bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 61: Histone Deacetylase 9 (HDAC9) Dysregulation Precipitates High-Fat-Diet--Induced Adipose Tissue Dysfunction and Systemic Metabolic Disorders
Authors: Chatterjee Tapan K , Knoll Ellen P , Tong Wilson , Blomkalns Andra L , Basford Joshua E , Hui David Y , Weintraub Neal L
Published: March 2022

During chronic overfeeding, adipose tissue expansion contributes to adipose inflammation, insulin resistance and obesity-related diabetes. Consequently, identifying mechanisms that promote “healthy” growth of adipose tissues could potentially improve obesity-related disease. We previously demonstrated that endogenous HDAC9 negatively regulates adipogenic differentiation in isolated preadipocytes. Here, we investigated the role of HDAC9 in regulating adipose tissue function and glucose intolerance in high fat fed mice. High fat feeding upregulated HDAC9 expression in adipose tissues, isolated preadipocytes and adipocytes, in conjunction with reduced expression of adipocyte differentiation-specific genes C/EBPα, PPARγ, FABP4, and adiponectin. HDAC9 gene knockout completely prevented these effects of high fat feeding on adipocyte differentiation-specific gene expression and adipogenic differentiation in vitro. HDAC9 knockout mice also gained less weight and exhibited less visceral adiposity, despite similar food intake, as compared to wild type mice during high fat feeding. In addition, HDAC9 gene deletion blunted elevations of plasma pro-inflammatory adipokines leptin and resistin, and resulted in improved systemic glucose tolerance, in the setting of high fat diet. Adipocytes were smaller in HDAC9 knockout mice, while ectopic lipid accumulation, as revealed by hepatosteatosis, following high fat feeding was completely prevented by HDAC9 gene knockout. Interestingly, adipose tissues of high fat fed HDAC9 knockout mice accumulated more anti-inflammatory M2 polarized macrophages and Treg cells, consistent with reduced adipose tissue inflammation. These findings suggest that HDAC9 disrupts adipose tissue function in high fat feeding, leading thereby to adipose tissue inflammation and glucose intolerance. Thus, HDAC9 could be a novel therapeutic target in the treatment of disorders associated with diet-induced obesity.
Abstract 64: Serum Amyloid A Augments Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in ApoE-Deficient Mice
Authors: Wroblewski Joanne M , De Beer Maria C , Witta Jassir , Rateri Debra L , Daugherty Alan , De Beer Frederick C , Webb Nancy R
Published: March 2022

Introduction and Objectives:
Abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Proteolytic enzymes, particularly matrix metalloproteinases (MMP’s), are hypothesized to be active participants in AAA pathogenesis. Serum amyloid A (SAA) is an acute phase reactant that, unlike C-reactive protein (CRP), correlates in humans with aortic dimensions before aneurysm formation. SAA induces MMP expression in cultured cells. In this study we determined whether SAA deficiency in mice altered AAA formation in the well-established angiotensin II (Ang II) infusion model.

Methods and Results:
We confirmed that SAA induced MMP-9 and MMP-13 mRNA abundance in J774 macrophage-like cells. Furthermore, we determined for the first time that SAA enhanced MMP-2 activity in cultured mouse abdominal aorta explants. Plasma SAA increased ∼400-fold in apoE
-/-
mice 24 hours after i.p. Ang II injection (100μg/kg) and ∼12-fold after 28-day Ang II infusion (1000ng/kg/hour). SAA was detected by immunostaining in human AAA tissues and in AAAs of AngII-infused apoE
-/-
mice. Based on
in vivo
ultrasound and
ex vivo
morphological analyses, apoE
-/-
mice with targeted deletion of SAA (SAAKO; n = 20) exhibited significantly reduced AAA 28 days after Ang II infusion compared to apoE
-/-
mice that were wild type for SAA (SAAWT; n = 20), despite a modest, but significant increase in the hypertensive response to Ang II. Although the incidence of aortic rupture in SAAKO mice (15%) and SAAWT mice (20%) were similar, the majority of ruptures (75%) in SAAWT mice occurred in the abdominal region, whereas all of the ruptures in the SAAKO mice occurred in the thoracic region. Atherosclerotic lesion area on the luminal surface of the aortic arch was similar in SAAKO and SAAWT mice after Ang II infusion.

Conclusion:
Our finding that SAA plays a role in experimental AAA may be pertinent to human studies correlating indexed aortic diameter with plasma SAA concentrations.
Abstract 77: CXCR7 Induction, Signaling and Phagocytic Function in Macrophages Linked with Atherosclerosis
Authors: Ma Wanshu , Liu Yiwei , Clark Brittany , Martin Bethany , Ellison Nicholas , Riggins Branden , Shen Jianzhong
Published: March 2022

The discovery of CXCR7 as a new receptor for SDF-1 places many previously described SDF-1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or non-signaling “decoy” receptor has been in debate. A recent study demonstrated that CXCR7 is not expressed in normal human or mouse blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed. Here, we show that CXCR7 was detected in macrophage-positive area of aortic atheroma of ApoE-null mice, but not in healthy arteries. Consistent with this, we found that during monocyte-to-macrophage differentiation, CXCR7 was dramatically upregulated at both mRNA and protein levels. In addition, CXCR7 induction was associated with a SDF-1 signaling switch from pro-survival to pro-inflammatory pathways including p38 and JNK, which was mimicked by a CXCR7-selective agonist and inhibited by CXCR7-blocking antibodies, but not by CXCR4-selective antagonist. Furthermore, we found that CXCR7 activation by SDF-1 or its selective agonist TC14012 significantly increased macrophage phagocytosis, which was suppressed by inhibiting either the p38 or JNK pathways. We conclude that CXCR7 is induced during monocyte-to-macrophage differentiation, which mediates signaling to p38 and JNK pathways, leading to enhanced cellular phagocytic function, thus possibly contributing to atherogenesis.
Abstract 82: TGF-β Regulates Matrix Production in Aortic Fibroblasts Through MicroRNA-29b
Authors: Spin Joshua M , Maegdefessel Lars , Azuma Junya , Toh Ryuji , Deng Alicia , Tsao Philip S
Published: March 2022

