bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 142: Actin Bundling Protein L-plastin Regulates Platelet Production, Spreading and Thrombosis
Authors: Guo Li , Jacob Shancy , Manne Bhanu Kanth , Bhatlekar Seema
Published: October 2023

Physiologically resting platelets circulate in the blood in discoid shape, supported by thick F-actin cytoskeleton. Upon vascular injury or during thrombosis, platelets become activated, extend filopodia, spread and expand surface area, aggregate and form thrombi, which process also relies on quick F-actin disassembly and reassembly. Although several actin-binding proteins in platelets have been identified, largely remain unknown. L-plastin is one actin bundling protein that has not been studied in platelets. It is predicted with two C-terminal actin binding domains that can cross-link actin filaments into thick bundles. It is critical for immunity, adhesion, and migration in leukocytes. We recently found it is critical in maintaining megakaryocyte cytoskeleton stiffness and inhibits platelet production. In our current study, we aim to investigate the role of L-plastin in platelet function and thrombosis
in vivo
using mice with L-plastin deficiency (KO, Lcp1
-/-
) and littermate controls (WT) of both genders. When stimulated with different concentrations of thrombin or 2MeSADP, washed platelets from both WT and KO mice expressed JONA and P-selectin without significant difference as detected by flow cytometry. This suggests that L-plastin does not alter early inside-out signaling pathway. When we measured the platelet spreading on immobilized fibrinogen, KO platelets showed significantly enlarged actin spreading area per platelet as compared to the WT (n=3, P<0.05), suggesting that L-plastin inhibits platelet spreading through outside-in signaling. Consistently, platelets from KO mice also have accelerated clot retraction. In addition, microfluidics assay using DiOC6 labeled whole blood from KO mice showed accelerated adhesion to both collagen and fibrinogen immobilized matrixes under a shear stress of 200/s (n=4, p<0.05). In a collagen/epinephrine induced pulmonary embolism (PE) model KO mice showed significantly accelerated mortality (n=8-9, P<0.01), suggesting an inhibitory role of L-plastin in thrombosis in vivo. In summary, we found an inhibitory role of L-plastin in platelet spreading
in vitro
and thrombosis
in vivo
.
Abstract 147: Spatially Resolved Metabolites In Stable And Unstable Human Atherosclerotic Plaques Identified By Mass Spectrometry Imaging
Authors: Seeley Erin , Liu Zhipeng , Yuan Shuai , Stroope Chad , Cockerham Elizabeth , Rashdan Nabil , Delgadillo Luisa , Finney Alexandra , kumar Dhananjay , Das Sandeep , Razani Babak , Liu Wanqing , Rom Oren , Pattillo Christopher B , Yurdagul Arif
Published: October 2023

Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. Acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.
Abstract 149: Manipulating Bile Acid Production Using Genome Editing To Modulate Cardio-metabolic Disease
Authors: Jarrett Kelsey E , Tarling Elizabeth J , Vallim Thomas A
Published: October 2023

Cardiovascular disease (CVD) remains the leading cause of death in the United States, in part due to consumption of a high cholesterol Western diet. Absorption of dietary cholesterol is facilitated by detergents, called bile acids, which are produced in the liver. Bile acids are a functionally diverse group of molecules with different capacities to affect lipid absorption. We hypothesize that specific changes in the bile acid pool will change cholesterol absorption and alter development of CVD.

To alter the bile acid pool, we disrupted three enzymes (
Cyp7a1
,
Cyp8b1
and
Cyp2c70
) using liver-directed AAV-CRISPR.
Cyp7a1
catalyzes the rate-limiting step of bile acid synthesis, whereas both
Cyp8b1
and
Cyp2c70
mediates production of specific bile acids (cholic acid and muricholic acid respectively). Using UPLC-MS/MS, we show that loss of CYP7A1 in adult mice reduces the size of the bile acid pool while loss of CYP8B1 or CYP2C70 alters the composition without changing total bile acid levels. To study atherosclerosis, we co-disrupted
Cyp7a1, Cyp8b1
and
Cyp2c70
concurrently with
Low Density Lipoprotein Receptor
(
Ldlr
) to induce hypercholesterolemia on a Western diet for 20 weeks. Loss of all three bile acid enzymes resulted in different atherosclerosis outcomes. Loss of CYP7A1 resulted in modest protection from atherosclerosis. Using a novel GC-MS-based technique, we demonstrate these mice have reduced cholesterol absorption. Loss of CYP8B1 caused modest reductions in plasma cholesterol and moderate atheroprotection, but did not alter cholesterol absorption. Finally, loss of CYP2C70 led to an early onset of hypercholesterolemia, which we hypothesize is due to enhanced cholesterol absorption, resulting in accelerated development of atherosclerosis.

We hypothesize that these differences in atherosclerosis observed with different bile acid profiles are due to systemic changes in cholesterol metabolism that are driven, in part, by changes in cholesterol absorption. Our studies suggest specific changes in bile acids have the potential to become a novel precision treatment for cardio-metabolic diseases.
Abstract 161: Ablation Of Intracellular Lipolytic Inhibitor G0S2 Promotes Vascular Lipolysis And Alleviates Diet-induced Atherosclerosis
Authors: Chen Yongbin , Johnson Scott M , Burr Stephanie D , Povero Davide , McMahon Cailin E , Liu Jun
Published: October 2023

