bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 211: Response Gene to Complement 32 Deficiency Protects Endothelial Cell From Inflammation and Attenuates Atheroslcerosis
Authors: Cui Xiao-Bing , Luan Jun-Na , Dong Kun , Wang Yongyi , Chen Sisi , Watford Wendy T , Chen Shi-You
Published: July 2021

Response gene to complement 32 (RGC-32) is involved in diet-induced obesity and hepatic steatosis. Therefore, we hypothesized that RGC-32 plays a role in atherosclerosis. Our current study showed that RGC-32 expression was dramatically induced in endothelial cells (ECs) of the atherosclerotic lesions from both apolipoprotein E-deficient (ApoE
-/-
) mice and diseased human arteries. RGC-32 deficiency (Rgc32
-/-
) significantly attenuated the high-fat diet-induced and spontaneously developed atherogenesis in ApoE
-/-
mice without affecting the serum lipid profiles. In addition, Rgc32
-/-
decreased the macrophage content without affecting fibrous cap size. Rgc32
-/-
in bone marrow cells had no effect on atherosclerosis development, and Rgc32
-/-
mice transplanted with wild-type (WT) bone marrow cells exhibited decreased atherosclerotic lesion area and macrophage infiltration, suggesting that RGC-32 in resident vascular cells plays a critical role in the atherogenesis. Rgc32
-/-
decreased the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in ECs both
in vivo
and
in vitro
, resulting in a decreased monocyte-EC interaction. Mechanistically, RGC-32 regulated ICAM-1 and VCAM-1 transcription via activation of nuclear factor (NF)-κB. It appeared that RGC-32 interacted with NF-κB, which facilitated NF-κB binding to ICAM-1 and VCAM-1 promoters. Indeed, blockade of NF-κB activity by its selective inhibitor ammonium pyrrolidinedithiocarbamate blocked RGC-32-induced ICAM-1 and VCAM-1 expression. In conclusion, RGC-32 promoted atherogenesis by inducing monocyte-EC interaction through NF-κB-dependent induction of endothelial ICAM-1 and VCAM-1 expression.
Abstract 223: CXCR4 Regulates B1 Cell Localization, Proliferation, Survival, and Atheroprotective IgM Production
Authors: Upadhye Aditi , Srikakulapu Prasad , Nguyen Anh , McSkimming Chantel , Tsimikas Sotirios , Taylor Angela , McNamara Coleen
Published: July 2021

Background:
B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory natural IgM antibodies that recognize oxidation-specific epitopes, such as MDA-LDL, present in diseased arteries. The bone marrow, spleen, and omental fat are known niches for B1 antibody production. However, the mechanisms underlying B1 localization to these sites and B1 antibody production are currently unclear.

Methods and Results:
To identify key immune mediators that may be relevant to atherosclerosis, our lab has correlated surface marker expression on peripheral blood mononuclear cells with clinical markers of atherosclerosis in a human cohort. Expression of the chemokine receptor CXCR4 on circulating B1 cells associates with decreased plaque burden in coronary arteries and increased plasma levels of anti-MDA-LDL IgM antibodies. To study the role of CXCR4 in modulating B1 cell function, we generated mice with B cell-specific loss of CXCR4 on the atherogenic ApoE
-/-
background (CXCR4
BKO
). Chow-fed, 8-week-old CXCR4
BKO
mice demonstrate fewer B1 cells (N=6-8, p<0.0001) and IgM antibody-secreting cells (N=6, p=0.004) in the bone marrow, and reduced plasma total IgM levels (N=6-8, p=0.04), relative to littermate controls (CXCR4
WT
). To determine the role of CXCR4 on the B1 cell subset specifically, we adoptively transferred CXCR4
WT
or CXCR4
BKO
B1 cells into lymphocyte-deficient Rag1
-/-
ApoE
-/-
mice. After 16 weeks of Western diet feeding, recipient mice given CXCR4
BKO
B1 cells demonstrate significantly reduced plasma IgM levels (n=5-7, p=0.02), and fewer donor B1 cells in the spleen, peritoneal cavity, and omental fat compared to recipients given CXCR4
WT
B1 cells. Few to no donor cells were detected in the bone marrow. To determine whether CXCR4 has a role in B1 cell proliferation or survival, BrdU incorporation and expression of the death receptor FasR were assayed in CXCR4
WT
and CXCR4
BKO
B1 cells. B1 cells from CXCR4
BKO
mice display heightened BrdU incorporation (n=4, p=0.005), yet have increased expression of FasR (n=4, p=0.01) relative to CXCR4
WT
B1 cells.

Conclusion:
Our data demonstrate novel roles for CXCR4 in regulating several processes in B1 cells, including proliferation and survival, consequently impacting production of IgM.
Abstract 233: Genetic Evidence for Overlap in the Pathogenesis of Peripheral Artery Disease and Coronary Artery Disease
Authors: Klarin Derek , Small Aeron , Huang Jie , Lynch Julie , Arya Shipra , Assimes Themistocles L , Natarajan Pradeep , Saleheen Danish , Kathiresan Sekar , Rader Daniel J , Concato John , Gaziano J. Michael , Cho Kelly , Sun Yan , Wilson Peter W , Chang Kyong-Mi , O’Donnell Christopher J , Tsao Philip S , Damrauer Scott M ,   VA Million Veteran Program
Published: July 2021

Introduction:
Peripheral artery disease (PAD) is thought to share atherosclerotic biologic mechanisms and traditional risk factors with coronary artery disease (CAD). Although significant progress has been made in identifying genetic contributors to CAD, genetic factors underlying PAD remain largely uncharacterized. The aim of our study was to evaluate the shared genetic architecture between PAD and CAD.

