bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 342: Analysis of Mechanism of a Novel Drug Candidate using an Organ-level Functional Microdevice that Reconstitutes Human Pulmonary Thrombosis
Authors: Jain Abhishek , Barrile Riccardo , Aisiku Omozuanvbo , Ceunynck Karen D , van der Meer Andries D , Otieno Monicah A , Louden Calvert S , Hamilton Geraldine A , Flaumenhaft Robert , Ingber Donald E
Published: June 2021

Pulmonary microvascular thrombosis is a catastrophic medical condition and yet, it is very difficult to predict response and study mechanism of action of potential drug candidates to humans. This is partly so because currently available
in vitro
assays do not recapitulate physiologically-relevant forces and animal models can also be very complex, making it impossible to analyze intercellular signaling within the lung that occurs under coagulation or drug administration. We designed a model of lung thrombosis in which human primary alveolar and endothelial cells are co-cultured and maintained up to 2 weeks. The device consists of a top chamber seeded with human alveolar epithelial cells (AE) and a lower chamber seeded with endothelial cells, separated by a porous matrix-coated membrane. Whole blood was perfused at a physiological shear stress through the vascular channel and clots were visualized in real-time. When healthy cells were cultured, no intravascular blood clotting was observed, even when lipopolysaccharide (LPS) endotoxin was administered. In contrast, when LPS was added to the AE channel, it caused a significant increase in platelet adhesion at the endothelium, demonstrating that the presence of alveolar epithelium is critical to LPS-induced intravascular thrombosis
in vitro
. We evaluated this device by analyzing a novel protease activator receptor-1 (PAR1) antithrombotic compound, termed parmodulin 2 (PM2). When the endothelium was cultured along with PM2 under the condition of LPS stimulated AE, we found inhibition of clotting, demonstrating the therapeutic effect of PM2 in the presence of epithelial-endothelial-blood cell signaling. Finally, to test if PM2 performs the therapeutic function of Activated Protein C (APC) that has been reported to stimulate its cytosolic effects via the β-arrestin pathway, we knocked down β-arrestin in the endothelium and analyzed clot formation again. We found that clotting reoccurred in vascular channel, thus showing that PM2 acts like an APC-like drug candidate. In conclusion, the lung alveolus-on-a-chip reconstitutes organ-level responses to blood clotting and may offer a valuable platform for drug development by allowing to dissect contributions of various cells in their mechanism of action.
Abstract 353: Chronic Polarization of Low-grade Inflammatory Monocytes During the Pathogenesis of Atherosclerosis
Authors: Li Liwu , Geng Shuo
Published: June 2021

Background:
Chronic inflammation mediated by low-grade inflammatory monocytes may serve as a key culprit for atherosclerosis. However, the cellular and molecular mechanisms responsible for the low-grade inflammatory polarization of monocytes are not well understood. We hypothesize that the selective clearance of homeostatic molecular switches may pre-dispose innate monocytes for the establishment of non-resolving low-grade inflammation.

Methods and Results:
By comparing high-fat-diet (HFD) fed ApoE deficient mice chronically challenged with either PBS or a subclinical dose endotoxin, we observed that subclinical endotoxin potently induced the establishment of low-grade inflammation, as manifested in elevated levels of systemic inflammatory mediators, accumulation of low-grade inflammatory circulating monocytes and neutrophils, as well as lipids. Immunohistochemical staining of liver and aorta tissues revealed significantly elevated steatosis and atherosclerosis in ApoE deficient mice chronically challenged with subclinical dose of endotoxin. At the mechanistic level, the polarization of low-grade inflammatory monocytes were due to the down-regulation and removal of key homeostatic molecules such as IRAK-M and Tollip. ApoE and IRAK-M double deficient mice had enhanced inflammatory polarization of innate monocytes, and developed severe atherosclerosis.

Conclusions:
Our data suggest that the clearance of homeostatic suppressors such as IRAK-M and Tollip may cause the memory establishment of low-grade inflammatory monocytes that are conducive for the chronic pathogenesis of atherosclerosis.

Key words:
Low-grade inflammation, monocyte polarization, innate memory, atherosclerosis
Abstract 36: Alpha 2-antiplasmin Prevents the Resolution of Deep Vein Thrombosis
Authors: Singh Satish , Houng Aiilyan K , Howard Samantha , Emerson B Tyler , Reed Guy L
Published: June 2021

Introduction:
Deep venous thrombosis is a major cause of death and disability. Despite anticoagulation treatment, venous thrombi persist for months, causing chronic venous obstruction, inflammatory remodeling and post-thrombotic syndrome. The mechanisms responsible for impaired clearance of venous thrombi are poorly understood. Alpha 2-antiplasmin (a2AP) is the primary physiological inhibitor of plasmin and a key regulator of the fibrinolytic pathway. The role of a2AP in the resolution of venous thrombosis has not been determined.

Hypothesis:
We tested the hypothesis that a2AP prevents the resolution of experimental deep venous thrombi.

Methods and Results:
Thrombus resolution and content were examined in a2AP
+/+
and a2AP
-/-
mice using a well-established, fibrinolytic-resistant, stasis model induced by ligation of the inferior vena cava (IVC). Thrombus weight and composition were determined after 7 days. Data was analyzed by one-way ANOVA with Neumann-Keul’s correction. Thrombus weight was reduced in a2AP
-/-
mice by >90% when compared to a2AP
+/+
mice (p<0.001); there was no significant difference between a2AP
-/-
mice and shams. Histochemical and immunofluorescence staining showed significant reductions in IVC fibrin content, neutrophil recruitment and matrix metalloproteinase-9 expression in a2AP
-/-
mice (p<0.001) vs. a2AP
+/+
mice. The relative effect of plasminogen activation and a2AP on resolution of preformed venous thrombi was examined in wild-type a2AP
+/+
mice treated 24 h after IVC ligation with tissue plasminogen activator (TPA) (1.2 or 5 mg/kg) or a monoclonal antibody inactivating a2AP (10 mg/Kg). By comparison to 7 day old venous thrombi in untreated mice, treatment with TPA at 1.2 mg/kg or 5 mg/kg did not decrease thrombus size after 7 days. In contrast, a2AP inactivation significantly reduced thrombus weight vs. untreated and TPA-treated mice (p<0.01 to p<0.001).