Adipose tissue plays a central role in the regulation of plasma triglyceride (TG) and fatty acid (FA) levels, whose elevation is associated with atherosclerotic cardiovascular disease (ASCVD). Circulating TGs are hydrolyzed by lipoprotein lipase (LPL), releasing FAs that are taken up by adipocytes. Conversely, lipolytic action of adipose tissue triglyceride lipase (ATGL) mobilizes intracellular TG storage, supplying FAs via circulation for utilization by other tissues. However, how intracellular lipolysis influences intravascular lipolysis and atherosclerotic development remains largely unknown. Herein, the current study aimed to explore the role of G0S2, a selective protein inhibitor of ATGL, in this particular context. When treated with a Western diet and antisense oligonucleotides (ASOs) targeting LDLR, mice with a global ablation of G0S2 exhibited a significantly reduced lesion formation in aorta and brachiocephalic artery than their wild type (WT) littermates. Despite of little to no impact on hypercholesterolemia, total plasma and lipoprotein-specific TG content were normalized in G0S2
-/-
mice. While it did not elicit any effects on intestinal lipid adsorption and hepatic TG secretion, the G0S2 ablation resulted in considerable elevation of LPL activity and protein levels in both plasma and adipose tissue. Accordingly, these G0S2
-/-
mice showed a greatly improved TG clearance upon an oral lipid challenge. Interestingly, transplantation of G0S2
-/-
adipose tissue increased circulating LPL in the WD-treated WT mice, which subsequently displayed a profound reduction of plasma TG levels as well as alleviation of hepatic steatosis. By studying adipose tissue explants and primary adipocytes, we observed that the G0S2 ablation led to markedly improved insulin sensitivity, resulting in increased FOXO1 phosphorylation and decreased expression of the LPL inhibitor ANGPTL4. Together, our data indicate that in the absence of G0S2, upregulation of adipose tissue LPL plays an important role in the clearance of circulating TGs and alleviation of diet-induced hypertriglyceridemia and atherosclerosis. We thus conclude that adipocyte G0S2 acts as a critical switch of both intracellular and vascular lipolysis.
Abstract 162: Multiancestry GWAS And Functional Genomics Analysis Of HNF1A Locus To Define Novel Pathogenic Mechanism For Ischemic Stroke
Authors: Hiwase Harsh , Yue Yunshan , Hoffman Eric K , Wu Haodi , Barndt Robert J , Chan Stephen Y , Hyacinth Hyacinth I. , Sun Wei
Published: October 2023

Background:
Ischemic stroke (IS) disproportionately affects populations of African descent due to genetic predispositions. A multiancestry Genome-wide Association Study (GWAS) identified non-coding SNPs in HNF1A locus highly associated with IS, exclusively to African ancestry. Analysis of this ancestry-specific genomic architecture can reveal novel pathogenic mechanism for IS.

Methods:
The IS-associated SNPs in HNF1A locus were analysed. Electrophoretic Mobility Shift Assay (EMSA) identified functional SNPs (fSNP) that allele-specifically bind to nuclear proteins from human vascular endothelial cells (ECs). Proteomics identified transcription factors (TFs) binding to fSNPs. siRNA knockdown was conducted to determine the function of TFs on HNF1A expression. The effects of stroke-like condition hypoxia on TFs and HNF1A were studied in various vascular cells.

Results:
GWAS-revealed non-coding tag SNP rs55931441 in HNF1A locus was associated with IS risk exclusively in African population. The frequency of risk allele A of tag SNP was 3.8% in African but absent in other races, supporting the causative role of this locus in determining the genetic risk of IS. EMSA revealed no nuclear protein binding to rs55931441, suggesting it is unlikely an fSNP with TF-mediated regulation on target genes. We expanded the screening to 7 haplotype SNPs in linkage disequilibrium with R
2
>0.8. Among them, rs144534697 presented with allele-imbalanced nuclear protein binding. Proteomics revealed ILF3, a vascular function-related TF, binds preferentially to non-risk allele C. Functional studies showed that ILF3 regulates HNF1A as a repressor. Less binding of ILF3 to risk allele A results in a loss of repression to HNF1A and augments its function as vascular pathogenic factor. Furthermore, vascular stressor hypoxia downregulated ILF3 and upregulated HNF1A in vascular ECs, smooth muscle cells and fibroblasts.

Conclusion:
Our functional genomics analysis of disease-associated SNPs identified by multiancestry GWAS offers a novel mechanistic approach for diseases with ancestry prominence. The newly identified ILF3-HNF1A pathway may serve as a general mechanistic explanation for the pathogenesis of IS, given their response to vascular stressor hypoxia.
Abstract 166: Memory Cd8 T Cells Contribute To Atherosclerosis In Aged Mice
Authors: Tyrrell Daniel J , Goldstein Daniel R
Published: October 2023

Aging is a strong risk factor for atherosclerosis and aging significantly impacts immune cell populations and function. Immune cells are a major contributor to development of atherosclerosis; however, most preclinical atherosclerosis studies are performed in young mice. The role of aging on CD8
+
T cells during atherogenesis remains unclear. Here we determined that depletion of CD8
+
T cells attenuated atherogenesis in aged, but not young, mice. Furthermore, adoptive transfer of splenic CD8
+
T cells from aged mice which contain a high proportion of memory T cells significantly enhanced atherosclerosis in young

Cd8
-/-

recipient mice compared with transfer of CD8
+
T cells from young donor mice. Finally, we characterized T cells in healthy and atherosclerotic young and aged mice by single-cell RNA-sequencing (scRNAseq) and T cell receptor sequencing (TCR-seq). We found that with aging effector memory, central memory, and granzyme K
+
effector memory CD8
+
T cells accumulate within atherosclerotic plaques, and aging induces transcriptomic changes related to CD8
+
T cell activation, migration, cytotoxicity, and exhaustion. Overall, our study identifies memory CD8
+
T cells as potential therapeutic targets for reducing atherosclerosis in older adults.
Abstract 216: Macrophage Efferocytosis Is Controlled By Epigenetic Modifications Mediated By RBPJ
Authors: Sadaf Samreen , Zhang Xinyi , Dutta Partha
Published: October 2023

