associated with atherogenesis may be a result of intercellular crosstalk between distinct myeloid subpopulations within the intima and may not originate directly from lipid loaded macrophages.
Abstract 413: Targeting Fundamental Mechanisms Of Aging To Attenuate Molecular Drivers Of Vascular Calcification
Authors: Antabi Mohamad Ali , Hagler Michael A , Zhang Heyu , Zhang Bin , Roos Carolyn M , Miller Jordan
Published: November 2022

Increases in vascular calcification (VC) and fibrosis are a near ubiquitous finding with aging. Here, we sought to test the hypothesis that Metformin (MET)—an AMPK activator entering large clinical trials that is thought to attenuate multimorbidity associated with aging—attenuates key molecular processes known to drive vascular calcification (VC) and fibrogenic signaling in vascular smooth muscle cells (VSMCs) in vitro. Given recent reports highlighting the importance of mitochondria and sirtuins with aging, we also tested the hypothesis that MET-driven reductions in osteogenic signaling are associated with upregulation of SIRT3. We used exogenous BMP2 (100 ng/ml) and TGFβ1 (10 ng/ml) as osteogenic and fibrogenic stimuli, respectively. Consistent with prior work from our lab, treatment with BMP2 for 24 hours robustly increased expression of the osteogenic genes Msx2 (3.9±1.5 fold-change, FC), Runx2 (3.5±1.2 FC), and osterix (17.2±5.2 FC) in VSMCs. Critically, co-treatment with Metformin dramatically reduced expression of these genes by 68 ± 22%, 58 ±20%, and 63 ± 4%, respectively. Subsequent experiments treating cells with exogenous TGFβ1 resulted in significant induction of the fibrogenic marker/known TGFβ1 target versican (1.7±0.4 FC), an effect that was largely attenuated by co-treatment with MET (58±3%). Treatment with MET did not, however, impact TGFβ1-induced increases in CTGF or fibronectin. Treatment with either BMP2 or TGFβ1 resulted in significant increases in mRNA levels of SIRT3 (2.5 ±0.11 and 1.4 ±0.17 FC, respectively), whereas treatment with MET resulted in modest and less consistent increases in SIRT3 mRNA (1.3 ± 0.2 FC). Interestingly, however, co-treatment with BMP2+MET or TGFβ1+MET resulted in uniform suppression of BMP2/ TGFβ1-induced increases in SIRT3 (54±19% and 25±12%, respectively). Collectively, these data suggest that treatment with MET may be a viable strategy to attenuate age-associated increases in osteogenic and fibrogenic signaling in conduit vessels, and suggest further in vivo investigation into therapeutic efficacy is warranted.
Abstract 414: Lipidomic Of Platelet Concentrate Dedicated For Transfusion
Authors: Duchez Anne-Claire , FAUTEUX-DANIEL Sebastien , SUT Caroline , EBERMEYER Theo , ARTHAUD Charles-Antoine , EYRAUD Marie-Ange , PRIER Amelie , AUDOUX Estelle , BERTRAND-MICHEL Justine , PAYRASTRE Bernard , GARRAUD Olivier , Boilard Eric , HAMZEH-COGNASSE2 Hind , COGNASSE Fabrice
Published: November 2022

Structural and biochemical changes in stored platelets are influenced by collection and processing methods. During platelet concentrate storage, lesions can appear of which certain can be involved in adverse reaction after transfusion. The aim of this study was to investigate the lipidome in the supernatant of platelet concentrates (PCs) dependent on processing and storage conditions comparing apheresis (SDA)
versus
buffy coat (BC) platelet concentrate (PC) methods. We investigated various lipid mediator accumulation (Lysophospholipids and Eicosanoids), in either platelet concentrates (SDA-PCs and BC-PCs) stored from 0 to 5 days. All components were produced following French Blood Establishment protocols according to European (EDQM-GTS) Standards; lipids were measured with LS/MS spectrometry. Our data showed no difference between SDA-PC and BC-PC regarding lysophosphatidic acid; however, lysophosphatidylcholine levels were higher in BC-PC than in SDA-PC. Other eicosanoids were affected by either collection or storage conditions of PC. Along the storage, some lipid mediator levels are modulated in SDA-PC and BC-PC. It could be defined as standard platelet components ready for transfusion. To what extent differences, even minimal, matter in clinical outcome remains to be evaluated.
Abstract 423: Imatinib Promotes Reverse Cholesterol Transport And Elevates Sr-bi
Authors: Sucharski Holly C , Dudley Emma K , Williams Jordan , Dewal Revati , Stanford Kristin I , Mohler Peter J , Koenig Sara N
Published: November 2022