Conclusions:
In experimental venous thrombosis, a2AP was required for the persistence of venous thrombi 7 days after formation. Venous thrombi resisted TPA, but were sensitive to resolution after a2AP inactivation. This suggests that a2AP may be responsible for the persistence of clinical venous thrombosis in humans.
Abstract 361: Oxidized Phospholipids Are Proinflammatory and Proatherogenic
Authors: Que Xuchu , Yeang Calvin , Hung Ming-Yow , Yamaguchi Fumihiro , Diehl Cody J , Gonen Ayelet , Prohaska Thomas A , Wang Shuling , Bowden Karen , Pattison Jennifer , Mellon Pamela L , Gaddis Dalia , Hedrick Catherine , Ley Klaus , Glass Christopher K , Peterson Kirk L , Binder Christoph J , Tsimikas Sotirios , Witztum Joseph L
Published: June 2021

Oxidized phospholipids (OxPL) are ubiquitously generated during inflammation, and found on apoptotic cells, OxLDL, and Lp(a). They facilitate uptake of OxLDL by macrophages (Mac) and mediate cellular inflammatory responses. The E06 natural IgM binds to the PC of OxPL, neutralizing biological effects and inhibiting OxLDL uptake by Mac. To determine the role of OxPL in atherogenesis, we generated transgenic mice expressing a single chain variant (scFv) of E06 in
Ldlr
background (E06-Tg). E06-scFv was secreted into plasma, bound to OxLDL and had sufficient titer to inhibit OxLDL uptake into Mac. E06-Tg or
Ldlr
mice were fed 1% Chol diet for 4, 7 or 12 months (n=12-15 mice/group). Plasma Chol was ~ 1600 mg/dL in all mice. Atherosclerosis decreased in E06-Tg mice:
En face
lesions decreased 57%, 34% and 28%, and aortic root lesions decreased 55%, 41% and 26% at 4, 7 and 12-months, respectively. OxLDL uptake by Macs was decreased: Thus, in E06-Tg mice, the uptake by peritoneal Mac of fluorescently-labeled OxLDL injected ip was decreased ~ 50%, as was peritoneal Mac Chol content. As Macs secrete E06-scFv, we performed BMT from E06-Tg donors into irradiated
Ldlr
recipients (n=10-12): This also decreased lesions 31% compared to BMT from control donors, even though plasma titers of E06-scFv were ~10% of Tg mice. Overexpression of E06-scFv was anti-inflammatory: Thus, in E06-Tg mice, both peritoneal and aortic wall resident macrophages exhibited decreased inflammatory gene expression, and phenotypic switches from M1 to M2 analyzed by RNAseq and FACS. Further, in E06-Tg mice, plasma SAA levels were reduced 32%, and hepatic steatosis was also decreased (-50% in both TG and Chol), as was hepatic inflammatory gene expression. Finally, E06-scFv attenuated both a progressive increase in aortic valve gradient (via echocardiography) and calcification in aged
Ldlr
mice. The E06-scFv lacks functional effects of an intact antibody other than the ability to “neutralize” OxPL. Thus, these data demonstrate that OxPL are profoundly proatherogenic and proinflammatory, which E06 counteracts
in vivo
. Neutralizing OxPL may therefore reduce the progression of atherosclerosis and cardiovascular events and more generally, represents a novel strategy to safely attenuate inflammation.
Abstract 370: Matrix Metalloproteinase-3 Regulates Arterial Calcification
Authors: Lin Tonghui , Wang Xue-lin , Cai Yujun , Zettervall Sara L , Guzman Raul J
Published: June 2021

Objective:
Medial artery calcification is associated with increased cardiovascular morbidity and mortality in patients with peripheral artery disease (PAD). It is highly prevalent in diabetes and chronic kidney disease, and it is an important risk factor for cardiovascular events. Accumulating evidences suggest that matrix degrading enzymes are prominently involved in this process. Clinical studies have shown that the matrix metallopeptidase3 (MMP-3, stromelysin-1) is correlated with prevalent arterial calcification in high risk human populations. In this study, we sought to determine whether reducing MMP-3 activity could decrease arterial calcification in smooth muscle cells, organ culture, and
in vivo
models.

Methods and Results:
Confluent human aortic smooth muscle cells (HASMCs) were cultured in calcification medium containing high phosphate (Pi) for 7 days in the absence or presence of an MMP-3 specific inhibitor. We found that adding MMP-3 inhibitor dose-dependently reduced Pi-induced calcium deposition as demonstrated by the O-cresolphthalein complexone assay. To further determine the importance of MMP-3 in arterial calcification, we utilized
ex vivo
aortic rings. In aortic rings from MMP-3 knockout mice, Pi-induced medial calcification was significantly reduced compared with wild-type mice. We next evaluated the effects of MMP-3 deletion
in vivo
. 12-week-old MMP-3 knockout and wild type mice were injected with 8mg/kg cholecalciferol and arteries were collected after 8 days, Calcium levels were significantly lower in MMP-3 knockout mice. Deletion of MMP-3 also reduced serum calcium levels, but not changed serum phosphate level. In concurrent studies, we showed by immunohistochemical staining that MMP-3 was highly expressed in calcified lesion from human tibial arteries.