Hypothesis:
DNA sequence variants associated with CAD risk are also associated with PAD.

Methods:
We analyzed the electronic health records from 239,621 U.S. Veterans of European ancestry with available imputed genetic data from the VA Million Veteran Program. Within this cohort we identified 16,364 participants with PAD, and 67,636 with CAD. We constructed additive weighted genetic risk scores (GRS) of 57 DNA sequence variants previously associated with CAD at genome-wide significance. We standardized the GRS to have a mean of zero and standard deviation (SD) of 1, and assessed the strength of its association with PAD through logistic regression. Models were adjusted for age, sex, and 7 principal components of ancestry. In a mediation analysis, we then included CAD status in the model to examine if these genetic associations remained significant after accounting for a CAD diagnosis.

Results:
The CAD GRS was significantly associated with clinical CAD [OR = 1.22 per SD increase in GRS, (95%CI: 1.21 -1.23, Z-Score = 44.5, P < 2 x10
-16
)]. The CAD GRS was also significantly associated with PAD [OR = 1.12 per SD increase in GRS, (95%CI: 1.10-1.14, Z-Score = 14.5, P < 2x10
-16
)]. This magnitude of association was attenuated when further adjusting for CAD in the regression model but remained strongly significant [OR = 1.05 per SD increase in GRS, (95%CI: 1.03-1.07, Z-Score = 6.2, P = 5.6x10
-10
)].

Conclusions:
In the largest single cohort of CAD and PAD cases worldwide, we found that a genetic risk score of DNA sequence variants associated with risk for CAD are also associated with risk of PAD, even after accounting for the presence of coronary atherosclerosis. These data suggest significant overlap in the mechanisms underlying development of atherosclerosis within both the peripheral and coronary arterial beds.
Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells
Authors: Barnhill Spencer , Arumugam Prakash , Matsuura John , Berceli Scott , Carroll Katie , Wrenshall Lucile
Published: July 2021

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.
Abstract 241: An Increased Response to Injury by Smooth Muscle Cells of Human Veins at Valve Sites
Authors: Kikuchi Shinsuke , Chen Lihua , Xiong Kevin , Saito Yukihiro , Azuma Nobuyoshi , Tang Gale , Sobel Michael , Wight Thomas , Kenagy Richard D
Published: July 2021

Objective:
Venous valves are prone to injury, thrombosis and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs non-valve sites to elucidate biological differences associated with vein valves.

Methods:
SMC migration was measured using 2.5 mm
2
explants of the intima/media of valve sinus segments (without valve leaflets) vs. non-valve segments of human saphenous veins. Proliferation and death of SMCs was determined by staining for Ki67 and TUNEL, respectively. Proliferation and migration of passaged valve vs non-valve SMCs was determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs non-valve intima/media was determined by RNA-Seq.

Results:
Valve SMCs demonstrated greater proliferation within tissue explants compared to non-valve SMCs (19.3±5.4% vs. 6.8±2.0% Ki67 positive nuclei at 4 days, respectively; mean ± SEM, 5 veins; P<.05). This was also true for migration (18.2±2.7 vs. 7.5±3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P<.0001). Cell death was not different (39.6±16.1% vs. 41.5±16.0% TUNEL positive cells, respectively, at 4 days, 5 veins). Cultured valve SMCs also proliferated faster than non-valve SMCs in response to PDGF-BB (2.9±0.2 vs. 2.1±0.2 fold of control, respectively; P<.001; N=5 vein’s paired cells). This was also true for migration (6.5±1.2 vs. 4.4±0.8 fold of control, respectively; P<.001; N=7 vein’s paired cells). Blockade of FGF2 inhibited the increased responses of valve SMCs, but had no effect on non-valve SMCs. Exogenous FGF2 increased migration of valve, but not non-valve SMCs. Unexpectedly, blockade of FGF2 did not block migration of valve or non-valve SMCs from tissue explants. 37 genes were differentially expressed by valve compared to non-valve intimal/medial tissue (11 veins).

Conclusions:
Valve, compared to non-valve, SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathological lesion formation in valves. While FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in valve wall tissue remain unidentified. Here, the newly identified differentially expressed genes may play a role.
Abstract 25: Ultrasound Cavitation Mediated Gene Delivery of ApoAI to the Artery Wall for Modulating Plaque Content
Authors: Ammi Azzdine , Christian Devon , Lindner Jonathan , Tavori Hagai
Published: July 2021

Gene therapy is a potential approach for inhibiting many of the processes that occur during atherogenesis, particularly when targeting key molecular regulators that have multiple adverse downstream signaling pathways. Local expression of human-apoAI in the artery wall has been shown to protect against atherosclerosis in mice, without affecting systemic HDL levels. We hypothesized that focal gene delivery in arterial plaque is possible using ultrasound (US)-mediated cavitation of acoustically-active cationic microbubble gene carriers. Lipid-shelled cationic microbubbles (MB) with a decafluorobutane gas core were prepared. Bicistronic plasmids (luciferase with either GFP or apoAI) were charge coupled to the MB and injected intravenously in 12 LDLR
-/-
mice fed high-fat diet for 8 weeks. The thoracic aortic root was exposed to US at 1.6MHz, a pulsing interval of 1Hz, and a mechanical index of 1.3 in order to produce repetitive cavitation of MB.
In vivo
optical imaging showed a focal luciferase activity only at the site of UMGD starting from a day after the procedure and lasting for 7-10 days. Activity was subsequently localized to the aortic root on
ex vivo
optical imaging. Immunohistochemistry of the aortic root confirmed luciferase transfection of both endothelial cells of the aortic root and base of the aortic cusps, and intramural cells in atherosclerotic lesions, likely macrophages. We further show that gene therapy with the luciferase-apoAI plasmid, results in migration of cells to the thymus 6-8 days after transfection. These results indicate that local US-mediated gene therapy approach for controlling atherosclerosis is feasible in this pre-clinical model.
Abstract 259: Phenotype Switch and Altered Mechanosignaling in Vascular Smooth Muscle Cells From Marfan Syndrome Mice
Authors: Nolasco Patricia , Fernandes Carolina G , Ferraz Mariana S , Soares Lazaro R , Alencar Adriano M , Laurindo Francisco R
Published: July 2021