Dyslipidemia is a cardiovascular risk factor for coronary artery disease and atherosclerosis that is characterized by elevated serum cholesterol and lipid levels. Although high-density lipoprotein-associated cholesterol (HDL-C) is associated with reduced risk of cardiovascular events, targeted therapy to increase HDL-C levels have been unsuccessful in altering outcomes of associated atherosclerotic disease. Single nucleotide polymorphisms in
SCARB1
, the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic cardiovascular disease. We were the first to identify inherited mutations in
SCARB1
that segregate with disease in a family with severe coronary artery disease and dyslipidemia, including elevated HDL. Our findings suggest that HDL function (vs. HDL-C concentration) may be a promising target for cholesterol-based therapy. Here, we performed an unbiased high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified five compounds that significantly increased endogenous HDL binding: imatinib, trimethoprim, eszopiclone, clemastine, and mepenzolate, of which, imatinib was the only compound to increase SR-BI expression. Imatinib is a tyrosine kinase inhibitor that is a chemotherapeutic agent designed to treat individuals with chronic myeloid leukemia. Limited clinical evidence suggests a reduction in total cholesterol with 400mg/day imatinib. Additionally, imatinib treatment (150mg/kg) in mouse models of atherosclerosis reduces total cholesterol. Yet, no data is available on the effects of imatinib on HDL and reverse cholesterol transport. We have found that imatinib promotes HDL binding and SR-BI expression
in vitro
. Furthermore, in wildtype C57Bl/6 mice on a high fat, high cholesterol diet, imatinib treatment (50mg/kg) was sufficient to decrease plasma total cholesterol, HDL-C and triglyceride levels and elevate hepatic SR-BI expression. In summary, our data supports the exploration of imatinib-mediated SR-BI regulation, HDL metabolism, and RCT pathway to identify new therapeutic targets for dyslipidemia.
Abstract 427: Extreme Low Cholesterol Efflux Is Linked More Strongly To Worse Anti-oxidative Function Than With Systemic Inflammation
Authors: Pahlavani Mandana , Saldanha Suzanne , Deodhar Sneha , Thenayan Lydia , Haley Robert , Wu Elaine , Gangwar Anamika , Rohatgi Anand
Published: November 2022