Conclusion:
Together these findings suggest that MMP-3 promotes medial artery calcification, and that it may serve as a potential therapeutic target aimed at improving the poor outcomes of our patients with PAD.
Abstract 379: Silencing of Fxyd1 Uncouples Enos in Human Umbilical Vein Endothelial Cells
Authors: Tang Owen , Huang Tom H , Li Jianmei , Tandy Sarah , Figtree Gemma
Published: June 2021

Introduction/Aims:
FXYD1 has been shown to be a master regulator of the sodium potassium pump activity in the caveolae of heart tissue, protecting the pump from glutathionylation and oxidative inhibition. However, very little is known about its expression and function in the endothelium, particularly in regard to its potential interaction with other caveolae proteins. Given the molecular mechanism of eNOS uncoupling is via glutathionylation, we examined whether FXYD1 was expressed in endothelial cells, and whether it had a functional partnership with eNOS.

Methods:
Co-immunoprecipitation was used to study the interaction of FXYD1 and eNOS. GSH antibody was used for immunodetection of eNOS glutathionylation. Silencing of FXYD1 using siRNA followed by DAF staining or spin trap were used to study the effects of FXYD1 on eNOS function. Enzyme-linked immunosorbent assay was also employed to quantify eNOS glutathionylation level in human erythrocyte samples post exposure to angiotensin II (0 to 500 nM) ex vivo.

Results:
FXYD1 co-immunoprecipitated with eNOS in human umbilical vein endothelial cells (HUVECs). Silencing of FXYD1 resulted in significantly increased eNOS glutathionylation, associated with significantly reduced NO bioavailability under baseline and acetylcholine stimulated conditions (1μM, 20mins). Furthermore, a dose-dependent increase in eNOS glutathionylation level was also observed in human erythrocyte exposed to angiotensin II.

Discussion:
Our findings demonstrated a novel functional partnership of FXYD1 with eNOS, protecting this vital enzyme from glutathionylation-mediated uncoupling. This has important implications for our understanding of ROS-signalling in the vasculature. Furthermore, FXYD1 expression/eNOS glutathionylation may serve as important biomarkers for oxidative stress.
Abstract 384: Integration of Aorta Network Models from Mouse Ath-HMDP with Human GWAS Reveals Novel Mechanisms of Coronary Artery Disease
Authors: Kurt Zeyneb , Bennett Brian J , Davis Richard C , Pan Calvin , Zhao Yuqi , Codoni Veronica , Trégouët David-Alexandre , Zhang Bin , Lusis Aldons J , Yang Xia
Published: June 2021

Coronary artery disease (CAD) remains one of leading causes of death worldwide. Although the genetic heritability of CAD has been estimated to be 40 to 60%, only ~10% can be explained by the genetic loci uncovered from recent large-scale human GWAS. To elucidate the missing mechanisms, we leveraged aorta transcriptome data from 372 mice profiled in an Atherosclerosis Hybrid Mouse Diversity Panel (ath-HMDP), human CAD GWAS from CARDIoGRAM+C4D, and human expression quantitative loci (eQTL) from the Genotype-Tissue Expression (GTEx) and the Cardiogenics transcriptomic studies. We first constructed aorta coexpression networks based on the ath-HMDP transcriptome data using MEGENA (Multiscale Embedded Gene Co-expression Network Analysis) and WGCNA (Weighted Gene Coexpression Network Analysis) to define coexpression modules with various levels of coherency and compactness. The coexpression modules from MEGENA and WGCNA were then correlated with the aortic lesion trait measured in ath-HMDP to reveal 29 coexpression modules demonstrating significant correlations with aortic lesions (p < 1e-3). We further integrated these aortic lesion-correlated modules with eQTLs from aortic and coronary arteries examined in the GTEx study and monocyte and macrophage eQTLs from Cardiogenics to map module genes to functional SNPs that likely affect the expression levels of these genes. When further incorporating the CARDIoGRAM+C4D CAD GWAS, 13 lesion-correlated modules in mouse ath-HMDP were enriched for functional SNPs demonstrating stronger association with CAD in humans. These modules were over-represented with genes in multiple biological pathways, including both known CAD processes (e.g., lipid & lipoprotein metabolisms, fatty acid triacylglycerol & ketone body metabolism, TCA cycle, vascular smooth muscle contraction, antigen processing, focal adhesion) and novel pathways (e.g., adipocytokine signaling, type I & II diabetes mellitus, neurotrophic factor signaling, lysosome). In conclusion, this cross-species integrative analysis that takes advantage of aorta profiling from a well-controlled mouse atherosclerosis study in conjunction with human genomics provides insights into the aorta-related mechanisms underlying CAD.
Abstract 389: Covalent Modifications of Apolipoproteins in High Density Lipoprotein by Oxidized Phospholipids Impair the Cholesterol Efflux Function of High Density Lipoprotein and are Detected in Hyperlipidemic Plasma
Authors: Gao Detao , Zhang Lifang , Podrez Eugene
Published: June 2021