Efferocytosis, phagocytic clearance of apoptotic cells, is essential for inflammation resolution and tissue homeostasis. A defect in efferocytosis is associated with advanced atherogenesis leading to the necrotic formation which can result in plaque rupture and abrupt thrombotic cardiovascular diseases. We observed that the recombination signal binding protein for immunoglobulin kappa J region (RBPJ), a transcription factor that normally involves in the canonical Notch signaling, was highly expressed by inflammatory macrophages. Our subsequent experiments revealed that although RBPJ did not affect inflammation, the transcription factor promoted apoptotic cell clearance by atherosclerotic plaque, alveolar, peritoneal, and bone marrow-derived macrophages. Inhibition of γ-secretase, which facilitates Notch intracellular domain release and RBPJ-mediated gene expression, significantly reduced efferocytosis. RNA sequencing analysis of RBPJ-deficient macrophages revealed Stard13 and Arsg as possible mediators of RBPJ-mediated efferocytosis. CUT&RUN and ChIP-qPCR experiments revealed that RBPJ diminished H3K9me3, a heterochromatin marking suppressing gene activity, on the Stard13 and Arsg promoters. Stard13 inhibited and activated Rho and RAC GTPases, respectively, promoting actin polymerization and efferocytosis. In sum, our findings indicate that RBPJ controls epigenetic modification of the Stard13 and Arsg promoters, encouraging apoptotic cell clearance.
Abstract 219: Translational Profiling Of Embryonic And Adult-derived Macrophages Exposed To Hypoxia
Authors: Yarovinsky Timur O , Wilcox Nicholas S , Pandya Prakruti , Ramgolam Vinod S , Moro Albertomaria , Wu Yinyu , Nicoli Stefania , Hirschi Karen K , Bender Jeffrey R
Published: October 2023

Cardiac-resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Post-transcriptional regulation at the levels of mRNA stability and translation may override transcriptional signals in response to hypoxia. We hypothesized that profiling of actively translated mRNAs in BMDM and FLDM after exposure to hypoxia would uncover functional differences in gene expression between these macrophage subsets. We used a translating ribosome affinity purification (TRAP) assay coupled with RNA-seq and validation by qRT-PCR, flow cytometry and immunoblotting to identify translated transcripts in macrophages exposed to hypoxia. We identified non-overlapping transcripts with increased translation in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2). Induction of most transcripts, including those encoding enzymes associated with glycolysis, was accompanied by increased translation. However, induction of the mRNA encoding complement receptor C5ar1 was accompanied by reduced translation efficiency. Furthermore, we determined the effects of deletion of the RNA-binding protein HuR on induction and translation of transcripts in response to hypoxia. Overall, these findings define translational differences in macrophage subset gene expression programs, highlighting potential macrophage-directed therapeutic targets in ischemic myocardium.
Abstract 226: Myeloid Deletion Of Rage Induces Insulin Resistance By Impairing Macrophage And Adipocyte Crosstalk In Obese Adipose Tissues
Authors: Arivazhagan Lakshmi , Gugger Paul F , Vakil Raveena , Ariyaratne Gallage H. D. Nipun , Ramasamy Ravichandran , Schmidt Ann Marie
Published: October 2023

Introduction:
Obesity, a major global health problem, is often associated with insulin resistance (IR). We previously showed that global or adipose tissue-specific
Ager
deletion protected from high fat diet (HFD) induced obesity and IR. We reported that upon HFD feeding, myeloid-specific deletion of
Ager
deleted (MDR) significantly impairs insulin sensitivity without differences in body mass vs. myeloid-
Ager
expressing controls. These adverse effects of myeloid-
Ager
deletion (body mass, IR) were rescued by simultaneous deletion of adipocyte
Ager.
The expression of inflammatory genes was significantly higher in the epididymal adipose tissue (eWAT) of MDR vs. controls. Here, we probed for the crosstalk mechanisms between macrophages and adipocytes in obese eWAT using Single nuclei RNA-sequencing technology.

Methods:
We generated mice with myeloid-specific (MDR) LyzMCre(+/+).Ager

flox/flox

and adipocyte and myeloid-specific (Double Knockouts (KO)) AdipoQCre(-/+)LyzMCre(+/+).Ager

flox/flox

deletion of
Ager
; LysMCre mice (Cre control) were controls. Mice were fed HFD (60% kcal/fat) for 3 months and assessed for body mass and glucose and insulin tolerance. After 12 weeks, eWAT were isolated and nuclei sequenced using 10x Genomics’ Single Nuclei RNA-sequencing technology and subjected to analyses.

Results:
Analysis of the snRNA-sequencing data showed a potential crosstalk between macrophages and adipocytes, which was impaired in the eWAT of HFD fed MDR mice. The significantly differentially regulated pathways between the groups identified in adipocytes and macrophages include: Autophagy, Lipid metabolism, Oxidative phosphorylation and Glycolysis.
In vivo,
the eWAT of MDR mice on HFD exhibited: larger adipocyte size with impaired autophagy of lipid filled adipocytes and fewer crown like structures vs. controls.
In vitro,
oxidative phosphorylation and lipid metabolism were impaired in the MDR macrophages vs control upon palmitate/oleate treatment, a mimic of HFD.