Abdominal aortic aneurysms (AAA) cause ∼15,000 annual deaths in the United States. The mechanisms of AAA expansion remain incompletely explored. MicroRNAs (miRs) regulate gene expression post-transcriptionally, and play crucial roles in vascular integrity. TGF-β can act as a profibrotic stimulus, is a known regulator of miR-29b expression in some cell types, and may play a protective role in AAA development. Numerous extracellular matrix genes are predicted targets of miR-29b. We have previously shown that miR-29b is decreased in both human AAA, and in aneurysmal aortic segments in murine AAA models. Further inhibition of miR-29b
in vivo
reduces abdominal aortic aneurysm progression in two murine AAA models: the porcine-pancreatic-elastase (PPE) model in C57Bl/6 mice, and the angiotensin II-infusion (AngII) model in ApoE
-/-
mice, and is associated with a pro-fibrotic response within the vessel wall. We performed vascular cell culture studies using human aortic adventitial fibroblasts (AAFs) and aortic smooth muscle cells (ASMCs) to evaluate the expression and regulation of miR-29b. Treatment with recombinant human TGFβ1 significantly decreased miR-29b levels in aortic adventitial fibroblasts, but not smooth muscle cells (vs. untreated cells). TGFβ1 treatment increased soluble collagen production by AAFs nearly 4-fold, and increased gene expression of
COL1A1
and
COL3A1
. In ASMCs, TGFβ1 primarily increased
ELN
expression, and
COL1A1
and
COL3A1
to a lesser extent. We altered miR-29b expression
in vitro
by transfecting AAFs and ASMCs with either an antagomir (anti-29b) to inhibit activity or a pre-miR (pre-29b) to enhance activity (vs. scrambled miR control). Successful transfection (>50% of all cells) was confirmed by visual fluorescent microscopic analysis and FACS for labeled tag. Anti-29b significantly augmented the effects of TGFβ treatment upon soluble collagen protein levels and collagen gene expression in AAFs, while pre-29b inhibited this response nearly to untreated-control levels. A similar gene expression response was seen in ASMCs. These data suggest that TGFβ regulation of miR-29b in aortic fibroblasts leads to a profibrotic response, partly explaining its protective effects in AAA development.
Abstract 84: Group V Secretory Phospholipase A2 Enhances the Progression of Angiotensin II-Induced Abdominal Aortic Aneurysms but Confers Protection Against Angiotensin II-Induced Cardiac Fibrosis in ApoE-Deficient Mice
Authors: Boyanovsky Boris B , Bailey William , Dixon Lauren , Webb Nancy R
Published: March 2022

Objective—
Abdominal aortic aneurysms (AAAs) and heart failure are complex life-threatening diseases whose etiology is not completely understood. We recently reported that deficiency of group X secretory phospholipase A2 (GX sPLA2) reduces abdominal aortic aneurysm (AAA) formation in apoE
-/-
mice infused with angiotensin II. In this study, we investigated whether deficiency of a related enzyme, GV sPLA2, also protects mice from experimental AAA. The impact of GV sPLA2 deficiency on Ang II-induced cardiac fibrosis was also assessed.

Methods and results—
ApoE
-/-
mice and apoE
-/-
mice lacking GV sPLA2 (GV DKO) were infused with 1000 ng/kg/min Ang II for up to 28 days. Increases in systolic blood pressure, urinary prostanoids, and plasma aldosterone were similar in apoE
-/-
and GV DKO mice after Ang II infusion. The incidence of aortic rupture in Ang II-infused GV DKO mice (10%) was significantly lower compared to apoE
-/-
mice (30%). Although the overall incidence of AAA in GV DKO mice (∼81.25%) and apoE
-/-
mice (100%) was similar, the mean percent increase in maximal luminal diameter of abdominal aortas was significantly smaller in GV DKO mice (68.5±7.7%) compared to apoE
-/-
mice (92.6±8.3%). Deficiency of GV sPLA2 resulted in increased Ang II-induced cardiac fibrosis that was most pronounced in perivascular regions. Perivascular collagen, visualized by picrosirius red staining, was associated with increased TUNEL staining and increased immunopositivity for macrophage and myofibroblast cells and NOX-2 and NOX-4.

Conclusion—
Our findings indicate that GV sPLA2 modulates pathological responses to Ang II, with different outcomes for AAA and cardiac fibrosis. These data have important implications for sPLA2 inhibitors that are currently being developed for clinical use.
Abstract 93: Natural CD4+CD25+ Regulatory T Cells Control the Development and Complications of Abdominal Aortic Aneurysm in Mice
Authors: Ait-Oufella Hafid , Wang Yu , Herbin Olivier , Potteaux Stephane , Bourcier Simon , Laurans Ludivine , Esposito Bruno , Tedgui Alain , Mallat Ziad
Published: March 2022

Abdominal aortic aneurysm (AAA) is characterized by a prominent inflammatory cell infiltrate and local destruction of extracellular matrix. Following the acute phase, the inflammatory infiltrate is predominately composed of T lymphocytes and macrophages. We have previously reported that inhibition of Th1 or Th2 cell responses had no effect on the development or the complications of experimental murine AAA. We hypothesized that another subset of T cell, natural Treg cells (Tregs), plays a role in the pathophysiology of AAA.

Male C57Bl/6 mice were infused with Angiotensin II (1μg/kg/mn) for 28 days using osmotic pumps. A control group received non-immune isotype-matched IgG antibody (n=20) and a group received anti-CD25 antibody (n=20) every 10 days that depleted more than 95% of Tregs in lymphoid organs. Treg depletion induced AAA rupture (15% vs 0%, P<0.05) and more severe AAA stages compared to control mice (P<0.01). Histological analysis showed an increase of elastin degradation (mean layer number 2.5 vs 5.6, P=0.01) and an increase of T cell accumulation in the adventitia of Treg-depleted mice (2816 vs 330 μm2, P<0.01).

