bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 595: Genomic, Transcriptomic and MiRNomic Analysis of Triglyceride and Cholesterol Distribution into Lipoprotein Fractions in the Rat
Authors: Hodúlová Miloslava
Published: July 2021

Background:
Dyslipidemia is central to the definition of metabolic syndrome (MS), one of most prevalent human diseases worldwide. We preformed genome-wide association study (GWAS) of triacylglycerols (TG) and cholesterol (C) distribution into lipoprotein fractions in the recombinant inbred strain panel PXO (segregating alleles of two MS models, SHR and PD strains, together with those of normolipidemic Brown Norway strain origin), followed by transcriptomic and miRNomic (microRNA profiling) analyses.

Methods:
We established morphometric and metabolic profile in adult male rats of 14 PXO strains and two progenitor strains (n=183) including glucose tolerance and TG and C concentrations in 20 lipoprotein fractions. GWAS utilizing >20,000 SNPs was performed using MapManager, the significance validated by 2000 permutations per trait. The hepatic transcriptome and miRNome profiles of the most contrasting strains were generated using Affymetrix GeneAtlas system followed by network analysis (Ingenuity Pathway Analysis).

Results:
We have identified 14 haplotype blocks showing suggestive or significant linkage to studied traits. Except for LDL-TG loci on chromosomes 3 and 12, PXO strains carrying the SHR allele displayed significantly higher values of the lipid linked traits, e.g. LDL-C (21.2±0.4 vs. 12.5±0.4 mg/dl in PXO strains with SHR allele vs. BXH2 allele). C concentrations in large, medium and very small LDL particles were significantly associated to a single gene (
Lrp1b
). Subsequent transcriptomic comparison of phenotypically most contrasting identified series of dysregulated metabolic and signaling pathways including cholesterol biosynthesis and the key upstream regulators such as
HNF1
,
HNF4
and
PPARA
.

Conclusion:
We identified several novel variants associated to TG and C concentrations in lipoprotein fractions together with their transcriptomic and biological network correlates.
Abstract 598: Irisin, a Novel Hormone, and Its Response to Increasing Exercise Intensity in Young Healthy Subjects
Authors: Cooke Alexandra , Gomez Yessica H , Mutter Andrew F , Mantzoros Christos S , Daskalopoulou Stella S
Published: July 2021

Introduction:
Irisin, a recently discovered hormone, has been shown to induce white adipose tissue browning, enhancing energy expenditure and possibly mediating some of the beneficial effects of exercise. We aimed to estimate a) the timeframe of changes in plasma irisin levels after acute maximal exercise, b) the effect of different exercise intensities on irisin levels immediately post-exercise, and c) the effect of smoking (chronic and acute) on exercise-induced irisin release.

Methods:
a) 4 healthy subjects (22.5±1.73 years) underwent maximal exercise to attain maximal oxygen consumption (VO
2max
). Blood was drawn at pre-specified intervals to define the time curve of irisin changes over a 24-hr period; b) 35 healthy, non-smoking (23.0±3.3 years) men and women (n=20/15) underwent three exercise protocols ≥48 hrs apart, in random order: 1) maximal intensity (VO
2max
), 2) relative intensity (70% VO
2max
, 10 mins); and 3) absolute intensity (75W output, 10 mins). Blood was drawn immediately pre- and 3 mins post-exercise; c) In 20 smokers irisin levels were measured pre- and 3 mins post-maximal exercise after 12 hrs abstinence from smoking on two separate days 1) continued abstinence (chronic smoking) and 2) after smoking one cigarette pre-exercise (acute smoking), compared to 10 non-smokers.

Results:
a) Irisin levels increased by 35% 3 mins post-exercise, then dropped and remained constant; b) Irisin levels post-exercise were significantly higher than pre-exercise after all intensities (all, P<.001). Post-pre exercise differences in irisin levels were significantly different between exercise intensities (P=.001), with the greatest increase after maximal intensity (P=.004 vs. relative, and absolute). Higher VO
2max
correlated with a greater post-pre exercise difference in irisin levels after maximal exercise; c) Smoking study analyses are underway.

Conclusions:
Plasma irisin levels were acutely elevated in response to exercise, with a greater increase after maximal exercise. Higher VO
2max
was associated with greater irisin release. These findings suggest that irisin release could be a function of muscle energy demand. Future studies need to determine the underlying mechanisms of irisin release and explore irisin’s therapeutic potential.
Abstract 600: Asymmetric Dimethylarginine is a Novel Regulator of mTOR Expression in Adipocytes
Authors: Dowsett Laura B , Boruc Olga , Lee Ben , Leiper James
Published: July 2021

The metabolic syndrome, in which cardiovascular disease and obesity play major roles, has become an important healthcare issue. Systemic concentrations of asymmetric dimethylarginine (ADMA) correlate with obesity and insulin resistance and it is considered to be an independent marker of cardiovascular disease. Currently, ADMA is understood to be an endogenous inhibitor of nitric oxide synthase; however, the direct effect of ADMA on adipocytes and in obesity has not been investigated. Here, we establish that ADMA in adipocytes regulates the expression and activity of the mTOR lipid synthesis pathway which results in adipocyte hypertrophy both in vitro and in vivo.

Treatment of 3T3-L1 adipocytes with ADMA (10μM) resulted in adipocyte hypertrophy (control 27μm2 ±1.4, 10μM ADMA 33μm2± 1.6). This could not be mimicked through the blockade of NO by L-NAME (28μm2 ± 1.5) and PB-ITU (25μm2± 1.2). Hypertrophy was driven by an increase in mTOR expression (x2.4 ± 0.4) which upregulates ACC (x4.6 ± 0.8) and FASN (x1.4 ± 0.1) suggesting ADMA increases lipid production in an NO-independent manner.

