Inhibition of VEGF receptor signaling attenuates intraplaque angiogenesis and plaque destabilization in a mouse model of advanced atherosclerosis

Abstracts / Atherosclerosis 263 (2017) e29ee110

Methods: Mice expressing a6b1-binding-deficient CCN1, Ccn1dm/dm, were resistant against the neointimal hyperplasia induced by carotid-artery-ligation. Results: We found that endothelial apoptosis was increased in the ligated arteries by TUNEL assay and proliferative endothelial cells (Ki67-positive) were increased as well in the wild-type mice. Endothelial activation reflected by the misregulated apoptosis and proliferation was repressed in the Ccn1dm/dm mice. Furthermore, VCAM-1, ET-1, vWF, and MCP-1 were upregulated, suggesting a dysfunctional endothelium, in the ligated wildtype arteries, however were not affected in the Ccn1dm/dm mice. Because integrin a6b1 is essential for CCN1 action, we examined the expression of the integrins. We found that a6b1was undetectable in the unligated arteries and was only induced after ligation in the endothelium. The induction of a6b1 by ligation was not observed in the Ccn1dm/dm mice. Conclusions: CCN1 is a critical pathophysiological regulator mediating the endothelial dysfunction induced by disturbed flow. The atherogenic activity of CCN1 is dependent on its receptor integrin a6b1, which is CCN1dependently upregulated by disturbed flow. CCN1 is a novel therapeutic target to restore endothelial function and prevent atherosclerosis.

SAG013. CHANGES OF MEMBRANE AND SOLUBLE ENDOGLIN LEVELS DURING EARLY DEVELOPMENT OF ENDOTHELIAL DYSFUNCTION IN MICE Barbora Vitverova1, Katerina Blazickova1, Jana Rathouska1, Ivana Nemeckova1, Matej Vicen1, Stefan Chlopicki2, Petr Nachtigal1. 1 Faculty of Pharmacy, Department of Biological and Medical Sciences, Charles University, Hradec Kralove, Czech republic; 2 Jagiellonian Centre for Experimental Therapeutics, Krakow, Poland Aim: Endothelial dysfunction (ED) is considered as the first and key step in the development of atherosclerosis. Our previous studies suggested potential role of endoglin in endothelial function and dysfunction. Aortic endoglin was suggested to regulate eNOS expression and soluble endoglin (sEng) was demonstrated to be cleaved from membrane endoglin in various cardiovascular pathologies. The aim of this study was to evaluate the changes in membrane endoglin expression in aorta and sEng levels in blood during early development of endothelial dysfunction in mice. Methods: Two-month-old female mice double knockout ApoE/LDLR- /mice and age-matched female mice C57BL/6J (control mice) were fed chow diet for 2 months. Western blot analysis of aorta and Luminex analysis of inflammatory and oxidative stress markers in blood were performed. Results: The membrane aortic expression of endoglin was significantly reduced in ApoE/LDLR- /- group as compared to control group. The same reduced expression was also demonstrated for p-eNOS (active form of eNOS) mediating NO-dependent vasodilation and pSmad2/3, which was shown to regulate eNOS expression. In addition, levels of sEng in blood were higher in ApoE/LDLR- /- group together with levels of soluble P-selectin. Conclusions: Our results suggest that early development of endothelial dysfunction is accompanied by reduced expression of aortic endoglin (affecting also p-eNOS expression) and increased levels of sEng. Prospective studies are now focused on the potential impact of reduced endoglin expression on the development of endothelial dysfunction and the mechanism of sEng cleavage from aorta.