Background:
Mechanisms whereby fibrillin-1 gene mutations promote aortic aneurysms in Marfan Syndrome (MFS) are complex. Vascular smooth muscle cells (VSMC) are crucial to aneurysm pathophysiology through (1) support of mechanical homeostasis, in which contractile VSMC connect extracellular matrix to shield synthetic VSMC against high mechanical loads and (2) key effects of synthetic VSMC on extracellular matrix organization. However, little is known about VSMC phenotypic adaptation and response to mechanical loading in MFS development.

Methods:
We investigated phenotypic patterns and response to cyclic stretch in VSMC cultured from aortae of wild-type (WT) mice or MFS mice bearing mgΔ
lpn
mutation, which develop aortic dilation/thickening from the first month of age, accelerated after 6 months. We studied VSMC from 8-9 week-old mice to focus on early disease stages. VSMC remained either static or underwent 10% cyclic stretching (1Hz, 8h). Protein expression/morphology were evaluated by Western and confocal analyses. We also performed Traction Force Microscopy to assess force distribution profile.

Results:
There were significant phenotypic changes in MFS-VSMC vs. WT, with loss of fusiform shape and enhanced spreading, promoting ≈7-fold increased in cell area vs. WT (p<0.001, n=5) and >60% decrease in calponin expression vs. WT (p<0.05, n=3). Also, MFS-VSMC had a 10-fold greater expression of endoplasmic reticulum stress marker Grp78 (n=2). Cyclic stretch promoted expected increase in synthetic phenotype markers in WT-VSMC but an opposing contractile phenotype switch in MFS-VSMC, with decreased calponin and pFAK expression and increased stress fiber buildup. Cyclic stretch promoted similar increase in Grp78 expression in WT vs. MFS-VSMC. MFS-VSMC showed ca.60% lower capacity to generate adhesive force and extracellular matrix contractile momentum (p<0.003, n=10), associated with lower deformation energy
in vitro
(9,16 vs. 1,67 pJ, p<0.004, n=6).

Conclusions:
MFS associate with important deregulation of VSMC phenotypic adaptations, which may impact on their force development and disrupt force distribution in aorta.
Abstract 263: Thrombin-Mediated Human Aortic Endothelial Barrier Dysfunction Alters Microrna Pathways Involved in Cell Survival, Injury, and Cancer
Authors: Wardell Christine M , Harlock John , Rapanos Theodore , Fox-Robichaud Alison , Gross Peter , Howe Kathryn L
Published: July 2021

Introduction:
Endothelial cells (EC) must maintain an effective physiologic barrier in a highly dynamic environment. Capable of regulating other cells within the vasculature (e.g. smooth muscle cells), ECs govern response to injury and inflammation. microRNAs (miR) are emerging as critical regulators of vascular disease, implicating EC miRs in homeostasis maintenance. Increased EC permeability has been documented in atherosusceptible regions of the aorta suggesting it plays a role in disease development, but whether this is a cause or consequence is unknown.

Hypothesis:
We hypothesized EC barrier dysfunction is a critical event that perpetuates chronic vascular disease via altered miR profiles.

Methods:
Human aortic endothelial cells (HAEC) cultured in transwells were exposed to thrombin (0.5, 1, 2U/ml) and permeability measured by fluorescence flux across monolayers at 90 minutes. Total HAEC RNA was isolated at various timepoints with individual miR transcripts counted using nanoString nCounter® (n=6 samples). miR that was altered >2-fold compared to controls were analyzed using nSolver
™
software and Ingenuity® Pathway Analysis (IPA).

Results:
Thrombin exposure increased EC permeability to 130±8.4% of untreated controls (2U/ml thrombin; p<0.05; n=16 transwells). Heatmaps from miR counts showed extensive miR profile changes in response to thrombin (n=6 samples (control, 90min, 4h, 24h)). Using IPA, miRs were clustered based on seed regions and matched with experimentally confirmed mRNA targets. At all timepoints, mRNA targets were shown to be most involved in cancer and injury disease pathways, with top cellular functions identified as cell movement, proliferation, growth, and survival.

Conclusions:
In response to a permeability insult, HAEC miR levels are significantly altered. Although our preliminary data need to be further substantiated, they highlight a possible mechanism whereby EC barrier dysfunction is a critical event that perpetuates chronic vascular disease. In this way, we might envision a scenario not unlike cancer (which has been compared to atherosclerosis), where an initial appropriate endothelial repair response becomes dysregulated in the context of ongoing injury (e.g. permeability) in atherosusceptible regions.
Abstract 299: Effect of Low-Dose versus Very-Low-Dose Rivaroxaban on Stroke and Major Bleeding Among Atrial Fibrillation Patients Undergoing Percutaneous Coronary Intervention: A Bivariate Analysis on the Net Clinical Benefit
Authors: Marszalek Jolanta , Mehrsefat Sara , Chi Gerald
Published: July 2021

Background:
Among atrial fibrillation (AF) patients undergoing percutaneous coronary intervention (PCI), both low-dose and very-low-dose rivaroxaban plus antiplatelets had similar rates of stroke and major bleeding compared to triple therapy.