Investigations in vivo were explored through the genetic manipulation of DDAH1, an enzyme responsible for ADMA breakdown. Specific deletion of DDAH1 within adipocytes increased intracellular ADMA concentrations. This in turn caused an upregulation of mTOR, ACC and FASN expression and resulted in adipocyte hypertrophy. Conversely, genetic overexpression of DDAH1 reduced ADMA production within adipocytes and caused a down-regulation in mTOR expression.

These data suggests that ADMA is a novel regulator of mTOR expression and activity at physiological concentrations. Pathological concentrations of ADMA are associated with adipocyte hypertrophy a known risk factor for insulin resistance and inflammation.
Abstract 607: Endothelin-1 Increases ß-Actin Expression During Osteochondrogenesis of Aortic Smooth Muscle Cells from Leptin-Deficient ob/ob Mice
Authors: Liberman Marcel , Carmo Luciana S , de Andrade Maria Claudina C
Published: July 2021

β-actin, a highly conserved gene/protein, which is usually not modulated by different stimuli, is routinely used to normalize target gene or protein expression in experimental biology, and so defined endogenous housekeeping gene. However, investigators showed that β-actin expression could change during cellular growth and differentiation. We hypothesized that endothelin 1 (ET-1), a cellular proliferative agonist, increases β-actin expression and stimulates calcification in vascular smooth muscle cells (SMC) from obob mice. Primary aortic SMC passage 4-6 isolated from obese leptin-deficient (obob) and from C57BL/6 (C57) mice were incubated without (Cont) or with ET-1 50nM (ET) for 0-72h. Cell lysates were assessed for β-actin and RUNX2 expression by western blotting and normalized by GAPDH. ObobET SMC increased β-actin expression after 24h, 48h and 72h (1.68±0.06, 1.66±0.05, 1.73±0.06 respectively) vs obobCont (1.01±0.07), p<.05 n=6. However, C57ET did not change β-actin expression (0.91±0.06, 1.04±0.03, 0.99±0.08) after 24h, 48h and 72h respectively vs. C57Cont (0.95 ± 0.04), p=NS n=6. Concomitantly, obobET SMC increased osteochondrogenic differentiation, by modulating RUNX2 (1.23±0.03 vs obobCont 1±0.01, p<.05 n=3) after 48h, which did not occur in C57 (C57ET 1.07±0.07 vs C57Cont 1.02±0.11), p=NS. Furthermore, obobET increased calcification vs obobCont (1.69±0.025 vs 1.03±0.03) after 14 days p<.05, n=3 and C57ET did not calcify (0.97±0.02 vs C57Cont 1±0.05), p=NS. We showed that ET-1 modulates β-actin expression and osteochodrogenenic differentiation only in obob SMC, thus increasing calcification. In conclusion, β-actin is not a consistent endogenous control in ET-1-stimulated SMC from obob mice during osteochondrogenic differentiation.
Abstract 614: Distinct Metabolism of Apo(a) and ApoB-100 Within Plasma Lp(a)
Authors: Diffenderfer Margaret R , Lamon-Fava Stefania , Marcovina Santica M , Barrett P H , Berglund Lars , Schaefer Ernst J
Published: July 2021

Lipoprotein(a) [Lp(a)] is a lipoprotein similar in lipid content and composition to LDL but different in having apolipoprotein (apo) (a) covalently linked to apoB-100. Apo(a) is a heavily glycosylated protein and highly polymorphic in size. The metabolism of apo(a) and apoB-100 within Lp(a) as reported in previous studies is not clear, due in part to differences in methodology and study design among the different studies.

We determined the kinetics of apo(a) and apoB-100 within plasma Lp(a) in five male subjects with dyslipidemia [Lp(a) concentration: 8.9-183.5 nmol/l] during a primed constant infusion of [5,5,5-
2
H
3
] L-leucine, in the constantly fed state. Lp(a) was immunoprecipitated directly from whole plasma, and apo(a) and apoB-100 in Lp(a) were separated by gradient gel electrophoresis. The kinetics of each apolipoprotein was determined by gas chromatography-mass spectrometry and multicompartmental modeling. Apo(a) was found to have a plasma residence time more than twice that of apoB-100 in Lp(a). The fractional catabolic rates of Lp(a)-apo(a) and Lp(a)-apoB-100 were 0.11 ± 0.02 and 0.29 ± 0.06 pools/day, respectively (mean ± SEM;
P
= 0.04). The median apo(a) production rate (0.248 nmol/kg·day
-1
) was significantly lower than that of Lp(a)-apoB-100 (0.514 nmol/kg·day
-1
;
P
= 0.03).

Our data support the concept that apo(a) and apoB-100 in plasma Lp(a) are catabolized differently in humans in the fed state. We hypothesize that apo(a) does not remain bound to a single apoB-100-containing lipoprotein but, rather, reassociates at least once with another apoB-100-containing particle during its plasma residence.
Abstract 626: The ABCA1 Agonist Peptides CS-6253 and ATI-5261 Replicate ApoA-I Function and Generate nHDL Particles that Are Remodeled and Maturated Within the Plasma Compartment, Resulting in Efficient Cholesterol SR-BI Delivery to Hepatic Cells
Authors: Hafiane Anouar , Bielicki John , Johansson Jan , Genest Jacques
Published: July 2021