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis and thrombosis. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interactions between oxPLs and proteins could provide important information regarding the type of proteins preferentially modified by oxPLs. However, to date, such studies are extremely limited due to significant technical challenges. We now carry out systematic studies on the protein adduction by oxPLs formed in murine and human plasma. Plasma samples were exposed to a physiologically relevant myeloperoxidase/H
2
O
2
/NO
2
–
oxidizing system of phagocytes. Protein adduction by the oxPLs generated in plasma was assessed using LC-MS/MS after tryptic digestion and peptide enrichment using a novel method that we developed. We found that HDL apolipoproteins are the major targets of modification by oxPLs in both murine and human plasma. For apoA-I, the most abundant apolipoprotein in HDL, the major modification sites of oxPLs were located in the region (AA144-186), which is critical for the ABCA1 mediated cholesterol efflux to HDL. We further demonstrated that human apoA-I was also heavily crosslinked by specific oxPLs via histidine and lysine residues located in the region (AA144-186) with apoA-I, or other apolipoproteins, including apoA-II, apoA-IV and apoC-I.
In vitro
experiments demonstrated that oxPLs modification on lipid free apoA-I or nascent HDL (HDL3) dramatically impairs their function as cholesterol efflux mediators. Using hyperlipidemic LDLr-/- mice, we detected a crosslink adduct of apoA-II with apoE by oxPL in murine plasma. To the best of our knowledge, this is the first report for the detection of endogenous protein adducts with oxPLs.
Abstract 392: The Plasma Proteome of Tangier Disease Patients Reveals Perturbations of Diabetic and Inflammatory Pathways
Authors: McParland James T , Pashos Evanthia , Rader Daniel J , Cuchel Marina
Published: June 2021

Aim:
The ATP-binding cassette transporter 1 (ABCA1) is a membrane protein well known for its role in cholesterol efflux and HDL formation. Recently, ABCA1 has been implicated as playing a key role in other processes, such as insulin secretion and inflammatory response. We sought to further investigate these potential roles through a quantitative proteomics approach. Specifically, we hypothesized that we could detect differential protein signatures in the plasma of Tangier patients that correspond to pathways involved in diabetes and inflammation.

Methods:
We used SOMAscan® technology (SomaLogic, Boulder, CO, USA) to analyze plasma collected from 5 Tangier disease patients (homozygotes or compound heterozygotes for functional
ABCA1
mutations) and 7 normolipidemic controls. We tested for differences in the levels of approximately 1,000 plasma proteins using a nonparametric test (KS). We then performed Ingenuity Canonical Pathway analysis to examine if proteins linked to diabetes and inflammation pathways were significantly more likely to be differentially abundant in the plasma. We corroborated the results using Gene Set Enrichment Analysis (GSEA).

Results:
We found an enrichment in differentially abundant proteins involved in type II diabetes mellitus signaling (p-value=0.0002) and inflammatory pathways, such as granulocyte adhesion and diapedesis (p-value=2.2*10
-12
). These results were also corroborated by GSEA, where gene sets corresponding to GO biological processes such as immune response (p-value=0.008) and inflammatory response (p-value=0.032) ranked at the top of the enrichment results.

Conclusions:
The results from this pilot study support the concept that ABCA1 is implicated in pathways affecting immune and inflammatory response and type II diabetes.
Abstract 398: LDL Receptor Signaling Mediates the Triglyceride-lowering Action of Akkermansia Muciniphila in Genetic Induced Hyperlipidemia
Authors: Shen Jing , Tong Xuedong , Sud Neetu , Su Qiaozhu
Published: June 2021

Akkermansia muciniphila
(
A. muciniphila
) is a mucin-degrading bacterium that resides in the mucus layer whose abundance inversely correlates with body weight and the development of diabetes in mice and humans. The objective of this study was to explore the regulatory effect of
A. muciniphila
on host lipoprotein metabolism, insulin sensitivity and hepatic metabolic inflammation. By establishing a novel mouse model that colonized the
A. muciniphila
in the gastrointestinal(GI) tract of the cAMP-responsive binding protein H (CREBH)-deficient mouse and in vivo chylomicron assay, we found that increased colonization of
A. muciniphila
in the GI tract of wild-type mice protected mice from an acute fat load-induced hyperlipidemia compared to vehicle-treated mice.
A. muciniphila
administration also significantly ameliorated chronic hypertriglyceridemia, improved insulin sensitivity and prevented overproduction of postprandial chylomicrons in the CREBH-null mice. Mechanistic studies revealed that increased
A. muciniphila
colonization induced expression of low density lipoprotein (LDL) receptors and apolipoprotein E (apoE) in the hepatocytes of CREBH-null mice, which facilitated the uptake of intermediate density lipoprotein (IDL) via the mediation of apolipoprotein B100 (apoB) and apoE, leading to the increased clearance of triglyceride-rich very-low-density lipoprotein (VLDL) particles, the precursors of IDLs, from the circulation. Treatment with
A. muciniphila
further improved hepatic ER stress and metabolic inflammation in CREBH-null mice.

Conclusion:
Increased colonization of the disease-protective gut bacteria
A. muciniphila
protected the host from acute and chronic hyperlipidemia by enhancing the LDL receptor expression and alleviating hepatic ER stress and the inflammatory response in CREBH-null mice.
Abstract 41: Platelet Dream Plays a Critical Role During Thrombogenesis in Mice
Authors: Kim Kyungho , Tseng Alan , Barazia Andrew , Italiano Joseph E , Cho Jaehyung
Published: June 2021