Reverse cholesterol transport is a main anti-atherosclerotic function of high-density lipoprotein (HDL), and cholesterol efflux capacity (CEC) is a key first step of this function. Inflammation and oxidation are metabolically linked to cholesterol transport but the relationships between these functions have not been well characterized. The objective of this study was to determine if extreme high or low CEC also had differences in inflammation and anti-oxidative functions. We hypothesized that inflammation and anti-oxidative functions of HDL are worse in those with extreme low vs. high CEC. Cholesterol efflux capacity was previously measured in 2,924 participants in the Dallas Heart Study, a multi-ethnic population-based cohort. Those below the 10
th
and above the 90
th
percentile were recruited 15-17 years later, and those with persistent extreme high and low CEC were studied (N=36). Several inflammatory markers such as interleukin-1(IL-1), IL-6, IL-10, tumor necrosis factor α (TNF- α), vascular cell adhesion protein 1(VCAM-1), and serum amyloid A (SAA) were measured using MAGPIX instrument on whole EDTA plasma. The antioxidant levels of paraoxonase-1 (PON1) were measured based on arylesterase activity using ELISA kit on whole citrated plasma. We analyzed data in whole group and stratified by sex, race/ethnicity, and diabetes status. Despite a two-fold difference in CEC, there were no significant differences in inflammatory markers between extreme high vs. low CEC. In those with diabetes, SAA levels were significantly higher in those with low vs. high efflux (P=0.02). High CEC group showed the inverse association with inflammatory marker (IL-1) while direct association with anti-inflammatory marker (IL-10) performing Pearson Correlation Coefficient, R=1, P<0.0001. Activity of PON1 was lower in those with extreme low vs. high CEC, median (IQR) low vs. high: 76.7 (62.2-104.5) vs. 90.1 (82.6-102.0) U/L, (P=0.03). In conclusion, these findings suggest that a key measure of reverse cholesterol transport is more strongly related to anti-Oxidative function than systemic inflammation. Future studies are warranted to determine the complementary information these metabolic markers contribute for cardiovascular risk.
Abstract 428: A Localized Giant Coronary Artery Aneurysm
Authors: Khan Muhammad S , Shailesh Fnu , Vitellas Michail , Ahmed Owais
Published: November 2022

Background:
Coronary artery aneurysm (CAA) incidence is thought to be as common as 0.3 to 5 % of the angiographically examined patients. Regardless of underlying pathogenic mechanism, it results in adverse long-term outcomes. The management of the CAA can be challenging because of lack of clearly defined guidelines.

Case Presentation:
A 75-year-old male without known history of tobacco abuse, DM, hypertension, OSA, CAD, PCI or connective tissue disorder presented with a chief complaint of 2 months of intermittent angina. He had an unremarkable serum troponin level and an EKG. Echocardiogram revealed moderate mitral regurgitation without any regional wall motion abnormality [1]. Because of equivocal exercise stress EKG test, he underwent a coronary angiogram which showed a proximal LAD stenosis followed by a localized fusiform aneurysm [2]. Given his significant symptoms, large size of the aneurysm, ostial LAD stenosis and proximity of diagonal arterial branch the patient underwent CABG surgery involving LIMA to LAD.

Discussion:
CAA patients may present with stable angina, ACS or it may just be an incidental finding on a coronary angiogram. The pathogenesis of CAA is not well known. However, some of the risk factors can be genetic predisposition, underlying atherosclerotic coronary artery disease, autoimmune and connective tissue disorders, trauma such as following PCI, and infections. Coronary CT can be of great utility in evaluating giant CAA as it can help to delineate mechanical complications of these aneurysms. In our patient it was unusual to see localized CAD and an aneurysm limited to one coronary artery territory when atherosclerotic CAAs usually affect more than one artery.

Conclusion:
Since pathophysiology of CAA is not well understood and there are no obvious society guidelines regarding their management, therefore, it becomes important to tailor treatment based on the location and size of the CAA, underlying risk factors and the clinical presentation.
Abstract 429: Loss Of Ten-Eleven Translocation 2 (TET2) Reduces RNA Expression Of Chemokine Receptor CCR6 And Increases Peritoneal B1 B Cell Number And Total Plasma IgM Level
Authors: Dennis Emily A , Bontha Sai Vineela , Marshall Melissa A , Srikakulapu Prasad , Garmey James C , Blackburn Cassidy M , McNamara Coleen A
Published: November 2022