SAG014. ROLE OF MICROPARTICLES AS MICRORNA MESSENGERS IN REESTABLISHING OF ATHEROSCLEROSIS-ASSOCIATED ENDOTHELIAL PROGENITOR CELL DYSFUNCTION

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origins on EPC dysfunctionality in atherosclerosis and involved mechanisms. Methods: late EPCs were obtained and expanded in culture from peripheral blood isolated from two animal groups: hypertensive-hyperlipidemic (HH) and control (C) hamsters. In parallel experiments, late EPC cultures from HH were incubated with MPs from C group. Results: late EPCs presented endothelial cell phenotype: (1) have the ability to uptake DiI-ac-LDL and UEA-1; (2) express CD34, CD133, KDR, CD144, vWF, Tie-2. Late EPCs from HH exhibited different morphological and functional characteristics compared to control: (1) are smaller and irregular in shape; (2) present decreased endothelial surface markers expression; (3) display reduced proliferation, migration and adhesion; (4) lose the ability to organize themselves into tubular structures and integrate into vascular network; (5) have diminished function of inward rectifier potassium channels. The incubation of late EPC with MPs improved the EPC functionality by transfer of miR-10a, miR-21, miR-126, miR-146a, miR-223 and IGF-1 expression activation. Conclusions: The data reveal that late EPCs from HH exhibit distinctive features and are dysfunctional, and their recovery can be supported by MP ability to transfer miRNAs. These findings bring a new light on the MP role in the cell-cell communication and EPC-mediated endothelial vascular repair. This work was supported by grants of the Romanian National Authority for Scientific Research, CNCS e UEFISCDI: project numbers PN-II-RU-TE-20144-0523 and PN-II-RU-TE-2014-4-0525.

SAG015. VEGF-A REGULATES SUBCELLULAR LOCALIZATION OF SCAVENGER RECEPTOR BI AND TRANSCYTOSIS OF HIGH DENSITY LIPOPROTEINS BUT NOT LOW DENSITY LIPOPROTEINS IN AORTIC ENDOTHELIAL CELLS Srividya Velagapudi, Antonio Piemontese, Lucia Rohrer, Arnold von Eckardstein. Institute of Clinical Chemistry, Schlieren, Switzerland Aim: Low and high density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and anti-atherogenic activities, respectively, within the vascular wall. However, the rate limiting factors that mediate transendothelial transport of lipoproteins are little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. Methods: High-throughput screening was performed, incubating human aortic endothelial cells (HAECs) with 141 kinase inhibiting drugs and Atto594 fluorescent labelled HDL and LDL. Biochemical assays were performed to verify screening hits using siRNA transfected or drug treated HAECs by incubating at 4
C (binding) or 37
C (internalization/ transport) with 125IHDL/LDL for 1h. Results: In the screen, inhibitors of VEGF receptors significantly decreased the uptake of HDL but not LDL. Silencing of VEGFR2 decreased binding/ association, and transport of 125I-HDL but not 125I-LDL. Binding/uptake and transport of HDL but not LDL were reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding/uptake and transport of HDL was abrogated by pharmacological inhibition of PI3K/Akt or p38MAPK as well as silencing of scavenger receptor BI (SR-BI). Moreover, the presence of VEGF-A was found to be a pre-requisite for localization of SR-BI in the plasma membrane of HAECs. Conclusions: The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and druggable barrier for the entry of HDL into vascular wall.

Nicoleta Alexandru1, Emanuel Dragan1, Violeta Ristoiu2, Loredan Niculescu1, Adriana Georgescu1. 1 Institute of Cellular Biology and Pathology - Nicolae Simionescu of Romanian Academy, Bucharest, Romania; 2 Faculty of Biology, University of Bucharest, Bucharest, Romania

SAG016. INHIBITION OF VEGF RECEPTOR SIGNALING ATTENUATES INTRAPLAQUE ANGIOGENESIS AND PLAQUE DESTABILIZATION IN A MOUSE MODEL OF ADVANCED ATHEROSCLEROSIS

Aim: (1) to obtain and functionally characterize the late endothelial progenitor cell (EPC) cultures from the animal blood; (2) to investigate the potential beneficial effects of circulating microparticles (MPs) of healthy