Novel apolipoprotein (apo) mimetic peptides are potent mediators of ABCA1-mediated cholesterol efflux, mimicking native apoA-I. We investigated CS-6253 (CS) and ATI-5261 (ATI) in their ability to promote lipid transfer between nascent (n)HDL particles and plasma lipoproteins and examined cholesterol influx from remodelled HDL-like particles to hepatic cells through the SR-BI receptor. nHDL-like lipoproteins nHDL-CS and nHDL-ATI were generated by incubating CS or ATI in the presence of BHK cells expressing ABCA1 labelled with
3
[H]cholesterol and
3
[H] choline; native apoA-I was used as control. These nHDL particles were incubated with plasma at 37°C. Both CS and ATI increased LCAT activity significantly, although were approximately 50% less efficient than nHDL-apoA-I (fractional esterification rate (FER) =22.32±0.86%/h; vs nHDL-CS and nHDL-ATI; FER=11.40±0.045%/h; p<0.05). The majority of
3
[H]cholesterol from nHDL mimetics was esterified by LCAT, resulting in an increase in α1-migrating HDL-like particles in plasma (as shown by 2D-PAGGE). The ability of nHDL mimetics to transfer
3
[H]-phosphatidyl choline to plasma apoB-containing lipoproteins is (76±20%) in HDL-apoA-I, (38±0.5%) in nHDL-ATI and (54±13%) in nHDL-CS. These data show that nHDL mimetics are actively remodelled in the presence of plasma lipoproteins. We then investigated cholesterol delivery from HDL-CS and HDL-ATI mimetic to hepatic tissue via SR-BI using Fu5AH cells, having HDL-apoA-I as control. Kinetic parameters for SR-BI-mediated cholesterol influx are as follows: HDL-CS (

K
m

= 0.30±0.05 ug/ml; p<0.05) efficient as HDL apoA-I at promoting cholesterol uptake into Fu5AH cells (

K
m

= 0.37±0.13 ug/ml), while HDL-ATI was least efficient (

K
m

= 1.03±0.20 ug/ml). The inhibition of SR-BI selective uptake with BLT-1 affected the uptake of cholesterol from apoB precipitated plasma containing either HDL-CS, HDL-ATI or apoA-I. Here we present an
in-vitro
model of nHDL-CS and nHDL-ATI that actively undergo remodelling and maturation in plasma and replicate the function of apoA-I. These mature HDL mimetics generated from ABCA1 agonist peptides deliver cholesterol efficiently to hepatocytes specifically through the SR-BI receptor.
Abstract 628: Hepatic Insulin Signaling Regulates ApoA-I Gene Expression Through the Type I Deiodinase.
Authors: Liu Jing , Hernandez-Ono Antonio , Galton Valerie , Ginsberg Henry
Published: July 2021

People with low levels of high density lipoprotein cholesterol (HDLC) and apolipoprotein A-I (ApoA-I) have a higher risk of cardiovascular disease. Low levels of HDLC are common in individuals who are insulin resistant (IR), e.g., with metabolic syndrome and type 2 diabetes mellitus (T2DM). Despite the high prevalence of these two disorders, very little work has been reported regarding the molecular pathways linking insulin signaling or action and the levels of either HDLC or ApoA-1. We reported previously that liver specific insulin receptor (InsR) knockout mice (LIRKO) have markedly reduced plasma HDLC levels that increase after restoration of hepatic Akt signaling. In the present study, we created acute LIRKO mice by injecting an albumin-Cre adenovirus (Ad) into InsR floxed mice and observed marked reductions in HDLC, the expression of ApoA-I, and the expression of the gene coding Type1 iodothyronine deiodinase1, a selenoenzyme expressed highly in the liver that converts thyroxine to 3,5,3’-triiodothyronine (T3) or reverse T3. Deiodinase 1 knockout mice also had significantly reduced hepatic ApoA-I mRNA levels. Overexpression of Dio1 in LIRKO restored HDLC and significantly increased the expression of ApoA-I mRNA. In vitro studies showed that the expression of ApoA-I was significantly reduced after knockdown of either InsR or Dio1 expression in HepG2 cells. Moreover, overexpression of Dio1 restored ApoA-I promoter activity that had been decreased by knockdown of InsR. Deletion analysis of ApoAI promoter regions showed that insulin signaling regulated ApoA-I expression by acting on a region which does not contain any thyroid response elements. Pulse-chase experiments in HepG2 cells showed that deficiency of insulin signaling resulted in decreased synthesis and secretion of ApoAI. Our results indicates that defective hepatic insulin signaling results in reduced expression of Dio1 which, in turn, leads to reduced expression of ApoA-I and decreased synthesis and secretion of ApoA-I from hepatocytes. We believe our studies have defined a novel pathway from insulin signaling to ApoA-I synthesis that may lead to new approaches for increasing HDL levels in people with defective insulin signaling.
Abstract 636: The Nuclear Receptor FXR Uncouples the Actions of Srebp2 and MiR-33
Authors: Vallim Elizabeth , Ahn Hannah , Vallim Thomas
Published: July 2021

The role of microRNAs in fine-tuning gene expression has now been well established, but the mechanisms for microRNA regulation at the transcriptional level remain understudied. The physiologic context in which microRNAs are acting is likely linked to the function of these same microRNAs and how they are regulated. Here we identify miR-33 as a direct target gene of the bile acid nuclear receptor FXR. We show that pharmacologic FXR activation induces miR-33 in wild-type, but not FXR knockout mice. MiR-33 is located in the Srebp2 locus, and we show that FXR activation also induces the expression of Srebp2 mRNA. Using ChIP-Seq, we identify at least one functional FXRE in an intronic region of Srebp2, demonstrating that miR-33 and Srebp2 are both direct FXR targets. Although FXR activation increases Srebp2 mRNA levels, we also demonstrate that FXR in fact decreases Srebp2 proteolytic processing, which determines its transcriptional activity. This decreased activity is a result of the FXR dependent induction of the integral ER membrane protein Insig2, which is itself also a direct FXR target. Together these results identify a molecular mechanism for the transcriptional regulation of Srebp2 and miR-33, as well as the mechanism for the decreased activity of Srebp2 processing in the ER by FXR. The best characterized target of miR-33 is ABCA1. We have previously demonstrated that FXR activation decreases ABCA1 protein levels via the induction of miR-144, a microRNA that also targets ABCA1. Our new findings suggest that FXR induces two independent microRNAs that function together to repress ABCA1 and reduce HDL levels.
Abstract 646: The Paradoxical Role of TREM-Like 1 in Atherosclerosis
Authors: Gonzalez Marieli , Collado Carlos J , Reyes Fiorella , Báez Mayra , Washington Anthony V
Published: July 2021

