bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 194: Pharmacologic Inhibition By Spironolactone Attenuates Experimental Abdominal Aortic Aneurysms
Authors: Ladd Zachary , Lu Guanyi , Su Gang , Hartman Joseph , Hensley Sara , Sharma Ashish , Upchurch Gilbert R
Published: November 2022

Introduction:
Abdominal aortic aneurysms (AAA) are characterized by vascular inflammation and remodeling that can lead to aortic rupture resulting in significant risk of mortality. Spironolactone has been recently described as a pannexin-1 (Panx1) channel modulator. Our hypothesis focuses on the role of spironolactone on Panx1 channels to attenuate AAA formation.

Methods:
A topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat inactivated elastase (control) AAA model was used in C57BL/6 (wild-type; WT) male mice. Mice were administered either a vehicle control (saline) or spironolactone (1.25 mg/kg) i.p. daily for 14 days. On day 14, the abdominal aortic diameter was measured by video micrometry and expressed as percentage increase over baseline. In a second model, mice were subjected to β-aminopropionitrile (BAPN) two days prior to elastase treatment and everyday thereafter. A vehicle control (saline) or spironolactone was given i.p. daily starting from day 14. Immunohistochemistry was used to analyze immune cell infiltration and elastic fiber disruption (Verhoeff Van Gieson staining). Groups were compared using ANOVA and data is displayed as mean ± SEM with p<0.05 as statistically significant.

Results:
Aortic diameter was significantly increased in elastase-treated WT mice compared to controls (202.5 ± 4.2% vs 0.5 ± 1.9%; n=9/group, p<0.001). Elastase-treated WT mice administered spironolactone had a significant, dose-independent decrease in AAA formation compared to elastase-treated WT controls (139.8 ± 9.9% vs 202.5 ± 4.2%; n=10/group, p<0.001). Elastase-treated mice given BAPN and administered spironolactone had significant attenuation of AAA formation compared to controls on day 28 (420.2 ± 30.5% vs 643.8 ± 41.3%; n=8/group, p<0.001). A significant decrease in immune cell infiltration and elastic fiber disruption occurred in all mice treated with spironolactone compared to respective controls.

Conclusion:
These results suggest spironolactone mitigates aortic remodeling to attenuate AAA formation as well as decrease growth of preformed aneurysms. Further studies aim to characterize the molecular mechanisms of specific intercellular interactions involving Panx1-mediated AAA formation by spironolactone.
Abstract 198: Robust Quantification Of Hdl Proteome For Mechanistic And Clinical Studies
Authors: Silva Amanda R , Souza Junior Douglas R , Ronsein Graziella E
Published: November 2022

Introduction:
Emerging studies have proposed novel roles of HDL in cardiovascular and noncardiovascular diseases. The understanding of how HDL participates in a pathophysiological process involves animal models, as well as clinical studies. A key issue involving HDL studies is the accurate quantification of its proteome. HDL proteome is complex, and its composition may be modulated by the subject’s health state. The main goal of this work is to establish a pipeline for accurate quantification of HDL proteome in clinical and animal studies.

Hypothesis:
The knowledge of the composition of HDL proteome in clinical and animal studies is critical to understand its biological functions.

Methods:
We first performed a deep proteomic analysis to identify proteins belonging to HDL of mice and humans. We then established a targeted, data independent acquisition (DIA) strategy that works for mouse and humans. Next, by using different software platforms, we developed a quantification methodology specific for each species.

Results:
Although mice and humans share many of their HDL proteins, the protein sequence is unique for each species, and thus, HDL proteome quantification methods must be species specific. Using recent advances in mass spectrometry, we employed a single acquisition method that worked for both, mice and human samples. Differentiation occurred after acquisition, using spectral libraries specifically built for each species. Using different software platforms, we compared four strategies of quantification for HDL proteome, showing that CVs depend on the strategy employed. Thus, using quality control samples (n=8), we showed that for the same protein, CVs may vary up to 50 %. The quantification strategy was thus optimized in order to provide the best quantification with the least variance, reaching CVs lower than 15 % for 90 % of the proteins.

Conclusions:
Using the same mass spectrometry acquisition method, it is possible to quantify HDL proteome of mice and humans in a consistent and accurate way. These advances may integrate animal models and clinical studies of HDL proteome.
Abstract 210: Susceptibility To Social Stress Modulates Hematopoiesis And Impairs Atherosclerosis Resolution
Authors: TUFANLI KIRECCIBASI Ozlem , Scolaro Bianca , Weinstock Ada , Torres Berrio Angelica , Parise Eric , Cathomas Flurin , Chan Kenny , Nestler Eric , Russo Scott , Fisher Edward
Published: November 2022

Background:
Depressive disorders frequently involve prolonged stress and are associated with increased risk of cardiovascular disease (CVD). A major hurdle in gaining mechanistic insight is the paucity of animal models that accurately recapitulate the symptoms of depression disorders. The major question we investigated was whether psychological stress impairs the benefits of the common therapy used to reduce CV risk, namely, lipid lowering.

Methods:
We employed a standard protocol of repeated social defeat stress (RSDS) that elicits a depression-like phenotype in mice to test the effects on atherosclerosis resolution. LDLr-deficient mice were kept on an atherogenic diet for 24 wks. Then RSDS was applied for 10 days. Mice that developed a depressive-like phenotype, termed stress susceptible (SS), were identified with behavior testing. SS and control mice were then placed on cholesterol-reducing therapy.