CD28 or B7 deficiency leads to a profound reduction in Treg cells. We infused Angiotensin II into C57Bl/6 CD28+/+ (n=17), CD28
-/-
mice (n=11) or B7
-/-
mice (n=16). CD28 or B7 deficiency did not alter the pressor response to Angiotensin II. CD28 and B7 deficiency induced an increase of AAA incidence compared to control mice, respectively 68%, 72%, and 10%, P<0.001. Moreover, CD28 and B7 deficiency induced 20% of AAA rupture whereas no rupture was observed in control mice. In another set of experiments, transfer of Tregs (3x106 cells) into CD28
-/-
mice abolished AAA rupture (0% vs 15%, P<0.05) and significantly reduced AAA severity (P<0.01). Moreover, Treg transfer decreased elastin degradation (mean layer number 3.7 vs 2.5, P=0.04) and T cell infiltration in the adventitia (390 vs 2747 μm2, P<0.01).

Conclusion:
We have shown for the first time that natural regulatory T cell protected against AAA development and rupture.
Abstract 94: Predicting Aortic Aneurysm Rupture: A Computational Fluid Dynamics Analysis
Authors: Boyd April J , Kuhn David C , Kulbisky Gordon P , Lozowy Richard J
Published: March 2022

Aortic size is the primary factor used to predict abdominal aortic aneurysm (AAA) rupture potential; however, this method fails to account for AAA that rupture at smaller sizes, or for those that reach extreme sizes without rupture. Currently there is no truly reliable way to evaluate the susceptibility of a particular AAA to rupture. Although computational fluid dynamic (CFD)methods have been used previously to evaluate AAA flow dynamics, these studies have yet to predict the rupture potential for specific AAA anatomy. We hypothesize that the site of maximal pressure and wall shear stress (WSS) within individual AAA will lead to biomechanical wall failure and will predict the site of aortic rupture.

In order to test this hypothesis we used commercially-available CFD software (ANSYS CFX v. 12.1) to solve the governing equations for mass and momentum for CTA-derived 3D images of ruptured AAA (RAAA) (n=5; 4 male, 1 female). Blood flow was considered to be laminar, Newtonian, and steady-state. The simulations were performed on a UNIX platform with 8 platform processors. The solution was considered to be converged when the maximum residuals for all governing equations were below 1.0 x 10
-5
. ANSYS software was used to generate a fully hexahedral mesh. Predicted intra-aortic pressure and WSS profiles were obtained.

The average AAA size at rupture was 8.3 +/- 1.52 cm. Three of the five RAAA ruptured at or near the site of maximal diameter. The maximal predicted intra-aortic pressure was 14.25 +/- 6.29 Pa and generally was localized on the anterior aortic wall. In most cases the site of actual rupture was the lateral wall of the AAA where the pressure was not significantly different from that at the site of maximal pressure (12.12 +/- 6.27 Pa, p >0.05, ns). In the normal aorta, velocity profiles are laminar to the aortic bifurcation, whereas in AAA there is significant vortex flow pattern within the aneurysm. In these RAAA the highest predicted WSS was on the anterior aortic wall and measured 0.184 +/- 0.02 Pa. At the actual site of rupture, WSS was significantly lower at 0.044 +/- 0.0006 Pa (p<0.0001). In all cases the rupture occurred in a low WSS region and was always associated with eddy formation or flow recirculation.

This CFD study was the first to model blood flow in the geometry of actual RAAA. Interestingly, the site of rupture was not in the region of maximal pressure or WSS as predicted. Rupture occurred in all cases in flow recirculation zones where low WSS predominated. This work will provide the basis for future research on a more precise prediction of rupture risk.
Abstract 99: Rosiglitazone Negatively Regulates C-jun N-terminal Kinase and Toll-like Receptor 4 Proinflammatory Signaling During Initiation of Experimental Aortic Aneurysms
Authors: Pirianov Grisha , Torsney Evelyn , Howe Franklyn , Cockerill Gillian
Published: March 2022

Objective:
Development and rupture of aortic aneurysms (AA) is a complex process involving inflammation, cell death, tissue and matrix remodeling. The thiazolidinediones (TZDs) including Rosiglitazone (RGZ) are a family of drugs which act as agonists of the nuclear peroxisome proliferator-activated receptors and have a broad spectrum of effects on a number of biological processes in the cardiovascular system. In our previous study we have demonstrated that RGZ has a marked effect on both aneurysm rupture and development, however, the precise mechanism of this is unknown.

Methods and Results:
In the present study, we examined possible targets of RGZ action in the early stages of Angiotensin II-induced AA in apolipoprotein E-deficient mice. For this purpose we employed immunoblotting, ELISA and antibody array approaches. We found that RGZ significantly inhibited c-Jun N-terminal kinase (JNK) phosphorylation and down-regulated toll-like receptor 4 (TLR4) expression at the site of lesion formation in response to Angiotensin II infusion in the initiation stage (6-72h) of experimental AA development. Importantly, this effect was also associated with a dramatic reduction in production of TLR4/JNK-dependent proinflammatory chemokines MCP-1 and MIP-1α.

Conclusion:
These data suggest that RGZ can modulate inflammatory processes by blocking TLR4/JNK signalling in initiation stages of AA development.
Abstract 102: Profound Exclusion of Apolipoprotein A-II from Selective Hepatic Cholesteryl Ester Uptake
Authors: Gillard Baiba K , Bassett G. R , Gotto Antonio M , Pownall Henry J
Published: March 2022