Atherosclerosis is a chronic inflammatory process of the vessel wall driven by an inflammatory state caused by the involvement of different cell populations, including platelets. The receptor Triggering Receptor Expressed on Myeloid Cells (TREM)-like transcript (TLT)-1 is prepacked in platelet α-granules and brought to the surface upon activation. TLT-1 has both membrane-bound and soluble forms (sTLT-1) and the latter has been found to enhance platelet activation as well as platelet-endothelial cell interactions. TLT-1 null mice (treml1-/-) have reduced platelet aggregation, are significantly more susceptible to lipopolysaccharide challenge compared to their wild type counterparts, and accordingly, hemorrhage in response to an acute inflammatory challenge. These results suggest that TLT-1 is a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms and in light of this, we hypothesized that TLT-1 plays an important role in atherosclerosis progression. To address this, we generated apoE-/-/treml1-/- double knockout mice [DKO]. We found that DKO mice fed an atherogenic diet (HFD) showed significantly increased weight gain when compared to apoE-/-. Accordingly, DKO mice showed increased total cholesterol and triglycerides compared to controls. Surprisingly, assessment of lesion size revealed that DKO mice have significantly smaller lesions in the aortic sinus at four and 12 weeks after HFD compared to apoE-/-. At 20 weeks, lesion differences are no longer significant, but compositional analysis revealed that DKO atherosclerotic lesions are less vulnerable as seen by decreased lesion calcification and increased smooth muscle cell content. Furthermore, q-rtPCR analysis of atherosclerosis-related genes revealed that TLT-1 differentially affects genes related to lipid metabolism (apo AI and B) and vascular inflammation (thrombospondin 4, PAI-1 and VGFR-2). While our data suggests that the smaller lesion size may be explained by lower platelet reactivity, differences in cholesterol levels suggest altered lipid metabolism which is supported by our metabolomics studies (González, M. et.al). Taken together TLT-1 plays a dual role in the progression of atherosclerosis.
Abstract 664: The Coronary Heart Disease--Associated Transcription Factor TCF21 Regulates Disease-Related Genes and May Contribute to the Migration of SMC Progenitors to the Fibrous Cap
Authors: Nurnberg Sylvia T , Cheng Karen , Kundu Ramendra K , Sazonova Olga V , Carcamo-Orive Ivan , Shankman Laura S , Raiesdana Azad , Kundu Soumajit , Owens Gary K , Quertermous Thomas
Published: July 2021

Introduction:
Recent genome-wide association studies have identified a number of genes that contribute to the risk for coronary heart disease, including TCF21, a bHLH transcription factor believed to serve a critical role in the differentiation of epicardial progenitor cells that give rise to coronary smooth muscle.

Methods and Results:
Results of immunolocalization and in situ hybridization studies showed that TCF21 is not expressed in medial smooth muscle cells (SMC) of adult human coronary arteries but primarily within the adventitia and within cells of unknown origin in atherosclerotic lesions. LacZ reporter animal studies with ApoE null background showed a shift in Tcf21 expression from adventitial and medial cells to the neointima, notably into the area below and within the forming fibrous cap, suggesting that Tcf21 expressing cells migrate into the forming atherosclerotic lesion. To investigate disease-related functions of TCF21 in SMCs, knockdown and overexpression studies were performed in human coronary artery smooth muscle cells, revealing an effect on migration, proliferation and SMC marker gene expression.

Genome-wide RNA-seq studies in these cells with siRNA treatment identified putative TCF21 downstream genes and pathways involved in vascular development as well as atherosclerosis. Supporting ChIP-seq data suggests that these - as over half of all differentially expressed genes - are direct transcriptional targets. To elucidate the behavior of Tcf21 expressing cells in the development of disease lesions, we are employing an in vivo Tcf21 lineage tracing model. Preliminary data from these studies suggests that Tcf21 expressing cells give rise to fibrous cap SMCs, and ongoing studies are aimed at the further characterization of molecular pathways that are active in these Tcf21 expressing cells.

Conclusions:
The gene regulatory and expression profile of Tcf21 during disease progression strongly implies a key role in Coronary Heart Disease. Tcf21 is likely to play a role in the migration of SMC progenitors from the adventitia into the forming atherosclerotic plaque. As a source of differentiated SMCs of the fibrous cap Tcf21 may therefore promote plaque stability and protect from lesion rupture.
Abstract 665: Activation of Histone Deacetylase 2: A Novel Strategy for Reversing Vascular Dysfunction in Atherogenesis
Authors: Pandey Deepesh , Sikka Gautam , Bergman Yehudis , Kim Jae H , Ryoo Sungwoo , Romer Lewis , Berkowitz Dan
Published: July 2021

Arginase 2 is a critical target in atherosclerosis as it regulates both endothelial NO, fibrosis and inflammation. The increase in Arg2 activity with endothelial cell activation is dependent on both early post-translational dependent mechanisms as well as a later increase in Arg 2 expression. The regulators of Arg2 transcription in the endothelium have not been characterized. The goal of current study is to determine the role of specific HDACs in the regulation of endothelial Arg2 transcription and thereby endothelial function.

The global HDAC inhibitor, trichostatin (TSA) both time and concentration-dependently increased Arg2 mRNA, protein levels and activity in both HAECs and mouse aortic rings, a process that leads to Arg2-dependent endothelial dysfunction. TSA and atherogenic stimulus enhances activity of common promoter regions of Arg 2. All non-selective Class I HDAC inhibitors (TSA, Scriptaid, varinostat) enhanced Arg2 expression, while only the, the HDCA 1 and 2 selective inhibitor, mocetinostat (MGCD) enhances Arg2 expression. Overexpression of HDAC 2, 3 or 8 in HAECs have no effect on Arg 2 expression while HDAC2 cDNA overexpression concentration-dependently suppresses Arg2 expression. Conversely, siRNA knockdown of HDAC2 enhances Arg2 expression. Additionally like TSA, mouse aortic rings pre-incubated with MGCD resulted in endothelial dysfunction. Finally HDAC inhibition with TSA decreased endothelial NO and increased ROS production in an arginase-inhibitable manner.