Results:
SS mice vs. control mice had expansion of bone marrow (BM) myeloid progenitors (~80% increase for GMP cells; P<0.001) and more circulating monocytes (~2X;
P
<0.05), which exhibited higher recruitment (2.5X;
P
<0.001) to atherosclerotic plaques. This resulted in increased (~50%;
P
<0.05) plaque macrophage content after 3 wks of cholesterol lowering. To investigate whether the effects of RSDS on inflammation and atherosclerosis were mediated by re-programming of the BM progenitors of plaque macrophages, we performed BM transplantation from control or SS mice into LDLr-deficient mice. After the recovery period, the mice were fed an atherogenic diet for 24 wks. At that time, we found that compared to control BM recipients, SS BM recipients sustained the increase in myeloid progenitors (~16% increase, P<0.05 for GMP population) and circulating inflammatory monocytes (50% increase;
P
<0.001), as well as enhanced atherosclerotic plaque inflammation (23% more macrophage content; P<0.001).

Conclusions:
Social stress impairs atherosclerosis inflammation resolution after cholesterol lowering, apparently by re-programming BM precursors of plaque macrophages. By applying molecular methods to this model, we hope to further understand the mechanistic basis for the increase in CVD risk in stressed people despite risk reducing therapies.
Abstract 218: Integration Of Coronary Artery Disease GWAS With Bulk And Single-cell Transcriptomics From Atherosclerotic Plaques By Deconvolution, Reveals Novel Smooth Muscle Cell Genes
Authors: Narayanan Sampath , Vuckovic Sofija , Wirka Robert , Lengquist Mariette , Quertermous Thomas , Hedin Ulf , Matic Ljubica
Published: November 2022

Background and Aim:
A central role of vascular smooth muscle cells (SMCs) in atherosclerosis has recently evolved that suggests causal genetic links to disease processes. Single cell sequencing studies of atherosclerotic plaques have identified multiple mesenchymal transition cell populations within the plaques. Here, we correlated cell fractions from plaques to coronary artery disease (CAD) related gene polymorphisms to identify novel SMC targets and study their influence on SMC function in atherosclerosis.

Methods:
Deconvolution analysis was performed on bulk microarray data from carotid plaques in the Biobank of Karolinska Endarterectomies (BiKE, n=127) using single cell sequencing data from coronary plaques (n=5). CAD-associated GWAS loci associated with mesenchymal cell fractions were identified, followed by functional analyses of these genes in SMC
in vitro
using migration, proliferation and apoptosis assays.

Results:
We identified 5 mesenchymal cell-specific genetic variants associated with CAD, BiKE patient symptomatology and gene expression eQTLs in BiKE plaque tissue and GTex normal arteries. These variants were harbored in genetic loci of
ARNTL, LDLR, MIA3, PAK1
and
ARHGAP15
. Microarray analysis revealed increased expression of
ARHGAP15
and
PAK1
and decreased levels of
LDLR
in carotid plaques compared with normal arteries (n=127
vs.
10 respectively, student’s t-test). Immunohistochemistry demonstrated increased expression of corresponding proteins in the fibrous cap of plaques compared to normal arteries (n=5). To investigate their function in SMCs, the genes were silenced using siRNAs followed by migration, proliferation and apoptosis assays. Preliminary results indicated that silencing of
MIA3, LDLR
and
ARNTL
inhibited SMC proliferation.

Conclusions:
The results of this project may reveal novel SMC-specific genetic links to the disease, which may serve as therapeutic targets to be explored for improved treatment of atherosclerosis.
Abstract 225: Lack Of Map Kinase Phosphatase-5 In Macrophages Protects Ldlr-null Mice Against Atherogenesis
Authors: Zhang Xinbo , Zhao Zhiqiang , Baldini Margaret , Zhang Cheng , Tao Bo , Zhang Lei , Bennett Anton M , Yu Jun
Published: November 2022

Objective:
Mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38 MAPK, are critical in regulating the expression of various pro-inflammatory and anti-inflammatory cytokines and chemokines. Previous work has suggested that the absence of MAP Kinase Phosphatase-5 (MKP-5) inhibits oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation without influencing the MAKP activation. The current study is to determine the role of macrophage MKP-5 in the pathogenesis of atherosclerosis and underlying mechanisms.

Approach and Results:
Nine-week-old male congenic MKP-5 deficient (MKP-5
-/-
) and C57Bl/6J control (WT) mice on an LDL receptor knock-out (LDLR
-/-
) background were fed with a high-fat diet containing 1.25% cholesterol (HFD) for 14 weeks. Global deficiency of MKP-5 attenuated atherosclerotic plaque formation without altering the lipid profile in vivo. To further elucidate the macrophage-specific effect of MKP-5 in atherogenesis, Lethally irradiated LDLR
-/-
mice were transplanted with WT or MKP-5
-/-
bone marrow and subjected to high-fat feeding. Mice transplanted with MKP-5
-/-
bone marrow developed smaller atherosclerotic lesions accompanied by decreased lipid deposition and macrophage content compared to WT. Lack of MKP-5 in macrophages reduced plasma levels of interleukin-1α (IL-1α) and IL-7, elevated anti-inflammatory cytokines IL-1 receptor antagonist (IL-1rn), and IL-4. Mechanistically, lack of MKP-5 in macrophages inhibited ox-LDL-induced foam cell formation through enhanced cholesterol efflux mediated by increased expression of ATP-binding cassette transporters ABCA1 and ABCG1.

Conclusions:
These data suggest that the myeloid MKP-5 deficiency reduces atherosclerosis progression and foam cell formation by ameliorating cholesterol efflux and inhibiting inflammation.
Abstract 228:
Trpm2
Deficiency In Macrophages Protects Mice Against Atherosclerosis
Authors: Zong Pengyu , Feng Jianlin , Yue Zhichao , Yue Lixia
Published: November 2022

Introduction:
Atherosclerosis and its major complications, ischemic heart diseases and stroke, are the leading causes of mortality worldwide. The prominent feature of atherosclerosis is the formation of foam cells, the infiltrated macrophages overloaded with lipids. Lipid uptake by macrophages is mainly mediated by CD36.