Reverse cholesterol transport (RCT), the transfer of cholesterol from peripheral tissues, including the subendothelial space of the arterial wall, to the liver for disposal, is a current model of HDL atheroprotection. The final RCT step, selective hepatic HDL-cholesteryl ester (CE) uptake, is mediated by scavenger receptor class B type I (SR-BI). The net receptor reaction of SR-BI vs. HDL is distinct from that of LDL vs. the LDL receptor. LDL holo particle uptake is succeeded by steps that breakdown apo B-100 and hydrolyze and recycle the CE. In contrast, HDL-CE uptake is selective, occurring without a concomitant net uptake of the major HDL protein, apo A-I and even though apo E and apo A-I bind equally well to SR-BI, apoA-I-containing particles mediate 2-fold more selective CE uptake. The reaction of HDL with SR-BI is similar to the activity of a streptococcal serum opacity factor (SOF) against HDL_both reactions selectively remove CE from HDL leaving remnants. In addition, SOF catalyzes the displacement of apo A-I leaving an apo A-II-rich neo HDL, an effect that was assigned to the greater lipophilicity of apo A-II vs. apo A-I. Thus, we tested the hypothesis that the same occurs during the interaction of HDL with SR-BI, i.e., that apo A-II vs. apo A-I is selectively excluded from cellular uptake via SR-BI. Herein, we compare the selective uptake of HDL-CE vs. HDL-apo A-I and apo A-II. Cellular uptake of HDL-[
3
H]CE labeled with [
125
I]apo A-I or [
125
I]apo A-II was compared in CHO-K1 and CHO-ldlA7 cells (LDL-R
-/-
) with and without over expression of mouse SR-BI, and Huh7 human hepatocytes. Cell-associated
125
I and
3
H were determined by γ- and β-counting respectively. Uptake of CE, apo A-I, and apo A-II SR-BI-over expressing CHO cells was 32,800 ± 4800, 9.3 ± 2.7, and 2.5 ± 0.2 nmol/mg cell protein. The corresponding values for Huh7 cells were 9,700 ± 1,800, 15 ± 2.4, and 7.6 ± 0.9 nmol/mg cell protein. Relative to CE, both apo A-I and apo A-II were excluded from uptake by all cells. However, relative to the apo A-I and apo A-II contents of HDL, uptake of apo A-I was twice that of apo A-II, thus supporting the hypothesis that the more lipophilic apo A-II is selectively excluded from cellular uptake via SR-BI and retained in the neo HDL remnant.
Abstract 103: Mass Spectral Analysis of Oxidative Stress Modification of Apolipoprotein E by Acrolein
Authors: Tran Tuyen N , Lee Yuan Yu (Kent) , Uchida Koji , Narayanaswami Vasanthy
Published: March 2022

Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. ApoE mediates this function via essential lysine residues that interact with the LDLr. Preliminary studies from our lab showed that rats exposed to environmental tobacco smoke displayed oxidative modification of apoE by acrolein (a highly reactive unsaturated aldehyde) and dissociation of lipoprotein-bound apoE; however, the details regarding the modification were not determined at the molecular level. The objectives of this project are to identify the specific sites of modification and the effect of modification on the structure and function of recombinant rat apoE. Acrolein modification was verified by Western blot analysis using an acrolein-lysine specific antibody. Matrix Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) analysis identified Lys64, 67, 68, 138 & 234 as likely modification sites by acrolein. Guanidine HCl-induced denaturation studies indicated that the overall fold of the modified protein was significantly altered as revealed by circular dichroism spectroscopy. Modified apoE also demonstrated a decrease in heparin binding affinity and lipid binding ability. The LDLr binding ability of modified apoE was significantly impaired. Loss of LDLr binding ability is attributed to acrolein modification of Lys138, an essential residue located on helix 4, that is directly involved in interaction with the LDLr as seen for human apoE3. Overall, we conclude that oxidative modification by acrolein disrupts the structural and functional integrity of apoE, which is likely to affect its role in maintaining plasma cholesterol homeostasis. Our data provide a molecular basis for the potential role of oxidative stress- (due to environmental factors or aging) mediated modification of apoE in altering lipoprotein metabolism, with direct implications in cardiovascular disease.
Abstract 108: Probucol Rescued Reproduction of LCAT Null Male Mice
Authors: Tsujita Maki , Akita Nobukatsu , Remaley Alan T , Yamazaki Takeshi , Yokoyama Shinji , Michikawa Makoto
Published: March 2022

Low plasma HDL mice, such as lecitin:cholesterol acyltransferase null mice (Lcat-null), Abca1- null mice and ABCA1 inactivator, probucol-fed mice have significantly low cholesterol content in their adrenal glands and ovaries. Cholesterylester content in brain of male LCAT null mice was significantly reduced while in female the content was not change compare to wild type mice. The reproduction of Lcat-null male pups was reduced to 31% of that expected by Mendelian genetics. Gender ratio in reproduction of wild type, ABCA1 null, and ABCG1null mice were 1 to1 in the same mating condition. Supplement of vitamin E in feeding chow increased vitamin E level in plasma and recovered reproduction defect of male LCAT null mice. Thus, plasma HDL is important lipoprotein to carry hydrophobic vitamin to steroidgenetic cells. Furthermore, although 0.2% probucol-fed mice reduced its plasma HDL only to 5% within 2 weeks, probucol-fed mice reproduced normally. This result suggesting some beneficial function may occur in this chow treatment. So that, we fed 0.2% probucol containing chow to LCAT (+/-) mice during their mating and found recovery of gender ratio in the weaned mice. HMGCoA reductase expression was increased in adrenal gland of probucol fed mice indicate cholesterol de novo synthesis may increased to support cholesterol requirement for steroid synthesis during reproduction. Probucol incorporated HDL (1.00 ug probucol / ug HDL-protein) derived probucol into NCI-H295 human adrenal cortex cell (0.73±0.12 ug probucol / mg cellular protein) and the expression of HMGCoA reductase was significantly increased compared to βactin housekeeping gene expression. Both FC and CE contents in the brain of probucol fed mice were significantly increased. Effect of probucol on cholesterol homeostasis in steroidgenic cells and neuronal cells will be examined in the future study.
Abstract 119: LCAT Activation in Combination with the CETP Inhibitor Anacetrapib Results in Enhanced Cholesterol Efflux and a Favorable Lipid Profile in Dyslipidemic Hamsters
Authors: Peier Andrea , Wang Sheng-ping , Krsmanovic Miha , Mendoza Vivienne , Shah Vinit , Castro-Perez Jose , Roddy Thomas P , Akinsanya Karen , Johns Doug
Published: March 2022

Lecithin cholesterol acyltransferase (LCAT) is a key enzyme involved in the metabolism of HDL. LCAT synthesizes cholesterol esters (CE) in plasma by catalyzing the transfer of the sn-2 fatty acid of phosphatidylcholine to cholesterol on HDL. The hydrophobic CE moves to the core of the HDL particle which results in HDL maturation and a progressive enlargement of the particle. Cholesteryl ester transfer protein (CETP) is a plasma protein that facilitates the transport of CE from HDL to ApoB-containing lipoproteins. CETP inhibition has been identified as a potential strategy for lowering LDL-c and raising HDL cholesterol levels for the treatment of CVD.