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain. Using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that hematopoietic and endothelial cell DREAM are required for platelet thrombus formation following arteriolar injury. DREAM deletion also prolonged tail bleeding times. In vitro flow chamber assays and in vivo adoptive transfer experiments indicated the importance of platelet DREAM in thrombogenesis. We found that deletion of platelet DREAM does not alter ultrastructural features but significantly impaired aggregation and ATP secretion induced by collagen-related peptide (CRP), ADP, or A23187, but not thrombin or U46619. Biochemical studies showed that platelet DREAM is required for phosphoinositide 3-kinase (PI3K) activation induced by GPVI-, A23187-, or integrin-mediated signaling. Studies using DREAM-null platelets and isoform-specific PI3K inhibitors revealed that platelet DREAM positively regulates granule secretion, Ca2+ mobilization, and aggregation induced by CRP or A23187 through PI3K class Iβ (PI3K-Iβ) activity. Genetic and pharmacological studies in megakaryoblastic MEG-01 cells showed that DREAM regulates A23187-induced Ca2+ mobilization and that the regulatory function of DREAM requires Ca2+ binding and PI3K-Iβ activity. Taken together, we have identified platelet DREAM as a novel regulator of PI3K-Iβ activity during thrombus formation.
Abstract 410: Link Between Hypertension and Insulin Resistance in South Asians: Implications for Prevention of Cardiovascular Complications
Authors: L'Italien Gilbert J , Nelson John R , Mathur Virendra S , Mason R. Preston , Chowdhury Palash R , Philip Sephy , Kulkarni Kris , Murthy Vemuri , Vemuri Dwarka , Shah Uday , Valencia Robert , Nanda Navin , Deedwania Prakash C , Chowdhury Sumita
Published: June 2021

Background:
The coexistence of hypertension (HTN) and insulin resistance (IR) substantially increases cardiovascular risk (CVR) and diabetes (DM) perhaps by endothelial dysfunction from low-grade chronic inflammation and oxidative stress. South Asian immigrants in United States (SA-USA) have a high salt consumption, IR, and salt sensitivity. The triglyceride (TG)/ HDL ratio is a known correlate of IR with ratios ≥3.5 associated with high risk. It is unknown whether SA-USA have a coexistence of HTN, IR, and DM resulting in high CVR.

Methods:
We reviewed the clinical history and Vertical Auto Profile technology (VAP) + lipid panel utilizing ultracentrifugation to directly measure lipid sub-fractions, for 348 participants (42% women) of the South Asian Cardiovascular Registry. Prevalent DM and Pre-DM were defined as HbA1c ≥ 6.5 and 5.7-6.4, respectively.

Results:
Of the 348 participants, mean age 55.3 years, 128 had HTN and were stratified into clinical risk groups based on fasting TG (mg/dL) levels: TG<150 (n=69), TG 150-199 (n=24), TG 200-499 (n=32), TG>500 (n=3). The corresponding TG/HDL ratios were: 1.7, 3.5, 6.2, and 20.4, respectively; the median TG (mg/dL) levels were: 100, 182, 268, and 572, respectively; the median LDL (mg/dL) levels were: 113, 144, 133, 102, respectively; the rates of DM were: 13%, 17%, 38%, and 67%; and the rates of Pre-DM/DM were: 62%, 63%, 78%, and 67%, respectively. Relative Risk for DM with TG/HDL≥ 3.5 was 3.6 (46 % had TG/HDL ≥ 3.5).

Conclusions:
South Asian immigrants participating in a community registry have: 1) A high rate of coexistence of HTN and IR portending a very high relative risk of diabetes. 2) A discordance of LDL levels and IR in patients with HTN. As IR gets higher, the relative risk of diabetes goes up but LDL levels decline.

Clinical Implications:
South Asian immigrants in the United States have a diet that is very high in salt, simple carbohydrates, and saturated fat. Many are vegetarian with no consumption of fish. Public health efforts to help reduce risk of CVD in South Asian immigrants should focus on low salt diet, with consumption of multigrain complex carbohydrates, poly-unsaturated fatty acids, omega-3 fatty acids, and lifestyle changes to reduce metabolic syndrome, diabetes, and hypertension.
Abstract 433: Liver Disease Alters HDL Metabolism and Function
Authors: Trieb Markus , Horvath Angela , Stadlbauer Vanessa , Taschler Ulrike , Curcic Sanja , Holzer Michael , Pasterk Lisa , Heinemann Akos , Marsche Gunther
Published: June 2021

Background/Aims:
High-density lipoproteins (HDLs) are important endogenous inhibitors of inflammatory responses. Dysfunctional HDL might contribute to the serious complications experienced by patients with acute and chronic liver failure, but the effect of cirrhosis on several metrics of HDL function is not understood.

Methods:
We measured metrics of HDL function in an explorative cross sectional study of 59 patients with compensated cirrhosis, 21 patients with acutely decompensated cirrhosis and 20 healthy controls. HDL particle size was assessed using native gel electrophoresis. HDL cholesterol efflux potential was assessed using [
3
H]-cholesterol labeled macrophages. Paraoxonase activity was determined by photometry using phenylacetate as substrate. Nuclear factor-κB activation in monocytes and cytokines were assessed by flow cytometry. Endothelial regenerative activity was determined using an electric cell-substrate impedance sensing system.

Results:
Sera of cirrhotic patients showed low HDL-cholesterol levels and profoundly suppressed activities of several serum enzymes involved in HDL maturation and metabolism. Native gel electrophoresis analyses revealed that cirrhotic HDL shifts toward the larger HDL
2
subclass. We observed that (i) HDL cholesterol efflux capability, (ii) paraoxonase activity, (iii) the ability to inhibit monocyte production of pro-inflammatory cytokines and (iv) endothelial regenerative activities were significantly reduced in stable cirrhotic patients, and markedly suppressed in acutely decompensated patients. Of particular interest, HDL cholesterol efflux capacity appeared to be strongly associated with mortality of cirrhotic patients, independently of HDL cholesterol levels.

