bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 214: Sumo2 Regulates Oxidative Function Of Adaptor Protein P66shc
Authors: Kumar Jitendra , Uppulapu Shravan kumar , kumari sujata , Sharma Kanika , Kumar Vikas , Kumar Santosh
Published: October 2023

SUMOylation is a highly dynamic post-translational modification which regulates protein function and/or stability. It involves conjugation of SUMOs (
S
mall
U
biquitin-Like
Mo
difier
s
) to the lysine residue/s of target proteins mediated via a highly coordinated enzymatic mechanism. Studies have shown that promoting SUMOylation (SUMO2/3) causes vascular endothelial dysfunction and accelerates atherosclerosis. We reported that SUMO2 overexpression in endothelial cells promotes oxidative stress and impairs endothelial function. However, the mechanism is not well understood. Adaptor protein p66Shc is a master regulator of oxidative stress and endothelial function. Genetic deletion of p66Shc protects mice against endothelial dysfunction due to metabolic dysregulation. Prior studies have shown that lysine modification of p66Shc regulates its oxidative function. As SUMOylation primarily occurs on lysine residue, we examined if p66Shc is a target of SUMO2. In this study, we show that p66Shc is a direct target of SUMO2. Using endothelial cells and HEK-293 cells, we show that overexpression of SUMO2 leads to p66Shc SUMO2ylation which is further increased in presence of E2-ligase Ubc9 and reduced by deSUMOylating enzyme SENP1. SUMO2ylation of p66Shc increases oxidative activation (serine-36 phosphorylation) of p66Shc which is essential for reactive oxygen production by p66Shc. Using mass spectroscopic study, we identified that lysine-81 is the only lysine present in CH2 domain which is getting SUMO2ylated. Rendering p66ShcK81 non-sumoylatable (p66ShcK81R) prevented the SUMO2ylation of p66Shc. In endothelial cells, overexpression of p66ShcK81R prevented the SUMO2-induced increase in serine-36 phosphorylation and mitochondrial translocation of p66Shc, which are essential for oxidative function of p66Shc. Collectively, we present a novel molecular mechanism via which SUMO2 regulates the oxidative function of p66Shc and identify a unique post-translational regulation of p66Shc in endothelial cells.
Abstract 220: Post-gwas Functional Genomics Analysis Of Abo Locus To Identify Novel Pathogenic Mechanisms For Chronic Thromboembolic Pulmonary Hypertension
Authors: Yue Yunshan , Hiwase Harsh , El Khoury Wadih , Kelly Neil , Chan Stephen Y , Sun Wei
Published: October 2023

Background:
Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and mysterious complication of pulmonary embolism, characterized by chronic pulmonary arterial thrombosis, occlusions and vasculopathy. Emerging evidence has suggested genetic relevance of CTEPH, particularly to ABO blood type. Genome-wide association study (GWAS) has identified ABO gene as the most significant genetic locus associated with CTEPH. This locus, however, consists only intronic SNPs and there is no mechanistic explanation on how these non-coding SNPs control the risk of CTEPH.

Methods:
GWAS-identified CTEPH-associated ABO locus was analyzed, and haplotype SNPs were identified based on linkage disequilibrium (LD). Electrophoretic mobility shift assay (EMSA) was applied to screen functional SNPs (fSNPs) based on allele-imbalanced nuclear protein binding. SNP-binding nuclear transcription factors (TFs) were identified by proteomics. Hi-C chromatin-interaction analysis was utilized to determine potential target genes affected by fSNPs. Knockdown of fSNP-associated TFs in human pulmonary artery endothelial cells (hPAECs) was applied to validate the regulation of TFs on target genes.

Results:
Based on GWAS, the minor allele of tag SNP rs2519093 is associated with higher risk of CTEPH (Odds ratio 2.22). It defines 11 haplotype SNPs in a LD with R
2
> 0.8. Screened by EMSA with hPAEC nuclear proteins, intronic SNPs rs579459 and rs550057 were identified functional given their allele-specific protein binding. Proteomics analysis identified 3 candidate TFs that allele-specifically bind to fSNP rs579459: PBX1, CTCF and SAFB2. Candidate target gene ADAMTS13 is chosen based on its prominent interaction with fSNP revealed by Hi-C analysis, and the potential role in thromboembolism through vWF cleavage. TF knockdown by siRNA downregulates ADAMTS13 in hPAECs, suggesting their regulatory function as activators.

Conclusion:
Leveraging our post-GWAS functional genomics approach, we identified fSNPs in ABO locus that define a genetic architecture controlling the susceptibility of CTEPH. This genetic architecture, along with the newly identified fSNP-associated TFs and target gene ADAMTS13, provides a biological explanation for the pathogenesis of CTEPH.
Abstract 225: Regulation Of Plasma Lipoproteins And Atherosclerosis By Microrna-541-3p And Transcription Factors Znf101 And Casz1
Authors: Ansari Abulaish , Yada Pradeep Kumar , Zhou Liye , Ijaz Laraib , christiano Amanda , Ahmad Sameer , Rimbert Antoine , Hussain M M
Published: October 2023