TET2 is an epigenetic regulator with an emerging role in regulating CVD severity. TET2 deficiency in mice (TET2
-/-
) was shown to increase atherosclerosis via upregulation of proinflammatory pathways in macrophages. While TET2 has been reported to regulate germinal center formation and class switch recombination in atherogenic B2 cells, its role in atheroprotective B1 cell subsets remains largely unexplored. To investigate the role of TET2 in B1 subsets, flow cytometry of cells from the peritoneal cavity (PEC), spleen and bone marrow of TET2
-/-
mice and wildtype littermate controls (n = 14/group) was performed. Results demonstrated increased B1a (p = 0.0162) and B1b (p = 0.0056) cells in TET2
-/-
mice in the PEC, their primary niche, but not in the spleen, while in the bone marrow only TET2
-/-
B1a cells were increased (p = 0.0021). An enzyme linked immunosorbent assay using the plasma from these mice demonstrated a significant increase in total IgM in the TET2
-/-
mice compared to the wildtype mice (p = 0.0287). In addition, sort purified PEC B1a, B1b and B2 cells from TET2
-/-
mice and wildtype littermate controls were analyzed for methylation status and RNA expression (n = 24). RNAseq analysis of TET2
-/-
PEC B1a, B1b, and B2 cells revealed significantly reduced chemokine receptor CCR6 expression in B1a (-4.88 fold change, 8.03E-08 padj.) and a trending reduction of CCR6 in B1b cells (-2.17 fold change, 0.3477 padj.) compared to wildtype. Further, methylation analysis showed significant hypermethylation of CCR6 in B1a and B1b TET2
-/-
cells compared to wildtype. This was corroborated by gene pathway analysis of the RNAseq data which showed significant downregulation of chemotaxis and migration pathways in B1a and B1b TET2
-/-
cells. We conclude that loss of TET2 increases hypermethylation of CCR6 and suppresses CCR6 RNA expression in B1 cells which may impair trafficking out of the PEC to the spleen, but not necessarily the bone marrow, leading to PEC accumulation of B1 subtypes and an increase in the total plasma level of atheroprotective IgM. Further study is needed to identify mechanisms of potential B1 cell-mediated atheroprotection in the case of TET2 loss.
Abstract 430: GFP-tagging Of Rabbit ApoAI Decreases Expression And Cholesterol-efflux Activity
Authors: Wacker Bradley K , Bi Lianxiang , Sorci-Thomas Mary , Ng Philip , Palmer Donna , Tang Chongren , Dichek David
Published: November 2022

Introduction:
Gene therapy using a helper-dependent adenovirus (HDAd) to express rabbit apolipoprotein (apo) AI in carotids of fat-fed rabbits reduces atherosclerosis. We hypothesize that transgenic apoAI promotes reverse cholesterol transport from the artery wall; however, this could not be tested because the transgenic rabbit apoAI is indistinguishable from endogenous rabbit apoAI. We therefore constructed an HDAd expressing GFP-tagged rabbit apoAI.

Methods:
We constructed HDAdGFPapoAI by fusing the GFP sequence to the N-terminus of the rabbit
APOA1
gene with a short linker. Bovine aortic endothelial cells (BAEC) were transduced with HDAdGFPapoAI, washed, and incubated in serum-free medium for 24h. Conditioned medium (CM) was collected and fluorescence measured. Cell extracts and CM were analyzed by western blotting for protein size, expression level, and presence of GFP and apoAI antigens. CM cholesterol-efflux activity was measured using cholesterol-loaded BHK cells expressing ABCA1.

Results:
CM of HDAdGFPapoAI-transduced BAEC had green fluorescence and contained a protein of the size predicted for the GFP-apoAI fusion (~56 kDa), which was detected with antibodies to both GFP and apoAI. A smaller protein (~30 kDa) with GFP and apoAI antigens was also detected. Compared to a vector expressing WT rabbit apoAI, GFP-tagging decreased expression of apoAI protein by ~50% and the percentage of secreted apoAI from 55% to 13%. In combination, this reduced secreted GFP-apoAI to only 12% of levels of secreted WT apoAI. While CM containing WT rabbit apoAI strongly stimulated cholesterol efflux from BHK cells (46%), GFP-apoAI-containing CM did not promote cholesterol efflux compared to CM from untransduced BAEC (7.8% vs 7.4% efflux), even when present in CM at a concentration similar to that of WT apoAI.