Methods:
To assess the risk-benefit profile of two rivaroxaban dosing strategies with a spectrum of non-inferiority margin, a previously described method of bivariate analysis was used. In the PIONEER AF-PCI trial, 2,124 patients were randomized to three groups and followed for 12 months: 1) low-dose rivaroxaban (15 mg once daily) plus a single P2Y
12
inhibitor (N=709); 2) very-low-dose rivaroxaban (2.5 mg twice daily) plus DAPT (N=709); and 3) warfarin plus DAPT (N=706). Risk differences in stroke and in TIMI major bleeding were simultaneously analyzed to arrive at net clinical benefit (NCB).

Results:
Compared to warfarin, both rivaroxaban dosing regimens had comparable risk of TIMI major bleeding (low-dose: -0.86% [95% CI: -2.48% to 0.86%], P=0.30 vs. very-low-dose: -1.17% [95% CI: -2.73% to 0.39%], P=0.14). Risk difference in all-cause stroke between rivaroxaban and warfarin was not significant (low-dose: 0.15% [95% CI: -0.94% to 1.23%], P=0.79 vs. very-low-dose: 0.41% [95% CI: -0.73% to 1.56%], P=0.48). With a non-inferiority margin of 80% or above (equivalent to a risk difference of 2.40%), both rivaroxaban regimens demonstrated similar, favorable NCB against warfarin (low-dose: -1.92% [-3.28% to -0.20%] vs. very-low-dose: -1.74% [-3.09% to -0.17%]).

Conclusions:
Bivariate analysis is a novel methodology that allows simultaneous display and assessment of both efficacy and safety outcomes. In the management of AF patients undergoing PCI, rivaroxaban (15 mg once daily or 2.5 mg twice daily) achieved a favorable benefit-to-risk tradeoff over warfarin at the non-inferiority margin of 80% or above.

Figure:
NCB of rivaroxaban dosing strategies. Shaded partition above the curve represents lack-of-benefit region.
Abstract 307: Circulating sRAGE is Associated With Aortic Dysfunction in Mice Models of Thoracic Aortic Aneurysm
Authors: Galatioto Josephine , Lai Eric , Bintanel-Morcillo Maria , Wang Tao , Ferrari Giovanni , Pyeritz Reed E , Bavaria Joseph E , Branchetti Emanuela
Published: July 2021

Backgound:
The Receptor for Advanced Glycation End products (RAGE) and its ligands are associated with vascular remodeling and trigger the release of a soluble receptor (sRAGE). We demonstrated that sRAGE is elevated in patients with thoracic aortic disease such as patients with bicuspid aortic valve and Marfan syndromes. Circulating sRAGE in these patients correlates with the presence of a dysfunctional aortic structure without linearly correlating with increasing aortic diameter. We hypothesized that elevated sRAGE levels are the result of structural degeneration during aortic aneurysm formation and that they are affected by pharmacological treatments aimed to inhibit vascular remodeling.

Methods:
Circulating sRAGE was tested by ELISA in two mouse models of thoracic aortic aneurysm (TAA): the AngII chronic infusion model and the hypomorphic FBN1
mgR/mgR
. C57BL6 mice were treated with AngII via osmotic pump for 28 days with or without losartan in drinking water. FBN1
mgR/mgR
mice were treated with losartan starting from postnatal day 16 (p16). Ascending aorta and blood were collected at day 28 of AngII infusion or at p60 in the hypomorphic model. Aortic dilatation and degeneration were tested by echocardiography and histological analysis.

Results:
Plasma sRAGE is significantly higher in AngII treated animals when compared to controls (118.1± 8.3 vs 45.6±5.01 pg/ml,
p
<0.0001) and is associated with aortic aneurysm formation and increased medial thickening. Similarly, sRAGE levels are higher in FBN1
mgR/mgR
mice compared to age-matched wild type (1939 ± 320.5 vs 45.6±5.01 pg/ml,
p
<0.0001) in concomitance with elastin degradation (media score 3.5± 0.8 vs 1.03.5± 0.2,
p
<0.05) and proximal aorta enlargement. Plasma sRAGE decreases in AngII and FBN1
mgR/mgR
mice treated with losartan when compared to age matched untreated or wild type mice (73±24.5 vs 118.1 ± 8.296 pg/ml, p<0.05 and 260.7 ± 4.333 vs 1939 ± 320.5 pg/ml, p<0.001) together with reduced aorta dilatation, medial thickening and elastin fragmentation.

Conclusion:
sRAGE is elevated in the presence of aortic dilatation in mouse models of TAA, as seen in human aneurysmal patients. These results suggest that sRAGE may be used as a circulating biomarker to assess disease severity in patients with TAA.
Abstract 31: Platelet Plasminogen Activator Inhibitor-1 is Regulated by a Major Chromosome 5 eQTL in Inbred Mice
Authors: Siebert Amy E , Verbeek Stephanie C , Johnston Alexander J , Brake Marisa A , Cleuren Audrey C , Johnsen Jill M , Banes-Berceli Amy , Battistuzzi Fabia U , Westrick Randal J
Published: July 2021