Hypothesis:
Previously we reported that: (1) global
Trpm2
deletion attenuates atherosclerosis; (2), TRPM2 and CD36 enhance the activation of each other in macrophages, thereby promoting foam cell formation. Thus, we hypothesized that macrophage specific
Trpm2
deletion protects mice against atherosclerosis.

Methods:
Macrophage-specific
Trpm2
deletion in was achieved by crossing

Apoe
-/-
/ Trpm2
fl/fl

mice with
Cd11b-cre
mice. High-fat diet (HFD) was used to induce atherosclerosis, and total serum cholesterol level was measured. Oil Red O (ORO) staining was used to evaluate atherosclerotic lesion size. Immunofluorescence staining of Mac-1, F4/80 and CD80 was used to examine the macrophage content in the plaques, which was further confirmed by flow cytometry analysis of the immune cell populations in digested aortas. Inflammation of the aortas was examined by WB analysis of expression of NLRP3, ASC, Caspase-1, IL-1β, macrophage chemoattractant protein-1 (MCP1) and migration-inhibitory factor (MIF).

Results:
Macrophage-specific
Trpm2
deletion: (1) did not influence the serum total cholesterol level; (2) decreased plaque lesion ratio as shown by en-face aorta ORO staining and reduced plaque size as shown by aortic root ORO staining; (3) reduced macrophage content and immune cell infiltration in the plaques; (4) inhibited NLRP3 inflammasome activation and inflammatory cytokines expression in the atherosclerotic aortas.

Conclusion:
Macrophage specific
Trpm2
deletion attenuates HFD-induced atherosclerosis in

Apoe
-/-

mice.
Abstract 236: Impact On Smsr, As A Pe-plc, On Nash And Liver Fibrosis
Authors: Chiang Yeunpo , Li Zhiqiang , Jiang Xian-Cheng
Published: November 2022

Sphingomyelin synthase-related protein (SMSr), a member in SMS gene family, has no SM synthase activity. Although SMSr is conserved throughout the animal kingdom and ubiquitously expressed in all tested mammalian tissues, its biochemical activity
in vivo
is unknown till very recently. We found that SMSr is a phosphatidylethanolamine-specific phospholipase C (PE-PLC) which is involved in tissue PE steady-state regulation. We also found that
Sms1/Sms2
double knockout (KO)-mediated glucosylceramide accumulation caused liver impairment, inflammation, and fibrosis. Importantly, these pathologic phenotypes could be reversed, at least partially, in
Sms1/Sms2/Smsr
triple KO mouse livers. Based on RNA-seq, we noticed that about 60 pro-inflammatory and fibrosis genes, including TGFb1, TNFa, IL-1a, IL-6, and collagen 1a1, were significantly upregulated in
Sms1/2
double KO mouse liver compared with wild type mouse liver, while the triple KO mice could reduce them. The observations were confirmed by real-time PCRs. Further, we found that
Sms1/Sms2/Smsr
triple KO mice had much less liver PE-PLC activity and higher PE levels compared with the double KO mice. We also measured plasma TGFb1 whose signaling in hepatocytes participated in NASH and liver fibrosis and found the triple KO mice had significantly lower active TGFβ1 in the circulation compared with the double KO mice. Moreover, we treated the double KO mice with PE (i.p.) (10 mg/kg) every day for 7 days to mimic the situation of SMSr deficiency and we found that the active form of TGFβ1 in the plasma was also dramatically reduced in PE-treated mice compared with vehicle. These findings implicated SMSr/PE-PLC deficiency-mediated PE accumulation can prevent the development of NASH and liver fibrosis. Thus, SMSr could be drug target for the treatment of both.
Abstract 237: Association Between The HDL-sized And Circulating Plasma Proteomes
Authors: Reasons Riley J , Hamid Zeeshan , Barber Jacob L , Kass Alec I , Dev Prasun K , Wallis Kit , Bouchard Claude , Robbins Jeremy , Gerszten Robert E , Olivier Michael , Sarzynski Mark A
Published: November 2022

Background:
Mass spectrometry (MS) profiling has identified over 250 proteins associated with HDL that are thought to underlie the diverse atheroprotective properties of HDL particles and thus may be important biomarkers of cardiovascular disease (CVD) risk. Likewise, recent studies have identified circulating plasma proteins as biomarkers of CVD risk factors and health outcomes. However, few studies have compared the HDL proteome with the circulating plasma proteome.

Purpose:
The purpose of this analysis was to examine the relationship between the abundance of individual proteins measured in whole plasma and the HDL-sized plasma fraction.

Methods:
We examined the HDL-sized and circulating plasma proteomes in 156 Black (30%) and White men and women (61%) from the HERITAGE Family Study. HDL was isolated from plasma via gel filtration chromatography and untargeted MS analysis was performed via nano-HPLC-MS/MS. The whole plasma proteome was measured using a modified aptamer (SOMAscan) assay. The correlations between protein abundances in HDL and whole plasma were examined for 101 HDL-associated proteins present in at least 40% of the sample.

Results:
The abundance of 56 proteins in HDL-sized and whole plasma were significantly (5% FDR) correlated with the strongest correlation for Haptoglobin levels (r=0.83, p=1.1x10
-40
) (
Table 1
). The remaining significant correlations ranged from weak to moderate (r= 0.18-0.64) and were found among a mix of frequently and occasionally observed HDL proteins.

