bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 206: An Obesity Paradox in Acute Decompensated Heart Failure
Authors: Khoshchehreh Mahdi , Malik Shaista
Published: July 2021

Background:
Prior studies on heart failure (HF) have shown that body mass index (BMI) is inversely associated with mortality. The aim of this study was to investigate the impact of morbid obesity (BMI > 40 kg/m2) on in-hospital mortality in patients presenting with Acute decompensated heart failure (ADHF).

Methods:
The Nationwide Inpatient Sample of the Healthcare Cost and Utilization Project was analyzed for acute HF hospitalizations across the United States. A total number of 966,167 hospitalized patients with ADHF in 2009 were reviewed.

Results:
Morbidly obese patients constituted 13.4% of all patients with ADHF. Analysis of the unadjusted data revealed that morbidly obese patients compared with those not morbidly obese were less likely to die during hospitalization (OR 0.55, %95CI 0.53-0.57, P<0.0001). Cox proportional hazards regression was used to estimate the overall probability of in-hospital death with adjustments for age, sex, race, Elixhauser comorbidities, primary payer, hospital location, hospital teaching status, hospital bed-size, and total hospital admissions. The adjusted hazard of in-hospital death (HR 0.87, p< <.0001) indicates that there was statistically significant difference in the risk of in-hospital death associated with being morbidly obese.

Conclusions:
In this cohort of hospitalized patients with ADHF, higher BMI was associated with lower in-hospital mortality risk. The relationship between BMI and adverse outcomes in HF appears to be complex and consistent with the phenomenon of the “obesity paradox.”
Abstract 211: The DPP-4 Inhibitor Linagliptan Reverses Endothelial Dysfunction of Mesenteric Arteries From Rats Fed a Western Diet
Authors: Salheen Salheen M , Nguyen Jason C , Jenkins Trisha A , Woodman Owen L
Published: July 2021

A high-fat ‘western’ diet (WD), a risk factor for the development of type 2 diabetes, may cause endothelial dysfunction one of the earliest events in atherogenesis. The dipeptidyl peptidase-4 (DPP-4) inhibitors are used to lower hyperglycemia in type 2 diabetes which is also associated with endothelial dysfunction. We tested whether consumption of a WD affected endothelium-dependent relaxation (EDR) of rat mesenteric arteries (MA) and whether the DPP-4 inhibitor linagliptin (1μM) improves EDR. Wistar Hooded rats were fed a standard diet (SD, 7% total fat) or WD (21% total fat) for 10 weeks. Consumption of the WD significantly increased superoxide release from MA assayed by lucigenin chemiluminescence (WD 1210±180 counts/mg versus SD 543±156 counts/mg, n=7-8, p<0.05) and linagliptin significantly reduced the vascular superoxide release (WD+linagliptin 432±102 counts/mg, p<0.05). Acetylcholine (ACh)-induced endothelium-dependent relaxation of MA was assessed using wire myography. WD significantly reduced the sensitivity to ACh (pEC50, SD 7.72±0.08, WD, 7.32±0.05 n=8, p<0.05) and treatment with linagliptin improved endothelial function (ACh pEC50 WD+linagliptin, 7.74±0.12, n=8, p<0.05). The contribution of EDHF to ACh-induced relaxation was determined in the presence of L-NNA and ODQ to block NOS and guanylate cyclase. EDHF-mediated relaxation was improved by linagliptin (pEC50, WD 6.24±0.06, WD+linagliptin 6.95±0.12, n=4-5, p<0.05). Linagliptin also significantly improved the contribution of NO (determined in the presence of TRAM-34 + apamin to block IKCa and SKCa) to relaxation (pEC50, WD 6.50±0.13, WD+linagliptin 7.30±0.10 n=4-6, p<0.05). Linagliptin significantly reduced vascular superoxide levels and improved the contribution of both NO and EDHF to preserve endothelium-dependent relaxation in rats fed a high fat diet. DPP-4 inhibition may have effects in addition to the lowering of plasma glucose to improve vascular function in diabetes.
Abstract 219: Tissue-Specific RNAseq Identifies Novel and Cardiometabolic Disease--Associated Long Intergenic Noncoding RNAs in Human Adipose
Authors: Zhang Xuan , Ballantyne Rachel , Ferguson Jane F , Xue Chenyi , Gosai Sagar , Gregory Brian , Li Mingyao , Reilly Muredach P
Published: July 2021

Objective:
The purpose is to identify novel long intergenic noncoding RNAs (lincRNAs) in human adipose, examine the association of their genetic variants with cardiometabolic traits, and explore their function in adipocytes.

Methods and results:
We performed deep high-throughput RNAseq (>200 million reads/sample) in adipose tissues of 13 volunteers. Of 54,944 human lincRNAs, 6,558 were detected in adipose. We utilized multiple GWAS meta-analysis of adipose-related cardiometabolic traits (e.g. BMI, triglyceride and HDL cholesterol) and interrogated 6,558 adipose LincRNAs for their overlap with GWAS SNPs. After performing Bonferroni correction for the number of adipose lincRNA SNPs tested, we identified 46 SNPs in 9 lincRNAs, 28 SNPs in 13 lincRNAs and 13 SNPs in 6 lincRNAs that were significantly associated with waist-hip ratio adjusted BMI, HDL and triglyceride respectively. Notably, RP11-392O17.1, more abundantly expressed in adipose, and linc-NFE2L3-1, predominantly detected in adipose and skeleton muscle, contain 22 and 4 SNPs reaching genome wide significance for BMI (lead SNPs rs12031603, P=2.15х10
-9
and rs10267498, P=2.73х10
-8
respectively). Linkage disequilibrium analysis confirmed RP11-392O17.1 and linc-NFE2L3-1 harbor the strongest GWAS signals in the loci, suggesting causality for GWAS association with BMI. Several adipose lincRNAs, including RP11-392O17.1, linc-NFE2L3-1 and linc-AQPEP, were induced during
in vitro
human adipocyte differentiation. As an example of adipose lincRNA functional impacts, Linc-AQPEP, an abundant adipose-specific lincRNA, was knocked down by 70% with lentiviral-based short hairpin RNAs, resulting in ~35% decrease of triglyceride content in human adipocytes. RNAseq analysis demonstrated differential expression (DE) in 3,291 of 13,653 adipocyte genes with Linc-AQPEP knockdown (FDR<0.01) and significant enrichments of DE genes in lipid biosynthesis, fatty acid metabolism and mitochondrion pathways. Additional studies are ongoing to further investigate biological roles of human lincRNAs in adipocytes.