Decreased fibrinolytic activity is strongly associated with cardiovascular disease. Plasminogen activator inhibitor-1, (PAI-1, Serpine1) is a major circulating fibrinolysis inhibitor, >80% of which is found in platelets. Platelet PAI-1 (pPAI-1) is produced by the megakaryocytes and packaged directly into platelets, thus it is distinct from plasma PAI-1. Plasma PAI-1 levels have been extensively studied and consistently linked with cardiovascular disease. However, variation in pPAI-1, either in the presence or absence of cardiovascular disease, is unknown. To study PAI-1 expression, we surveyed ten mouse strains for pPAI-1 antigen. In particular, the LEWES/EiJ mouse strain had significantly increased pPAI-1 (and Serpine1 mRNA), with an average concentration of 3.1 pg/μg total platelet protein compared to C57BL/6J (B6), which had an average pPAI-1 concentration of 0.4 pg/μg total protein (q=0.0018). Outcrossing LEWES/EiJ x C57BL/6J produced F1 mice with average pPAI-1 levels of 1.6 pg/μg total protein (q=0.0018), suggesting a semidominantly-inherited Serpine1 regulatory effect. To identify pPAI-1 regulatory regions, we produced 48 F2 mice, measured pPAI-1 levels and genotyped 12 (with <0.4 pg/μg total protein) and 12 (with >1.0 pg/μg total protein) using the Mouse Universal Genotyping Array (MegaMUGA). QTL analysis revealed several candidate regions including a major significant 14.4 megabase locus (128.2-142.6 megabases) on Chromosome 5 (LOD score = 4.81). The Serpine1 gene resides within this candidate interval, strongly suggesting that a Serpine1 cis-eQTL is responsible for pPAI-1 expression differences between LEWES/EiJ and C57BL/6J. Identifying pPAI-1 expression control elements offers insights into platelet-specific gene expression and identifies a putative therapeutic target for modulating hemostasis.
Abstract 317: Satellite Cells Influence Collateral Vessel Formation via Paracrine Effects
Authors: Hansen Laura , Liu Wenxue , Joseph Giji , Weiss Daiana , Taylor W. Robert
Published: July 2021

Satellite cells are myogenic cells that play a critical role in skeletal muscle repair. They serve as stem cells for muscles, remaining dormant in healthy muscle but activating upon injury resulting in increased proliferation and differentiation into myoblasts. Another key aspect of muscle regeneration is reestablishing vascular supply, but the role of satellite cells in this process is not well established though they are known to produce a number of potential paracrine signals. Thus we hypothesized that satellite cells promote vascular growth through paracrine signaling induced by activation following muscle injury or ischemic damage from diseases such as peripheral artery disease. Using a murine model of hind limb ischemia, we showed that satellite cells increased 3.4 fold (p<0.01) in response to ischemia. To determine if satellite cells produce paracrine factors, we used a co-culture system for migration and proliferation. Satellite cells freshly isolated from the ischemic limb led to a 3.5 fold increase in smooth muscle migration (p<0.0001) and a 1.3 fold increase (p<0.01) in smooth muscle proliferation. Additionally, cultured satellite cells increased endothelial cell migration 2.8 fold. These results demonstrate the satellite cells produce paracrine factors which can drive both smooth muscle and endothelial cell migration and proliferation which are required for the development of collateral vessels. To test the potential therapeutic capability of satellite cells, alginate encapsulated satellite cells were delivered in the hind limb ischemic model. Using a whole animal in vivo imager to track luciferase expression of the cells, we found the encapsulated cells were viable for up to 2 weeks. The mice that received satellite cells also had significantly increased perfusion (28%, p<0.05) at 2 weeks as measured by Laser Doppler imaging. In conclusion our studies have shown that satellite cells increase in response to ischemia, produce paracrine factors that increase vascular cell migration in vitro, and lead to functional increases in perfusion in vivo. We believe these results demonstrate the critical role satellite cells play in collateral vessel formation and may be a potential new therapeutic approach for treating peripheral artery disease.
Abstract 323: Utilization and Outcomes of Carotid Artery Stenting vs. Carotid Endarterectomy
Authors: Joy Parijat S , Kumar Gagan
Published: July 2021

Introduction:
Carotid artery stenting is an alternative to carotid endarterectomy in average surgical-risk symptomatic patients and asymptomatic patients with ≥60% stenosis. We wanted to compare utilization and peri-procedural mortality between these procedures.

Methods:
The 2000-2013 National Inpatient Sample (NIS) was analyzed for admissions when procedures for carotid artery stenting (CAS) or carotid endarterectomy (CEA) were performed. Admissions when both procedures were performed were excluded. Trend of procedures and death during index admission was compared depending on prior cerebrovascular symptoms.

Results:
During the study period, 1991941 patients underwent CEA of which 9.12% were symptomatic and 343,741 patients underwent CAS of which 10.8% were symptomatic. Mean age for CAS vs CEA group was lower among both symptomatic (68.6 vs 69.6 yrs, p<0.001) and asymptomatic patients (70.7 vs 71.2 yrs, p<0.001). More males than females underwent CAS (57% vs 43%) and CEA (58% vs 42%). Both CAS and CEA during same admission was carried out in 20,875 (0.89%) patients. There was a rising trend of both CEA and CAS procedures in symptomatic and asymptomatic patients (ptrend < 0.001)(Figure A1 & B1). Trend of mortality has not changed significantly in all groups except for CEA in asymptomatic patients wherein mortality rate has decreased (ptrend <0.001)(Figure A2 & B2). On multivariable logistic regression analysis, associated conditions significant for mortality in symptomatic patients were atrial fibrillation (OR 2.05, p<0.001), myocardial infarction (OR 1.61, p=0.001) heart failure (OR 1.39, p=0.021) and malnutrition (OR 3.58, p<0.001). Adjusted likelihood of mortality after CAS vs CEA was higher in symptomatic (OR 3.78, p<0.001, C statistic 0.74) and asymptomatic patients (OR 2.00, p<0.001, C statistic 0.80).