Discussion:
We found that protein abundance measured in HDL-sized and whole plasma were moderately to strongly correlated for several proteins, whereas 45% of protein levels showed no association between HDL-sized and whole plasma fractions. Given the inherit differences in measurement techniques and sources of proteins, it appears that the plasma HDL-sized proteome is mostly distinct from the circulating whole plasma proteome as measured by the SOMAscan assay.
Abstract 240: Role Of Smooth Muscle-derived Vascular Progenitor Cells In Atherosclerosis
Authors: Dubner Allison M , Lu Sizhao , Mutryn Marie , Jolly Austin , Strand Keith , Navarro Natalie , Moulton Karen , Nemenoff Raphael , Weiser-Evans Mary C
Published: November 2022

Purpose:
Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Emerging evidence implicates the outer layer of the blood vessel, the adventitia, in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis progression by facilitating inflammatory cell infiltration. Our group previously identified a unique population of multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and reside in the vessel adventitia, where they are poised to respond to vascular injury. We hypothesized that in the setting of atherosclerosis, AdvSca1-SM cells contribute to VV expansion to drive disease progression.

Methods:
We generated a highly specific lineage tracing mouse model in order to track AdvSca1-SM cells in vivo even if they differentiate into other cell types. Lineage tracing mice were placed on either normal chow or Western diet for 8 or 16 weeks, then vascular tissue was analyzed using IF microscopy, scRNA-Seq, and flow cytometry.

Results:
scRNA-Seq and flow cytometry revealed that AdvSca1-SM cells in atherosclerosis primarily differentiate into mature SMCs, modulated SMCs, and myofibroblasts. Contrary to our preliminary data, AdvSca1-SM cells very rarely differentiate into endothelial cells. Additionally, despite previous evidence of SMCs gaining a macrophage-like phenotype in atherosclerosis, we identified only rare instances of AdvSca1-SM cells contributing to macrophage populations.

Conclusions:
As in our findings in acute vascular injury, AdvSca1-SM cells in atherosclerosis predominantly differentiate into SMCs and myofibroblasts. Future studies on advanced lesions will define the functional role of AdvSca1-SM cells in atherosclerotic plaque progression.
Abstract 244: Differentially Expressed Genes Mediated By Erk5 S496 Phosphorylation In Hypercholesterolemia-induced Macrophage Reprogramming
Authors: Li Shengyu , Kotla Sivareddy , Imanishi Masaki , Ko Kyung Ae , Samanthapudi Venkatasubrahman , Savage Hannah , Schadler Keri , Deswal Anita , Lin Steven , Reyes-Gibby Cielito , Yeung Sai-Ching , Pownall Henry J , Fujiwara Keigi , Le Nhat-Tu , Wang Guangyu , Abe Jun-ichi
Published: November 2022

The crucial role of ERK5 S496 phosphorylation in reprogramming macrophage phenotype to pro-inflammatory senescent phenotype (PISP) has been reported, but the exact molecular mechanism remains unclear. This study focused on identifying the dysregulated molecular pathways and core genes that are differentially regulated in bone marrow derived macrophage (BMDMs) isolated from wild type and ERK5 S549A knock-in (KI) mice under normal or hypercholesterolemia (HC) after high-fat diet (HFD) and AAV-PCSK9 injection. The extent of atherosclerosis was inhibited in ERK5 S496A KI mice. We sequenced RNA-seq for wild type and ERK5 S549A KI mice and used Top Hat program (v2.0.12) with default parameters to map all reads to the mouse genome (Mus musculus GRCm38). Gene expression and significance of differential expression were calculated by Cuffdiff (v2.0.12). Differentially expressed genes (DEGs) were defined by Cuffdiff according to
Q
value ≤0.05 as a threshold. Hallmark analysis was performed by Gene Set Enrichment Analysis (GSEA v4.2.1). We used the R package “GOplot” to perform GO bubble plot, GO circle plot, and GO chord plot. We identified 784 DEGs regulated by HC-induced ERK5 S496 phosphorylation, and the GO analysis revealed that they are involved in critical senescent processes including cell cycle, cellular response to DNA damage stimulus, protein transport, and negative regulation of apoptotic process. Gene-annotation enrichment analysis (GOCircle) showed that Z-scores of both cell cycle and cellular response to DNA damage stimulus were negative in ERK5 S496A KI mice, suggesting the role of cell cycle and DNA damage response in inducing PISP. Interestingly, we only found 40 DEGs in BMDMs isolated from normal chow diet and HFD-fed wild type mice, and 15 out of 40 DEGs were significantly regulated by ERK5 S496 phosphorylation, supporting the critical role of ERK5 S496 phosphorylation in HC-mediated macrophage reprogramming. Our study identified 10 core genes (Ahr, Gclm, H3C3, H4c11, Lpar1, Megf9, Nfe2, Ppih, Rpl22l1, and Tpt1) that are regulated by HC-mediated ERK5 S496 phosphorylation, which might be crucial for HC-induced PISP. However, functional analysis is further needed to validate their roles in PISP induction.
Abstract 245: Antibody Responses To ApoA-I Rely On An Interplay Between B And T Cells
Authors: Pitts Michelle , Nardo David , Venditto Vincent J
Published: November 2022