Conclusion:
Our data suggest human LincRNAs may play specific roles in adipocyte biology and that their genetic variations may contribute to cardiometabolic traits.
Abstract 231: ABCG1 Null and SR-BI Null Mice Reduced Plasma HDL Uptake by the Liver and Small Intestine
Authors: Tsujita Maki , Akita Nobukatsu , Hossain M Anwar , Yokota Tomo , Remaley Alan T , Kumamoto Natsuko , Ueda Takashi , Ugawa Shinya , Yokoyama Shinji
Published: July 2021

Aim:
To reveal cholesterol transport via non-liver excretion pathway of plasma HDL-cholesterol, we focused on small intestinal and kidney in hyper- and hypo-lipoproteinemia model mice.

Method:
Mouse plasma was collected within EDTA coated tubes. Plasma lipoprotein profiles were measured by size exclusion HPLC and plasma preβHDL level was detected by immno-blotting of 2D PAGE by anti-mouse apoA-I peptide polyclonal antibody. Mouse HDL was co-incubated with 3H-cholesteryl oleyl ether (CEt) presence of CETP containing human plasma proteins for 48 hrs. The (100μL) of 3H-CEt-HDL was injected trough tail vein in anesthetized mice. 30μL of blood was collected each time point to chase the radioactivity, Mice organs were harvested after 3 hrs of the injection. Since lack of cellular catabolic pathway, CEt within the cells remained. For the immunohistochemical study, mouse was anesthetized and perfused thoroughly with PBS/5mM EDTA followed by 4% PFA/0.1M PB for the fixation. Leica CM1850 Cryostat was used for sectioning (thickness 16 μm). The sections were incubated in PBS containing 5% skim milk, 0.5% Triton X-100 and 5% inactivated normal horse serum for 3 hrs at room temperature, then incubated with primary antibodies (anti-SR-BI antibody diluted to 1:10,000) for overnight at 4°C. Secondary antibodies (Alexa fluor594) dissolved in the same blocking solution were applied to the sections for 90 min at room temperature. The fluorescence samples were observed by Nikon A1RSi confocal microscopy system.

Results and conclusion:
HDL derived CEt in intestinal cells were significantly reduced in ABCG1 null and SR-BI null mice during this experimental period. The other hand, kidney radioactivity showed no difference. Interestingly, preβHDL in SR-BI null mice was reduced by 2D western blotting of anti-mouse apoA-I peptide. Indicates that reduced speed of the plasma apoA-I recycling in SR-BI null mice, it may also cause reduced uptake of HDL-CEt by alternative pathway. Here we show that strong SR-BI-immunoreactivity was also found in the basolateral membrane and even higher intensity in some region. SR-BI located at the basolateral membrane may function as HDL-CE receptor in the small intestine.
Abstract 238: Incubation of MDCO216 With Human Serum or Plasma Potentiates ABCA1-Mediated Cholesterol Efflux Capacity, Increases Prebeta-1 HDL and Causes a Shift From Small to Large Alpha HDL Particles
Authors: Kempen Herman J , Schranz Dorota B , Asztalos Bela , Jeyarajah Elias , Otvos James , Drazul-Schrader Denise , Collins Heidi L , Adelman Steven J
Published: July 2021

MDCO216 is a complex of dimeric apoA-IMilano and POPC, shown to reduce atherosclerotic plaque burden. Here we studied the effect of incubation of human plasma or serum with MDCO216 on cholesterol efflux capacity from J774 cells, on prebeta-1 HDL and various HDL subfractions. At clinically relevant concentrations MDCO216 by itself markedly stimulates global and ABCA1-mediated cholesterol efflux. When incubated with human serum a time-dependent synergistic increase of the ABCA1 mediated efflux capacity is observed. Using the Sekisui ELISA for prebeta-1 HDL, MDCO216 itself is poorly detected. Prebeta-1 HDL is rapidly lost when human plasma alone is incubated at 37
o
C. However, incubation of human plasma with MDCO216 at 37
o
C causes a robust generation of new prebeta-1 HDL. 2D-Electrophoresis followed by immunoblotting with a general apoA-I antibody that also detects apoA-IM confirms the increase in prebeta-1 HDL (having a different mobility than pure MDCO216 particles), and shows a concomitant disappearance of alpha-3 HDL, alpha-4 HDL and MDCO216, and an increase in alpha-1 and alpha-2 HDL. NMR analysis of plasma incubated with MDCO216 at 47
o
C confirms very rapid disappearance of small HDL and increase of medium and large HDL particles. In conclusion, incubation of human plasma or serum with MDCO216 strongly enhances ABCA1-mediated cholesterol efflux capacity, causes a strong increase of prebeta-1 HDL and drastically changes HDL subfraction distribution, consistent with anti-atherosclerotic activity.
Abstract 241: Anti-Inflammatory Properties of Cynomolgus Monkey and Rabbit High-Density Lipoprotein Following Treatment With Recombinant Lecithin Cholesterol Acyltransferase
Authors: Peach Matthew , Cai Feng , Zhou Mingyue , Tang Chongren , Shaywitz Adam J , Fitzpatrick V D , Xu Ren
Published: July 2021

Introduction:
Lecithin cholesterol acyltransferase (LCAT) modulates HDL formation and increases HDL-C. Anti-inflammatory properties of HDL may be atheroprotective. We evaluated HDL from rabbits and cynomolgus monkeys treated with recombinant LCAT (rLCAT) to determine its anti-inflammatory properties.