High apoB-containing low-density lipoproteins (LDL) and low apoA1-containing high-density lipoproteins (HDL) are associated with atherosclerotic cardiovascular disease. MicroRNAs (miRs) are small non-coding RNAs that modulate multiple pathways. We screened a miR library in human liver cells and identified miR-541-3p that decreases apoB and increases apoA1 by inducing the degradation of mRNAs of two different transcription factors, Znf101 and Casz1. Molecular and physiological studies revealed that Znf101 enhances apoB expression and that Casz1 represses apoA1 expression. Thus, miR-541-3p differentially regulates apoB and apoA1 expression by reducing the expression of an enhancer and a repressor. Moreover, the hepatic knockdown of orthologous genes in mice reduced plasma lipids and atherosclerosis. Mechanistic studies revealed that lower hepatic lipoprotein production and increased HDL cholesterol efflux capacity might contribute to reductions in atherosclerosis. In addition, these livers showed reduced expression of genes involved in lipogenesis. Furthermore, knockdown of these genes and overexpression of miR-541-3p in human liver cells reduced lipogenesis. Thus, in addition, to apolipoprotein/lipoprotein production, miR-541-3p also regulates hepatic lipid synthesis. By screening human genetic datasets, we found that genetic variants in
MIR541
,
ZNF101
and
CASZ1
loci are significantly associated with plasma lipid and lipoprotein levels. This study suggests miR-541-3p, Znf101 and Casz1 as potential therapeutic agents/targets to reduce plasma lipids, lipoproteins and atherosclerosis without causing steatosis.
Abstract 227: Measuring HDL-apolipoprotein A-I Exchange By Subclass In Reconstituted And Human HDL
Authors: Ong Carlos , Bacha Shumylla , Vaisar Tomas , Borja Mark S
Published: October 2023

Objective:
Levels of high density lipoprotein (HDL) are inversely associated with cardiovascular disease risk. HDL particles contain a diverse array of lipids, cholesterol, and proteins, and exist over a range of sizes ranging from about 8 nm to about 17 nm diameter, but it is not well understood how HDL size affects its function. In this study we examine the effect of HDL particle size on the rate of HDL-apolipoprotein A-I (apoA-I) exchange in reconstituted and human HDL particles.

Method and Results:
Reconstituted HDL particles were synthesized from 16:0-18:1n-9 PC, cholesterol, and recombinant apoA-I. Human HDL was isolated from plasma from healthy volunteers by density gradient ultracentrifugation. In both cases, the HDL was further separated by subclass using size exclusion chromatography. ApoA-I exchange was measured by incubating fluorescently-labeled, lipid-free apoA-I with HDL and quantifying the fluorescent signal appearing in the HDL region versus the lipid-free region of a nondenaturing gel over time. ApoA-I exchange rates increased inversely with particle size in both reconstituted and human HDL, with medium-sized HDL particles exhibiting twofold faster exchange rates compared to the largest HDL particles.

Conclusions:
Smaller HDL particles exhibit faster rates of HDL-apoA-I exchange in both reconstituted and human HDL particles. Reconstituted HDL particles provide an accurate approximation of apoA-I exchange in human HDL particles.
Abstract 232: Searching for Novel Regulators of the Mevalonate Pathway
Authors: Chuecos Marcel A , De Giorgi Marco , Walkey Christopher , Nguyen Bryan , Islam Nelufa Y , Jain Antrix , Jung Sung Yun , Malovannaya Anna , Lagor William R
Published: October 2023

The mevalonate pathway is an important metabolic pathway that provides cells with vital bioactive molecules. The pathway converts mevalonate into various sterol and nonsterol isoprenoids required for cellular maintenance and health, including, cholesterol, ubiquinione, dolichol, heme A, farnesyl, and gernaylgeranyl protein anchors. However, these molecules are energetically expensive to synthesize and toxic in excess, so tight regulation of the pathway is necessary at multiple steps. In order to identify novel proteins that help regulate the mevalonate pathway, we performed unbiased proteomic screens to detect protein-protein interactions in Huh7 cells, a human hepatocellular carcinoma cell line. Proximity labeling with a promiscuous biotin ligase (BioID), and affinity purification were performed using multiple enzymes from the pathway as bait (MVK, MVD, FDFT1, FDPS, SQLE, and LSS). Several proteins were detected using both techniques at statistically significant levels compared to control. An extensive literature review revealed that two hits, NAB1 (NGFI-A-binding protein 1) and KHDRBS1 (KH RNA Binding Domain Containing, Signal Transduction Associated 1), could have relevant and understudied interactions with mevalonate kinase (MVK). Therefore, to determine if NAB1 or KHDRBS1 regulate MVK, they were independently knocked down using siRNAs
in vitro
. Knockdown of NAB1 led to decreased mRNA expression and protein levels of MVK under sterol depleted conditions, and decreased expression of high-density lipoprotein (HDL) associated genes (
ABCA1
,
ApoA1
, etc). Conversely, when MVK was knocked down under sterol replete conditions, NAB1 protein levels were similarly reduced. Thus, these two proteins undergo a previously unknown interaction which warrants further study. NAB1-MVK interaction might help explain the association between MVK single nucleotide polymorphisms (SNP) and circulating HDL levels in humans, and the increase of circulating HDL in NAB1 knock out (KO) mice.
Abstract 238: Prediabetes Increases Platelet Activation Via Sod2 In Young And Middle-aged Veterans
Authors: Ahmed Azaj , Eustes Alicia , Geasland Katharine , Swamy Jagadish , Spitz Douglas , Jalal Diana , Dayal Sanjana
Published: October 2023