The purpose of the current study was to test the hypothesis that CETP inhibition in the context of LCAT activation might yield an unfavorable lipoprotein profile (e.g. dysfunctional HDL). We measured plasma lipoprotein (via FPLC), particle size and
ex vivo
cholesterol efflux from dyslipidemic hamsters chronically treated with a small molecule LCAT activator (20 and 60 mpk/day) in the presence or absence of the CETP inhibitor anacetrapib (20mpk/day). As expected, anacetrapib significantly reduced LDL-c (-45%) and increased HDL-c (+63%) after chronic treatment. LCAT activation reduced TG (-35%) and increased HDL-c (+40-68%) but had no effect on LDL-c. Both treatments led to an apparent increase in HDL particle size as measured by FPLC. Additionally, LCAT activation showed a significant reduction in ABCA1-mediated efflux, presumably due to the resulting increase in HDL particle size. Animals that received both anacetrapib and the LCAT activator maintained the favorable lipid profile observed with single treatment. Moreover, HDL from hamsters treated with both LCATa and CETPi demonstrated a significant increase in ABCG1- and SRB1-mediated efflux whereas single treatment did not exhibit a measurable effect. We provide evidence that LCAT activation in the context of a CETP inhibitor, maintains a favorable lipid profile (lower LDL-c) and appear s to have a synergistic effect on cholesterol efflux.
Abstract 120: Effects of Anacetrapib Treatment on CETP Metabolism
Authors: Reyes-Soffer Gissette , Millar John S , Lassman Michael E , Jumes Patricia , Wagner John A , Gutstein David E , Ramakrishnan Rajasekhar , Holleran Stephen , Rader Daniel J
Published: March 2022

Background

Cholesteryl ester transfer protein (CETP) is required for the exchange of core lipids, cholesteryl esters and triglycerides (TG), between lipoproteins. This results in net exchanges of TG in very-low-density (VLDL) or low-density lipoproteins (LDL) for cholesteryl esters in high-density lipoproteins (HDL). Treatment with anacetrapib, a CETP inhibitor in phase 3 development, is associated with an increased HDL-C and apo AI, and reduced both LDL-C and triglycerides. There are no published data on the regulation of CETP levels in humans. CETP mass has been reported to increase following CETP inhibition which prompted us to examine CETP kinetics. We developed a novel method using stable isotopes and LC-MS analysis to study the effects of anacetrapib on CETP turnover.

Methods

Thirty-nine moderately hyperlipidemic participants were enrolled in a fixed-sequence study: 75% (N=29) were on atorvastatin (20mg/day) plus placebo for four weeks followed by atorvastatin plus anacetrapib (100 mg/day) for 8 weeks (S-ANA). Twenty-Five percent (N=10) of participants received double placebo for four weeks followed by placebo plus anacetrapib for 8 weeks (P-ANA). At the end of each period, we measured CETP mass, and kinetic studies were performed using D3 leucine to determine the fractional clearance and production rates of CETP.

Results

Eight weeks of anacetrapib treatment was associated with 129% increase in CETP plasma levels and an 18% decrease in CETP activity (RFU/sec) in both groups. The increase in CETP mass was associated with a significant reduction in the fractional clearance of CETP from plasma (0.48 vs. 0.23 pools/day) p <.001. The decrease in fractional clearance of CETP was similar in Panel A and Panel B (Panel A 57%, Panel B 55%). There were no significant changes in the production rate of CETP.

Conclusions

Using a novel method, we demonstrated significant effects of CETP inhibition with anacetrapib on the metabolism of CETP in plasma.
Abstract 124: Increased Selective Clearance of Cholesteryl-Ester in Patients with Rheumatoid Arthritis is Reversed by Treatment with Tofacitinib
Authors: Turner Scott M , Beysen Carine , Charles-Schoemann Christina , Fleischmann Roy , Davignon Jean , Schwartz Howard , Milad Mark , Luo Zhen , Bradley John , Kaplan Irina , Riese Richard , Zuckerman Andrea , Hellerstein Marc K
Published: March 2022

Inflammation is associated with dysfunctional HDL. Rheumatoid arthritis (RA) is a chronic inflammatory disease with significantly elevated cardiovascular risk in spite of low LDL and HDL cholesterol (C) levels. Using stable isotope methods, we examined underlying metabolic mechanisms for the reduced cholesterol levels in active RA and for the changes with tofacitinib (tofa) treatment.

Apolipoprotein and cholesterol kinetics were assessed in 36 RA patients at baseline and following 6 weeks of tofa treatment compared to baseline values in 33 non-RA controls matched for age, gender, and menopausal status. All subjects received constant IV infusions of [3,4]
13
C
2
cholesterol and U-
13
C
6
leucine with collection of fasting blood samples hourly for 22 hours. Whole-body cholesterol efflux and cholesteryl ester (CE) and apolipoprotein production and fractional catabolic rates (FCR) were determined by modeling isotope enrichments.