Methods:
HDL from serum was isolated by ultracentrifugation. HUVECs were treated with HDL, stimulated with TNFα, and mRNA expression of VCAM-1, E-Selectin, and ICAM-1 was measured.

Results:
TNFα induced adhesion molecule expression in HUVECs treated with HDL (0.3 mg/mL) from rLCAT treated rabbits was reduced (E-Selectin) or similar (VCAM-1, ICAM-1) compared to pre-dose HDL. Using HDL (0.3 mg/mL) from rLCAT treated cynomolgus monkeys reduced expression of E-Selectin, VCAM-1, and ICAM-1 compared to control (pre-dose or vehicle) HDL in a rLCAT dose-dependent manner. In HUVECs treated with cynomolgus monkey HDL at concentrations normalized to serum HDL-C, adhesion molecule expression was reduced but showed a different profile. HDL after treatment with 1 or 5 mpk rLCAT reduced expression more than HDL from controls, while HDL after treatment with 20 or 150 mpk rLCAT blocked expression less than controls.

Conclusions:
HDL from rabbits and cynomolgus monkeys after rLCAT treatment was equally or more anti-inflammatory compared to control HDL when tested at a fixed concentration, a measure of HDL quality. To account for treatment changes to both HDL quality and quantity, HDL was tested relative to serum HDL-C. In this case, HDL from the 1 mpk group proved most efficacious in reducing TNFα stimulated adhesion molecule expression compared to vehicle HDL or HDL from higher doses.
Abstract 254: A Novel Role of Endothelial and Macrophage Epsins in Atherosclerosis
Authors: Chen Hong
Published: July 2021

Background:
Epsins are a family of ubiquitin-binding endocytic clathrin adaptors. We recently published that endothelial epsins function as critical regulators of tumor angiogenesis by controlling VEGF signaling (JCI, 2012; ATVB, 2013). Our goal is to define the novel role of epsins in endothelial cells (EC) and macrophages in regulating atherogenesis.

Methods and Results:
We engineered mice with specific deletion of epsins in EC (EC-DKO) or myeloid cells (MΦ-DKO). Strikingly, either EC-DKO or MΦ-DKO mice on ApoE-/- background fed western diet significantly reduced atherosclerotic lesion formation and foam cell accumulation. FACS analysis revealed that epsin deficiency greatly reduced TNFα and LPS-induced adhesion molecule expression (ICAM-1, VCAM-1, P- and E-selectins, CCR2 and MCP-1) in aortic EC and leukocyte recruitment in aorta. Mechanistically, EC epsins promote TNFR/TLR signaling and NF-κB and MAPK activation by recruiting NEMO, an essential NF-κB activator. In macrophages, epsin deficiency did not alter LDL scavenger receptors, CD36, Lox1 or SRB1, or reverse cholesterol transport proteins, ABCA1 or ABCG1, but did significantly reduce Lucifer Yellow pinocytosis, indicating a major defect in lipid uptake. Oil Red O staining of isolated ApoE-/-/MΦ-DKO macrophages showed little lipid accumulation, suggesting a mechanism in which epsin deficiency impairs foam cell formation. Epsin deficiency also significantly suppressed the pro-inflammatory M1 macrophage phenotype found in plaques thus suggesting an important pro-inflammatory role for epsins in macrophages. Loss of macrophage epsins significantly inhibited TNFα-stimulated activation of NF-κB and MAPK signaling pathways. We also observed a synthetic peptide comprising the epsin ubiquitin-interacting motif (UIM) and lesion homing sequence potently disrupted association of epsins with TNFR/TLR signaling complex in vitro, and inhibited atherosclerotic plaque in vivo.

Conclusions:
We demonstrate epsins promote atherogenesis by potentiating endothelium activation, leukocyte recruitment, foam cell formation and maintaining pro-inflammatory macrophages within the atherosclerotic plaque, thus suggesting epsins as a novel therapeutic target to combat atherogenesis.
Abstract 265: Platelet C-Type Lectinlike (CLEC)-2 Receptor Plays a Protective Role Against Development of Atherosclerosis in Atherosclerosis-Prone Mice
Authors: Lordkipanidze Marie , Harrison Matthew J , Watson Steve P , Rainger G E
Published: July 2021

Background:
Platelets can influence progression of plaque formation by facilitating recruitment of inflammatory cells at the sites of atherosclerotic lesions. A C-type lectin-like receptor, CLEC-2, abundantly expressed on the platelet surface, has been shown to regulate lymphatic development in utero though an interaction with Podoplanin. Interestingly, lymphatic vasculature is increased in ischemic and inflamed hearts, and in cholesterol-rich atherosclerotic lesions. Moreover, Podoplanin expression is up-regulated on inflammatory macrophages and on T-helper 17 cells. However, the role of the Podoplanin - CLEC-2 interaction in atherosclerosis remains unknown.

Aim:
We sought to investigate the role of CLEC-2 on atherosclerotic development in ApoE-deficient mice.