Aging is a risk factor for thrombotic cardiovascular diseases and presence of a risk factor may aggravate the outcome. Due to the increase in overweight/obese people in the aging population, the incidence of prediabetes is increasing. Recent studies suggest that prediabetes is not only a prelude to diabetes, but also it is an independent risk factor for thrombotic events. However, the prothrombotic mechanisms are not known. We hypothesized that loss of superoxide dismutase 2 (SOD2) during aging promotes mito-oxidant accumulation in platelets, leading to platelet hyperactivity, and that this phenotype is accentuated by prediabetes. From the Iowa City VA Healthcare System, we recruited 15 young (18-49 year) and 12 middle-aged (50-64 year) Veterans with prediabetes (FBG 100-124 mg/dL and/or HbA1c 5.7-6.4). We also recruited age- and BMI- matched 14 healthy young and 23 middle-aged Veterans. Washed platelets were prepared from whole blood and markers of platelet activation (fibrinogen, activated α
II
bβ3, and Annexin V) were measured via flowcytometry.
Ex vivo
, thrombus formation was assessed in a collagen coated microfluidic chamber by measuring accumulation of platelets under arterial shear stress. Expression of SOD2 in platelets measured by western blot was decreased in all three groups vs. young healthy Veterans. Upon agonist activation, platelets from healthy middle-aged Veterans showed only moderate increase in activated αIIbβ3 or fibrinogen and Annexin V binding (P=0.09, P=0.08, and P=0.2 vs. healthy young). Prediabetes caused significant increases in what in both young and middle-aged (main effect of prediabetes, P<0.05 to P<0.01) Veterans. In the flow chamber, platelets from young or middle-aged prediabetics showed greater accumulation (P<0.0001 vs. healthy young or healthy aged). Preincubation of platelets with mitochondria targetable SOD-mimetic, decreased activated αIIbβ3, fibrinogen binding, as well as thrombus formation in both prediabetic groups. We conclude that a prothrombotic state exists in young and middle-aged prediabetics that causes platelet activation and thrombus growth in SOD2-dependent manner. These findings suggest a therapeutic potential of SOD-mimetic in decreasing thrombotic burden in prediabetes.
Abstract 242: A Novel Strategy To Combat The Procoagulant Phenotype In Immune Thrombotic Thrombocytopenia Using 12-LOX Inhibition
Authors: Michael James , Renna Stephanie A , Zhao Xuefei , Kunapuli Satya P , Ma Peisong , Holinstat Michael A , Boxer Matthew , Maloney David , McKenzie Steven E
Published: October 2023

The risk of stroke or other thrombotic complications following vaccination of SARS-CoV-2 has been extensively reported since widespread vaccination efforts began in early 2021. This disorder, termed vaccine-induced immune thrombotic thrombocytopenia (VITT), has remarkable overlap in clinical manifestations to that of heparin-induced thrombocytopenia (HIT). HIT and VITT are pathologies caused by IgG immune complexes that activate platelets via Fc Receptor for IgG IIA (FcγRIIA), which are collectively categorized as immune thrombotic thrombocytopenia (ITT) disorders. Thrombotic events remain high following the standard of care treatment with concurrent risk of bleeding complications. This study sought to investigate a novel approach to treatment of ITT. Recent reports demonstrate increased procoagulant activity in ITT. However, these reports required
ex vivo
analysis, and
in vivo
relevance remains unclear. Using human and mouse model systems, we investigated the cooperativity of protease activated receptors (PARs) and FcγRIIA in ITT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4
+/+
or IIA-Par4
-/-
, respectively) with a well-established model IgG immune complex (αCD9). IIA-Par4
+/+
mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4
-/-
mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in ITT
in vivo
. In human platelets, we observed that PAR1, PAR4, and FcγRIIA each contributed to the procoagulant phenotype, via receptor-specific increases in cytosolic Ca
2+
. As inhibition of 12(S)-lipoxygenase has been reported to decrease platelet activation downstream of FcγRIIA and PAR4, we used inhibitor VLX-1005 and observed significant reduction in both platelet procoagulant phenotype
ex vivo
, and thrombocytopenia and thrombosis in our humanized mouse model of HIT
in vivo
. These results extend our understanding of ITT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in ITT.
Abstract 243: Local Periadventitial Delivery Of Dexamethasone Improves Murine Deep Vein Thrombosis Resolution
Authors: Vijay Aatira , Goudot Guillaume , Jha Prabhash K , Mauskapf Adam , Henke Peter K , Aikawa Masanori , Seward Kirk P , Jaffer Farouc A
Published: October 2023

Background:
Post deep vein thrombosis (DVT), up to 30-50% of patients will develop post-thrombotic syndrome (PTS), despite anticoagulation. Currently no preventive therapies, including catheter-based thrombectomy/thrombolysis (CDT), have shown to reduce PTS. Local steroid therapy adjunct to CDT is being tested clinically for DVT patients in the DEXTERITY trials, but minimal mechanistic data exists on DVT resolution. Here, we investigate effects of local dexamethasone (DEX) therapy on murine DVT resolution.

Method:
C57/BL6 male mice (n=146) underwent inferior vena cava (IVC) complete ligation to develop stasis DVT on day 0 (D0) followed by mechanical de-ligation on D2 to spur restoration of blood flow (RBF). On D2, mice were injected with PBS/DEX (0.1, 0.2, or 0.5 mg/mouse, 60μl volume, across four zones) in the periadventitial tissue of thrombosed IVC, with or without de-ligation (Fig. A-B). Mice underwent D4&D8 serial ultrasound imaging of IVC blood flow to assess RBF-defined as IVC mean flow velocity > 0 mL/sec. D8 thrombus burden was measured followed by histological/picrosirius red assessment of vein wall fibrosis.

Results:
N=6-11 mice per group underwent IVC DVT creation. Low-dose (0.1 mg) but not higher doses of DEX reduced thrombus burden (p=0.02) and weight (p=0.01) on D8 (Fig. C-D). Low-dose DEX increased D4 RBF rates and mean flow velocity (MFV) (Fig. E-F). Complete IVC stasis without de-ligation but with DEX non-significantly reduced thrombus burden and weight (Fig. G-H). Low-dose DEX further decreased vein wall fibrosis (Fig. I; p=0.001). D8 thrombi exhibited reduced macrophage content with DEX (Fig. J; n=15-18 sections/group, p=0.007).