At baseline, total-C, HDL-C, LDL-C, CE and apoA-I concentrations were lower and CE FCR was elevated in RA. No differences were seen in CE production rate, whole body cholesterol efflux or HDL-apoA-I and LDL-apoB production and FCRs. RA patients had elevated serum HDL-SAA, myeloperoxidase and reduced
in vitro
LCAT activity and mass. Following 6 weeks of tofa therapy, cholesterol and apoAI approached levels of controls and apoB concentrations increased. Tofa treatment reduced CE FCR, increased HDL-apoA-I production rate with no changes in LDL-apoB production rates or LDL-apoB and HDL-apoA-I FCRs. Increased CE FCR in the absence of changes in HDL-apoA-I or LDL-apoB particle clearance suggest increased selective CE uptake, presumably by SR-B1, which was normalized by tofa. Change in CE FCR with treatment was negatively correlated with changes in HDL size and concentration of large HDL particles, but not with LDL size or composition. Change in CE production rate with treatment was positively correlated with change in LCAT mass.

This is the first study to evaluate the effect of chronic inflammation on
in vivo
cholesterol and lipoprotein kinetics in humans. Inflammation increases selective CE uptake explaining the reduced cholesterol levels in RA. Reducing inflammation with tofa restored selective CE uptake to normal rates.
Abstract 132: Simulated Intraplaque Hemorrhage Stimulates Plaque Progression in an Animal Model
Authors: Chiu Stephanie E , Zhan James Q , Moody Alan R
Published: March 2022

Intraplaque hemorrhage (IPH) is a feature of advanced plaques and a risk factor for clinical events. Several studies have demonstrated a link between carotid IPH and subsequent cerebrovascular events. Deposited RBCs contribute ingredients likely to promote plaque instability.

In this study, a catheter-based approach was used for intramural injection of RBCs into atherosclerotic plaques. We explored the hypothesis that an animal model of atherosclerosis demonstrates increased macrophage infiltration and neovascularization in plaques injected with RBCs.

Rabbits (n=4) were administered high cholesterol diet starting 2 weeks prior to endothelial denudation. Five weeks after denudation, a microinfusion catheter with a balloon-actuated microneedle delivered 70-100 μl of RBC and iodinated contrast mixture at multiple sites along the aorta. X-ray fluoroscopy and CT identified plaque injection sites and aortas were harvested for histopathology 5 weeks later.

Only sections from plaque injection sites (n=14) were positive for Perl’s iron stain. In the 3 aortas analyzed with immunohistochemistry (1 excluded due to plaque variability), plaque macrophage area proportion was significantly higher in plaque injection sites vs. non-injected vessel sections. Increase in plaque neovessel density was not significant. In conclusion, intraplaque deposition of RBCs in the atherosclerotic rabbit aorta increases measures of plaque instability.

Micrographs show Perl’s iron (a), macrophage (b), and neovessel (c) staining in an RBC-injected plaque. Scale bars represent 250 μm. Mean macrophage proportion and microvessel density and associated SEM are shown in bar graphs.
Abstract 137: Reversal of Endothelial Dysfunction Using Polyphenol Rich Foods and Supplements Coupled with Avoidance of Major Dietary Lectins
Authors: Gundry Steven R , Epstein Jean
Published: March 2022

Endothelial dysfunction (ED) is highly correlated with the development of coronary artery and vascular disease in general. While dietary factors have been previously implicated in the causation of endothelial dysfunction, a purposeful change in dietary habits, coupled with the addition of several polyphenol-containing supplements, has not been studied for their effects on endothelial function, as measured by Peripheral Arterial Tonometry (PAT).

200 consecutive pts, aged 51-86, M:F ratio 3/2, with known vascular risk factors of HTN, DM, Hypercholesterolemia, hx of MI, Stent, CABG, were enrolled in a dietary program, which emphasizes large amts of leafy green vegetables, olive oil, radical reduction of grain, legumes, nightshades, and fruits; and generous amts of grassfed animal proteins, emphasizing Shellfish and avoiding commercial poultry (Diet Evolution). All pts were instructed to take 2-4,000 mg of high DHA fish oil, 200mg of Grape Seed Extract, and 50 mg of Pycnogenol per day. All pts had Endothelial Reactivity (ER) using PAT before and after a 5-minute arm occlusion using the EndoPAT 2000 (Itamar, Israel) at baseline and at 6 months.

Baseline Endothelial Reactivity was 1.88+/-0.7 (range 1.0-3.3), with 145/200 pts (72%)having endothelial dysfunction (less than 1.60). At 6 months, ER increased to 2.25+/-0.5 (range 1.2-3.6) (p<0.01). Only 40/200 (20%) remained with ED, but all had increased ER numbers. Ten pts stopped the polyphenols after a normal PAT; all developed ED on repeat PAT.

We conclude that a limited grain, legume, nightshade, and fruit diet with emphasis on greens, olive oil and grassfed animal proteins, particularly Shellfish, with avoidance of commercial poultry, with supplemental fish oil and polyphenols dramatically improves endothelial function in a group of high risk patients.
Abstract 14: Small Molecule Inhibitors of the CD40-TRAF6 Interaction Reduce Atherosclerosis by Inducing Hypo-inflammatory Myeloid Cells
Authors: Seijkens Tom , Zarzycka Barbara , Soehnlein Oliver , Hoeksema Marten A. , Beckers Linda , Smeets Esther , Meiler Svenja U. , Gijbels Marion J. , Grommes Jochen , Schrijver Roy , Boon Louis , Hackeng Tilman M. , Vriend Gert , Nabuurs Sander B. , Gerdes Norbert , de Winther Menno P.J. , Weber Christian , Nicolaes Gerry A.F. , Lutgens Esther
Published: March 2022

The co-stimulatory CD40-CD40L dyad is well known for its pro-inflammatory role in atherosclerosis. Inhibition of CD40(L) in hyperlipidemic mice drastically reduces atherosclerosis. However, long term inhibition of CD40(L) results in immunosuppression and/or thromboembolic events. Therefore more targeted inhibition of the CD40L-CD40 dyad is required. To elicit intracellular signalling upon activation, CD40 needs to recruit adaptor proteins: the tumour necrosis factor receptor-associated factors (TRAFs). Using mice with a mutation in the CD40-TRAF binding site, we previously showed that CD40-TRAF6, and not CD40-TRAF2/3/5, are pivotal in atherosclerosis.