Methods:
CreERT.CLEC-2fl/fl x ApoE-/- and litter-matched ApoE-/- mice were treated with tamoxifen at the age of 9 weeks and were put on a high-fat diet for 6 weeks. Animals were killed at the age of 16 weeks, when platelet function assays and atherosclerosis assays were carried out.

Results:
Expression of CLEC-2 was abolished in tamoxifen-treated CreERT.CLEC-2fl/fl x ApoE-/- mice (n=8), whereas normal levels were seen in ApoE-/- controls (n=10) also treated with tamoxifen. CreERT.CLEC-2fl/fl x ApoE-/- and ApoE-/- mice had similar baseline characteristics, comparable levels of platelet glycoprotein expression (GPIb, GPIIbIIIa and GPVI) and normal platelet responses to platelet agonists (collagen-related peptide, PAR-4 peptide, ADP and arachidonic acid). Blood lipid levels were comparable between CreERT.CLEC-2fl/fl x ApoE-/- and control animals. Atherosclerotic plaque burden was significantly higher in the aortas of CreERT.CLEC-2fl/fl x ApoE-/- mice fed a high-fat diet for 6 weeks compared with their ApoE-/- counterparts. The higher plaque burden was seen consistently throughout the aorta, but reached significance at the level of whole aorta, abdominal aorta, outer curvature and the left subclavian region. This was seen in both male and female animals.

Conclusions:
The platelet-borne CLEC-2 receptor appears to have a protective role against atherogenesis in atheroprone mice. Further research to investigate the underlying mechanisms is warranted.
Abstract 296: Elevated Nox1 in the Endothelium Determines Oxidant Load Obesity and is Reduced with Increases in Muscle Mass
Authors: Qiu Shuiqing , Mintz James , Salet Christina , Han Weihong , Su Yunchao , Chen Feng , Yu Yanfang , Fulton David , Stepp David
Published: July 2021

Increases in muscle mass, as observed with exercise, are positive influences of cardiovascular health. Recent studies from our lab have shown that increased muscle mass via deletion of myostatin improves vasodilation but the underlying mechanisms are unknown. Improving insulin resistance has been shown to improve oxidant stress, specifically via expression of NOX1.

Hypothesis:
Increasing muscle mass by deletion of muscle growth negative regulator myostatin, improves vascular function in mesenteric arteries from obese
db/db
mice via reducing NOX-mediated oxidant stress. Myostatin deletion increased muscle mass in both lean (gastrocnemius 57.93%, gluteus maximus 60.95%) and obese mice (gastrocnemius 79.64%, gluteus maximus 112.32%). Fasting glucose, HbA1c and glucose tolerance are improved in obese myostatin null mice. Obese mice demonstrate superoxide-mediated impairment of acetylcholine (Ach)-induced vasodilation compared to lean mice. Deletion of myostatin in obese mice improved Ach-induced vasodilation in mesenteric arteries without effects in lean mice. This improvement was blunted by L-NAME. PGI2 and EDHF mediated vasodilation were unaffected by obesity or myostatin deletion. Treatment with sepiapterin (1х10
-6
mol/L, a precursor of the nitric oxide synthase cofactor tetrahydrobiopoterin) or GKT(1х10
-6
mol/L, NADPH oxidase inhibitor) restored impaired vasodilation in obese mice. Elevated oxidized DNA was observed in the endothelium of obese mesenteric arteries, and this elevation was blunted by myostatin deletion. NOX1 expression was increased in the endothelium of obese mice mesenteric arteries, which was reversed by myostatin deletion. Taken together, these data suggest that increasing muscle mass by deletion of myostatin improves NO-mediated vasodilation in obese mice; this vasodilation improvement is mediated by down regulation of NOX1in the endothelium.
Abstract 356: Serum Amyloid A Augments Aortic Aneurysm Formation Induced by Mineralocorticoid Receptor Agonists in the Presence of High Salt
Authors: Webb Nancy R , Wroblewski Joanne M , Lutshumba Jenny , De Beer Maria C , Noffsinger Vicky P , Ji Ailing , Guo Zhenheng , De Beer Frederick C
Published: July 2021

Objectives:
The annual mortality in the United States from ruptured aortic aneurysms is ~15000. Therapeutic interventions that prevent AAA progression and rupture remain to be identified. In humans, plasma concentrations of the acute phase reactant serum amyloid A (SAA) correlates with aortic dimensions before aneurysm formation. We have shown that endogenous SAA augments AAA in the well-established angiotensin II (AngII) infusion mouse model (unpublished data). Here we investigated whether endogenous SAA impacts aneurysm formation induced by deoxycorticosterone acetate (DOCA), a mineralocorticoid receptor agonist, in the presence of high salt.

Approach and results:
DOCA pellets (50mg, 21 day release) were implanted subcutaneously in the lateral dorsal region of 8-month old male C57BL/6 (SAAWT) mice and C57BL/6 mice lacking both acute phase SAA isoforms, SAA1.1 and SAA2.1 (SAAKO). The mice were also provided drinking water containing 0.9% NaCl and 0.2% KCl for 21 days (n = 7-8). As expected, DOCA + salt resulted in significantly increased systolic blood pressure, which was not affected by the absence of SAA. Unexpectedly SAAKO mice displayed a reduced urine output, accompanied by a reduced water intake. Plasma sodium and potassium concentrations in SAAWT and SAAKO mice were similar after treatment. The maximal luminal diameter of the abdominal aorta, as determined by ultrasound, was significantly lower in SAAKO mice compared to SAAWT mice after a 3-week DOCA + salt regime. Aneurysm incidence, determined by ultrasound and ex vivo analyses, was 67% for SAAWT mice and 25 % for SAAKO mice. Notably, plasma SAA was markedly increased in the SAAWT mice that formed aneurysms compared to those that did not. In SAAWT mice, immunohistochemical staining and in situ zymography identified SAA in aneurysmal aortic tissue, but not control aortas, that co-localized to regions of enhanced matrix metalloproteinase (MMP) activity, suggesting a role for SAA in MMP activation.