Conclusion:
These experiments reveal for the first time protective roles for RAGE in macrophages in HFD feeding; unveiling a novel role for RAGE in adipocyte/macrophage intrinsic and cross-talk communications in obesity and IR.
Abstract 231: Classification Of Sitosterolemia-associated Mutations In ABCG5 And ABCG8
Authors: Chauhan Isha , Poole Brittney , Zelows Mikala , Walton Bonnie , Mutch Megan E , Graf Gregory A
Published: October 2023

Background:
Sitosterolemia is a rare, recessive form of familial hypercholesterolemia and is caused by mutations in ABCG5 and ABCG8. ABCG5 and ABCG8 form an obligate heterodimer that promotes biliary secretion and opposes intestinal absorption of cholesterol and phytosterols. Whole genome and exome sequencing has revealed over 2000 pathogenic or likely pathogenic loss of function mutations of ABCG5 and ABCG8. Missense mutants in clinically confirmed Sitosterolemia are of particular interest for their potential to reveal structure-function relationships in ABCG5/ABCG8. Recent advancements demonstrate efficacy of two classes of compounds that enhance activity (Potentiators) or rescue (Correctors) disease-causing mutants of multiple ABC transporters. This presents the opportunity to classify missense mutants of ABCG5 and ABCG8 and examine their rescue by these compounds.

Methods:
We mapped 57 missense mutants of ABCG5 and ABCG8 onto the primary protein structure of each half transporter. Mutants were generated through site-directed mutagenesis and confirmed with Sanger sequencing. Plasmids encoding normal and mutant proteins were co-transfected into human Huh-7 hepatocytes and analyzed for maturation by the appearance of the higher molecular weight forms of each glycoprotein in SDS-PAGE. Subcellular localization was determined by immunofluorescence microscopy and cell-surface biotinylation.

Results:
Mutations in ABCG5 and ABCG8 cluster in the nucleotide binding domain and transmembrane spanning domain of each half-transporter. Of the mutants analyzed, 57% were maturation-incompetent and potentially capable for rescue by Correctors. Most maturation-competent mutants were located on the cell surface suggesting they may be amenable to enhanced activity by Potentiators. However, several maturation-competent mutants displayed intracellular staining patterns and are unlikely to benefit from either class of compounds.

Conclusion:
Formation and trafficking of the ABCG5 ABCG8 heterodimer provides insight into ABC transporters and diseases associated with loss of function alleles. Partial rescue of disease-causing mutants within ABCG5 and ABCG8 may be feasible with regulators of proteostasis and small molecule chaperones.
Abstract 263: Prothrombotic State Exists In Long-COVID Even After Mild Acute Infection
Authors: Patil Gokul , Eustes Alicia S , Ahmed Azaj , Alpana Garg , Dayal Sanjana
Published: October 2023

Long-term consequences of COVID-19 (long-COVID) are becoming a significant burden to health care. Many people remain symptomatic for months or years after recovery from COVID-19, including some who had only mild symptoms during acute infection. Sustained increases in pro-thrombotic markers have been reported in patients who were initially hospitalized with acute COVID-19. However, whether patients with less severe acute infection develop a prothrombotic-state during long-COVID is unclear. Our
objective
was to measure prothrombotic factors in long-COVID patients with mild disease (non-hospitalization) during acute infection. We recruited 47 consecutive adult patients with long-COVID symptoms seen in the Post-COVID clinic. All patients had a confirmed COVID-19 diagnosis by RT-PCR or antibody test during acute infection and had at least one of the symptoms of long-COVID per WHO criteria. Twenty asymptomatic healthy subjects not previously diagnosed with COVID-19 were recruited as controls. Markers of platelet activation and platelet-neutrophil aggregates (PNA) were quantified using whole blood flow cytometry. Serum levels of cell-free DNA (cfDNA) and citrullinated histones (H3Cit) and thrombin generation potential in plasma was measured. At the time of recruitment (6 weeks to 2 years after acute infection), the most common post-COVID symptoms included fatigue, chest pain, shortness of breath, headache, and dizziness. In whole blood, activation with TRAP caused increased surface expression of P-selectin without inducing activation of integrin αII
b
β
3
on platelets (P<0.001 and P=0.5 respectively vs control). Concurrently, there was increased PNA in the long-COVID cohort (P<0.01 vs. control). H3Cit and cfDNA were elevated in long-COVID (P<0.01 and P<0.05 respectively vs. control) and thrombin generation potential was increased, reflected by shorter lag-time, higher peak thrombin and increased velocity index (P<0.01, P<0.05 and P<0.0001 respectively vs control). Within the long-COVID cohort, these markers were not different for age, sex, BMI or time since infection. We conclude that a prothrombotic state persists for months to years in long-COVID patients who exhibited mild disease at the time of initial infection.
Abstract 265: Coronavirus Infection Induced Interferon Signaling Promotes Atherosclerotic Plaque Vulnerability
Authors: Zhang Hanming , Zhang Xinbo , Jovin Daniel , Sun Jonathan , Chaube Bal K , Esplugues Enric , Suarez Yajaira , Fernandez-Hernando Carlos F
Published: October 2023