Conclusion:
Cirrhosis affects HDL metabolism and several metrics of HDL function. HDL cholesterol efflux capacity appears to predict 1-year mortality independent of HDL-cholesterol levels.
Abstract 436: Smooth Muscle Specific Knock-out of the Zinc-Finger Protein 148(ZFP148) Attenuates Atherosclerotic Lesion Formation via Regulation of Apoptotic Signaling Pathways
Authors: Salmon Morgan , Fashandi Anna Z , Spinosa Michael D , Sharma Ashish K , Owens Gary K , Merchant Juanita L , Zehner Zendra E , Upchurch Gilbert R , Ailawadi Gorav
Published: June 2021

Objective:
Zinc-finger protein 148 (ZFP148) plays a profound role in the modulation of aortic aneurysm formation in part via modulation of smooth muscle (SMC) genes. The current study objective was to determine whether smooth muscle specific knock-out of ZFP148 is critical in atherosclerotic lesion formation.

Methods:
ZFP148 was examined via immunohistochemistry and confocal microscopy in human atherosclerotic lesion samples (n=12/group). 6-8 week male (n=12/group) ZFP flx/flx Myh11 Cre+ ApoE-/-(SMC tamoxifen ZFP148 KO), Myh11 ZFP148 flx/wt Cre+ ApoE-/- and Myh11 ZFP wt/wt Cre+ ApoE-/- underwent tamoxifen injections followed by western diet feeding for either 13 or 25 weeks. A separate set of mice were fed western diet for 18 weeks and then administered tamoxifen injections. Aortic samples were evaluated with histology for α-actin, macrophages, neutrophils, TER119, caspase3, Ki67, picosirus red and movat staining.
In vitro
ZFP148 was knocked down using siRNA in smooth muscle cells and stimulated with the oxidized phospholipid POVPC.

Results:
ZFP148 expression was elevated in human atherosclerotic lesion samples and localized to smooth muscle cells. Lesion size was significantly reduced in SMC ZFP148 KO mice compared with controls in 25 week western diet fed mice(p<0.0357). SMC ZFP148 KO demonstrated reduced macrophage, Caspase3, and TER119 staining. Conversely, SMC ZFP148 KO increased SMα-actin coverage. Lesion size was also decreased in mice that were administered tamoxifen injections following 18 weeks of western diet feeding(p<0.0415). There were no significant changes in lesion size at 13 weeks of western diet feeding; however, macrophage staining was decreased. Knock-down of ZFP148 followed by treatment with POVPC attenuated the down-regulation of SM22α, SM-MHC, and SMαA. Knock-down of ZFP148 followed by POVPC treatment also prevented the up-regulation of Bax and BAD in vascular smooth muscle cells.

Conclusions:
While earlier studies documented a role for ZFP148 in aneurysm disease, the present study suggests that SMC ZFP148 KO attenuates atherosclerotic lesion formation in early and late atherosclerotic disease. ZFP148 represents a key regulator of multiple types of vascular disease.
Abstract 440: Non-Invasive Quantification of Rat Vasculature Biomechanical Properties Under a Cardiovascular Challenge: Implications in Atherosclerosis Progression
Authors: Castle Paige E , Greve Joan M
Published: June 2021

Introduction:
Measurements of cyclic strain give insight into vessel wall properties, offer a better understanding of the pathogenesis of atherosclerosis, and can be used in quantifying loss of vessel compliance. Using dobutamine stimulation to increase heart rate and cardiac output provides a clinically relevant means to begin to understand alterations in cyclic strain during a vascular challenge which mimics some aspects of exercise. Similar work has been completed in murine models, but translating these methods to a species that is an order of magnitude larger allows for a better understanding of how these results could allometrically scale to the human condition. We hypothesize that using a pharmacological cardiovascular stimulant will cause cyclic strain shifts that vary between species.

Materials and Methods:
Cyclic strain was non-invasively quantified using MRI. Young rats and mice were anesthetized using isoflurane and imaged at 7T. 2D and 3D gradient echo data were used to plan ECG-gated acquisitions that included 12 CINE frames across the cardiac cycle, in the infrarenal aorta pre- and post- dobutamine infusion. Vessel strain was quantified in each CINE frame.

Results:
Baseline strain in rats was 17% and 16%, for males and females respectively (A, C). In mice, baseline strain was 22% in males and 18% in females (B, D). After infusion of dobutamine, strain in rats increased to 26% in males and 27% in females, while in mice strain increased to 36% in males and 32% in females.

Conclusions:
In conclusion, there are evident variations in cyclic strain under a cardiovascular challenge between species. With this insight, a clearer understanding of the biomechanical forces experienced by the vasculature in different species may lead to improved extrapolation from mouse to human in understanding the effects of atherosclerosis on vessel compliance and developing therapeutic approaches.
Abstract 443: Constitutive Bone Morphogenetic Protein 2-dependent Osteogenic Signaling in M1 Macrophages
Authors: Dube Prabhatchandra
Published: June 2021