Conclusions:
We conclude that endogenous SAA augments aortic aneurysm formation induced by mineralocorticoid receptor agonists in the presence of high salt. Thus, SAA contributes to pathological processes leading to aortic aneurysm in two robust and mechanistically distinct animal models.
Abstract 378: Macrophage ß3 Integrin is an Important Player in Inflammation and Cellular Plasticity
Authors: Wolf Cristina A , Bobak Izabela , Su Xinming , Damm Ellen , Weilbaecher Katherine N , Schneider Jochen G
Published: July 2021

Communication between cells and the surrounding environment is a crucial mechanism for survival. Integrins are membrane-bound molecules that are involved in signaling between the cells and the extracellular matrix, thereby influencing cytoskeletal stability and intracellular signaling. β3 integrin and its binding partner αv form the αvβ3 heterodimer that is expressed in various cells. We and others have described the consequences of its absence in inflammation, atherosclerosis and cancer in vivo. However, the distinct role of this integrin as a signaling molecule and the consequences of its absence for macrophage structure remain mostly elusive.

Our aim is to further characterize the phenotype of β3-deficient (β3-/-) bone marrow-derived macrophages (BMDM) under stimulatory conditions (LPS and LDLs) compared to control cells in vitro.

qPCR, WB, ELISA, migration, proliferation assays were used to investigate β3-/- BMDM and controls (wt BMDM and Raw 264.7).

LPS was described to be not only pro- but also anti-inflammatory in a time-dependent manner. We show that LPS stimulation leads to high expression of pro-inflammatory cytokines (IL-1β and TNFα) shortly after treatment, while expression of anti-inflammatory cytokine (IL-10) arises at a later stage (12h post stimulation). Interestingly, β3-/- BMDM express more IL-1β than controls. IL-10 expression appears much earlier in β3-/- BMDM (6h post stimulation) but is reduced after 12h, indicating a faster and higher cellular response in the absence of the β3 integrin.

OxLDL, the leading cause to foam cell formation, stimulates the expression of IL-1β in controls and β3-/- BMDM with the latter expressing significantly less of this cytokine indicating that lack of β3 causes differential cellular responses after LPS and oxLDL stimulation. Other LDL forms tested (nLDL, acLDL, cLDL) did not have any effect on IL-1β expression.

In addition, we identified a higher proliferation rate in the β3-/- BMDM when cultured with M-CSF and a migration deficit in response to LPS, M-CSF and VEGF.

Taken together, our results show that macrophage β3 deficiency causes differential cellular plasticity depending on the stimulus, with functional consequences that could be essential in inflammation and atherosclerosis.
Abstract 386: Circulating Endothelial Microparticles, Elevated Blood Pressure and Endothelin-1 Mediated Vasoconstrictor Tone
Authors: Beckstrom Collin A , Bammer Tyler D , Dow Caitlin , Lincenberg Grace , Diehl Kyle J , Greiner Jared J , Stauffer Brian L , DeSouza Christopher A
Published: July 2021

Clinical interest in circulating microparticles originating from both endothelial cells and platelets has increased due to their putative role in inflammation, endovascular function, angiogenesis and thrombosis. Elevated blood pressure is associated with profound endothelial dysfunction, particularly enhanced endothelin (ET)-1-mediated vasoconstrictor tone. There is some evidence to suggest that circulating microparticles are influenced by blood pressure and may contribute to associated vascular abnormalities. As part of an ongoing study, we are determining: 1) whether circulating endothelial (EMP) and platelet (PMP) microparticles are higher in adults with elevated blood pressure (SBP >130 mmHg) and if so; 2) if these microparticles are associated with ET-1 mediated vasoconstriction. To date, 22 sedentary, non-obese middle-aged adults have been studied: 11 normotensive (age: 55+2 yr; 7 M/4 F; BP: 118/74+3/2 mm Hg) and 11 prehypertensive/hypertensive (age: 55+2 yr; 7 M/4 F; BP: 140/85+2/3 mm Hg). All subjects were free of overt cardiometabolic disease. EMPs and PMPs were measured in platelet-poor plasma by flow cytometry. EMPs were defined as CD31+/CD42b- events and PMPs were defined as CD31+/CD42+ events. Forearm blood flow (FBF: plethysmography) responses to intra-arterial infusion of BQ-123 (100 nmol/min; for 60 min), a selective ETA receptor antagonist. EMPs were ~70% higher (p<0.01) in the prehypertensive/hypertensive (39072+3951 MP/μL) compared with normotensive (22726+2552 MP/μL). There was no difference in EMPs with the elevated blood pressure group between the prehypertensive (n=5) and hypertensive (n=6) adults. PMPs were not significantly different between the groups (503+132 vs 431+80 MP/μL). Resting FBF increased ~40% (p<0.01) in response to BQ-123 in the prehypertensive/hypertensive group only. EMPs were significantly correlated with systolic blood pressure (r=0.68) and peak FBF response to ETA receptor blockade (r=0.61). These initial results indicate that circulating EMPs, but not PMPs, are elevated in prehypertensive/hypertensive adults. Moreover, circulating EMPs are associated with systolic blood pressure and enhanced ET-1 mediated vasoconstrictor tone.
Abstract 398: Tocomin Restores Endothelium-Dependent Relaxation in Diabetic Rat Aorta
Authors: Ali Saher F , Woodman Owen L
Published: July 2021