Conclusion:
Utilization of CAS and CEA has increased over time. Mortality after CAS vs. CEA during index admission, remains high.
Abstract 324: Pathological Quantitative Assessment of Plaque Instability in Patients Undergoing Carotid Endarterectomy
Authors: Konishi Takao
Published: July 2021

Introduction:
Instability of carotid atherosclerotic plaques leads to cerebral thromboemboli and ischemic symptoms. However, there has been no specific pathological quantification for the instability of carotid atherosclerotic plaque. The purpose of this study was to quantify atherosclerotic plaque instability in patients undergoing carotid endarterectomy (CEA).

Methods:
Carotid plaques were collected after CEA from 67 symptomatic and 15 asymptomatic patients between May 2015 and August 2016. Samples were stained with hematoxylin/eosin and Elastica-Masson (E-Masson). Immunohistochemistry was performed by using an endothelial specific antibody to CD31, CD 34 and PDGFRβ. Plaques were assessed for histopathological characteristics.

Results:
Multivariable logistic regression analysis demonstrated that plaque instability was independently associated with the presence of plaque rupture (odds ratio [OR], 9.75, 95% confidence interval [CI]: 1.62 to 58.6, p = 0.013), the minimal fibrous cap thickness (FCT) (OR per 10 μm 0.70, 95% CI: 0.51 to 0.96, p = 0.025), the presence of microcalcification in the fibrous cap (OR 7.82, 95% CI: 1.35 to 45.4, p = 0.022) and the intraplaque microvessels (OR 1.91, 95% CI: 1.02 to 3.57, p = 0.043). If these four independent parameters were combined to a score using the equation derived from the multivariable logistic regression model (Logit(Score) = 0.179 + 2.277 * (insert 1 if plaque rupture present; else 0) - 0.355 * (insert minimal FCT in multiples of 10 μm) + 2.057 * (insert 1 if microcalcification in the fibrous cap present; else 0) + 0.646 * (insert intraplaque microvessels /mm
2
), the diagnostic efficiency could be improved to an AUC 0.92 (95% CI: 0.85-0.99, optimal cut-off value 0.814, sensitivity 89.6%, specificity 86.7%, PPV 96.8%, NPV 65.0%, diagnostic accuracy 89.0%).

Conclusions:
This study suggested the diagnostic scoring was useful for the quantification of carotid plaque instability in patients undergoing CEA.
Abstract 344: Comparative Metabolomic Profiling of Thrombotic Myocardial Infarction Reveals a Metabolic Signature Distinct From Non-Thrombotic Myocardial Infarction and Stable Coronary Artery Disease in Human Participants
Authors: DeFilippis Andrew P , Trainor Patrick J , Hill Bradford G , Amraotkar Alok R , Rai Shesh N , Hirsch Glenn A , Rouchka Eric C , Bhatnagar Aruni
Published: July 2021

Background:
Current non-invasive diagnostics for acute myocardial infarction (MI) identify myocardial necrosis rather than the primary cause and therapeutic target—plaque disruption and resultant thrombosis.

Aim:
The aim of this study is to identify change specific to plaque disruption and pathological thrombosis.

Methods:
We quantified 1,032 plasma metabolites by mass spectrometry in 11 thrombotic MI, 12 non-thrombotic MI and 15 stable CAD subjects at two acute phase [time of catheterization (T0), six hours (T6)] and one quiescent (>3 months follow-up) time points. A statistical classifier was constructed utilizing (T0) abundances of a parsimonious set of metabolites that demonstrated a significant change between quiescent phase and the acute phase that was distinct from any change seen in non-thrombotic MI or stable CAD subjects. Classifier performance as estimated by 10-fold cross-validation was suggestive of high sensitivity and specificity for differentiating thrombotic from non-thrombotic MI and stable CAD subjects.

Results:
Nineteen metabolites (Table 1) with an intra-subject fold change from time of acute thrombotic MI presentation to the quiescent state were distinct from any change measured in both the non-thrombotic MI and stable CAD subjects undergoing cardiac catheterization over the same time course (false discovery rate <5%).

Conclusions:
We have identified a candidate metabolic signature that differentiates acute thrombotic MI from quiescent state and from acute non-thrombotic MI and stable CAD. Further validation of these metabolites is warranted given their potential as diagnostic biomarkers and novel therapeutic targets for the prevention or treatment of acute MI.
Abstract 345: Detection of Hypercoagulant Condition Caused by Elevated Prothrombin With a Thrombin Generation Assay
Authors: Chang William C , Machlus Kellie R , Wolberg Alisa S , Ovanesov Mikhail V
Published: July 2021

Hypercoagulability resulting from elevated prothrombin is associated with increased thrombosis risk. Patients with elevated prothrombin include those with the G20210A mutation that represents the 2nd most common risk factor for venous thrombosis in European Caucasians. We hypothesized that the commercial thrombin generation assay (TGA), which measures the rate of synthetic thrombin substrate consumption, may detect procoagulant condition caused by elevated prothrombin.

Normal pooled plasma was treated with prothrombin or other coagulation factors to increase their levels to 200% or 400% of normal. TGA was performed using the Calibrated Automated Thrombogram (CAT) platform (Stago, USA) and analyzed with and without correction for substrate consumption. As expected, addition of elevated coagulation factors resulted in elevated TGA as measured by thrombin peak height (TPH) and endogenous thrombin potential (ETP). ETP allowed detection of up to 83% of spiked samples with 200% prothrombin but TPH has failed to distinguish these samples from the procoagulant samples obtained with other coagulation factors.