Apolipoprotein A-I (ApoA-I), the major protein of high-density lipoprotein (HDL), has been investigated as the target and regulatory element of immune responses in both patients and mice susceptible to atherosclerosis. Previous work by our lab has focused on investigating the immune responses to ApoA-I in wild-type mice as a strategy to understand their role in models of atherosclerosis and other inflammatory conditions. We hypothesize that peripheral immunologic tolerance is responsible for regulating the dynamic immune response to ApoA-I. To examine this, we first evaluated antibody profiles to ApoA-I in both wild-type C57BL/6 and LDL receptor knockout mice and found that anti-ApoA-I antibodies naturally develop to the protein. To determine whether this response was T cell dependent or independent, we examined whether nu/nu mice on a BALB/c background, and the parent strain, displayed the phenotype. These studies showed that both nu/nu, or wild type BALB/c mice fail to mount antibody responses to ApoA-I suggesting a regulatory mechanism might be responsible for suppressing this antibody response. Ex vivo stimulation of immune cells with ApoA-I provide evidence of both B cell and T cell activation, suggesting that peripheral tolerance mechanisms may be responsible for regulating the humoral and cellular immune response to ApoA-I. These findings agree with previous studies where ApoA-I has been shown to regulate T cell activity in the context of atherosclerosis by inducing the development of regulatory T cells, which assist in suppressing the development of plaque formation; as well as studies showing a higher propensity toward Treg development in BALB/c mice. Ongoing experiments, which more clearly characterize the role of B cells and T cells in response to ApoA-I, will be presented with the goal of clarifying the immune mechanisms responsible for anti-ApoA-I autoimmunity. These data will help guide continued understanding of the role of immune responses to ApoA-I in atherosclerotic disease progression.
Abstract 257: Heavy Clot Burden Despite Enoxaparin Therapy In The Setting Of Covid Pneumonia
Authors: Triggs Connor , Mandava Sri
Published: November 2022

Case:
A 71-year-old male with a past medical history of hypertension, tobacco use, and hyperlipidemia was admitted to the hospital for COVID-19 pneumonia. Initial CT scan was negative for a pulmonary embolism. He was intubated on day 4, and his follow-up CT showed bilateral pulmonary emboli despite prophylactic Enoxaparin 40 mg subcutaneously daily since admission. He was subsequently put on a therapeutic dose of 1mg/kg every 12 hours. The following day he was found to have a pneumothorax that necessitated a chest tube, and that same evening, he developed acute lower limb ischemia in the setting of therapeutic anticoagulation for 24 hours. Arterial doppler ultrasound revealed extensive occlusive thrombus in the major arteries of the left lower extremity. Emergent thromboembolectomy of the left iliac artery, superficial femoral artery, popliteal artery, and tibial artery and a 4-compartment fasciotomy on the lower leg was performed, but 2 days later he necessitated an above knee amputation. Thereafter, the patient’s right lower extremity became cool, but an arterial doppler showed collateral flow suggestive of chronic arterial disease.

Discussion:
This patient with no known history of prior blood clots or arrhythmias sustained heavy clot burden and limb amputation despite prophylactic and eventual therapeutic anticoagulation and not to mention barotrauma. Notably, his right lower extremity became cool concerning for micro-coagulopathy in the setting of an unremarkable ultrasound. Little guidance is available regarding anticoagulation strategy on individuals with COVID, let alone those at risk of blood clots. The question remains as to whether practitioners need to anti-coagulate all critically ill patients with COVID-19 more aggressively, anti-coagulate individuals at risk for atherosclerotic disease more aggressively, or consider agents such as antiplatelet agents for arterial thrombosis prophylaxis.
Abstract 259:

BMPR2
+/r899x

Mutation Contributes To Sex-linked Disparity In Hypoxia-induced Pulmonary Hypertension
Authors: Erewele Ejehi , Oliveira Suellen D , Castellon Maricela , Chen Jiwang , Minshall Richard D
Published: November 2022

Introduction:
Dysfunctional bone morphogenetic protein receptor 2 (BMPR2) and endothelial nitric oxide synthase (eNOS) have been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), a life-threatening disease characterized by pulmonary vascular remodeling, elevated right ventricular systolic pressure (RVSP), and right ventricular hypertrophy (RVH). Although the incidence of this disease is three times higher in females, males with PAH usually have a worse prognosis. The mechanism behind this sex-associated difference remains undetermined.

Objective:
Determine whether

BMPR2
+/R899X

loss-of-function mutation contributes to sex-associated differences in PAH, including the degree of vascular remodeling, RVSP, and RVH.

Methods:
Wild-type (WT) and

BMPR2
+/R899X

mice (male/female) were exposed to hypoxia (Hx) or normoxia (Nx) for 4 weeks, when the RVSP and RVH were measured. Lung tissue was used for western blot and histological analysis.

Results/Discussion:
Hx exposure elevated RVSP, inducing RVH in both groups, with a greater effect in

BMPR2
+/R899X

mice compared to WT (RVSP: 45.0+/- 2.2 and 34.9 +/- 1.3 mmHg, respectively). A higher RVSP was observed in

BMPR2
+/R899X

female mice, with males exhibiting more pronounced RVH, suggesting females might have a protective mechanism against severe RVH. Lung histology revealed no differences in vessel thickness/area between sexes, which may indicate that the RVSP differences are unlikely a response to vascular remodeling. Hx exposure increased the amount of α-SMA+ microvessels and eNOS expression in

BMPR2
+/R899X

females compared to males, but further experiments are required to determine whether elevated eNOS reflects higher function or an oxidative stress-driven endothelial cell phenotype.

Conclusion:
This work is uncovering the translational relevance of

BMPR2
+/R899X

mutation in the sex-linked differences in PAH incidence, severity, and outcome.
Abstract 265: Defining Microrna-125 As A Key Determinant In Inflammatory And Mitochondrial Oxidative Stress Of Cardiomyocytes In Myocardial Infarction
Authors: Duan Qihua , Wade Henry , Su Qiaozhu
Published: November 2022

MicroRNAs (miRNAs) have been demonstrated to regulate more than 60% of human genes by complementary base pairing between their seed sequences and the targeted mRNAs, resulting in decay and/or translational repression of the mRNAs. A unique mechanistic action of miRNAs is that a single miRNA can simultaneously target a cluster of genes that are involved in functionally associated signalling cascades to generate synergistic impact on cellular process. The objective of this study is to define miR-125a and miR-125b as key determinants in cardiac mitochondrial injury upon myocardial infarction (MI).