Increased superoxide in blood vessels can lead to impaired endothelial function. Tocotrienols, components of vitamin E are effective antioxidants that may have beneficial effects on vascular function particularly in pathologies involving oxidative stress such as diabetes. Tocomin is an extract of palm oil with high tocotrienol content. This study investigated the effect of 4 week treatment with tocomin (composition: tocotrienol rich fraction: 40%, α-tocopherol: 11% and palm olein: 38%) treatment on endothelium-dependent and endothelium-independent relaxation in the diabetic rat aorta. Male Wistar rats were randomly assigned to 4 groups (control, control+tocomin, diabetic and diabetic+tocomin). Diabetes was induced by a single injection of streptozotocin (STZ, 50 mg/kg iv). Rats were treated with tocomin (40 mg/kg per day s.c.) or vehicle (100% peanut oil) for 4 weeks commencing 6 weeks after induction of diabetes. Endothelium-dependent (EDR) to ACh, endothelium-independent relaxation (EIR) to sodium nitroprusside and superoxide production were measured using standard organ bath techniques and lucigenin enhanced chemiluminescence. Diabetes increased blood glucose (control 6.8±0.3 vs diabetic 32.1±1.2 mmol/L, n=7-9) and glycated hemoglobin (HbA1c control 5.6±0.2% vs diabetic 13.3±0.3%, n=7-9). Neither parameter was affected by tocomin treatment (blood glucose control+tocomin 7.3±0.7 mmol/L, diabetic+tocomin 26.3±4 mmol/L, HbA1c control+tocomin 5.5±0.2%, diabetic+tocomin 11.8±1.3%, n=8-10). Diabetes impaired EDR (ACh Rmax control 93±4% vs diabetic 76±4%, n=6-8, p<0.05) without affecting EIR. Tocomin treatment significantly improved EDR (ACh Rmax diabetic 76±4% vs diabetic+tocomin 89±3%, n=6-8, p<0.05) in diabetic rat aortae. Diabetes also significantly increased superoxide production (control 573±75 vs diabetic 1113±90 counts/mg dry tissue, n=4-9, p<0.05), which was alleviated by tocomin treatment (761±71 counts/mg dry tissue, n=4-9, p<0.05, in comparison to diabetic). These findings demonstrate that 4 week treatment of diabetic rats with tocotrienol rich tocomin significantly reduces superoxide production and improves EDR without affecting blood glucose levels.
Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis
Authors: Dunn Jessilyn , Qiu Haiwei , Kim Soyeon , Jjingo Daudi , Hoffman Ryan , Kim Chanwoo , Jang Inhwan , Jordan King , Jo Hanjoong
Published: July 2021

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.
Abstract 403: Metabolic Characteristics of Spontaneously Hypertensive Rats Compared With Normotensive Controls
Authors: Clark Jaime , Zahradka Peter , Taylor Carla
Published: July 2021

Objectives:
To differentiate the metabolic responses of spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto (WKY) rats.

Methods:
Blood pressure and pulse wave velocity (PWV) measurements were conducted on 25 week old SHR and WKY rats. At the time of termination, vital organs were harvested and weighed, and serum was collected for analysis of blood lipids, liver enzymes, and urea levels.

Results:
SHR exhibited higher readings for mean arterial pressure (MAP) (p< 0.0001), peak velocity (PWV) (p=0.017), heart/body weight (BW) ratio (p<0.0001), and left ventricular wall/BW ratio (p<0.0001) compared to WKY. WKY displayed higher weight gain and blood lipid levels (serum triglycerides, total cholesterol, LDL-C, and HDL-C) compared to SHR (p<0.0001). Blood lipid levels were positively correlated with weight gain, and negatively correlated with MAP and left ventricular wall/body weight ratio. Weight gain and MAP were negatively correlated (r=-0.60, p<0.0001). Serum AST levels were higher among SHR compared to WKY (p=0.026), and serum AST was positively correlated with left ventricular wall/BW ratio (r=0.35, p=0.030). There was no correlation between AST levels and liver/BW ratio or MAP. Serum urea levels were higher among SHR compared to WKY (p=0.0032), and positively correlated with MAP (r=0.42, p=0.011) and heart/BW ratio (r=0.43, p=0.0096). There were no differences in kidney/BW ratios, however, the peri-renal adipose/BW ratio was lower in SHR compared to WKY (p=0.001), and negatively correlated with MAP (r=-0.41, p=0.018).

Conclusion:
The positive relationship between serum AST and left ventricular wall/BW ratio, but not liver/BW ratio, suggests a possible response to changes in cardiac muscle, independent of blood pressure. Serum urea levels responded to MAP and heart/BW ratio. These metabolic responses of SHR compared to WKY invite further investigation of possible mechanisms underlying the link with cardiovascular dysfunction.
Abstract 414: In Hyperalphalipoproteinemic Subjects, the Presence of Coronary Artery Disease is Associated With Marked Changes in the High-Density Lipoprotein Phosphosphingolipidome
Authors: Hancock-Cerutti William , Lhomme Marie , Dauteuille Carolane , Lecocq Sora , Chapman John , Rader Daniel , Kontush Anatol , Cuchel Marina
Published: July 2021