In conclusion, HDAC2 is critical regulator of Arg2 expression thereby regulating endothelial NO and ROS production, and consequently endothelial function. Overexpression or activation of HDAC2 thus represents a novel therapy for the prevention and treatment of endothelial dysfunction and atherosclerosis.
Abstract 68: Activation of Brown Adipose Tissue Reduces Development of Atherosclerosis
Authors: Berbée Jimmy , Boon Mariëtte R , Khedoe Padmini P , Bartelt Alexander , Kooijman Sander , Vazirpanah Nadia , Brouwers Linda P , Gordts Philip L , Esko Jeffrey D , Hiemstra Pieter S , Havekes Louis M , Heeren Joerg , Rensen Patrick C
Published: July 2021

Objectives:
Brown adipose tissue (BAT) recently emerged as a novel player in lipoprotein metabolism. BAT combusts high amounts fatty acids into heat resulting in reduced plasma triglyceride (TG) levels as well as obesity. However, the precise role of BAT in cholesterol metabolism and atherosclerosis development remains unclear. We aimed to assess the effect of stimulating BAT activity by ß3-adrenergic receptor (ß3-AR) agonism on lipoprotein metabolism and atherosclerosis development in
APOE*3-Leiden.CETP
mice, a well-established model for human-like lipoprotein metabolism.

Methods and results:
Mice were fed a Western-type diet to induce dyslipidemia and were treated subcutaneously with the selective ß3-AR agonist CL316243 (3x20 μg/week) or vehicle for 10 weeks. ß3-AR agonism reduced total fat mass (-59%) and white adipose tissue (WAT) pad size (-40%). ß3-AR agonism decreased the size of intracellular lipid vacuoles in BAT, and increased UCP-1 expression in WAT, indicating activation of brown adipocytes (
i.e.
BAT) as well as their precursors (
i.e.
WAT). These effects were accompanied by increased uptake of glycerol tri[
3
H]oleate from VLDL-like particles by BAT (+125%) and by WAT (+120%) as well as fatty acid oxidation (+25%) as determined by indirect calorimetry. In line with these data, activation of BAT decreased plasma levels of TG (-46%) and (V)LDL-cholesterol (-24%) and increased HDL-cholesterol (+23%), accompanied by increased reverse cholesterol transport (+2.5-fold). As a consequence of improving lipoprotein metabolism, BAT activation reduced atherosclerosis development (-43%) as well as lesion severity, indicated by more mild type I-III lesions (+64%) and reduced severe type IV-V lesions (-66%), in the aortic root. These effects were dependent on a functional hepatic apoE-LDLr clearance pathway, as BAT activation in

apoe
-/-

and

ldlr
-/-

mice lowered plasma TG levels but did not attenuate hypercholesterolemia and atherosclerosis

Conclusion:
We demonstrate that activation of BAT via the ß3-AR is a powerful tool to reduce dyslipidemia and protect against atherosclerosis. These findings indicate that activation of BAT is a promising novel strategy to combat dyslipidemia and cardiovascular disease.
Abstract 71: AAV-Mediated Expression of Human IDOL in Mouse Liver Increases LDL Cholesterol in a Dose-Dependent Fashion
Authors: Ibrahim Salam , Somanathan Suryanarayan , Billheimer Jeffrey , Wilson James M , Rader Daniel J
Published: July 2021

IDOL (inducible degrader of the low-density lipoprotein receptor, LDLR), originally known as Mylip, is an E3 ubiquitin ligase that promotes the ubiquitination and degradation of the LDLR. Adenoviral overexpression of mouse IDOL was shown to increase LDL cholesterol by decreasing LDLR. Human genetics support a role for genetic variation in IDOL influencing LDL-C levels and support the concept that IDOL inhibition could be a novel strategy to reduce LDL-C levels. In this study we examined the effects of stable expression of human IDOL in mouse liver on LDLR protein and plasma lipids. Using an adeno-associated virus serotype 9 (AAV9)-mediated delivery, we expressed hIDOL (AAV-hIDOL) under the liver-specific TBG promoter to achieve liver-specific expression in adult mice. Five doses of AAV-hIDOL (1x10
10
, 3x10
10
, 1x10
11
, 3x10
11
, 1x10
12
GC) were administered by intra-peritoneal injection and compared to a group injected with AAV-Null as control. AAV-hIDOL produced a dose-dependent increase in hIDOL expression in liver as shown by RT-PCR. Analysis of liver LDLR protein by western blotting revealed a dose-dependent decrease of the LDLR that was completely abolished starting at the dose of 3x10
11
GC. A significant dose-dependent increase in total and LDL cholesterol was observed. At the highest vector dose, LDL-C levels increased more than two-fold. Plasma triglyceride (TG) levels were unchanged through the dose range except at the highest dose where they increased by about 75%. Finally, we used a “humanized” mouse model of heterozygous familial hypercholesterolemia (LDLR
+/-
/Apobec1
-/-
/hApoB-Tg) and expressed hIDOL using AAV-hIDOL at 2x10
11
GC. In this model TC and LDL-C levels were increased by about 60% starting from one week and were sustainable for at least 4 weeks post-injection. In conclusion, our study demonstrates a dose-dependent physiological effect of human IDOL on LDL metabolism in mice and provides a potential model for preclinical testing of IDOL inhibitors for reduction of LDL-C levels.
Abstract 104: NMR Analyses of the Structural and Oligomeric Properties of the C-terminal Transmembrane Domain of SR-BI in Detergent Micelles
Authors: Chadwick Alexandra C , Jensen Davin R , Peterson Francis C , Volkman Brian F , Sahoo Daisy
Published: July 2021