To discover small drug-like molecules that inhibit the CD40-TRAF6 interaction an
in silico
approach of virtual ligand screening was used; this resulted in the identification of 40.000 compounds. Analysis of the top 800 compounds identified 7 compounds that dose-dependently reduced NFκB activation and IL1β and IL6 expression in RAW cells and CD40-stimulated bone marrow-derived macrophages, respectively. Surface plasmon resonance experiments confirmed direct binding of the compounds to the TRAF6 C-domain.

To analyze the effects of the top two compounds on atherosclerosis, Apoe
-/-
mice were daily treated with compounds 6877002 or 6860766 for 6 weeks. Compound treatment reduced atherosclerosis in the aortic arch by 47.1% (6877002) and 66.8% (6860766). The number of plaque monocytes/macrophages was decreased by 41.4% (6877002) and 53.0% (6860766), and granulocytes by 62.5% (6877002) and 41.5% (6860766). Intravital microscopy showed that the compounds reduced monocyte recruitment to the endothelium by 40.1% (6877002) and 51.2% (6860766), neutrophil adhesion was reduced by 40.2% (6877002) and 51.2% (6860766). In accordance, expression of CCL2-CCR2 and CCL5-CCR5 was remarkably reduced in compound treated macrophages. Moreover, compound treatment abolished CD40-induced expression of TNFα, IL1β, IL6, IL10, and IL12.

These results establish the importance and possibilities of long-term therapeutic inhibition of CD40-TRAF6 interactions in atherosclerosis and other inflammatory diseases and harbor the potential to overcome the current limitations of CD40(L) blocking therapies.
Abstract 146: Endothelin-1 Inoculation Attenuates Aortic Atherosclerosis in Rabbits under Cholesterol Rich Diet
Authors: Pineda Mauricio , Pineda Andres M , Maldonado Luz A , Uribe Ana M
Published: March 2022

Introduction

The atherogenic potential of Endothelin-1 (ET1) has been extensively recognized and studied as a possible therapeutic target. However, the effects of an immunologic blockade of ET1 are unknown.

Hypothesis

Our hypothesis was that the induction of an immunological response through subcutaneous inoculation of Endothelin-1 prevents atherosclerosis in rabbits exposed to a cholesterol rich diet (CRD) for 36 weeks.

Methods

Eighteen New Zealand rabbits were divided in 4 different groups. Group A: 4 rabbits received CRD only. Group B: 5 rabbits were pre-treated with subcutaneous inoculation of ET1 (20 mg) plus Freund’s adjuvant (administered in 3 doses, 15 days apart) and then received CRD. Group C: 5 rabbits were pre-treated with subcutaneous inoculation of ET1 (20 mg) plus Montanide ISA 16 adjuvant (same schedule) and then received CRD; and group D: 4 rabbits received normal diet and no ET1 injection (control group). After 16 weeks, one rabbit in each group (A, B and C) and 2 rabbits in the control group were sacrificed. The rest of the animals were sacrificed after completing 36 weeks. An aortic extraction was done and pathologic slices prepared for hystomorphologic measurements, including vessel, lumen, and plaque area. Plaque/vessel area, plaque/lumen area, and plaque/lumen perimeter ratios, were also calculated.

Results

Compared with rabbits that received CRD alone, rabbits that were pre-treated with subcutaneous inoculation of ET1 plus Freund’s adjuvant had an statistically significant difference in the plaque area (0.9 ± 0.9 vs. 0.2 ± 0.3 mm2, p=0.03), plaque perimeter (2.4 ± 2.0 vs. 0.8 ± 1.1 mm2, p=0.04), plaque width (0.4 ± 0.3 vs. 0.2 ± 0.2 mm2, p=0.02), and calculated ratios (p<0.04) at 16 weeks. The difference was more significant after 36 weeks, with plaque area (1.3 ± 1.1 vs. 0.27 ± 0.5 mm2, p<0.001), plaque perimeter (4.6 ± 2.4 vs. 1.4 ± 1.5 mm2, p<0.001), plaque width (0.4 ± 0.2 vs. 0.2 ± 0.1 mm2, p<0.001), and calculated ratios (p<0.014). This gruop of rabbits also had the highest anti-ET1 antibody titers (1/12800).

Conclusions

Our findings suggest that the pre-treatment with subcutaneous inoculation of Endothelin-1 significantly attenuates aortic atherosclerosis in New Zealand rabbits exposed to a cholesterol rich diet for 16 to 36 weeks.
Abstract 150: Carotid Artery Atherosclerosis: Biomarkers of Plaque Instability
Authors: Simon Bartholomew V , Roztocil Elisa , Kumar Neil G , Caliste Xzabia , Doyle Adam J , Bandyopadhyay Gautam , Miller-Graziano Carol , Gillespie David L , Cullen John P
Published: March 2022

Objective

To identify intraplaque and plasma biomarkers of carotid atherosclerotic plaque instability. We hypothesized that comparison of plaques and plasma from symptomatic and asymptomatic patients would show differences in proteolytic and inflammatory proteins, identifying them as potential markers of an unstable plaque.

Methods

42 symptomatic and asymptomatic patients (22 women, 20 men, 52-89 years) presenting for carotid endarterectomy had blood samples drawn and carotid plaques processed for protein extraction. The intraplaque expression of proteolytic enzymes (uPA, tPA, PAI-1 and MMP-2) and inflammatory markers (TF, TSP-1, and TNF-α) were evaluated by Western blot analysis. Plasma analyte concentrations were assayed with LUMINEX CVD1 and Cytokine panels using LUMINEX technology.

Results

Intraplaque expression of tPA and uPA was 5.9x and 4.5x higher (n=15, p< .05) respectively in the symptomatic group when compared to the asymptomatic group. The symptomatic group also demonstrated a 4.3x increase in active MMP-2 and a 3.6x increase in PAI-1 expression. Investigation of inflammatory proteins demonstrated 2.7-, 4.7-, and 3.3-fold increases in the expression of TNF- α, TSP-1, and TF, respectively. Plasma levels of MPO, MMP-9, IL8, MCP-1 and TNFα were significantly elevated in both the symptomatic and asymptomatic groups when compared to healthy controls (n=7, p<0.05). While there was no change in ICAM-1 levels between asymptomatic patients and healthy controls, symptomatic patients demonstrated a 35% decrease (n=7, p<0.05) when compared to controls. Also, plasma PAI-1 levels in asymptomatic patients were 55% lower than in symptomatic patients and 75% lower than in healthy controls.