Atherosclerosis is the leading cause of carotid and coronary artery disease. Calcification often complicates atherosclerotic plaques and contributes to plaque instability.Mechanisms calcification are somewhat reminiscent of bone formation, involving interplay between endothelium, smooth muscle cells and plaque macrophages. Macrophages contribute to vascular calcification through signals that stimulate the osteogenic program in smooth muscle cells and/or by becoming osteoclast-like cells. Yet, the specific roles of polarized M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages, predominant in plaques, in lesion calcification remain poorly understood. Recent studies from our laboratory show that the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) is a key component of mechanisms linked to ER stress signaling in M1, but not M2 macrophages. Because ER stress has a recognized effect in inducing vascular calcification, we speculated that TRPC3, by virtue of its roles in M1 macrophages, might have an effect on the osteogenic potential of these cells. To address this question we utilized bone marrow derived macrophages from mice with macrophage-specific loss of TRPC3 function (MacTRPC3KO) or from their littermate controls (TRPC3
lox/lox
) and polarized
in vitro
to the M1 and M2 phenotypes. Osteogenic proteins and factors along with signaling mechanisms from the two groups were examined under basal and ER stress conditions. The results showed reduced expression of BMP-2 and Runx-2 at mRNA level in MacTRPC3KO M1 macrophages but not at the protein level between the groups. We also examined the phosphorylation status of SMAD1/5 in M1 macrophages, which is an early indicator of signaling downstream the BMP-2 receptor. Although no differences were observed between groups, SMAD1/5 was significantly phosphorylated, even under basal conditions. BMP-2 levels in supernatants from cultures of M1 macrophages was also significantly elevated regardless of the treatment condition. Notably, phosphorylation levels of SMAD1/5 were markedly reduced when cells were exposed to the BMP-2 receptor blocker dorsomorphin. These results indicate that BMP-2 is involved in activating osteogenic signaling in M1 macrophages.
Abstract 446: EphA2 Regulates Vascular Smooth Muscle Fibroproliferative Remodeling in Atherosclerosis
Authors: Finney Alexandra C , Green Jonette M , Rana Mohammad A , Funk Steven D , Traylor James G , Orr A. Wayne
Published: June 2021

Eph receptors, the largest mammalian subfamily of receptor tyrosine kinases, regulate inflammation and tissue remodeling. The EphA2 receptor shows enhanced expression in atherosclerosis, and deletion of EphA2 in atherosclerosis-prone ApoE knockout mice attenuates lesion formation. We now show that EphA2 knockout mice exhibit reduced late stage plaque progression associated with diminished smooth muscle content. While EphA2 is absent in quiescent vascular smooth muscle cells
in vitro
and
in vivo
, dedifferentiation to a synthetic phenotype significantly upregulates EphA2 expression. Deletion of EphA2, a known oncogene, reduces markers of proliferation in atherosclerotic plaques, and EphA2 knockdown similarly reduces vascular smooth muscle proliferation in culture with associated reductions in serum-induced ERK and Akt signaling. In addition to proliferation, EphA2 knockout mice show significantly reduced collagen content in their atherosclerotic plaques, and EphA2 knockdown reduces smooth muscle matrix deposition
in vitro
. Together these data suggest a potential role for EphA2 in smooth-muscle driven vascular fibroproliferative remodeling, representing the first link between EphA2 signaling and smooth muscle function.
Abstract 447: Atherosclerosis-driven Treg Plasticity Results in the Formation of a Dysfunctional Subset of Plastic IFNg
+
Th1/Tregs
Authors: Butcher Matthew J , Filipowicz Adam R , Waseem Tayab C , McGary Christopher M , Magilnick Nathaniel , Boldin Mark , Lundberg Patric S , Galkina Elena V
Published: June 2021

Foxp3
+
T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Emerging evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. Here, we addressed whether atherosclerosis impacts Treg plasticity or functionality in

Apoe
-/-

mice and what effect Treg plasticity might have on the pathology of atherosclerosis. We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3
+
Tregs, and the accumulation of an intermediate Th1-like IFNγ
+
CCR5
+
Treg subset (Th1/Tregs) within the arterial wall. Using

Foxp3
Gfp/Gfp

and

Foxp3
Yfp-cre/Yfp-cre
Rosa
tdTomato/tdTomato

lineage tracing mice, we demonstrate that Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than T effector cells. Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Importantly, using

Mir146a
-/-

mice, we demonstrate that plasticity-prone

Mir146
-/-

Tregs fail to reduce atherosclerosis, arterial Th1, or macrophage content in

Apoe
-/-

recipients. Lastly, we utilized single cell RNA-sequencing to gain whole transcriptome information on Th1/Tregs, Treg, and Th1 cells. Th1/Tregs yielded a unique transcriptional phenotype characterized by co-expression of Treg and Th1 lineage genes, and down-regulation of Treg-related genes, including
Ikzf2
,
Ikzf4, Tigit,
and
Lilrb4
. Additionally, an ingenuity pathway analysis further implicates IFNγ, IFNα, IL-2, IL-7, CTLA4, T cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. In conclusion, atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ
+
Th1/Tregs that permit further arterial inflammation and atherogenesis.
Abstract 452: Longitudinal Visualization of Calcification Genesis and Growth
in vivo
: Novel Implications for Plaque Vulnerability
Authors: Hutcheson Joshua D , Goettsch Claudia , Pieper Brett , Pham Tan , Choi Jung , Mlynarchik Andrew , Aikawa Masanori , Aikawa Elena
Published: June 2021

Background:
Clinical evidence links arterial calcification and cardiovascular risk. Fibrous cap microcalcifications can promote atherosclerotic plaque failure, and large calcifications can stabilize the plaque. Therefore, calcification morphology can determine cardiovascular morbidity, but temporal patterns of calcific mineral deposition and growth remain unknown.

Results:
Apolipoprotein E-deficient (
Apoe-/-
) mice on an atherogenic diet develop plaque calcification. Longitudinal studies were performed using two different fluorescent calcium tracers injected intravenously into
Apoe-/-
mice: calcein injection following 18 weeks of atherogenic diet (n=7) and alizarin red S injection into the same mice 1 (n=4) or 3 (n=3) weeks later. Imaging green (calcein) and red (alizarin red S) fluorescence provided snapshots of aortic calcification at 18, 19, and 21 weeks. Observations within histological sections revealed green microcalcifications at 18 weeks embedded within alizarin red stained larger calcifications that were formed by 19 weeks (a). These data demonstrate that microcalcifications present at the start of calcification become the core of the larger calcifications that develop over time. Serial histological sections from aortic root to arch (b) were digitally reconstructed into 3D volumes (c) to reveal total calcific burden and localization within the aortic wall (d). Total calcification volume increased at a significant rate of 6.0x10
6
μm
3
per week (R
2
=0.99, p=0.007) and progressed from aortic arch to aortic root over time (p<0.001). Observations closely match calcification morphologies found by micro-computed tomography of human coronary arteries.