Approaches and Results:
To this aim, a MI mouse model was established by ligation of the left anterior descending coronary artery. MicroRNA profiling of heart tissues from MI and Sham mice revealed that miR-125a/b were robustly upregulated in the MI hearts. TargetScan analysis identified multiple putative targeting sites of miR-125a/b within the 3’-UTR of genes involved in inflammatory signaling, mitochondrial and endoplasmic reticulum (ER) stress, mitochondrial fusion and fatty acid β-oxidation. These genes include A20 in NFκB-mediated inflammation, ITPR1 in ER calcium channel, PACS2 and MFN1 in mitochondrial membrane remodeling as well as PGC1á/å and PPARα that regulate mitochondrial biogenesis and energy homeostasis. Q-RT-PCR analysis validated the significantly reduced expression of these genes, which confirmed the specific-interaction between miR-125a/b and the genes. Immunoblotting analysis further revealed activation of ER stress in the MI heart, indicated by the activation of ER markers, Grp78 and phosphor-JNK, in the MI cardiomyocytes. The adverse consequences from multiple compromised signalling led to the development of cardiac apoptosis and heart failure. In vitro, exposure the human cardiomyocytes AC16 to hypoxia or Lipopolysaccharides treatment upregulated miR-125a/b expression which was accompanied with impaired mitochondrial biosynthesis and oxidative metabolism, leading to apoptotic cell death.

Conclusion:
This novel finding defines miR-125a/b as key regulators of cardiac mitochondrial and ER homeostasis, which may lend support to the development of therapeutic strategy for MI by manipulating miR-125a/b abundance.
Abstract 266: Distinct Satellite Cell Trajectories Characterize Regenerative And Non-regenerative Responses In The Ischemic Limb
Authors: Xu Yueyuan , Peters Derek , Lin Xin , Wei Xiaolin , Southerland Kevin , Diao Yarui
Published: November 2022

Introduction:
Skeletal muscle regeneration is a critical determinant of outcomes in the ischemic limb. The precise mechanisms of skeletal muscle recovery in the ischemic limb remain unknown. A major hindrance to fully understanding the regenerative process in limb ischemia is determining the heterogeneity and cell fate decisions of muscle satellite cells (MuSCs).

Methods:
We analyzed muscle regeneration in C57BL/6 (regenerative phenotype) and BALB/c (non-regenerative phenotype) mice undergoing limb ischemia using single-cell RNA sequencing spanning 4 distinct time points (no injury, days 1, 3, and 7). We also performed trajectory inference to observe stage-specific regulatory programs during this dynamic process.

Results:
We identified 9 distinct MuSC populations in both strains (Figure A). Pseudotime analysis presented an organized regeneration trajectory of cells from quiescent to proliferative to differentiated MuSCs (Figures B-C). Further analysis demonstrated disparate MuSC fate decisions between C57BL/6 and BALB/c mice (Figure D-E). C57BL/6 mice displayed an organized progression of cells from a quiescent sate to proliferating and committed myoblast. In contrast, BALB/c mice demonstrated aberrant MuSC regeneration, highlighted by precocious differentiation (Figure E). Furthermore, gene set enrichment analysis (GSEA) confirmed impaired MuSC proliferation in BALB/c mice (Figure F).

Conclusions:
These findings advance our understanding of MuSC fate decisions in the ischemic limb and provide a potential mechanism for myopathy observed in PAD patients.
Abstract 289: Utilization Of Intra-Arterial Contrast For Computed Tomography Aortography In A Swine Model
Authors: Treffalls Rebecca N , Poe Kelly , Abdou Hossam , Stonko David P , Edwards Joseph , Ptak Thomas , White Joseph M , Scalea Thomas M , Morrison Jonathan J
Published: November 2022

Background:
Intraoperative computed tomography (CT) imaging with endovascular catheter delivery of intra-arterial (IA) contrast could potentially provide higher signal attenuation in directed anatomic locations with lower contrast volumes. We compared image quality and attenuation timing of IA versus IV contrast protocols for abdominal imaging.

Methods:
Five anesthetized swine had internal jugular access and ascending aortic pigtails placed percutaneously via the brachial artery. The IV contrast protocol utilized 100 mL of iodinated contrast at 5 mL/sec over 20/sec. The IA protocol delivered 50 mL at 5 mL/sec over 10/sec. A 16-slice CT scanner with a 10 mm detector acquired static serial images at 1 image/second for 45 secs. Region-of-interest markers were used to select the aorta and portal vein to capture arterial and venous attenuation via Hounsfield Units (HU) per second. Attenuation curves were assessed using a Pearson’s correlation. Adequate attenuation was defined as >100 HU a priori and image quality assessed using Contrast-to-Noise Ratio (CNR).

Results:
Both contrast protocols achieved adequate image attenuation with an aortic peak of 665 ± 226 (Mean HU ± SD) for IA and 414 ± 141 for IV. IA contrast achieved faster peak aortic attenuation compared to IV contrast [8 vs 20 sec; p<0.001] (Fig 1). Portal values (attenuation and time to peak) were similar for IA vs IV: [146 ± 46 vs 169 ± 39; 34 vs 42 sec; p<0.05]. IA administration achieved a superior CNR in less time compared to IV [10 vs 23 sec; p<0.001]. All curves were modeled using non-linear least squares regression and achieved an R
2
>0.94; p<0.001.