Conclusion:
Following stasis DVT induction, locally delivered low-dose DEX improves DVT resolution by reducing thrombus burden, vein wall injury, macrophage content, and increases RBF rate. These data provide a scientific foundation for ongoing DEXTERITY trail testing adjunct DEX for DVT patients undergoing CDT.
Abstract 244: Evaluation Of Lipid, Lipoprotein, And Apolipoprotein Analysis By Nuclear Magnetic Resonance
Authors: Diffenderfer Margaret R , Fernandes Marianna , He Lihong , Flowers Sarah E , Schaefer Ernst J
Published: October 2023

Serum lipoproteins play a causative role in cardiovascular disease (CVD). Lipoproteins vary in size and density, as well as apolipoprotein (apo), total cholesterol (TC), free cholesterol (FC), triglyceride (TG), and phospholipid (PL) content. High resolution nuclear magnetic resonance (NMR) can simultaneously assess these characteristics within very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). We sought to validate the use of specialized NMR analysis and software for the measurement of serum TC, TG, LDL-C, small dense LDL-C, HDL-C, apoA-I, apoB, and LDL particle number (LDL-P). Fasting serum concentrations of TC, TG, direct LDL-C, HDL-C, apoA-I, and apoB were measured in 1,458 men and women (mean age 58.2 years) by 600 MHz NMR using Bruker methods and software and were compared with concentrations obtained by validated automated standardized Roche assays. LDL-P obtained using the Bruker software was compared with that obtained using Numares software. NMR-obtained concentrations were highly correlated with Roche assay results: TC 0.988, TG 0.992, direct LDL-C 0.972, HDL-C 0.969, apoA-I 0.953, and apoB 0.969 (all
P<
0.00001). In some cases, corrections were introduced to match absolute concentrations. Correlations between LDL-P analyses were 0.920 (
P<
0.0001). Bruker software provides for assessment of a total of 114 parameters including particle number and TC, FC, TG, PL, and apoB levels in total VLDL, VLDL1-5 (large-small), IDL, and total LDL, LDL1-6 (large-small), and TC, FC, TG, PL, apoA-I, and apoA-II levels in total HDL and HDL1-4 (large-small). However, in our reporting we have focused on the lipid, lipoprotein, and apolipoprotein parameters that we could validate using standard methods, as well as particle number for VLDL, IDL, total LDL, LDL1-6, and HDL and cholesterol and triglyceride values in these particles. We observed substantial gender differences with regard to HDL particles and their constituents. In conclusion, NMR lipoprotein analysis allows for complete lipoprotein analysis except for lipoprotein(a) and holds great promise for CVD risk assessment and for the detection of lipoprotein disorders.
Abstract 254: Apolipoprotein B Mediates Hepatic Proliferation And Cell Survival Signalling: A Crispr/cas-9 Gene Editing ApoB-knockout Study
Authors: Su Qiaozhu , Wade Henry
Published: October 2023

Introduction:
Apolipoprotein B (ApoB) is a key structure protein in very-low-density lipoprotein (VLDL). Ample research has been focusing on its crucial role in lipoprotein metabolism and the onset of cardiovascular disease (CVD). However, in vivo research on the broader pathophysiological function of ApoB has been hindered by the embryonic lethality of ApoB knockout (ApoB-KO) animal models. Thus, the objective of this study is to establish ApoB-KO cell models and demonstrate ApoB as a key determinant in governing hepatocyte proliferation and apoptosis.

Approaches and Results:
the Crispr/Cas-9 gene editing technology was applied to genetically disrupt ApoB expression in both human and mouse hepatocytes. Total RNAs of the parental and ApoB-KO cells were subjected to RNA sequencing profiling for whole transcriptome. Subsequent bioinformatics analysis revealed that ApoB-KO significantly altered expression of more than 2000 genes in hepatocytes. More importantly, expression of genes involved in DNA replication, cell cycle, cell proliferation and apoptotic signaling were the genes mostly affected, which were either up- or down-regulated by more than 5-folds. These included the histone associated genes H2ac10-12, cyclin Ccna2, Ccnb1 and Cdc20, cell proliferation gene Fosb and apoptosis gene P53. A BrDU cell proliferation assay detecting the actively proliferating cells further demonstrated that depletion of ApoB greatly inhabited cell proliferation, indicated by the markedly reduced cell numbers and the prolonged cell doubling time in ApoB-KO cells compared to their parental cells. Mechanistic study revealed that cAMP signaling, and the insulin associated PI3K kinase pathway that medicated gene transcription, cell growth and proliferation were profoundly impaired upon ApoB depletion. Reconstitution of ApoB in the KO cells by expressing a truncated ApoB protein, ApoB72, was able to promote cell growth rate and improve cell survival to a level comparable to the parental cells.

Conclusion:
This is the first study that characterizes a novel function of ApoB as a key determinant in hepatic cell proliferation and survival pathways. This novel finding greatly advances our knowledge of ApoB beyond the boundary of VLDL metabolism and CVD.
Abstract 261: Tissue Factor Pathway Inhibitor Prolongs The Initiation Of Thrombin Generation And Is Associated With Acute Kidney Injury In Covid-19
Authors: Eustes Alicia S , Lentz Steven R , Dayal Sanjana
Published: October 2023

COVID-19 can cause profound inflammation and coagulopathy, yet the mechanisms underlying the thrombo-inflammatory state are not fully elucidated. Patients with COVID-19 have elevated plasma levels of not only procoagulant factors but also anticoagulant proteins, such as tissue factor pathway inhibitor (TFPI), and very little research has been done to test the role of COVID-19 infection on anti-coagulant pathways. We tested the mechanistic contribution of TFPI to thrombin generation and its association with renal failure during the initial wave of COVID-19 (April 2020 to January 2021). We utilized blood plasma collected from a clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. We first confirmed that there was increased thrombin generation potential in the COVID-19 cohort, reflected by greater peak thrombin (
p
= 0.020), velocity index (
p
= 0.022), and endogenous thrombin potential (ETP) (
p
= 0.006); however, the time to initiation of thrombin generation (lag time) was delayed (
p
= 0.037). Plasma levels of TFPI in were elevated in COVID-19 patients compared to controls (
p
< 0.0001) and TFPI concentration was positively associated with lag time (
p
= 0.002). Treatment of plasma with anti-TFPI blocking antibody shortened the lag time in both controls (
p
= 0.001) and COVID-19 patients (
p
< 0.0001) and normalized the difference in lag time between healthy subjects and COVID-19 patients without affecting peak thrombin, velocity index, or ETP. Interestingly, the levels of TFPI in the plasma of COVID-19 patients positively correlated with acute kidney injury (
p
= 0.026) but not with mortality, admission to the intensive care unit, or thrombosis. Together these data indicate that TFPI affects the pro-thrombotic state and is associated with clinical outcome in COVID-19.
Abstract 264: In-hospital Antiplatelet Use Is Associated With Decreased Thromboembolic Events In Covid-19 Patients
Authors: Yan Crystal L , Rivera Rodriguez Beatriz , Andrade-Bucknor Sharon
Published: October 2023