Atherosclerosis is a chronic inflammatory vascular disease characterized by the formation of lipid-rich plaques inside arteries. The rupture of vulnerable plaques and consequent acute complications, such as myocardial infarction and stroke, are the leading causes of morbidity and mortality worldwide. It is known that pre-existing cardiovascular comorbidities pose a risk for severe outcomes in COVID-19, though the underlying pathophysiology is not fully understood. To investigate the role of coronavirus infection in established atherosclerotic plaques, we developed an atherosclerotic model of natural murine beta coronavirus infection with mouse hepatitis virus strain-A59 (MHV-A59). We fed
Ldlr-/-
mice a Western diet for three months prior to MHV-A59 intranasal inoculation and assessed plaque morphology at day post infection (dpi) 7 and dpi 30. Respiratory infection in mice recapitulated many aspects of COVID-19 pathophysiology. More strikingly, virus was detected in atherosclerotic aorta and induced multiple features of vulnerable plaques, a prerequisite for plaque rupture, characterized by thinner fibrous caps, less collagen content, and larger necrotic cores. The phenotype was persistent at dpi 30 when the mice were fully recovered. To further explore the underlying mechanism, we performed single-cell RNA sequencing (scRNA-seq) on infected aortas isolated at dpi 7. Our data revealed that interferon γ was exclusively upregulated in activated T cells and elicited various responses across multiple cell populations within infected aortas. The drastic responses that potentially contribute to plaque instability include activation of a more proinflammatory endothelial cell phenotype, downregulation of TGF- β mediated fibroblast to myofibroblast transition, and enhanced degradation of extracellular matrix and smooth muscle cell senescence. Moreover, macrophages in infected plaques activate inflammasomes, release IL-18, and undergo pyroptosis, thereby further contributing to the hyperinflammatory state of the atherosclerotic plaques. Taken together, our data shed light on a molecular mechanism for coronavirus infection induced plaque instability and provide a small animal model to explore future therapeutics.
Abstract 266: The Outcome Of Aortic Dissection In Patients With Protein Energy Undernutrition, A Nationwide Analysis.
Authors: Amoah Joseph , Fatuyi Michael , Somoza-Cano Francisco , Noureldin Sara , Dekowski Sammir
Published: October 2023

Objective:
Acute Aortic Dissection is a life-threatening emergency associated with increased morbidity and mortality. Data on the effect of protein energy undernutrition(PEU) on the clinical outcome of patients admitted for aortic dissection are limited.

Methods:
Using the 2019 National Inpatient Sample Database, NIS was searched for adult patients with aortic dissection as the principal diagnosis with or without PEU as a secondary diagnosis using ICD-10 codes. The primary outcome was Inpatient mortality with secondary outcomes being acute kidney injury, cardiogenic shock, CVA, myocardial infarction, cardiac tamponade, acute hypoxic respiratory failure, intraoperative and post-procedure complications, and acute mesenteric ischemia

Results:
17290 patients were admitted for acute aortic dissection. Of those, 8% had a history of PEU. Patients with PEU were older when compared with patients without PEU (mean age, 66.1 95% CI, 64.5-67.8 vs 63.8, 95% CI, 63.2- 64.3). There was no statistically significant difference in in-hospital mortality (p=0.1852) and acute mesenteric ischemia (p=0.4808). However, Patients with PEU had increased rate of acute kidney injury (p<0.0001), myocardial infarction(p=0.0171), cardiogenic shock (p<0.0001), CVA (p<0.0001), cardiac tamponade (p=0.0171), acute hypoxic respiratory failure (p<0.0001) and intraoperative and post-procedure complications (p=0.001). Patients with PEU also had increased length of hospital stay (23 days, 95% CI 21-25 VS 8 days, 95% CI 8 -9) and total hospital charges ($ 646967.2, 95% CI $533697.8 - $771236 VS $234639.3, 95% CI $218237 - $251040.8)

Conclusion:
This study shows no significant difference in mortality between patients with PEU admitted for aortic dissection compared to the general population. However, there were increased AKI, myocardial infarction, length of hospital stay, and charges in a patient with a history of PEU
Abstract 268: Exercise Modulates Inflammatory, Cytokine, And Growth Factor Pathway Dysregulation In A Mouse Model Of Aortic Dissection And Aneurysm
Authors: Cahn Matthew D , Zhang Jackie M , Ni Fenge , Lal Brajesh , Strickland Dudley , Ucuzian Areck
Published: October 2023

Aerobic exercise can prevent aortopathy in mouse models. The objective of this study was to better understand the mechanisms underlying this finding. Mice treated with β-aminopropionitrile (BAPN), an irreversible inhibitor of lysyl oxidase (LOX) which induces thoracic aortic aneurysm and dissection (TAAD), underwent either a forced moderate treadmill regimen or maintained conventional cage activity without forced exercise. RNA sequencing was utilized to determine the transcriptomic profile of the ascending and thoracic aorta (n=7 per group). BAPN induced overexpression of 357 genes and decreased expression of 77 genes compared with controls. Causal analysis in Ingenuity Pathway Analysis® software using a fold change |2| and FDR < 0.05 revealed that BAPN activates phagosome formation, leukocyte extravasation, phagocytosis, hepatic fibrosis signaling, neuroinflammation signaling, production of nitric oxide and reactive oxygen species in macrophages, leukocyte migration and inflammatory response, among other pathways, in the aorta (z-score > 2, p-value < 0.05). Pathways of the overexpressed upstream regulators TYROBP, IL6, SPI1, IL1B, CCR2, and TGFβ1, among others, were predicted to be activated (z-score > 2, BH p-value of overlap < 0.05). A comparison analysis between the two groups sorted by trend and score demonstrates that moderate aerobic exercise reverses the predicted activation of these pathways. Next, we investigated whether activated TGFβ1 signaling, predicted to be normalized with exercise compared with unexercised mice, is required for the aortopathy caused by LOX inhibition. We administered SB431542, a selective small peptide inhibitor of the TGF-β1 receptor (TGF-β1R), to a separate cohort of BAPN-treated mice. Histologic analysis demonstrated that the addition of the TGF-β1R inhibitor reverses many of the histologic changes characteristic of BAPN-induced aortopathy, including reversal of wall thickening (p < 0.001), elastin degradation (p < 0.01), and cystic medial degeneration (p = 0.04) in the thoracic aorta. While further investigation is necessary, our study suggests that the prevention of TAAD and its complications by moderate aerobic exercise may in part be due to deactivation of the dysregulated TGF-β1 pathway.
Abstract 272: A Possible Role Of Apolipoprotein B In Degenerative Ascending Aortic Aneurysm Formation And Progression
Authors: Freiholtz David , Bergman Otto , Österholm Corbascio Cecilia , Lång Karin , Olsson Christian , Franco-Cereceda Anders , Eriksson Per , Gisterå Anton , Björck Hanna M
Published: October 2023

Background:
Aortic valve regurgitation (AR) is associated with degenerative changes in the ascending aorta and the occurrence of ascending aortic aneurysm (AscAA). The molecular mechanism and initiating factors remain largely unknown, but leukocyte infiltration and inflammation are known characteristics. The aim of this study was to investigate the molecular signature of AR-associated degeneration and identify factors associated with degenerative AscAA formation.