Conclusion:
IA contrast achieves adequate opacification and a superior CNR compared to IV contrast, while using a smaller volume for intraoperative directed imaging. The incorporation of intraoperative CT with IA contrast could radically change imaging protocols for endovascular aortic repair.
Abstract 298: Ketosis Prevents Abdominal Aortic Aneurysm Progression And Rupture In Male Rats
Authors: Elizondo Benedetto Santiago , Zaghloul Mohamed , Arif Batool , Sastriques Dunlop Sergio , Zayed Mohamed A
Published: November 2022

Abdominal aortic aneurysms (AAA) are common in aging populations, and its rupture is associated with high mortality. There is no effective medical therapy for the prevention of AAA expansion and rupture. Ketone bodies (KB) are argued to produce novel responses against injury. Serum KB are elevated through a ketogenic diet (KD) or the intake of exogenous KB (EKB). We hypothesize that ketosis can reduce AAA expansion and risk of rupture. Male Sprague-Dawley rats underwent intraluminal exposure to porcine pancreatic elastase (PPE) to induce AAA formation. Rats were also administered daily β-aminopropionitrile (BAPN) to promote AAA rupture. Control rats (N=12) were fed a standard diet (SD). Treated rats were given either a KD (N=9) or EKB (N=10) starting 3 days post AAA induction. Ketosis was verified by blood BHB level >0.5mmol/L. In vivo aortic diameter was evaluated by USG. After 2 weeks, survival animals were harvested, and AAA tissue analysis was performed. Rupture AAA was determined by necropsy. Rats treated with KD consistently remained in ketosis, while rats treated with EKB remained in ketosis for only 8-hour per day. AAA size was significantly reduced (p<0.01 and p<0.05) in both, KD and EKB groups at week 1 and 2 respectively. Rupture rate was also reduced in the KD and EKB groups (22% and 40%, respectively) compared to 67% in the SD group (p=0.03 and 0.12, respectively). AAA trichrome staining demonstrated greater content of collagen in KD and EKB groups when compared to SD group (p=0.08 and 0.02, respectively). AAA gelatin zymography demonstrated significantly decreased active MMP 9 levels in KD and EKB groups (p<0.05) while total MMP 2 was attenuated in the KD group (p<0.01). Ketosis appears to reduce AAA expansion and risk of rupture. This may have resulted from AAA increased collagen deposition as well as decreased MMP activity. These findings provide initial impetus to investigate whether ketosis in patients with small AAAs can prevent expansion and risk of rupture.
Abstract 301: Inhibition Of Tlr-7 Signaling Attenuates TAAD Formation
Authors: Qi Xiaoyan , Liao Mingmei , Hung Alex , Arnaoutakis George , Upchurch Gilbert R , Jiang Zhihua
Published: November 2022

The critical role for chronic inflammation in the development of thoracic aortic aneurysms and dissections (TAADs) has been recognized in both experimental and clinical settings. However, challenges remain on translating this knowledge to clinical applications. In this study, we tested the hypothesis that TLR-7 signaling triggered by self-RNAs substantiates chronic inflammation, promoting TAAD formation. A mouse TAAD model induced by SMC-specific deletion of
Tgfbr1
(

Tgfbr1
iko

) was used. In this model, the expression of TLR-7 was progressively upregulated during the early stages, resulting in a two-fold differential at two weeks compared to control aortas (
P
=0.003). RNAScope assays showed that cells located in the media and adventitia were responsible for the upregulation. Additionally, immunofluorescence (IF) staining showed that TLR-7 is induced in human TAADs. Treatment with hydroxychloroquine, which inhibits TLR-7 receptor function, significantly reduced the number of intimal/medial tears (
P
=0.026) and mitigated intramural hemorrhage (
P
=0.007) compared with vehicle controls (n=7/group). Further assays with bone marrow derived dendritic cells demonstrated that RNAs, particularly small RNAs, extracted from TAADs induced significantly higher IFNα/β levels compared with normal aortas (
P
=0.025). Similarly, RNAs extracted from

Tgfbr1
iko

SMCs induced higher levels of IFNα, IP-10, and MCP1 compared with those harvested from wild type SMCs. Necroptosis (labeled with RIPK3 and pMLKL) and RNA-oxidation (labeled with 8-OHG) were evident in the media of TAADs, and might serve as the source for the endogenous TLR-7 ligand. Finally, treatment with the TLR-7 specific inhibitor, M5049, at a dose of 1.0 or 5.0 mg/kg/day via oral gavage prevented dilation of TAADs at four weeks compared with vehicle controls (9% vs. 8% vs. 23%, P<0.001). In conclusion, self-RNAs released from stressed and dying cells is associated with chronically inflamed aortic tissue and promotes TAAD development via triggering TLR-7 signaling. Blocking TLR-7 signaling may represent a novel strategy to treat human TAADs.
Abstract 310: MRI Characterization Of Thrombus For Differentiating Acute And Chronic Dvt
Authors: Sinha Kavya , Karmonik Christof , Roy Trisha , Lumsden Alan B
Published: November 2022

Background:
Optimal patient selection for the endovascular treatment of iliofemoral DVTs remains a significant clinical challenge.
2
Symptom onset is used as a gross surrogate for determining the composition of the clot to determine if it is amenable to endovascular treatment. As thrombus ages, collagen deposition increases, and the bulk of fibrin within the thrombus becomes crosslinked and resistant to lysis
3
. Symptom onset has poor correlation with collagen content
4
and is not a predictor of endovascular success. The goal of this study is to relate MRI characteristics to clot composition to guide patient selection for endovascular treatment of iliofemoral DVT in the future.
5

Methods:
2 patients with popliteal deep venous thrombus that presented within a week of symptom onset underwent Inari thrombectomy. Patient 1 was imaged in the 7T MRI and patient 2 was imaged with 3T Both patients had T2-weighted (T2W) and UTE sequences.