Background:
COVID-19 infection has been associated with a prothrombotic state. Previous studies found conflicting results regarding the association of in-hospital antiplatelet use and mortality in patients with COVID-19. The purpose of our study was to determine whether in-hospital antiplatelet use was associated with lower morbidity or mortality in patients hospitalized with COVID-19.

Methods:
We conducted a two-center retrospective study of 692 adult patients hospitalized from March 2020 to October 2021 with COVID-19 confirmed by RT-PCR. Groups were stratified based on in-hospital antiplatelet use. Baseline characteristics, biomarkers, and in-hospital outcomes were described. A multivariate regression model was used to estimate the association of the primary outcome of interest with in-hospital antiplatelet use. Antiplatelet use was defined as aspirin and/or P2Y12 inhibitor therapy. Statistical analyses were performed using SPSS 28.

Results:
Antiplatelet use was present in 40.2% (278/692) of patients. These patients were older (68 vs. 62 years old, p<0.001) and had higher rates of hypertension (74.8% vs. 59.9%, p=0.001), type 2 diabetes (48.6% vs. 34.5%, p=0.001), coronary artery disease (23% vs. 7%, p<0.001), and heart failure (19.4% vs. 8.5%, <0.001). There was no significant difference in peak d-dimer levels between groups. Patients with in-hospital antiplatelet use had lower adjusted odds ratios of venous thromboembolism (0.62 [0.40-0.95], p=0.03), ICU admission (0.69 [0.49-0.96], p=0.03), and intubation (0.62 [0.44-0.88], p=0.006). There was no difference in all-cause mortality.

Conclusion:
Hospitalized COVID-19 patients treated with antiplatelet therapy had more cardiovascular risk factors, yet they had lower adjusted odds of venous thromboembolism, ICU admission, and intubation. Antiplatelet therapy during hospitalization with COVID-19 was not associated with lower mortality risk.
Abstract 273: Smooth Muscle Cell Specific Beclin-1 Deficiency Accelerates Aortic Aneurysms In Male And Female Normolipidemic And Hypercholesterolemic Mice
Authors: Frony Ana Clara , Javidan Aida , Jiang Weihua , Yang Lihua , Subramanian Venkateswaran
Published: October 2023

Background and Objective:
Ascending and abdominal aortic aneurysms (AAs) are permanent dilations of the aorta with 80% mortality after rupture. Vascular smooth muscle cell (SMC)-rich aortic media is disrupted in AAs. Autophagy is a process by which cell recycles their cytoplasmic materials for energy purposes under stress. Recent clinical studies highlighted that Beclin-1, an autophagy induction gene, is upregulated in AA tissues from patients. Using Angiotensin II (AngII) infusion model of AAs, we examined the functional role of SMC-derived Beclin-1 during AA development in normolipidemic and hypercholesterolemic mice.

Methods and Results:
Mice with inducible deletion of Beclin-1 in SMCs were produced by breeding male Acta2-Cre
ERT2
hemizygous mice female Beclin-1 floxed mice in normolipidemic and hypercholesterolemic (LDLR-/-) background. At 8 weeks of age, Beclin-1 x Acta2-Cre
ERT2
(Cre+) and non-Cre littermates (Cre-) mice were injected with tamoxifen (75 mg/kg, i.p.) for 5 days. After 2 weeks, Western blot analyses showed depletion of Beclin-1 protein in the aortic media from Cre+ mice compared to Cre- littermates. To study the role of SMC-Beclin-1, normolipidemic or hypercholesterolemic male Cre+ and Cre- (N=7 per group) mice were fed either chow or fat-enriched diet (21% fat; 0.15% cholesterol) for 5 weeks. After 1 week of diet feeding, mice were infused with saline or low dose of AngII (500 ng/kg/min) by osmotic mini-pumps for 4 weeks. SMC-Beclin-1 deficiency in either normolipidemic or hypercholesterolemic mice spontaneously but significantly accelerated ascending (Cre-: 1.2 ± 0.07 vs Cre+: 1.8 ± 0.04 mm;
P
<0.001), arch (Cre-: 1.1 ± 0.04 vs Cre+: 1.8 ± 0.03 mm;
P
<0.001), descending (Cre-: 1.1 ± 0.04 vs Cre+: 1.5 ± 0.04 mm;
P
<0.001), and abdominal (Cre-: 1.0 ± 0.02 vs Cre+: 1.5 ± 0.14 mm;
P
<0.001) aortic expansion. AngII-infusion had no additional influence on SMC-Beclin-1 deficiency accelerated aortic expansion. In addition, SMC-Beclin-1 deficiency significantly increased aortic medial hypertrophy and elastin breaks (P<0.001 Cre- vs Cre+) associated with complete loss of medial alpha-SMC actin positive area.