Material and Methods:
Patients undergoing elective open-heart surgery for AscAA- and/or aortic valve surgery were included. Control samples were attained from organ donors. Histomorphology and protein expression/localization was assessed using immunohistochemistry, and gene expression in aortic intima-media was analyzed for totally n=16 non-dilated AR, n=19 non-dilated aortic stenosis (AS) and 36 dilated patients, as well as in n=6 controls.

Results:
Gene expression analysis showed that already prior to aortic dilatation, AR was associated with biological processes related to extracellular matrix turnover, as well as degenerative histological changes. In differential gene expression analysis of non-dilated vs dilated aortic tissue from AR-patients, genes with the greatest fold-change were PRPF40B, FMNL3, OLR1, MS4A7, MS4A14, of which OLR1 has a role in atherosclerosis and vascular inflammation. Apolipoprotein B 100 (ApoB) showed a different expression pattern between AR vs. AS patients with a high degree of medial infiltration in AR-patients. In AS-patients ApoB was almost exclusively localized to the endothelium. Further, the SRBI protein, a protein mediating LDL-transcytosis, was significantly increased in the endothelium of AR-patients compared with AS patients.

Conclusions:
The prevalence of AR-associated AscAA may involve an early infiltration of ApoB in the ascending aortic media, with possible down-stream implications for disease development. Further, this may explain the rarity of AscAA presenting with AS.
Abstract 276: High Mobility Group Box 1 Inhibition By Antisense Oligonucleotide Attenuates Angiotensin Ii-induced Abdominal Aortic Aneurysm
Authors: Mohammadmoradi Shayan , Sawada Hisashi , Ito Sohei , Mullick Adam E , Howatt Deborah A , Franklin Michael , Lu Hong S , Daugherty Alan
Published: October 2023

Introduction:
Vascular inflammation is a hallmark of abdominal aortic aneurysm (AAA). However, underlying mechanisms that initiate inflammatory pathways in the aorta are not clearly elucidated. Here, we explore the role of high-mobility group box 1 (HMGB1), a highly conserved nonhistone DNA-binding nuclear inflammatory molecule in AAA.

Methods and Results:
To determine the molecular signature and biological processes involved in AAA, we analyzed RNA sequencing (RNA-seq) data of patients with AAA (GSE57691) and mice with angiotensin-II (AngII) infusion (GSE17901) downloaded from the GEO database. Gene ontology analysis revealed that the major differences in enriched genes between the two groups involved inflammatory responses and cytokine-cytokine interaction. Both human and murine gene expression profiles indicated a marked upregulation of HMGB1 in the diseased abdominal aorta. AngII infusion also indicated a marked increase of HMGB1 in the abdominal aorta of male LDLr-/- mice after seven days. mRNA expression of genes related to the HMGB1 release mechanism was altered in diseased tissue relative to the control by RNA-seq. To explore the role of HMGB1 in AAA formation, we performed mouse in vivo studies with genetic manipulation in HMGB1, utilizing a novel antisense oligonucleotide (ASO) approach to inhibit HMGB1. Proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced hypercholesterolemic male mice fed a Western diet were infused with AngII (1,000 ng/kg/min) for 4 weeks to induce AAA. Mice (N=15/group) were subcutaneously injected with phosphate-buffered saline (PBS) or HMGB1 ASO (50 mg/kg) at days 0 and 3 in the initial week and then once a week during the remainder of the study. There was no difference in blood pressure between the two groups in response to AngII. However, the aortic ex-vivo analysis indicated that the HMGB1 ASO profoundly attenuated AngII-induced AAA. Of note, ascending aorta area was also markedly reduced in the ASO group.

Conclusion:
Our data indicated the marked upregulation of HMGB1 in human and murine aneurysm. ASO-driven HMGB1 inhibition also resulted in profound attenuation of the AngII-induced AAA.
Abstract 279: Intricate Roles Of Myeloid-derived Thrombospondin-1 In Aortic Aneurysm And Rupture
Authors: Zhou Ting , Yang Huan , Burkel Brian , Ponik Suzanne , Lu Hong S , Daugherty Alan , Liu Bo
Published: October 2023

Introduction:
Abdominal aortic aneurysm (AAA) is the progressive weakening and dilation of the aorta. Aneurysm rupture is the major cause of mortality among AAA patients, but its molecular mechanism is still unclear. We previously reported elevated expression of thrombospondin-1 (TSP1), a matricellular protein, in human and mouse AAA. We also identified Mφs as the major source of TSP1. In this study, we investigated the role of myeloid-derived TSP1 in AAA and rupture.