Results:
Patient 1 had a thrombus with high collagen content indicated by low signal intensity on T2W imaging and high signal intensity on UTE MRI. The extracted clot had gross chronic characteristics and was extremely challenging to extract despite multiple passes. Patient 2 had a thrombus with low collagen content indicated by low signal intensity on T2W imaging and isointense on ultrashort echo time imaging. The clot had acute gross characteristics and was easy to extract.

Conclusion:
Symptom onset is not always reflective of the composition of deep venous thrombus. MRI characterization of thrombus can aid patient selection for endovascular treatment by differentiating patients with high and low collagen content.
Abstract 311: Different Oral Antithrombotic Therapy For The Treatment Of Ventricular Thrombus
Authors: Yang Qing , Lang Xinyue , Quan Xin , Liang Yan
Published: November 2022

Aim of the study:
To evaluate the utilization of oral antithrombotic agents - NOACs, VKAs or antiplatelet therapy (APT) - in ventricular thrombus (VT) patients and to analyze factors for thrombus resolution and clinical outcomes.

Methods:
This retrospective observational study was conducted from 2010 to 2019 in National Center of Cardiovascular Diseases of China. We included patients with VT confirmed by imaging. The primary outcome was the rate of thrombus resolution. HR was calculated with or without adjustment for covariates using Cox proportional hazards regression models.

Results:
298 patients were included. 45.3% (135 of 298) of them received VKAs, 20.5% (61 of 298) received NOACs, and 34.2% (102 of 298) received APT. NOACs were more likely to have the thrombus resolution within 12 weeks follow-up comparing VKAs or APT (HR 1.97, 95% CI 1.31 to 2.98, p=0.001; HR 2.65, 95% CI 1.67 to 4.20, p<0.001) with or without adjustment. In univariable analysis, patients who were diagnosed as dilated cardiomyopathy or had a history with heart failure had a greater risk to have the thrombus resolved (HR 1.89, 95% CI 1.26 to 2.85, p=0.002; HR 2.27, 95% CI 1.59 to 3.24, p<0.001). In subgroup analysis, patients with coronary artery diseases, the rate of thrombus resolution favored NOACs in comparison with VKAs or APT (HR 2.17, 95% CI 1.08 to 4.39; HR 2.16, 95% CI 1.05 to 4.43)). No significant differences were identified in bleeding rate, thromboembolism rate or all-cause death in 12 months follow-up.

Conclusion:
Our findings showed that patients who were diagnosed with dilated cardiomyopathy, or with a heart failure medical history had a greater thrombus resolution. Within 12 weeks follow-up, patients with NOACs had a higher resolution and a similar safety profile comparing VKAs or APT, which persisted after adjusting for other factors. Large randomized controlled trials are required urgently.
Abstract 313: TGF-beta Signaling Mediates Endothelial-to-mesenchymal Transition During Arteriovenous Fistula Remodeling In The Chronic Kidney Disease Environment
Authors: ZHANG WEICHANG , Gonzalez Luis , Keyuree. Satam , Ohashi Yuichi , Aoyagi Yukihiko , Setia Ocean , Jodhel Destina , Xin Li , Chang Shu , Dardik Alan
Published: November 2022

Objectives:
Arteriovenous fistulae (AVF) are the optimal conduit for hemodialysis access in patients with end-stage chronic kidney disease (CKD); however, AVF have high rates of failure due to aggressive neointimal hyperplasia(NIH). NIH in the CKD environment is characterized by endothelial-to-mesenchymal transition (EndMT). We hypothesized that endothelial TGF-β signaling promotes EndMT to cause AVF failure.

Methods:
Aortocaval AVF were created in C57BL/6J mice, with control mice having sham procedures. 3 weeks prior to AVF, CKD was created via 5/6 nephrectomy (Nx); five groups were tested, including sham, sham+5/6Nx, AVF+0/6Nx, AVF+3/6Nx, AVF+5/6Nx. Endothelial cell (EC)-and smooth muscle cell (SMC)-specific TGFβRI II knockout mice were treated with tamoxifen 2 weeks prior to AVF; Ultrasound was used to determine AVF diameter and patency at postoperative days 7, 14, and 21. AVF were harvested at days 7 or 21 and examined with histology, Immunofluorescence and Western blot.

Results:
There was no difference between mice treated with 0/6Nx or 3/6Nx regarding AVF diameter, AVF wall thickness and accumulation of extracellular matrix (ECM) components fibronectin, collagen1, and collagen3; however, AVF diameter was significantly decreased in mice treated with 5/6Nx compared with 0/6Nx or 3/6Nx, whereas there was increased AVF wall thickness as well as immunoreactivity of fibronectin (p=0.031), collagen1 (p=0.022), collagen3 (p=0.005), and TGF-β1 (p=0.022), compared with 0/6Nx. Endothelial cells in the AVF of mice treated with 5/6Nx had significantly increased immunoreactivity of the mesenchymal markers notch3 (p<0.0001), vimentin (p<0.0001) and FSP-1 (p=0.02) compared to 0/6Nx. However, blocking TGFβ signaling using either EC-KO or SMC-KO mice had significantly decreased immunoreactivity of mesenchymal markers and wall thickness (p<0.0001), consistent with reduced EndMT, and increased diameter (p=0.035). Interestingly, the AVF wall thickness in EC-KO mice was thinner than in SMC-KO mice.

Conclusion:
Both EC- and SMC-specific TGF-β signaling promote EndMT and wall thickening during venous remodeling in the CKD environment. These data suggest that the TGF-β signaling pathway may be a potential therapeutic target to prevent AVF failure.