Conclusion:
These findings demonstrate that SMC-Beclin-1 plays a critical role in suppressing aortic aneurysmal formation in mice.
Abstract 277: The Effect Of Phosphodiesterase Inhibitors On Bapn Induced Aortic Dissection And Aneurysm
Authors: Tyagi Sam , Hubbuch Jacob , Franklin Michael , Howatt Deborah A , Sawada Hisashi , Lu Hong S , Daugherty Alan
Published: October 2023

Introduction:
Aortic dissection (AoD) is manifested by bleeding into the medial layers of the aortic wall. Aortic aneurysm (AA) is a dilation of the aorta. One fatal consequence of AoD and AA is rupture of the aortic wall. There are no pharmacological or therapeutic agents to decrease the risk of AA or to promote positive remodeling of the aorta after AoD. Phosphodiesterase inhibitors (PDEi) have diverse effects on cardiovascular diseases. There is experimental evidence that cilostazol (PDE3i) protects against AA, and alternatively there are clinical reports of sildenafil (PDE5i) inducing AoD. However, the role in PDEi’s in aortic pathology is still unclear. Our hypothesis is that sildenafil will exacerbate AoD, and cilostazol protects against AoD and AA. Our aim was to determine the effects of PDE3i and PED5i in β-aminoproprionitrile (BAPN) AoD and angiotensin II- (AngII) induced AAA models, respectively.

Methods and Results:
BAPN was administered to induce AoD formation in male C57BL/6J mice. We analyzed ascending and descending aortic tissue to determine if there was increased expression of known PDE related genes using RNA sequencing in mice administered either vehicle or BAPN for 7 days mice. We found increased PDE3 mRNA abundance in the ascending aorta by 19% (P=0.006), and increased of PDE5 mRNA abundance in the descending aorta by 21% (P=0.048). Subsequently, BAPN was administered in drinking water in 4-week-old male C57BL/6J mice for 12 weeks. There were three groups of mice: regular diet, and diets containing cilostazol or sildenafil. There had no difference on death rate due to aortic rupture between the groups. After termination, the ascending, descending and abdominal aortas were measured in situ, and there was no difference in aortic diameters. In a separate study, AngII osmotic pumps were inserted in male ApoE-/- mice to induced aortic aneurysm using the three there was no difference in the mortality rates. After 4 weeks of AngII infusion, the aorta was measured in situ, and there was no difference in aortic diameters.

Conclusion:
Inhibition of either PDE3 or PDE5 has no effects on either BAPN-induced AoD or AngII-induced AAA.
Abstract 283: Outcomes Among Heart Failure Patients Hospitalized For Aortic Dissection: Insight From The National Inpatient Sample
Authors: Casipit Bruce Adrian , Al-Sudani Hussein
Published: October 2023

Background:
Aortic dissection is a significant cause of morbidity and mortality among hospitalized patients. Previous studies suggested that heart failure (HF) is a significant complication of aortic dissection which oftentimes lead to poor outcomes due to delays in diagnosis and overall increase disease burden. However, there is paucity of data regarding the outcomes of hospitalized aortic dissection patients with comorbid HF.

Methods:
We utilized the National Inpatient Sample (NIS) Database of the years 2018-2020 in conducting a retrospective cohort study. All analyses were done through StataBE 17.0. Our outcomes of interests were risk for in-hospital mortality, acute kidney injury (AKI) and hospital length of stay (LOS) among hospitalized aortic dissection patients with comorbid HF.

Results:
A total of 149,026 hospitalized aortic dissection patients were identified in our study, of which 10.27% (n= 15,305/149,026) had HF. Among those, 6390 met our inclusion criteria. The overall in-hospital mortality among aortic dissection patients was 4.14% (n=6,170/149,026). Among those with concomitant HF, the mortality rate was significantly higher at 6.37%(n=407/6390) (p=0.00). After adjusting for possible patient and hospital level confounders, concomitant HF among aortic dissection patients was deemed to be an independent predictor of overall in-hospital mortality (aOR 2.21; 95% CI, 1.46-3.36; p=0.00) and it was found that systolic (aOR 2.47; 95% CI, 1.42-4.29; p=0.00) and diastolic HF (aOR 2.11; 95% CI, 1.08-4.13; p=0.03) were associated with higher in-hospital mortality compared to those with combined HF (aOR 1.48; 95% CI, 0.44-4.95; p=0.52). Further, concomitant HF increased the risk for the development of in-hospital AKI (aOR 3.16; 95% CI, 2.35-4.24; p=0.00) and longer LOS (aOR 4.16; 95% CI, 2.92-5.40; p=0.00), regardless of HF subtype.

Conclusion:
Our analysis showed that comorbid HF increased the risk for in-hospital mortality, risk for AKI and longer hospital LOS. On subgroup analysis, systolic and diastolic HF were associated with increased risk for in-hospital mortality and all three HF subtypes increased the risk for AKI and were associated with a longer hospital LOS.
Abstract 288: Development Of A Phenotypic Screen With 56 Marfan Syndrome Patient-derived Fibroblasts
Authors: Mieremet Arnout , van Andel Mitzi , Morcos Yousef , Sengle Gerhard , Reinhardt Dieter , Mulder Barbara , Zwinderman Aeilko , Groenink Maarten , Driessen Antoine , Micha Dimitra , de Waard Vivian
Published: October 2023

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the extracellular matrix gene coding for fibrillin-1 (FBN1). MFS leads to aortic aneurysms as key feature. Due to the high genetic diversity within the MFS patient population, genotype-phenotype predictions on disease progression and response to therapy remain challenging. Furthermore, current preclinical murine models represent only a subset of FBN1 mutations, which limits translational studies. In this study, we developed a preclinical model using primary cells of MFS patients that represent genetic and clinical variability in order to develop improved drug strategies. Human primary fibroblasts were isolated from skin biopsies derived from MFS patients (n=56) participating in the COMPARE trial. Human aortic smooth muscle cells (AoSMCs) were isolated from aortic tissue derived from MFS patients undergoing prophylactic aortic surgery (n=4). Long-term cultures in 384-well plates were assessed for the extracellular matrix and disease associated pathways. Analysis of all MFS fibroblasts showed either a reduced or abolished fibrillin-1 extracellular matrix network. Moreover, substantially reduced secretion of fibrillin-1 and asprosin in the culture medium was measured, as compared to healthy control fibroblasts. MFS fibroblasts with an abolished fibrillin-1 matrix were associated with patients with more rapid aortic root diameter growth. Exploring disease-relevant pathways revealed reduced nuclear localization of transcription factor KLF4 in MFS fibroblasts, which associated with the level of fibrillin-1 matrix. As a marker for myofibroblasts, α-smooth muscle actin (ASMA) staining was increased in these MFS fibroblasts. These findings were validated in AoSMC cultures, showing similar MFS fibrillin-1 matrix, nuclear KLF4, and ASMA expression profiles. In conclusion, phenotypic screening of patient-derived cells contributes to improved understanding of disease development and is valuable as preclinical tool for risk assessment and drug development for MFS and related conditions.
Abstract 291: Thrombin-activated Platelet Releasate Reduces Myoblast Fusion Capacity In Muscle Cells Affected By Peripheral Artery Disease
Authors: Assaf Edwyn , Xie Bowen , Shea Susan M , Steinman Richard , Sachdev Ulka
Published: October 2023