For samples with 400% prothrombin, CAT failed to return values for TPH for 40% of samples and ETP for 100% of samples but the remaining 60% of TPH values were detected as the procoagulant samples. By analyzing the raw fluorescence data directly, we found that elevated prothrombin, but not other coagulation factors, resulted in thrombin substrate depletion which allowed us to correctly distinguish all samples within our dataset regarding whether or not they had added prothrombin.

We conclude that the commercially available TGA has limited utility in distinguishing samples with elevated prothrombin but additional analysis of data can address this problem. With this additional analysis, TGA may be developable into new point-of-care test to diagnose patients with hypercoagulant conditions undetectable by currently used clotting assays.

Disclaimer:
This is an informal communication and represents authors’ best judgment. These comments do not bind or obligate FDA.
Abstract 35: Predictors and Etiologies of 30-Day Readmission Rate in Patients With Pulmonary Embolism: A National Population-Based Cohort Study
Authors: Setareh-Shenas Saman , Lemor Alejandro , Castaneda Mayorga Daniel , Alvarado Alonso , Herzog Eyal
Published: July 2021

Background:
The hospitalization rates of patients with pulmonary embolism (PE) has been on the rise. However, there is limited data recognizing the etiologies and predictors of early readmission rate in this patient population.

Methods:
We utilized the National Readmission Database (NRD) 2013, subset of the Healthcare Cost and Utilization Project (HCUP) sponsored by the Agency for Healthcare Research and Quality (AHRQ). PE was identified using ICD 9 code (415.1X) as primary diagnosis. Co-morbidities identified by “CM_” variables provided by NRD. Primary outcome was identified as predictors of 30-day readmission rates and secondary outcomes as the leading etiologies and trends of readmission rate. To identify predictors of outcome, a two level hierarchical logistical model was used.

Results:
We analyzed 76,994 patients with primary diagnosis of PE. Total of 11.6% (8,934) patients were readmitted within 30 days of index hospitalization. Significant predictors of readmission were associated with end stage renal disease, drug abuse, chronic lung disease, congestive heart failure, and gastrointestinal bleed during primary admission. In addition, female gender, those admitted to teaching hospital and with longer length of stay (LOS) had higher 30-day readmission rate. Remarkably, elderly age (age>75 years) was not associated with increase in readmission rates. The leading etiologies besides DVT/PE for readmission were sepsis/septic shock, cancer, heart failure and pneumonia.

Conclusion:
The rising hospitalization rate of patients with pulmonary embolism, imposes a higher burden on the cost of health care. We identified important predictors and etiologies of 30-day readmission rate. These findings may help in prevention of hospital readmissions and may decrease the cost of care.
Abstract 351: Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation
Authors: Zhao Xiao-Min , Wang Yun , Qin Shu-Cun , Jiang Xian
Published: July 2021

Hypothesis:
Phospholipid transfer protein (PLTP) has a direct effect on platelet aggregation, since PLTP knockout mice have longer bleeding time.

Methods and Results:
Platelets from humans or mice were prepared as were mouse platelet-rich plasma and human recombinant PLTP (rPLTP). In mice, we assessed ADP- and collagen-induced platelet aggregation, phosphatidylserine (PS) externalization, and photothrombosis-induced cerebral infarction. We found that human platelets produce PLTP. Platelet aggregation increased upon PLTP overexpression whereas it decreased with PLTP deficiency in a gene dose-dependent manner. Human rPLTP increased mouse or human platelet aggregation in a dose-dependent manner. PS externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild type controls, platelets from PLTP transgenic mice had significantly greater amounts of PS on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less on the surface, thus influencing fibrinogen binding. Moreover, rPLTP together with ADP significantly increased PS exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. Importantly, PLTP overexpression significantly accelerated the incidence of photothrombosis-induced infarction, whereas PLTP deficiency reduced the incidence.

Conclusions:
PLTP promotes PS externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. Thus, PLTP is involved in hypercoagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.
Abstract 355: Photoluminescent Polylactones Based Nanoscaffolds for Enhancing Reendothelialization
Authors: Kuriakose Aneetta E , Rajanikant Priyesh , Mali Upasana , Shan Dingying , Tang Liping , Banarjee Subhash , Yang Jian , Nguyen Kytai T
Published: July 2021

Percutaneous coronary intervention is commonly employed for revascularization. These strategies, however, injure the arterial wall initiating a cascade of inflammatory responses that culminates in restenosis. In our research work, we proposed to use a platelet mimicking fluorescent nano-scaffold system that can cloak an injured artery site from inflammatory cells, capture the endothelial progenitor cells using specialized targeting agents, and promote
in situ
endothelial regeneration. We used photo-luminescent polylactone (BPLP) copolymers such as BPLP-co-poly(L-lactide) (BPLP-PLLA) and BPLP-co-poly(lactic-co-glycolic) acid (BPLP-PLGA50:50 and BPLP-PLGA75:25) to prepare nanoscaffolds; their intrinsic fluorescent properties help us localize and monitor the scaffold’s degradation
in vivo
as endothelial regeneration ensues. Our initial studies were focused on optimizing a suitable polymer to be used to make a novel nano-scaffold system.