Conclusion:
GFP-rabbit apoAI protein has reduced expression and secretion (likely due to misfolding/degradation) and does not promote cholesterol efflux (n-terminal GFP may disrupt the phospholipid/cholesterol organization needed for efflux via ABCA1 to this dynamic apoprotein). It will not be useful to investigate mechanisms of atheroprotection by vessel wall-expressed apoAI. Alternatives include use of a smaller tag or expression of human apoAI.
Abstract 431: MaxSpliZer: A Tool To Predict Effects Of
LDLR
Splice Variants Based On The Maximum Entropy Model
Authors: Saadatagah Seyedmohammad , Shahnazar Reza , Sherafati Alborz , Alhalabi Lubna , Miller Alexandra A , Marwan Hamed , Kullo Iftikhar J
Published: November 2022

Background:
The Clinical Genome Resource Consortium (ClinGen) recommends MaxEntScan (MES) model to predict the effects of
LDLR
splice variants. We developed “MaxSpliZer”, a software tool to automate the implementation of MES, and validated it using ClinVar and UK-Biobank (UKBB) data.

Method:
We tested concordance of MaxSpliZer predictions with ClinVar classifications of benign/likely benign (B/LB) and pathogenic/likely pathogenic (P/LP) for
LDLR
variants with potential effect on splicing. We also annotated
LDLR
splice variants in 200,618 UKBB participants, categorizing them using MaxSpliZer as deleterious (n=90) and non-deleterious (n=7,404). Low-density lipoprotein cholesterol (LDL-C) levels were compared in these two groups after adjustment for lipid-lowering medication use.

Results:
MaxSpliZer prediction was concordant with the ClinVar classification in 128 of 138 P/LP variants (sensitivity 93%) and 432 of 436 B/LB variants (specificity 99%). In the UKBB, splice variants predicted as deleterious by MaxSpliZer had higher LDL-C than non-deleterious splice variants (158.7±47.4 vs. 146.0±34.8mg/dL, p-value = 0.014). Compared to manual curation time of 12±7 min per variant, MaxSpliZer took 0.52±0.11 min for single entries and 1.5 s per variant for biobank-scale data.

Conclusion:
MaxSpliZer, a software tool that implements MES based on the ClinGen guideline, can accurately classify
LDLR
splice variants in a rapid automated fashion.
Abstract 437: Carnitine Palmitoyltransferase 1a Modulates Lipoprotein Metabolism
Authors: Zelows Mikala , Kaur Rupinder , Williams Kori , Graf Gregory A , Helsley Robert N
Published: November 2022

Background:
Nonalcoholic fatty liver disease (NAFLD) affects almost 1 billion people worldwide and is associated with risk factors such as obesity and dyslipidemia. Studies show linked variants and methylation status of carnitine palmitoytransferase 1a (CPT1a) to disrupt very low-density lipoprotein (VLDL) cholesterol and triglyceride (TG) levels. We exhibit liver-specific deletion of CPT1a in mice lowers plasma cholesterol and TG while exacerbating NAFLD and inflammation.

Methods:
Eight-week old
Cpt1a
floxed mice with the human apoB100 transgene (Cpt1a
fl/fl
/B100
Tg
) were administered control adenoassociated virus (AAV) or AAV encoding Cre-recombinase under control of a liver specific promoter (TBG-Cre). Control and
Cpt1a
liver-specific knock out (LKO) mice were placed on low-fat control or western-type diet (WTD; 42% kcal fat, 0.2% cholesterol) for 16 weeks. Body weights were recorded weekly and body composition by MRI was performed at the study midpoint and end. Tissues and plasma were collected and analyzed for lipid composition and gene and protein expression by QPCR and immunoblotting, respectively.

Results:
CPT1a LKO mice had lower plasma cholesterol and TG irrespective of diet. The reduction in plasma cholesterol was limited to the LDL pool in FPLC-fractionated plasma. Hepatic TG was elevated in mice fed WTD and LKO
Cpt1a
mice. Loss of hepatic
Cpt1a
had no effect on hepatic cholesterol in male mice, but increased total and free cholesterol by 2- and 2.5-fold, respectively, in females. The rise in hepatic free cholesterol in female
Cpt1a
LKO mice associated with increases in Kupffer cell (
Clec4f
) and collagen (
Col1a1
) gene expression. Free cholesterol levels were not related to differences in transcripts for enzymes involved in cholesterol synthesis or secretion (
Srebp2, Hmgcr, Hmgcs, Soat2, Mttp
), hepatic clearance (
Ldlr
), metabolism (
Cpy7a1, Cyp8b1
), or biliary secretion (
Abcg5, Abcg8
).