Introduction:
In the absence of injury, direct interactions between platelets (Plt) and skeletal muscle myoblasts (Mb) are limited. However, ischemic muscle injury increases vascular permeability resulting in potential interactions between Mb and Plt. In PAD, Mb have poor differentiation capacity and Plt have increased activation. We hypothesize that Plt activation directly deters myogenic potential of ischemic Mb in PAD.

Methods:
Ischemic and perfused muscle was obtained during amputations for PAD. Mb were isolated using sequential plating and cell sorting. Mb from healthy donors (PAD-) were purchased. Isolated Plt from healthy donors without PAD were resuspended in physiologic and supraphysiologic concentrations, with some being thrombin-activated (TA) under shear conditions. Acellular Plt releasate (PR) from resting or TA Plt were added to proliferation or differentiation media (10% volume) and incubated 5 days with Mb. Proliferation was measured by CellTiter Glo. Multinucleated cells positive for myosin heavy chain were counted to obtain myocyte fusion index (MFI), a surrogate for differentiation. Resting and TA Plt were aggregated with Mb under shear conditions and their binding was measured by immunofluorescence.

Results:
Proliferation was higher in ischemic PAD Mb exposed to supraphysiologic TAPR compared to those in proliferation media (p=0.0022) or physiologic TAPR (p=0.0005) (Fig 1A - N=4). However, TAPR treatment also significantly and consistently decreased MFI across cell groups in a dose-dependent manner (Fig 1B). In the presence of thrombin and shear stress, Plt-Mb were prominently expressed (Fig 1C).

Conclusion:
Released mediators from TA Plt enhance Mb proliferation at the expense of terminal differentiation, potentially leading to defective muscle repair. Inhibiting the interactions between Plt and Mb is a potential target to improve response to ischemic injury, reduce myopathy and a consideration for best medical therapy for PAD.
Abstract 295: Anti-coagulation Choice in Patient with Atrial Fibrillation and Acute Ischemic Stroke
Authors: Ho Huy , Eddib Ahmed , Ahmed Mohammed , Sanchez Saramarina , Pasupuleti Suman , Nerella Nishant K
Published: October 2023

To prevent embolic stroke, patients with atrial fibrillation are risk stratified, and started on anticoagulation accordingly. This poses a management challenge when these patients present to the hospital with ischemic stroke. Often, anticoagulation is held due to risk of hemorrhagic conversion. The optimal choice of anticoagulation to reduce the risk of hemorrhagic transformation of ischemic stroke in patients with atrial fibrillation is poorly studied. This study evaluates the risk of hemorrhagic conversion between different anticoagulation therapies in patients with atrial fibrillation who present to the hospital for ischemic stroke. Retrospectively, 8,596 acute stroke patients with atrial fibrillation were studied. Patients were subdivided into three groups depending on their home anticoagulation: apixaban, rivaroxaban, and warfarin. Binary logistic regression was used to predict the likelihood of an outcome for a binary variable. The outcome measured in this study was incidents of hemorrhagic conversion and ICU admission. Patients who were taking apixaban and rivaroxaban were less likely to have hemorrhagic conversion compared to the warfarin group (Odds Ratio 0.608 [0.467, 0.493], p<0.0001 and 0.734 [0.054, 0.493] p=0.0471). There was no significant difference when comparing the incidence of hemorrhagic conversion between apixaban and rivaroxaban. Patients who were taking apixaban were 0.787 times as likely to be admitted to the ICU compared to rivaroxaban group [0.679, 0.911], p=0.0003. Patient who were taking apixaban were 0.758 times as likely to be admitted to the ICU compare to Warfarin [0.662, 0.868], p= <0.0001. This study suggests that hemorrhagic conversion risk is higher in the warfarin group compared to apixaban and rivaroxaban groups. Additionally, patients who were taking apixaban experienced significantly less ICU admissions. Because this is a retrospective study, we cannot conclude a causative relationship. However, based on the results of this study, it is reasonable to consider alternatives to warfarin (such as apixaban and rivaroxaban) in patients with atrial fibrillation who present with ischemic stroke.
Abstract 296: Role Of Endothelial Cell-specific Knockout Of The Aryl Hydrocarbon Receptor In Ischemic Limb Myopathy In Mice Subjected To Hind Limb Ischemia
Authors: Palzkill Victoria R , Tan Jianna , Morcos Juliana , Ryan Terence E
Published: October 2023

Introduction:
Chronic kidney disease (CKD) is a risk factor for peripheral arterial disease (PAD) that accelerates disease evolution and worsens limb outcomes, however the molecular mechanisms linking the pathobiologies are ill-defined. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, has been previously shown to regulate angiogenesis. Notably, CKD results in the accumulation of tryptophan-derived metabolites that are endogenous ligands for the AHR. On this basis, we tested the hypothesis that endothelial cell-specific knockout of the AHR would enhance limb perfusion recovery and capillarity in mice with CKD subjected to hindlimb ischemia.