Conclusion:
Temporal and spatial understanding arterial calcification growth is crucial given the link between mineral morphology and cardiovascular risk, and these techniques provide a method for testing therapeutic approaches to control calcification morphology over time
in situ
.
Abstract 463: Linoleate Epoxides, Epomes, Are Potent Anti-inflammatory Fatty Acid Epoxides and Nearly Equivalent to Arachidonate-derived Epetres
Authors: Singh Vishal , Saha Piu , Vijay-Kumar Matam , Shearer Gregory
Published: June 2021

Background:
Epoxides of arachidonic acid (EpETrEs, also commonly EETs) are potent anti-inflammatory lipid mediators and are associated with hypotensive and protective vascular actions. Marine omega-3 versions (epoxides of eicosapentaenoic and docosahexaenoic acids) have similar action. Surprisingly, none of these epoxides are as abundant in tissues or in plasma as epoxides of linoleic acid (EpOMEs) are. EpOMEs are up to one-hundred times more abundant than EpETrEs. Hence, EpOMEs could exert physiologic effects even with potency two-orders of magnitude higher, provided they have comparable efficacy. Few studies consider EpOMEs when assessing outcomes.

Objective:
To compare the potency and efficacy of EpOMEs to EpETrEs in a model of inflammation relevant to vascular disease, the RAW 264.7 macrophage.

Approach and results:
The suppression of inflammatory cytokines (IL-6 and TNFa) in RAW 264.7 macrophages responding to LPS treatment (500 nM, 24 hrs.) was measured in the presence of 1 hour pretreatment with 9(10)-EpOME and 11(12)-EpETrE over 4-orders of magnitude (0.03 nM to 100 nM). The resulting dose-response curves were used to estimate potency (IC
50
), and efficacy (suppression as percent of maximal LPS activation). Least-square-mean and 95% CIs are reported. The IC
50
for suppression of cytokines by EpOME and EpETrE were not different, regardless of cytokine, yielding an estimate of 4.2 [2.6, 6.8] nM and indicating that EpOMEs are equipotent to EpETrEs in cytokine suppression. While EpOMEs had the same effectiveness in suppressing IL-6 to 59% [54, 64] of max, they were not as effective in suppressing TNFa, to 83% [80, 86] of max versus 78% [75, 81] for EpETrEs (p<0.01).

Conclusions:
Given their great abundance in tissue and plasma, linoleate epoxides would need to be more than 100 times less potent than arachidonate epoxides to be physiologically irrelevant. Here, we find they are equipotent for suppression of two pro-inflammatory cytokines, with moderately lower efficacy for one cytokine, TNFa. We find little reason to ignore linoleate-derived EpOMEs and conclude they are a physiologically relevant pool of epoxides whose levels should be measured along with EpETrE and other epoxides when estimating overall epoxide effects.
Abstract 471: Lifestyle Interventions Stop Metabolic Syndrome
Authors: Ramirez Francisco E , Siebold Jill , Antuna Kathelyn , Sanchez Albert , Nedley Neil
Published: June 2021

Background:
Remission of metabolic syndrome has been documented with gastric banding surgery. We document a clinical case of remission with lifestyle interventions.

Hypothesis:
A long term whole food plant based diet (WFBD) and lifestyle changes could potentially stop the metabolic syndrome.

Methods:
A 58 year old Caucasian from the US participated of a program in Weimar California. He had a history of diabetes mellitus type 2 for 5 years with peripheral neuropathy for 1.5 years, he took metformin 500 mg bid. He was also hypertensive, he used losartan 50 mg bid and furosemide 40 mg qd. The medical lifestyle program included a WFPBD, exercise, two meals a day, sleeping hygiene, cooking classes together with psychological, medical and spiritual consultations. The program lasted 18 days. The patient continued with the program at home. Laboratory values are reported in mg/dL

Results:
At baseline blood pressure was 158/78 mm hg, he claim to be compliant with medication, fasting blood glucose (FBG) was 230 and weighed 381.4 pounds. At the end of the residential 18 days he had lost 16.2 pounds, blood pressure was 116/72 mm hg, FBG of 110. The patient continued with the learned lifestyle at home. 142 days later he is seen by a physician who stops all medication, he had lost 83.4 pounds, blood pressure was 116/68 mm hg, FBG was 89 mg/dl, hemoglobin A1c was 5.8%. In 286 days he is seen again by a physician, he is now 270 pounds, the patient continues with no medication. 326 days after the start of the program he does lab work, he had a total cholesterol of 140, triglycerides of 56, HDL of 68, LDL of 61 and hemoglobin A1c of 5.5%. 470 days after the program a physician recorded that he had lost 139.4 pounds from the start of the program. He is not using any medication. 524 days after the program he now weighs 240 pounds, having lost 141.4 pounds. 917 days after the end of the program labs are taken: cholesterol 139, triglycerides 55, HDL 73, LDL 55, FBG 99, hemoglobin A1C 5.3%, CHL/HDL ratio of 1.9, blood pressure of 118/70 mm hg. The patient reports feeling well.

Conclusions:
A change in lifestyle that includes a plant based diet was an effective technique to stop the metabolic syndrome without the need of surgery.