Plasma concentrations of HDL-C have long been shown to correlate inversely with risk of coronary artery disease (CAD), however the causal nature of this association has not been established. Here we examine the chemical composition and phosphoshingolipidome of HDL in a cohort of subjects with premature CAD, despite having HDL levels ≥90th percentile. We hypothesized that HDL from hyperalphalipoproteinemic subjects with CAD would have distinct compositional changes compared to healthy hyperalphalipoproteinemic subjects, which may relate a reduction in HDL functionality and a pro-atherogenic state. Subjects with HDL ≥90th percentile and CAD (HHDL+CAD, n=25) were compared to healthy subjects with HDL ≥90th percentile (HHDL, n=23). A group of healthy controls with HDL between the 25th and 75th percentile (n=11) was used as reference. HDL subfractions were isolated from EDTA plasma using isopycnic density gradient ultracentrifugation and their chemical composition was assayed using commercial enzymatic assay kits. Quantification of >160 molecular species of total HDL phospho- and sphingolipids was accomplished via a novel method using LC-ESI/MS/MS analysis. The most striking differences were observed in total HDL phospho- and sphingolipid subclasses between groups. When expressed as a percentage of total phosphosphingolipid, phosphatidylcholine (PC) and phosphatidylinositol (PI) were depleted in HHDL+CAD group compared to HHDL, while sphingomyelin (SM) was increased, resulting in a lower PC/SM ratio. The HDL PC/SM ratio has previously been shown to correlate with HDL surface fluidity and antioxidative activity. Additionally, enrichment of PI, a minor, negatively charged phospholipid, has been shown to stimulate cholesterol efflux to reconstituted HDL. Reduction in these two metrics, observed in the HHDL+CAD group, may indicate functionally deficient HDL and may represent pro-atherogenic, HDL-associated biomarkers of CAD in hyperalphalipoproteinemia.
Abstract 415: Role of Proteolysis in the Remodeling of High-Density Lipoprotein
Authors: Jayaraman Shobini
Published: July 2021

Introduction:
Both quantity and quality of the circulating HDL determine their optimal anti-atherogenic potential. During atherogenesis, various cell types in the arterial intima release enzymes into the intimal fluid, which modify HDL proteins and lipids that adversely affect HDL functionality.

Hypothesis:
The emerging paradigm for the
in vivo
proteolytic inactivation of HDL is centered on pre-beta-HDL. Over 90% of the major HDL proteins, apoA-I and apoA-II, circulate on mature HDL. Although binding to HDL protects these proteins from proteolysis, such proteolysis cannot be completely excluded, and its effects on HDL functionality remain unknown.

Methods:
Human plasma HDL were subjected to mild proteolysis with plasmin, a protease active in atherosclerotic lesions. The proteolytic products were analyzed by SDS-PAGE and Western blotting. HDL remodeling was monitored under near-physiological conditions by size-exclusion chromatography and gel electrophoresis.

Results:
HDL treatment with plasmin caused no significant structural remodeling of lipoprotein particles. Interestingly, plasmin cleaved apoA-I and apoA-II on HDL. The major protein fragments were observed in the 10-12 kDa range. Western blotting indicated that these fragments were derived from both apoA-I and apoA-II. Next, intact and plasmin-treated HDL were incubated at 37
o
C, pH 7.5 for 6-12 h. Intact HDL showed dissociation of a fraction of lipid-free apoA-I without significant changes in the particle size. In contrast, plasmin-treated HDL underwent fusion with release of full-length and fragmented apoA-I and apoA-II, indicating lipoprotein destabilization.

Conclusion:
Our results reveal that plasmin, can cleave HDL-bound forms of apoA-I and apoA-II and thereby destabilize HDL under near-physiological conditions, resulting in HDL disintegration and dissociation of lipid-free proteins. For the first time, we demonstrate that proteolysis can render not only lipid-free but also HDL-bound proteins dysfunctional. Destabilization of HDL via proteolytic modifications may contribute to the recently observed excessive accumulation of lipid-free apoA-I in the arterial intima, which probably contributes to the progression of atherosclerosis.
Abstract 424: Phospholipid Transfer Protein Deficiency Promotes Inflammatory Factors' Expression via TLR4-TAK1-NFκB Pathway in Macrophage
Authors: Yu Yang
Published: July 2021

Phospholipid transfer protein (PLTP) plays important roles in macrophage mediated inflammation. In the current study we observed that endogenous PLTP modulated pro-inflammatory pathways in macrophage. With the presence of LPS, peritoneal derived macrophage (PDM) or bone marrow derived macrophage (BMDM) from PLTP deficient mice (PLTP-/-) expressed significantly higher level of pro-inflammatory cytokines compared with PDM or BMDM from wild-type mice (WT), respectively. LPS induced TNFα expression in PLTP-/- BMDM or PLTP knockdown RAW264.7 were suppressed by (5Z)-7-Oxozeaenol, a TAK1 inhibitor, suggesting PLTP deficiency enhanced the expression of pro-inflammatory cytokines via TAK1-NFκB pathway in macrophage. Furthermore, abundance of TLR4 on the membrane was dramatically increased in BMDM from PLTP-/- compared with WT. In addition, inhibition of ABCA1 by chemical inhibitor, glyburide, did not reduce nuclear levels of active STAT3 of BMDM, which indicated that no autocrine PLTP triggered ABCA1-JAK2-STAT3 pathway in this study. In conclusion, PLTP deficiency or low expression may enhance LPS induced pro-inflammatory cytokines expression via TLR4-TAK1-NFκB pathway in macrophage.
Abstract 456: Enhanced Expression of Aortic and Plasma Vascular Endothelial Growth Factor-C in Experimental Atherosclerosis
Authors: Hii Chung Shii
Published: July 2021

Background:
Members of VEGF family such as VEGF-A has been extensively studied given its association with plaque stability through its modulation on angiogenesis. However, contribution of VEGF-A in angiogenesis during atherosclerosis remains controversial as circulating VEGF-A is shown to have a minor impact on atherosclerosis development. Thus another member of VEGF family, VEGF-C, has been implicated in atherosclerosis as VEGF-C displays dual functions being both angiogenic and lymphangiogenic. In contrast to VEGF-A, little information is available on VEGF-C. Moreover, few studies that investigated VEGF-C expression in atherosclerosis were limited so far to advanced plaques since most of the studies have been performed in post-mortem human aortic tissue. Thus, we conducted an in depth analysis of the temporal and spatial distribution of VEGF-C in aortic tissue from

apoE
-/-

mice as the disease progresses from fatty streaks (or early lesions) to more advanced lesions and also to identify the cellular source of VEGF-C in atherosclerotic lesions.