In the reverse cholesterol transport pathway, high density lipoprotein (HDL) carries cholesterol from peripheral tissues to the liver for excretion via bile formation following interaction of HDL with its receptor, scavenger receptor BI (SR-BI). Due to the absence of a high-resolution structure of SR-BI, our understanding of receptor-ligand interactions between SR-BI and HDL that lead to whole body cholesterol removal remain limited. Our laboratory has demonstrated that oligomerization of SR-BI, likely mediated by its C-terminal transmembrane domain (C-TMD), facilitates delivery of HDL-cholesterol into cells. In this study, we describe the use of novel nuclear magnetic resonance (NMR) strategies to determine the structure and dimerization properties of SR-BI(405-475), a peptide that encompasses residues 405-475 and includes a portion of the extracellular domain, as well as the entire C-TMD of SR-BI. [U-15N,13C]-SR-BI(405-475) was bacterially expressed and purified, and alpha-helical secondary structure was identified by circular dichroism. The ability of SR-BI(405-475) to dimerize was confirmed by chemical crosslinking strategies. Next, shifts in the monomer-dimer equilibrium of [U-15N]-SR-BI(405-475) were assessed by 1H-15N-HSQC analysis upon altering the ratio of peptide to detergent micelles. By comparing the appearance or disappearance of peaks in our NMR spectra, we determined that 1 mM SR-BI(405-475) in 5% 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-[1’-rac-glycerol] (LPPG) micelles in water at pH 6.8 yielded a mixture of monomer and dimer, while 100 μM peptide in 5% LPPG micelles shifted the oligomeric status to primarily monomer. Using 1H-15N-13C 3D NMR residue assignments, we also determined that these appearing/disappearing peaks represented the residues in a GXXXG dimerization motif (G420, A421, M422, G423, and G424), as well as several leucine residues in a putative leucine zipper dimerization motif. Together, we have made significant strides towards acquiring the first high-resolution structural information for SR-BI and have obtained convincing evidence of SR-BI dimerization via its C-TMD. These novel findings may ultimately help in designing therapeutics needed to enhance whole body cholesterol removal.
Abstract 13: Development of Functionalized Nanoparticle Platform for Reducing Atherosclerosis
Authors: Ghosh Shobha , Wang Jing , Bie Jinghua , Yuan Quan , Zolotarskaya Olga , Yang Hu
Published: July 2021

No therapy is currently available to enhance the removal of cholesteryl esters (CE) from existing atherosclerotic plaques to facilitate plaque regression. Such a strategy is crucial to reduce the burden of existing disease in addition to preventing the progression targeted by the current therapeutics. Earlier studies from our laboratory have established the anti-atherogenic role of CE hydrolase (CEH)-mediated CE mobilization from macrophage foam cells and final elimination of cholesterol by the liver. While transgenic expression of CEH was used in pre-clinical animal studies, increase in human CEH by activation of Liver-X-receptor (LXR) was also established. Increased lipogenesis induced by LXR ligands precludes their use. The current studies focused on the development of mannose-functionalized dendrimer nanoparticles (DNPs) for the delivery of LXR ligand (TO901317) or CEH expression vector to plaque associated macrophage foam cells. As shown in the Figure, mannose functionalization restricts the uptake of DNPs to macrophages and minimal uptake was seen with primary hepatocytes (
A
). Western diet fed LDLR-/- mice were injected (iv) with DNPs and tissues harvested 48 later to monitor gene expression by QPCR. DNP-mediated delivery of LXR ligand (DNP-LXR) increased the target gene expression (ABCA1, ABCG1) in plaque associated macrophage foam cells in the aortic arch with no effects on target gene expression in the liver (
B
) demonstrating the specific delivery of LXR ligand. Comparable increase in CEH activity was seen following exposure of macrophages to free LXR ligand and DNP-delivered LXR ligand (
C
) and DNP-mediated delivery of CEH expression vectors driven either by CMV or SR-A promoter induced dramatic increase in CEH expression (
D
). These data establish functionalized DNP as a suitable platform for specific and functional delivery of drugs or DNA to plaque associated macrophages to enhance processes involved in cholesterol removal and plaque regression.
Abstract 145: Endothelial Dysfunction: A Key Determinant of Ventricular Dysfunction in End-Stage Renal Disease
Authors: Dubin Ruth , Guajardo Isabella , Mills Claire , Donovan Catherine , Beussink-Nelson Lauren , Ganz Peter , Shah Sanjiv
Published: July 2021

Objectives:
Mechanisms underlying the high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) are poorly understood. We sought to determine whether endothelial dysfunction is associated with left ventricular (LV) and right ventricular (RV) dysfunction in ESRD.

Methods:
Stable patients with ESRD (n=75) underwent measurement of: (1) flow-mediated dilation (FMD), using upper arm brachial occlusion, and (2) cardiac mechanics, using speckle-tracking echocardiography (STE). Microvascular function was measured as the velocity time integral (VTI) of hyperemic blood flow following cuff deflation. Eight participants returned for repeat endothelial testing at 1-week intervals.

Results:
The mean±SD age was 54±11 years, 38% were diabetic, and 17% were on peritoneal dialysis. FMD median (IQR) was 4.7% (2.7-6.9%) and VTI was 0.62m (0.45-0.72m). After adjustment for age, gender, diabetes and systolic blood pressure, lower VTI was associated with worse RV longitudinal free wall strain (β=6.3% per 1m VTI; 95% CI [1.8, 11]; p=0.007). In patients with ejection fraction ≥50%, lower FMD was associated with worse LV global longitudinal strain (β=0.36% per 1% FMD; 95% CI [0.11,0.61]; p=0.005). Mean absolute differences at one week for FMD and VTI were 1.8% and 0.19m.