Methods and Results:
Male myeloid-specific TSP1 deficient mice (

Thbs1
Δ

Mφ
) and wildtype littermates (

Thbs1
wt

) were challenged with angiotensin II (Ang II) coupled with hypercholesterolemia. Sixty-one percent of

Thbs1
Δ

Mφ
died from AAA rupture, an incidence that was 2.6 times higher than

Thbs1
wt

(n=23 for

Thbs1
wt

, n=30 for

Thbs1
Δ

Mφ
). Intriguingly,

Thbs1
Δ

Mφ
that survived to the end of 28-day Ang II infusion had less aortic dilation than

Thbs1
wt

. Majority of the rupture occurred between 7 to 14 days of Ang II infusion. Ultrasound imaging at early time points (baseline, 3 or 7 days after AAA induction) showed no difference in the aortic diameters between genotypes. To understand the molecular mechanisms underlying the rupture-prone phenotype of

Thbs1
Δ

Mφ
, we conducted single-cell RNA sequencing on the aortic tissues 7 days after AAA induction.

Thbs1
Δ

Mφ
showed increased percentages of smooth muscle cells (SMCs), and decreased Mφs and fibroblasts. Six functional SMC subclusters were identified, which were contractile, fibroblast-like, proliferative, macrophage-like, hemoglobin positive, and intermediate SMCs.

Thbs1
Δ

Mφ
specifically increased the intermediate SMCs, all the other subclusters were decreased. Pathway analysis revealed an extended reduction of extracellular matrix-related genes. Histological analysis confirmed the predicted matrix weakening caused by the myeloid-specific TSP1 deficiency.

Conclusions:
Myeloid TSP1 is critical for Mφ infiltration, which fuels aneurysm growth. However, Mφs, likely through signaling to SMCs and fibroblasts via TSP1, may also contribute to the vascular remodeling at least in the Ang II-induced aortopathy. Therefore,

Thbs1
Δ

Mφ
provides a unique model to investigate the complex interplays between Mφs and vascular cells in AAA and rupture.
Abstract 281: A20 (tnfaip3) Knockdown Protects From Abdominal Aortic Aneurysms By Limiting Extra-cellular Matrix Degradation And Maintaining Aortic Smooth Muscle Cell Mass
Authors: Aridi Tarek , Gigioli John , Patel Nyah , Wade Jacqueline , Brennan Phillip , Roshan Keyan , Angolano Cleide , Ferran Christiane
Published: October 2023

Background:
Our lab has recently demonstrated that haploinsufficiency of A20/TNFAIP3, a potent anti-inflammatory/NFκB inhibitory gene, protects mice from abdominal aortic aneurysms (AAA) by enhancing TGFβ signaling and modulating smooth muscle cell (SMC) differentiation and apoptosis. Here, we explored the impact of A20 knockdown on TGFβ-mediated SMC proliferation and extra cellular matrix (ECM) degradation, both involved in AAA pathogenesis.

Methods:
AAA was induced by periadventitial elastase application to the infrarenal aorta of both A20 wild-type (WT) and A20 heterozygous (Het) 10-week-old mice who received 0.2% BAPN in their drinking water. Aortae were harvested 28 days after for immunohistochemistry (IHC) analysis of SMC proliferation and ECM markers.
In vitro
, human aortic SMC cells (HAoSMC) were transduced with rAd.ShRNA A20 (siA20), resulting in 50-70% A20 knockdown, or control rAd.ShRNA Scramble (siScr). HAoSMC were starved for 24 hours, then treated with TGFβ. Proliferation was analyzed by MTS assay, and cell extracts assayed by Western blot (WB) for cell-cycle markers.

Results:
A20 haplo-insufficiency protected from AAA formation by: 1) decreasing ECM degradation, as gauged by significantly lower MMP-9 expression (n=3, p<0.01), and 2) promoting vessel wall thickening by increasing SMC proliferation (ki67+ nuclei/vessel wall, n=4, p<0.01). Increased SMC proliferation in Het vs. WT aortae correlated with significantly lower expression of the cell-cycle brake, p21 (p21+ nuclei/vessel wall, n=3-7, p<0.05). Akin mouse aortae, A20 knockdown significantly increased TGFβ-mediated HAoSMC proliferation, assessed by the MTS assay (siA20 vs. siScr, n=5, p<0.05). This associated with significantly higher protein levels of the proliferation regulator phospho-ERK1/2 in siA20 vs. siScr-transduced HAoSMC (WB, n=4, p<0.001).

Conclusion:
Our findings qualify the protective effect of A20 knockdown against AAA development by preserving ECM integrity and promoting TGFβ-mediated SMC proliferation. These targets of A20 knockdown were validated in HAoSMC, further supporting our pursuit of A20-siRNA based therapies to prevent or limit progression of AAA.
Abstract 286: Identification Of Microrna Regulators Of Cell Signalling In Marfan Aorta Using Machine Learning
Authors: Dong Charlotte X , Malecki Cassandra , Robertson Elizabeth N , Hambly Brett D , Jeremy Richmond W
Published: October 2023