Methods and results:
Compared to wild-type mice, expression of VEGF-C was increased in the aorta, more specifically in intimal lesions, of

apoE
-/-

. Within early lesions, monocyte-derived cells were the main VEGF-C producers. The spatial analysis of VEGF-C expression as plaque progresses from fatty streaks to more advanced plaque revealed that intimal VEGF-C correlated with plaque progression and preferentially localized in the fibrous cap and shoulder regions of advanced plaques. Moreover, this increase in tissue VEGF-C was accompanied by an increase in plasma VEGF-C during disease progression. Finally, aortic and plasma VEGF-C levels were markedly reduced in atherosclerotic mice treated with the cholesterol-lowering drug ezetimibe in which plaque regressed. We also showed that macrophages and smooth muscle cells express VEGFR-2 and -3 implying that VEGF-C has other cellular targets and potentially could have effect on these cells. The potential function(s) of VEGF-C in atherosclerosis will be discussed.

Conclusions:
This study provides the evidence for the close association between VEGF-C and progression of atherosclerosis.
Abstract 482: Attachment Site Curvature of Thoracic Endografts is Correlated to Adjacent Aortic Wall Stress and Stress Distribution
Authors: Couper Nathan , Richards Michael , Chandra Ankur
Published: July 2021

INTRODUCTION:
TEVAR has been seen to cause acute and chronic stent-induced tears of the adjacent aortic wall after treatment in 10-25% of cases with increasing frequency as the stent is placed closer to the aortic valve. The underlying cause for these tears and the ability to predict their occurrence is poorly understood. We hypothesize the cause of these tears is related to stent-induced changes in the adjacent aortic wall which could be quantified and predicted through finite element analysis (FEA) of stent-aorta interface.

METHODS:
Abaqus
TM
was used to resolve the FEA model of the stent-aorta interface in three configurations. The maximum principal stress in the vessel wall was averaged over the volume around the stent attachment point and the curvature of the stent was calculated at both the distal and proximal ends. (Figure 1).

RESULTS:
As the curvature of the attachment site increased, an increase in adjacent aortic wall stress was noted. These ranged from mean curvature (1/m) of 0.1 with wall stress of 49kPa for the distal attachment, position #2 to mean curvature of 6.7 and wall stress of 82kPa for the distal attachment site in position #3. There was an increase in maximum stress distribution as the TEVAR approached the aortic root of 104kPa, 109kPa, and 112kPa for positions 1-3 repectively.

CONCLUSIONS:
An increase in adjacent aortic wall stress and stress distribution was noted as TEVAR were placed closer to the aortic root which corresponds to the increase in stent-induced aortic tears observed in clinical series. This approach provides the basis for a predictive clinical tool to allow for patient-specific TEVAR planning with associated aortic wall stress analysis to minimize adjacent aortic trauma and assist in future stent design.
Abstract 495: Calpain Inhibition in Bone Marrow--Derived Cells Attenuates Atherosclerosis but Not Aortic Aneurysms in Hypercholesterolemic Mice Infused With Angiotensin II
Authors: Subramanian Venkateswaran , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Rateri Debra L , Baud Laurent
Published: July 2021

Background and Objective:
Calpains are calcium-dependent neutral cysteine proteases that are essential for multiple cellular functions such as cytoskeletal remodeling, cell cycle, and apoptosis. Angiotensin II (AngII)-infusion into mice profoundly increased both aortic protein and activity of calpains. In addition, pharmacological inhibition of calpain attenuated AngII-induced aortic medial macrophage accumulation, atherosclerosis, ascending and abdominal aortic aneurysm (AAs) in mice. Using mice that overexpress calpastatin, the endogenous inhibitor of calpains, the purpose of this study was to determine whether calpains in bone marrow-derived cells contribute to AngII-induced atherosclerosis and AAs in hypercholesterolemic mice.

Methods and Results:
Bone marrow cells were harvested from either calpastatin (CAST) overexpression transgenic (Tg) or wild type (WT) mice and injected (7.5 x 10
6
cells/mouse) into age-matched male LDL receptor -/- recipient mice that had been irradiated with a total of 900 Rads from a cesium source. Four weeks after bone marrow repopulation, recipient mice were fed a saturated fat-enriched diet and infused with either saline or AngII (1,000 ng/kg/min) for 4 weeks. The CAST Tg or WT genotypes used for repopulation had no effect on body weight, white blood cell counts and plasma cholesterol concentrations during AngII infusion. Baseline systolic blood pressure was not different, and AngII infusion increased systolic blood pressure equivalently in both genotypes. Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in WT and Tg mice (1.28 ± 0.11 versus 1.32 ± 0.08 mm, P = NS). Calpain inhibition showed no discernible effects on AngII-induced ascending AA development (11.7 ± 0.47 versus 11.1 ± 0.54 mm
2
, P = NS). Interestingly, overexpression of CAST in bone marrow-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches by en face measurement (Intimal lesions: WT = 19.9 ± 1.8 % versus Tg = 11.0 ± 1.2 %; n=14, P<0.001).

Conclusion:
Calpain inhibition in bone marrow-derived cells attenuates AngII-induced atherosclerosis but does not influence formation of ascending and abdominal AAs in hypercholesterolemic mice.