Conclusions:
Liver-specific deletion of CPT1a reduces plasma LDL-cholesterol and increases cholesterol levels in female, but not male mice. The increase in hepatic free cholesterol did not alter expression of cholesterol-responsive genes, suggesting cholesterol accumulation outside of the regulatory pool.
Abstract 440: Heat Shock Proteins Prevent Environmental Chemicals-induced Endothelial Activation And Injury
Authors: Malovichko Marina V , Wickramasinghe Nalinie S , McFall Samantha , Taylor Breandon S , Zelko Igor N , Sithu Srinivas D , Srivastava Sanjay
Published: November 2022

Environmental pollution accounts for nine million premature deaths worldwide every year and cardiovascular disease is the leading cause environmental pollution-associated deaths. Our previous studies have shown that exposure to combustion-derived chemicals (e.g., acrolein), petroleum products-derived chemicals (e.g., benzene) and metalloid such as arsenic exacerbates atherosclerosis in mice.
In vitro
studies suggest that these chemicals endothelial activation and injury, at least in part, by inducing endoplasmic reticulum stress. To examine how endothelial cells cope with environmental chemicals-induced toxicity, we performed transcriptomic analyses of human aortic endothelial cells (HAEC) exposed to acrolein (10 μM),
t,t
-muconaldehyde (MA, reactive metabolite of benzene, 10 μM), and arsenic (20 μM). Our data show that exposure of HAEC to these chemicals significantly induced the expression (log 2FC = 2-13-fold) of heat shock proteins -
Hspa1a, Hspa1b, Hspa6
, and
Hspa7
, the molecular chaperones which keep the proteins in their native structures. MA-induced transcription of HSPs in HAEC was accompanied by the induction (log 2FC = 1-3) of oxidative stress-responsive genes superoxide dismutase 1, heme oxygenase-1, cyclooxygenase-2, and
Akr1b10
; ER-stress responsive gene
Atf3; and
adhesion molecules
Icam-1
and
P-selectin;
and downregulation of endothelial nitric oxide synthase (
Nos
3, log 2FC = -0.4). Like HAEC, MA also significantly induced
Hspa1b1
and
Atf3
(log 2FC= 2.3 and 0.7 respectively). siRNA-mediated knockdown of
Hspa1b
in HAEC significantly increased MA-induced transcription of
Icam-1
and leukocyte adhesion to endothelial cells, abundance of ATF3 protein, endothelial cell apoptosis, and attenuated MA-induced downregulation of
Nos
3 gene. The reactome analysis of MA-treated HAEC suggested that heat shock factor-1 (HSF-1) is the transcriptional regulator of
Hspa1a
and
Hspa1b
. Western blotting of MA-treated HAEC showed that MA upregulates as well as phosphorylates HSF-1, which attenuates IL-6 expression by activating ATF3. Collectively, these data suggest that HSPs prevent environmental chemicals-induced endothelial toxicity by preventing oxidative stress, ER-stress, inflammation, and depletion of
Nos3
.
Abstract 453: Investigating The Role Of Glutathione During Macrophage Apoptotic Cell Clearance
Authors: Delgadillo Luisa F , Mackay Ryan P , Rashdan Nabil A , Yurdagul Arif , Pattillo Christopher B
Published: November 2022

Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD.

Methods:
Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis.

Results:
Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression.