Methods:
Twelve-week-old conditional AHR knockout mice (AHR
fl/fl
, Cre-negative) and endothelium-specific AHR knockout mice (AHR
ecKO
) were fed 0.2% adenine to induce CKD or casein control diet (n=10/group/sex). Femoral artery ligation (FAL) was performed on the left limb using the right limb as a non-ischemic control. Limb perfusion was measured using laser Doppler flowmetry. Skeletal muscle function was measured using nerve stimulus. Gastrocnemius mitochondrial respiratory function and H
2
O
2
production were assessed.

Results:
Regardless of genotype, mice with CKD had significantly lower renal function. Interestingly, male AHR
ecKO
mice with CKD displayed a significant increase in percentage of perfused capillaries within the ischemic tibialis anterior muscle (AHR
ecKO
73.41% ± 4.80% vs AHR
fl/fl
64.75% ± 8.325%;
p= 0.0074
) and improved laser Doppler perfusion recovery in the paw and gastrocnemius muscle compared to male AHR
fl/fl
mice with CKD. AHR
ecKO
mice had a larger mean myofiber size (
p=0.0258
), although muscle weight and contractile function were not different. Mitochondrial respiratory capacity was significantly higher
(p=0.0085)
in AHR
ecKO
than AHR
fl/fl
mice.

Conclusion:
In mice with CKD, endothelium-specific deletion of the AHR improved blood perfusion and increased myofiber size in the ischemic limb muscle. These changes conferred improvement in muscle mitochondrial function, but not muscle strength. Interestingly, these changes were observed in male, but not female, mice.
Abstract 298: Roles Of Tet2 In Vascular Regeneration For Peripheral Artery Disease
Authors: Lee Bob S , Liu Renjing
Published: October 2023

Peripheral artery disease (PAD) is the manifestation of atherosclerosis in the extremity, causing blockage of blood vessels that reduces blood flow to the lower limb (ischemia). End-stage PAD patients require limb amputation, with increased risk of morbidity and mortality from heart attacks. Although PAD affects >200 million individuals worldwide, surgery remains the only endpoint option that itself is associated with significant morbidity and mortality. Novel treatment for PAD is therefore urgently needed. Despite vascular smooth muscle cells (VSMC) being essential for normal vascular formation and repair, their role in PAD pathogenesis has been largely ignored. Our lab has long been specialised in studying VSMC biology, and was the first to identify a DNA demethylase, Tet2, as a master regulator of VSMC functions (
Liu et al, Circulation. 2013
). We found without Tet2, VSMCs form pathological and weakened arteries that lead to restenosis, the precursor of atherosclerosis. Given the vital role VSMC in vessel formation, we

hypothesise

that restoring Tet2 expression to ischaemic tissues is critical for promoting angiogenesis, blood flow recovery and to prevent tissue caused by PAD.

Using a VSMC-specific Tet2 knockout (VSM-
Tet2
KO
) mouse line generated by the Liu lab at VCCRI, we studied the vasculo-protective role of TET2 in PAD by mimicking PAD through femoral artery ligation. Our preliminary data demonstrates: (1) severe tissue necrosis and amputation in the knockout mice following surgery that is not observed in controls (2) Loss of Tet2 reduces blood flow to the foot as suggested by laser doppler measurements. (3) Significantly retarded neovascularisation in ischemic muscle regions displayed by micro-computer tomography scans .
These are the first data to demonstrate Tet2’s role in PAD.
Abstract 305: Dietary β-carotene Accelerates Atherosclerotic Resolution By Promoting Treg Expansion In The Atherosclerosis Lesion
Authors: Blanco Cirer Amparo H , Pinos Ivan , Coronel Johana , Albakri Asma'a , McQueen James , Molina Donald , Sim JaeYoung , Fisher Edward , Amengual Jaume
Published: October 2023

Forkhead box P3 (
FoxP3
)
+
regulatory T cells (Tregs) mediate atherosclerosis resolution and regression by promoting an anti-inflammatory environment in the lesion. FoxP3 is upregulated by retinoic acid, the active form of vitamin A, but whether vitamin A status influences atherosclerosis resolution remains unexplored. We hypothesized that β-carotene, the main vitamin A precursor in human diet, accelerates atherosclerosis resolution by upregulating FoxP3 expression to promote Treg expansion. We induced atherosclerosis in

FoxP3
EGFP

mice fed Western diet control (WD-Control) and injected with an anti-sense oligonucleotide targeting the LDLR (ASO-LDLR) for 16 weeks. After this period, we harvested a subset of mice as Baseline, while the remaining mice underwent atherosclerosis resolution upon the interruption of ASO-LDLR infusions to recover LDLR expression and normalize plasma lipids for a period of three weeks. During this time, mice either continued on the same WD-Control or were switched to a Western diet supplemented with 50 mg of β-carotene/kg of diet (WD- β-carotene). To examine the implication of Tregs, we injected a subset of WD-β-carotene with anti-CD25 (WD-β-carotene + anti-CD25), while WD-Control and a second subset of WD- β-carotene mice were injected with IgG control. We estimated atherosclerosis resolution by quantifying macrophage and collagen contents in the lesions present at the level of the aortic root. WD-β-carotene-fed mice injected with IgG showed a reduction in macrophage content accompanied by an increase in collagen in comparison to all the remaining groups, while WD-β-carotene + anti-CD25 lesions resembled those present in WD-Control mice. We examined Treg the relative number of CD25
+
FoxP3
+
and CD25
-
FoxP3
+
cells in the spleen, blood, and the lesion. CD25
+
FoxP3
+
numbers decreased in response to anti-CD25 infusions in all the tissues examined. Dietary β-carotene favored an increase of 269% and 653% in CD25
-
FoxP3
+
Tregs in comparison to the Baseline of WD-Control groups, respectively. These effects occurred independently of changes in plasma lipids or changes in body weight. Our data show that β-carotene supplementation results in Treg expansion and acceleration of atherosclerosis resolution by increasing Treg number.