We fabricated ~200 nm sized BSA-loaded BPLP-polylactone based nanoparticles by standard double emulsion method. BPLP-PLGA based particles released BSA within 14 days, however, BPLP-PLLA showed slow and continued release for 28 days. BPLP-PLGA degraded completely within 4 weeks of study, whereas BPLP-PLLA degraded only 20% during the same time. The cyto-compatibility study showed that the BPLP-PLGA50:50, especially at high concentrations (> 1000μg/ml), is significantly toxic to the human umbilical vein endothelial cells; however, no such toxicity was observed for BPLP-PLGA75:25 and BPLP-PLLA. Hemo-compatibility of the particles demonstrated that they didn’t cause any significant lysis of red blood cells, and neither affected blood clotting kinetics compared to that of the control. Furthermore, we observed a minimal number of platelet attachment and activation on BPLP-PLGA materials compared to that of the glass surface. Based on these initial results we selected BPLP-PLGA 75:25 to fabricate nanoscaffolds. Currently, we are optimizing the effective particle size to enhance their margination towards the arterial wall and other factors, including specialized capturing agents to capture EPC, and growth factor cocktail to improve EPC homing and differentiation at the injured site.
Abstract 362: Knockout of NADPH Oxidase 2 Improves Global Metabolism and Endothelial Function in Aging Mice
Authors: Fan Lampson M , Geng Li , Li Jian-Mei
Published: July 2021

Oxidative stress attributable to the activation of a Nox2-containing NADPH oxidase has been suggested to play a crucial role in the development of aging-associated vascular diseases. However, the mechanism of endothelial Nox2 activation in normal aging process remains unclear. In this study, we investigated the therapeutic potential of targeting Nox2 in improving global metabolism and endothelial function at old age by using age-matched wild-type and Nox2 knockout mice at 3-4 months (young); 11-12 months (middle aged) and 21-22 months (aging). Compared to young mice, middle-aged and ageing wild-type mice had significantly higher blood pressure, hyperglycaemia, hyperinsulinaemia. These were accompanied by oxidative stress in multiple organs including the lung, the liver, the heart and the vessels. The vessel motor function was examined in an organ bath using aortas isolated from these mice. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in aortas of wild-type aging mice, and this was accompanied by increased expressions of Nox2 and markers of inflammation, activation of MAPK and Akt and decreased insulin receptor expression and function. However, these aging-associated disorders in aortas were significantly reduced by knocking out Nox2 in mice. In response to high glucose plus high insulin challenge, coronary microvascular endothelial cells isolated from wild-type mice displayed significantly increased Nox2 expression, oxidative stress and cell senescence, e.g. increased p53 expression and β-galactosidase activity. However, these responses were absent or significantly reduced in the endothelial cells isolated from Nox2 knockout mice. In conclusion, metabolic disorders in particular hyperglycaemia and insulin resistance play an important role in mediating Nox2 activation and oxidative stress in multiple organs in aging. Nox2 is involved in normal aging process-associated vascular inflammation and oxidative damage of endothelial dysfunction.
Abstract 363: Lysosomal Lipid Accumulation and Dysregulation of Downstream Cholesterol Homeostasis in Human Arterial Smooth Muscle Cells
Authors: Dubland Joshua A , Pryma Collin S , Allahverdian Sima , Wang Ying , Chan Teddy , Francis Gordon A
Published: July 2021

Background and Hypothesis:
Previously we reported that ≥ 50% of the foam cells in human coronary artery atherosclerosis are smooth muscle cell (SMC) derived, and that the rate-limiting cholesterol exporter, ATP-binding cassette transporter protein A1 (ABCA1), has reduced expression in SMCs compared to leukocytes in the intima. Upregulation of ABCA1 expression is dependent on normal flux of lipoprotein-derived cholesterol out of lysosomes and the subsequent generation of 27-hydroxycholesterol (27-OHC). Excess lipoprotein-derived cholesterol is also converted to cholesteryl esters (CE) through the actions of acyl-coA cholesterol acyltransferase (ACAT). Processes by which macrophages store lipoprotein-derived cholesterol in cytosolic and lysosomal compartments are well described, whereas less is known about SMCs.

Hypothesis:
We hypothesize that preferential SMC foam cell formation and the observed derangement in ABCA1 expression are associated with increased lysosomal sequestration of atherogenic lipids.

Methods and Results:
Human aortic SMCs and human monocyte-derived macrophages (HMMs) were loaded with aggregated LDL (agLDL) for 24 hrs, followed by a 24 hr equilibration without lipids to allow processing of lipoproteins. Data are presented as mean±SEM. In response to agLDL, CE content (nmol/mg protein) measured by LC/MS/MS increased from 2.2±1.3 to 277.0±18.2 in HMMs, and 3.5±5.3 to 46.8±14.5 in SMCs (n=9). Confocal microscopy indicated elevated lysosomal lipid accumulation in SMCs compared to HMMs. Levels of 27-OHC (ng/mg protein) as measured by LC/MS/MS increased from 80.9±9.5 to 281.4±22.7 (3.5-fold) in HMMs, and 0.7±0.2 to 1.5±0.3 (1.4-fold) in SMCs (n=9). ACAT activity, measured by the incorporation of
14
C-labelled oleate (pmol/mg protein), increased from 21.0±1.7 to 678.1±110.9 (32.2-fold) in HMMs, and 55.6±3.3 to 133.7±15.2 (2.4-fold) in SMCs (n=16). By Western blot, fold change in ABCA1 with exposure to agLDL was 2.3±0.3 in HMMs and 1.3±0.2 in SMCs (n=7-10).

Conclusions:
Accumulation of atherogenic lipids in the lysosomes of SMCs provides a potential mechanism for the reduced ABCA1 expression and preferential formation of foam cells by arterial SMCs.