Conclusions:
In a diverse cohort of patients on hemo- or peritoneal dialysis, worse endothelial function was associated with LV and RV mechanics after adjustment for clinical factors. Repeatability of FMD and VTI were in accordance with current guidelines. Future studies are needed to investigate whether therapies that improve endothelial function could improve cardiac function in ESRD.
Abstract 15: Proteomic Analysis of HDL from Inbred Strains of Mice Implicates APOE in Reduced Cholesterol Efflux Capacity via the ABCA1 Pathway
Authors: Pamir Nathalie , Hutchins Patrick , Ronsein Graziella , Vaisar Tomas , Reardon Catherine , Getz Godfrey , Lusis Aldons J , Heinecke Jay W
Published: July 2021

Background:
Human HDL’s efflux capacity-the ability to promote cholesterol efflux from macrophages-associates strongly and negatively with risk of future cardiac events. However, the molecular factors that regulate efflux capacity remain poorly understood.

Methods and Results:
We investigated the relationships between HDL’s size, protein cargo, and sterol efflux capacity, using HDLs from five inbred mouse strains with different HDL-cholesterol levels and susceptibilities to atherosclerosis. Like human HDL, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. Hierarchical cluster analysis recapitulated the genealogy of the mouse strains, indicating that the HDL proteome is under genetic control. HDL particle size from the different strains correlated strongly and positively with APOA2 content. Analysis of HDL isolated from
Apoa2
-/-
or
Apoa2
transgenic mice confirmed that APOA2 is a major determinant of HDL particle size. Unexpectedly, APOE correlated strongly and negatively with
in vitro
efflux capacity, suggesting that it impairs the efflux capacity of HDL. Moreover, the efflux capacity of HDL isolated from
Apoe
-/-
mice was significantly greater than that of HDL from wild-type mice. This difference resulted from a marked increase in sterol efflux by the ABCA1 pathway when we tested HDL from the mutant strain.

Conclusions:
Our observations demonstrate that APOA2 determines(or correlates with) HDL particle size and that APOE regulates HDL’s efflux capacity by a pathway selectively involving ABCA1. These findings may be clinically relevant, because HDL’s APOE content is a strong predictor of cardiovascular disease risk and because ABCA1 deficiency promotes unregulated sterol accumulation in human macrophages.
Abstract 154: Atherosclerosis-specific CD4 T Cells Use the Chemokine CCL5 and Its Receptor CCR5 to Home to Mature Atherosclerotic Lesions in Mice
Authors: Li Jie , Ley Klaus
Published: July 2021

Introduction:
T cell adaptive immunity is involved in the pathogenesis of atherosclerosis, but the phenotype of T cell subset and chemokine receptor that regulates effector T cell trafficking to atherosclerotic aortas is unknown. By multi-color flow cytometry, we discovered that >40% of T cells in the atherosclerotic aortas express CCR5. We hypothesize that CCR5 plays an important role in T cell recruitment to atherosclerotic aortas and CCR5+ T cells play an important roll in the pathogenesis of atherosclerosis.

Methods:
T cell phenotype in the aorta of
Apoe
-/-
mice on western diet 3-5 months was analyzed by multi-color flow cytometry. T cell homing assay were done
in vitro
by incubating 400,000 T
eff
from
CD45.1Apoe
-/-
mice with explanted

Apoe
-/-

aortas 12 to 18 hrs with media alone, CCL5 neutralizing antibody (0.5 ug/ml) or PTX (300ng/ml), and
in vivo
by adoptively transferring splenocytes from
Apoe
-/-
Ccr5
-/-
and
dsRedApoe
-/-
mice at 1:1 ratio into
CD45.1Apoe
-/-
recipients. 24 hours later, recipient aortas were digested and analyzed by flow cytometry. To visualize T cell proximity to APCs in the aorta by 2 photon imaging, T
eff
from
Apoe
-/-
mice were labeled with SNARF before incubation with explanted aortas from
CD11c-YFP+Apoe
-/-
mice in the above 3 conditions. In vitro T cell suppression assay was done by incubating 10
4
CD4+CD25- T cells with α-CD3 mAb and 5x10
4
irradiated splenic APCs and decreasing amount of Treg or CCR5Teff. Transcriptomic analysis is done following SMARTseq II prortocol from Illumina.

Results:
43±6% of αβ T cells in the atherosclerotic aortas express CCR5, significantly higher than that in aortas of wild-type C57BL/6 mice (10±7%) or
Apoe
-/-
mice on normal diet (7±6%). CCR5 and its ligand CCL5 play the major role in regulating T cell homing to atherosclerotic aortas
in vitro
and
in vivo
. Interestingly, at late stage of disease, the CCR5+ T cells in the atherosclerotic plaques are all FoxP3
+
IFN-γ
+
T-bet
+
CD25
-
CD44
hi
CD62L
lo
(CCR5Teff) and do not suppress T cell proliferation. CCR5Teffs are only found in the aorta and para-aortic lymph nodes of
Apoe
-/-
mice but not in the spleen or other organs. Transcriptomic analysis shows that CCR5Teff is more similar to Teff rather than Tregs.
Abstract 175: Lack of Macrophage GLUT1-Mediated Glucose Metabolism Increases Atherosclerotic Lesion Instability
Authors: Zhao Liyang , Freemerman Alex , Johnson Amy R , Sundaram Sneha , Christensen Taylor , Bennett Brian J , Makowski Liza
Published: July 2021