Conclusion:
These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.
Abstract 461: Antiphospholipid Antibodies In Covid-19 Convalescent Plasma
Authors: Becicka Wyatt , Berreby Gil , Wang Jintao , Moitra Jaideep , Panicker Sumith , Tourdot Benjamin , Norris Philip , Kelly Kathleen , Zouo Yu , Dumont Larry , Knight Jason , Kanthi Yogendra
Published: November 2022

Background:
Although convalescent plasma has historically yielded significant benefits in the treatment of infectious diseases, numerous clinical trials have not shown a consistent benefit in treating either moderately or severely ill COVID-19 patients with plasma from convalescent patients. We previously reported increased levels of antiphospholipid antibodies (aPL) in acutely ill COVID patients, with high aPL titers correlating with more neutrophil extracellular traps (NETs) and poorer patient outcomes. We hypothesized that persistence of potentially deleterious autoantibodies in previously hospitalized, convalescent plasma donors may activate immune cells and add context for the lack of clinical efficacy in COVID.

Methods:
116 samples of convalescent plasma from donors previously hospitalized with COVID were obtained from Vitalant. Antibody titers (IgG, IgM, IgA) of anti-2-glycoprotein 1 (a2GP1) and anti-cardiolipin (aCL) and anti-phosphatidylserine/prothrombin (IgG, IgM) were quantified. NET formation by healthy human neutrophils treated with convalescent plasma was evaluated in vitro.

Results:
High aPL titers >40 U/mL) were observed in 10/116 (8.6%) convalescent samples, and 6/10 were positive for either IgG or IgM aCL. The aPL positivity rate exceeded the expected background population rate of ~3%. Six of 116 convalescent samples increased NET-associated MPO activity in purified, primary neutrophils from healthy donors compared with control plasma (mean increase of 23% +/- 11, p<0.01), where only 2 of these were aPL-positive samples.

Conclusion:
aPL were elevated in a larger proportion of surveyed convalescent plasma samples than reported in the healthy population. aPL positivity did not correlate with purified neutrophil activation, suggesting aPL in donor plasma does not have a direct, activating effect on healthy neutrophils. Considering the complex interactions among circulating blood cells, ongoing experiments are examining the impact of convalescent plasma on platelet and endothelial cell function to further elucidate potential autoantibody-mediated vascular perturbations. The results of these studies will be important in informing the vascular implications and safety of convalescent plasma in COVID.
Abstract 463: The Smooth-Muscle-Cell-Angiotensin II-Sensitive LncRNA Controls Cell Division Fidelity And Mitochondrial Organization
Authors: Espinosa-Diez Cristina , Liu Mingjun , Mahan Sidney , Du Mingyuan , An Wenxi , Hahn Scott , Straub Adam C , Shiva Sruti , Gomez Delphine A
Published: November 2022

Reactivation of the cell cycle and increase in proliferation rate (hyperplasia) is a common response of vascular smooth muscle cells (SMC) to modifications of their environment during remodeling. Although SMC hyperplasia is a predominant feature of many vascular diseases, SMC can also increase their mass within the remodeled vessel wall by enlarging their size and becoming hypertrophic. Hypertrophy is usually accompanied by cell cycle defects, cell polyploidy and binucleation, and senescence. However, the molecular mechanisms favoring SMC hypertrophy and their repercussion on SMC phenotype are not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression, and they have been identified as modulators of cell division. We recently discovered a novel lncRNA, SAS (SMC-Angiotensin II-Sensitive), whose expression was markedly decreased in multiple models of SMC dedifferentiation, suggesting that SAS could regulate SMC phenotype and function. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation, cell arrest and migration in aortic and renal artery-derived SMC treated with Platelet Derived Growth Factor. SAS knockdown was also associated with distinct SMC morphological changes including increase in cell size and binucleation demonstrating hypertrophy. Together, these observations suggest that decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion and cytokinesis. Interestingly, SAS expression is decreased in response to Angiotensin-II in cultured VSMC and in the aorta of hypertensive mice (2 Kidney-1 Clip model), suggesting a role in mediating hypertension induced SMC hypertrophy. Similarly, to Angiotensin-II treatment, SAS knockdown promoted senescence. Furthermore, SAS deficient cells present mitochondria hyperfusion and increased oxygen consumption that correlates with the observed exacerbated senescence. Altogether, our results indicate that SAS is a potent regulator of VSMC morphology and is required for proper cell division and mitochondria organization.
Abstract 467: The Histone Acetyltransferases P300 And Cbp Coordinate Distinct Chromatin Remodeling Programs In Vascular Smooth Muscle Plasticity
Authors: Chakraborty Raja , Ostriker Allison C , Xie Yi , Dave Jui M , Chatterjee Payel , Gallagher Patrick , Sessa William C , Hwa John , Martin Kathleen A
Published: November 2022

Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methyl cytosine dioxygenase TET2 acting as a master regulator of SMC phenotype. The histone acetyltransferases (HATs) p300 and CBP are highly homologous and often considered to be interchangeable, and their roles in SMC phenotypic regulation are not known. We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (
p300

iKO

or
CBP

iKO

), and in human intimal hyperplasia. P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in injury-induced vessel remodeling. Knockdown experiments revealed opposing effects of p300 and CBP in VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300
iKO
mice exhibited severe intimal hyperplasia following arterial injury while CBP
iKO
mice were entirely protected. In normal aorta, p300
iKO
reduced, but CBP
iKO
enhanced, contractile proteins and contractility. Mechanistically, we found that these HATs oppositely regulate histone acetylation, DNA hydroxy methylation, and RNA PolII binding to promoters of differentiation-specific contractile genes. We report p300 and TET2 function together, as p300 was required for TET2-dependent hydroxy methylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 co-immunoprecipitated with p300 and this interaction was enhanced by rapamycin but repressed by PDGF, with p300 promoting TET2 protein stability. CBP did not associate with TET2, instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile gene promoters. Immunostaining revealed, p300 expression is repressed but CBP is induced in human intimal hyperplasia. This work reveals that p300 and CBP serve non-redundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific HATs may hold therapeutic promise for cardiovascular diseases.
Abstract 468: Expression Qtl Analysis And Fine-mapping In Human Coronary Artery Prioritize Candidate Genes For Functional Characterization.
Authors: Hodonsky Chani J , Turner Adam , Barrientos Nelson , Lopez Nicolas , Kovacic Jason C , Bjorkegren Johan , Leeper Nicholas , Miller Clint
Published: November 2022

Background:
Atherosclerotic plaque progression to coronary artery disease is the leading cause of death worldwide. The basic pathophysiology of atherosclerosis is well described, but underlying genetic mechanisms of contributing cell types remain incompletely characterized. We utilized human coronary artery RNA sequencing data to identify differentially expressed genes and genetic variants that affect expression.

Methods:
We used bulk RNA sequencing data from coronary artery samples from 138 American adults (30% female) representing a broad phenotypic range of atherosclerosis. To account for ancestral diversity, we incorporated local ancestry into permutation-based eQTL analyses using a combination of published methods. We also used mixQTL to evaluate the contribution of allele-specific expression to identifying eQTLs. All analyses adjusted for age, sex, and ancestry. We utilized CAD GWAS summary statistics, coronary artery ENCODE annotations, and activity-by-contact (ABC) scores to fine-map eGenes.

Results:
In local-ancestry eQTL analyses, we report 467 eGenes (FDR<0.05), 75 of which had no significant coronary artery eQTLs in GTEx. Additionally, we identify over 4,000 eGenes using mixQTL. 25 GWAS signals colocalized using a European LD reference panel, 9 of which colocalized uniquely to 1 of 4 datasets analyzed. Fine-mapping demonstrated multiple independent associations at over 20% of loci, potentially representing ancestry-specific associations. Population-specific analyses are ongoing.

Conclusion:
Understanding changes in the gene expression program at all disease stages will help characterize coronary artery disease processes as well as identify potential therapeutic targets for functional examination. This work represents a step toward characterizing CAD-related gene expression programs and highlight the importance of inclusive, deliberate study design.