Marfan syndrome (MFS), due to
FBN1
mutations, is associated with thoracic aortic aneurysm (TAA). The TGFß and other signalling pathways have been implicated in TAA formation, however, regulators of cell signalling in MFS are yet to be defined. We hypothesized that distinct microRNA (miRNA) expression profiles occur in MFS aorta and that these can illuminate novel cell signalling targets contributing to TAA. MiRNAs were extracted from MFS (n=15) and controls (n=11) ascending aortic tissue, for discovery open-array panel profiling of miRNA expression. Individual miRNA expression profiles were compared between MFS and controls by random forest machine learning algorithm (Python). The miRNAs identified with a significant importance value were selected for gene target prediction (score >90) through miRDB, followed by KEGG analysis. Any miRNA with more than 2000 predicted targets in the genome was excluded. P=0.05 was used as a cut-off value for KEGG analysis. The random forest classifier identified 53 miRNAs with differential expression between MFS and controls, contributing to the model (predictive accuracy 73%). The five most significant miRNAs were miR-181a-3p, miR-144-3p, miR-545-5p, miR-27a-5p and miR-181c-3p. 2984 unique target genes were identified from 53 significant miRNAs. KEGG analysis of these target genes identified multiple pathways as having potential differential regulation by miRNAs between MFS and controls. The most significant and relevant pathways identified were PI3K/AKT (p<0.00001) and the downstream FOXO (p<0.0004) pathways. Others include the MAPK (p<0.00001), mTOR (p<0.00002), RAP1 (p<0.005), ErbB (p<0.003) and RAS (p<0.001) pathways, in addition to TGFß (p<0.01). Mapping miRNA targets to KEGG pathways identified potential regulatory points, e.g., miR-29c-3p affecting
FBN1, AKT3, PTEN
(within the TGFß and PI3K/AKT pathways); miR-19a-3p affecting
RAF1
,
FMR1,
RIN2
(within the MAPK, fragile X and adherens junction pathways); miR-27a-5p affecting
LTBP1
(within the TGFß pathway). In conclusion, machine learning analysis identifies differential expression of multiple miRNAs in aortic tissue from MFS, which map to cell signalling pathways implicated in the pathogenesis of TAA in MFS.
Abstract 292: Platelet Hyperreactivity Characterization by Aggregation and RNA-seq Informs Cardiovascular Risk
Authors: Barrett Tessa J , Cornwell Macintosh , Xia Yuhe , Smilowitz Nathaniel , Newman Jonathan D , Muller Matthew , Rockman Caron , Ruggles Kelly , Voora Deepak , Hochman Judith , Berger Jeffrey S
Published: October 2023

Background:
Patients with peripheral artery disease (PAD) are at increased risk of major adverse cardiovascular and limb events (MACLE), many of which are platelet mediated. In this study, we investigated the association between platelet hyperreactivity and 30-day MACLE in patients undergoing lower extremity revascularization (LER) and derived and validated a gene signature of platelet hyperreactivity.

Methods:
Patients with symptomatic PAD undergoing LER were recruited into the Platelet Activity and Cardiovascular Events in PAD (PACE-PAD) study. Platelet RNA was collected and platelet aggregation to 0.4 μM epinephrine was measured by light transmission aggregometry (LTA) immediately prior to LER. Patients were followed for 30 days post-procedure MACLE. Integration of platelet RNA-seq and aggregation responses was used to generate a
P
latelet
R
eactivity
E
xpre
S
ion
S
ignature (
PRESS
) geneset.

Results:
A total of 289 patients were included (mean [SD] age 70 [11] years; 94 [33%] female). Patients with platelet hyperreactivity (>60% agg) had an increased incidence of 30-day MACLE post-LER (37.2% vs. 15.3%, P<0.01; adjusted hazard ratio [aHR] 2.80, 95% CI 1.5 to 5.1, P=0.002), which persisted across age, sex, race/ethnicity, antiplatelet therapy, and clinical risk categories. We derived and validated a platelet hyperreactivity geneset, PRESS, that associates with incident cardiac and limb events and can discriminate platelet hyperreactivity across disease states (
Fig. 1
).

Conclusion:
Platelet hyperreactivity measured before LER was independently associated with 30 day MACLE. Our transcriptomic signature of platelet hyperreactivity, PRESS, accurately identified patients with hyperreactive platelets. Use of our PRESS geneset as a diagnostic tool of platelet hyperreactivity opens the possibility of personalized intervention approaches to prevent platelet-mediated complications.
Abstract 294: Therapeutic Effects Of Ginseng Gintonin In A Mouse Model Of Focal Ischemic Stroke Via Inhibition Of Lysophosphatidic Acid Receptors
Authors: Choi Sun Hye , Nah Seung-Yeol , Kim Dong-Eog E
Published: October 2023

Background & Objective:
Gintonin is a ginseng-derived lysophosphatidic acid receptor (LPAR) ligand. Previous studies showed beneficial roles of gintonin in mouse models of neurogenerative diseases such as Alzheimer disease and Parkinson disease. We aimed to investigate if gintonin has neurovascular protective effects in a mouse model of focal ischemic stroke.

Method:
C57BL/6N mice were treated orally with saline (n=50) or gintonin 150 (n=65) or 300 (n=20) mg/kg once daily for 1 week. Among the 150 mg/kg group animals, 15 randomly selected mice were pretreated with intraperitoneal administration of an LPAR antagonist (Ki16425, 30 mg/kg) before the oral treatment. Then, all mice underwent common carotid artery occlusion (CCAO)-related hemispheric ischemia after inducing cerebral autoregulatory dysfunction (CAD). We compared post-ischemic survival rates (Log-rank test) and final infarct volumes (Studen’s t-test) at 1 week.

Results:
Gintonin 150mg/kg (vs. saline) increased the 1-week survival rate (35/50 [70%] vs. 23/50 [46%], p=0.011) and reduced the infarct size (92.20±14.53mm
3
vs. 138.70±16mm
3
, p=0.038). Gintonin 300mg/kg tended to increase the survival rate (12/20 [60%], p=0.11 vs. saline) and did not significantly reduce the infarct size (40±36 mm
3
, p=0.97). Pretreatment with the LPA receptor antagonist abolished the gintonin 150mg/kg-mediated improvement in post-stroke survival (6/15 [40%], p=0.61 vs. saline) and the reduction of infarct size (197.81±31.97mm
3
, p=0.88 vs. saline).

Conclusion:
This study suggests that gintonin could improve post-stroke survival and reduce infarct volume through LPAR inhibition in mice.