Macrophages play a key role in the pathogenesis of atherosclerosis. Metabolic programs powered by glucose or lipid enable macrophages to elicit pro- or anti-inflammatory responses, respectively. Although the chronic inflammatory feature of atherosclerosis has been well-established, the role of macrophage substrate metabolism in atherogenesis remains unclear. We previously demonstrated that macrophages with elevated GLUT1-mediated glucose metabolism have an increased inflammatory response. Therefore, we created a novel macrophage GLUT1-deficient murine model by crossing GLUT1 floxed to LysM-Cre mice. Recent work suggests that lack of GLUT1 reduces the pro-inflammatory response and the ability for GLUT1-/- macrophages to polarize to the classically activated state in vitro. Therefore, the objective of this study was to examine how macrophage metabolic reprogramming affects the development of atherosclerosis. We hypothesized that macrophages with restricted glucose metabolism due to lack of glucose transporter GLUT1 will have reduced pro-inflammatory activation during atherogenesis. We transplanted bone marrow from Glut1MΦfl/fl or Glut1MΦ-/- mice into Ldlr-/- mice and fed mice a Western diet for 12 weeks. Glut1MΦfl/fl Ldlr-/- or Glut1MΦ-/- Ldlr-/- chimeric mice did not exhibit significant differences in body weight, body composition, blood pressure, fasting blood glucose, or triacylglycerol and LDL and HDL cholesterol. Digital histology analysis of Oil Red O stained slides indicated that deleting macrophage GLUT1 did not affect total lesion area in aortic root; however, mice with blunted glucose metabolism displayed more and larger necrotic cores. Ongoing studies are investigating apoptosis and phagocytic capacity of macrophages with and without GLUT1 to elucidate the roles of macrophage glucose metabolism on the morphology of atherosclerotic lesion. In summary, we observed that mice lacking macrophage GLUT1 developed increased necrotic cores in lesions of the aorta root relative to mice with wild type macrophage GLUT1 which suggests that maintenance of atherosclerotic lesion stability may be regulated by glucose-dependent mechanisms.
Abstract 18: Adipose Specific Microsomal Triglyceride Transfer Protein Deficient Mice Are Resistant To High Fat Diet Induced Obesity
Authors: Bakillah Ahmed , Hussain M. Mahmood
Published: July 2021

Microsomal triglyceride transfer protein (MTP) transfers neutral lipids and is essential for the assembly of apolipoprotein B (apoB)-containing lipoproteins by the liver and the small intestine. To determine whether MTP plays a role in adipocyte maturation and lipid metabolism, we used 3T3-L1 preadipocytes. MTP expression and activity transiently increased during early stages of differentiation, and declined later. The increases in MTP expression preceded the expression of PPARγ. Overexpression of MTP enhanced lipid droplet formation in 3T3-L1 cells, and its knockdown attenuated cellular lipid accumulation. These studies indicated that MTP positively affects cellular adipocyte differentiation and adipogenesis. Adipose-specific
Mttp
gene ablated (

A-Mttp
-/-

) mice were lean and had reduced white adipose tissue after feeding a high-fat diet (HFD) compared with wild-type (

Mttp
fl/fl

) mice. The adipose tissue of

A-Mttp
-/-

mice had increased number of smaller size adipocytes and had less macrophage infiltration as compared with

Mttp
fl/fl

mice. Further, these mice were protected from HFD-induced fatty liver. The

A-Mttp
-/-

mice had moderate increase in plasma triglyceride, but normal cholesterol, glucose and insulin levels. Gene expression analysis showed that the adipose tissue of the

A-Mttp
-/-

mice had significantly lower mRNA levels of PPARγ and its downstream gene targets FAS and FABP4 and lipins. These data suggest that MTP might modulate adipogenesis by influencing PPARγ expression, and plays a role in the accretion of lipids to form larger lipid droplets. Thus agents that inactivate adipose MTP may be useful anti-obesity drugs.
Abstract 183: Genetic Determinants of CD39 Expression and Platelet Aggregation
Authors: Flynn John M , Haddad Elias V , Cleator John H , Novitskaya Tatiana , Oates John A , Boutaud Olivier G , Gumina Richard J
Published: July 2021

Introduction:
CD39 is an ATP and ADP hydrolyzing ectonucleotidase found on the endothelium, leukocytes and platelets. Prior work has shown that the genotype at the single nucleotide polymorphism rs10748643 correlates with CD39 expression (GG:high, AG:intermediate, AA: low) in lymphocytes. However the influence on CD39 expression in platelets is unresolved.

Objective:
The aim of this study was to determine whether the rs10748643 genotype associates with low-dose ADP-induced platelet aggregation
ex vivo
.

Methods:
Whole blood was obtained from healthy volunteers not on aspirin (baseline) and then treated with aspirin for 14 days (81 mg). Serum thromboxane levels were also measured to demonstrate ASA adherence. Platelet rich plasma was generated and platelet aggregation in response to 2.5 uM ADP was measured. DNA from whole blood was isolated and used for genotyping at rs10748643 by the Vanderbilt DNA Resources Core.

Results:
The study population included 65 subjects.Genotyping revealed 23 GG, 28 AG and 14 AA at rs10748643 subjects. There was no difference in age (Years: AA: 32.4±11.7, AG: 34.2±9.5, GG: 33.5±9.4;
P
=0.72) or sex (Male: AA: 57%, AG: 76%, GG: 60%;
P
=0.34). Stimulation of platelets from untreated subjects with 2.5 uM ADP showed no difference in the percent aggregation (AA: 77.8±7.05%; AG: 75.9±4.85%, GG: 78.7±4.31%;
P
=0.233). Following aspirin treatment there was a significant decrease in thromboxane levels (
P
<0.001 compared to baseline) and an expected significant decrease in the percent aggregation in all genotypes compared to the pre-treatment aggregation (
P
<0.001). Analysis of “on aspirin” platelet aggregation by genotype demonstrated a nonsignificant trend of lower aggregation in AA individuals compared to AG and GG individuals (AA: 46±3.37%; AG: 52.4±3.13, GG: 56.2±3.1;
P
=0.148).

Conclusions:
These data suggests that the rs10748643 genotype does not associate with differences in platelet aggregation at baseline or following aspirin therapy. However, the small size of the cohort examined is a clear limitation of the study. The trend for lower aggregation in AA individuals on aspirin is intriguing and studies in a larger cohort will determine if there is significance of these findings.