Conclusion

Elevated expression of certain inflammatory markers and proteolytic enzymes in carotid plaques was demonstrated in symptomatic patients. Marked differences were also observed in ICAM-1 and PAI-1 expression in the plasma of symptomatic patients relative to asymptomatic patients and healthy controls. This leaves promising avenues for further inquiry into the roles of these proteins in atherosclerotic disease and their potential as clinical biomarkers of an unstable atherosclerotic plaque.
Abstract 155: Contribution of Foamy Monocytes to Nascent Atherosclerosis
Authors: Xu Lu , Foster Greg , Mukherjee Aparna , Perrard Xiaoyuan Dai , Perrard Jerry , Simon Scott I , Ballantyne Christie M , Wu Huaizhu
Published: March 2022

Infiltration of monocytes into the arterial wall and subsequent differentiation into macrophages, which take up lipoproteins to become foam cells, is a key step in atherogenesis. We reported that apoE
-/-
mice on high fat diet (HFD) for 12 weeks had foamy monocytes, characterized as intracellular lipid droplets, in blood. However, atherosclerosis starts early in apoE
-/-
mice after HFD, and it is unknown when foamy monocytes first appear in blood of apoE-/-mice after HFD and whether foamy monocytes contribute to nascent atherosclerosis. In the current study, apoE
-/-
and wild-type (WT) mice were fed HFD or normal diet (ND) for 1-5 weeks. Monocyte numbers increased in apoE
-/-
mice from 1 week on HFD. Importantly, foamy monocytes began appearing in blood of apoE
-/-
mice within 1 week on HFD. The proportion of foamy to total monocytes increased from ~20% at 1 week to ~40% at 5 weeks on HFD. The majority (~85%) of foamy monocytes were CD11c
+
while the majority of nonfoamy monocytes were CD11c
-
in apoE
-/-
mice on HFD. Foamy monocytes also expressed higher levels of TNF-α and scavenger receptors including CD36 and CD204 than nonfoamy monocytes. Cholesteryl-ester-rich VLDL were isolated from apoE
-/-
mice on HFD, labeled with DiI and intravenously injected into apoE-/- mice on ND (recipients). At 3 hours post injection, both CD11c
+
and CD11c
-
monocytes in recipients took up VLDL, becoming DiI
+
. At 24 hours post injection, ~95% of the DiI
+
monocytes became CD11c
+
, suggesting that VLDL uptake, which would cause foamy monocyte formation, induced monocyte phenotypic change to CD11c
+
. Along with the early appearance of CD11c
+
foamy monocytes, atherosclerosis started early in apoE
-/-
mice on HFD. Proportions of CD11c
+
/CD11b
+
cells examined by flow cytometry and mRNA levels of CD11c and inflammatory markers such as IL-6 and IL-1β increased in aorta of apoE
-/-
mice within 3 weeks on HFD compared to those of apoE
-/-
mice on ND or of WT on ND or HFD. In vitro assay revealed that foamy monocytes adhered more avidly to VCAM-1 under fluid shear stress. In summary, appearance of foamy monocytes and aortic atherosclerotic lesions began early in apoE
-/-
mice fed HFD. Foamy monocytes may contribute to nascent atherosclerosis via infiltration into arterial wall and production of inflammatory markers.
Abstract 161: Alternate Day Fasting With a High Fat and Low Fat Diet: Is Brachial Artery Flow Mediated Dilation Mediated by Adipokine Physiology?
Authors: Klempel Monica C , Kroeger Cynthia M , Phillips Shane A , Varady Krista A
Published: March 2022

Background

Calorie restriction (CR) with variable macronutrient distributions has yielded inconsistent effects on brachial artery flow mediated dilation (FMD) related to weight loss and adipocyte physiology. Alternate day fasting (ADF) is a modified form of CR that has already elicited favorable changes in body weight and adipokine levels in both high fat (ADF-HF) and low fat (ADF-LF) background diets. However, the degree to which adipokines mediate vascular biology mechanisms on an ADF regimen is still unclear. This study examined if beneficial changes in body weight and adipokines influences endothelial function in ADF-HF and ADF-LF diets.

Methods

Thirty-two obese subjects were randomized to an ADF-HF (45% fat) or ADF-LF diet (25% fat), which consisted of two phases: 1) a 2-week baseline weight maintenance period, and 2) an 8-week ADF weight loss period. All food was provided to subjects.

Results

Body weight was reduced (P < 0.0001) by ADF-HF (5 ± 1%) and by ADF-LF (4 ± 1%). Fat mass decreased (P < 0.0001) by ADF-HF (5 ± 1 kg) and ADF-LF (4 ± 1 kg). Fat free mass remained unchanged. Waist circumference decreased (P < 0.001) by ADF-HF (7 ± 1 cm) and ADF-LF (7 ± 1 cm). FMD decreased (P < 0.05) in ADF-HF (2 ± 2%) and increased (P < 0.05) in ADF-LF (2 ± 2%). Adiponectin increased (P < 0.05) in ADF-HF (43 ± 7%) and in ADF-LF (51 ± 7%). Leptin decreased (P < 0.05) in ADF-HF (32 ± 5%) and in ADF-LF (30 ± 3%). Resistin decreased (P < 0.05) in ADF-HF (23 ± 5%) and ADF-LF (27 ± 4%). Increases in adiponectin were associated with augmented FMD post-treatment in the ADF-LF group only (r = 0.34, P = 0.03). Leptin and resistin were not correlated with changes in FMD.

Conclusion

Thus, an ADF-LF diet produces more favorable increases in FMD which are correlated with increased adiponectin as compared to the ADF-HF diet. These findings are important in terms of developing future behavioral modifications in energy restriction and macronutrient distribution that will improve vascular health.