Bieke Van Der Veken, Guido De Meyer, Wim Martinet. Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

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Abstracts / Atherosclerosis 263 (2017) e29ee110

Aim: Intraplaque (IP) microvessels have a major causative effect on atherosclerotic plaque development. An increase in microvessel density in ruptured versus nonruptured human plaques suggests that VEGF and other angiogenic factors promote atherosclerosis and potentially destabilize plaques. Because apolipoprotein deficient mice with a heterozygous mutation (C1039G+/-) in the fibrillin-1 gene (ApoE-/-Fbn1C1039G+/-) manifest substantial IP neovascularization, they represent a unique tool to investigate angiogenesis and its role in atherosclerosis. Here, we examined whether administration of axitinib (inhibitor of VEGF receptor-1,-2 and -3) inhibits IP neovascularization and stabilizes atherosclerotic plaques. Methods: ApoE-/- Fbn1C1039G+/- mice were fed a western diet (WD) for 20 weeks. After 14 weeks WD, mice received axitinib (35 mg/g) or solvent i.p. 4x/week for 6 weeks. Cardiac function was monitored to evaluate the effect of axitinib on atherosclerosis-driven complications such as myocardial infarction. Results: Axitinib reduced IP neovascularization by 50%. The smooth muscle cell content doubled, whereas the amount of macrophages decreased by 30%. Overall cardiac function was improved in axitinibtreated animals (fractional shortening: 19±3% vs. 27±2%, p¼0.033; LVIDd 4.6±0.2mm vs. 4.0±0.2mm, p¼0.02; LVIDs 3.7±0.2mm vs. 3.0±0.2mm, p¼0.04). Moreover, the number of animals with myocardial infarction was decreased by 40%. Coronary plaque formation was observed in almost all control animals whereas treated animals showed a 30% reduction in the occurrence of coronary plaques. Conclusions: Inhibition of VEGF receptor signaling by axitinib attenuates intraplaque angiogenesis and plaque destabilization in mice. Epigenetics, micrornas, and gene-environment interactions SAG017. GENETIC AND FUNCTIONAL INTERACTION OF THE CORONARY ARTERY DISEASE RISK GENE ADAMTS7 WITH LDL-CHOLESTEROL Patrick Schindler1, Lingyao Zeng1, Simon Von Ameln1, Jeanette Erdmann2, Thorsten Kessler1, Heribert Schunkert1, 3. 1 Deutsches Herzzentrum München, Klinik für Herz- und Kreislauferkrankungen, €t München, Munich, Germany; 2 Institut für Technische Universita €t zu Lübeck, and Integrative und Experimentelle Genomik, Universita Deutsches Zentrum für Herz-Kreislauf-For, Lübeck, Germany; 3 Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK) e.V., Partner Site Munich Heart Alliance, Munich, Germany Aim: A chromosomal locus encompassing the ADAMTS7 gene has been associated with coronary artery disease (CAD) in genome-wide association studies (GWAS). Mouse studies suggest that Adamts-7 contributes to vascular remodeling and atherosclerotic plaque formation. Here, we sought to investigate the interaction of ADAMTS7 and LDL-cholesterol (LDL-C). Methods: We used a LDL-C-genetic risk score (GRS) as a surrogate for LDLC to investigate the association of ADAMTS7 (rs7173743) and CAD in individual-level data from two German GWAS. Gene expression was investigated in vascular smooth muscle cells (VSMC) treated with oxidized LDLcholesterol (oxLDL), solubilized cholesterol (sChol), or the respective controls using quantitative PCR. Expression of the oxLDL-receptor 1 (OLR1) was silenced by RNA interference. Results: Studying 3,204 cases and 1,780 controls revealed an inverse relationship between LDL-C-GRS and a relative increase of CAD risk related to ADAMTS7 (P<0.05, R¼-0.88) which was strongest in the first LDL-C-GRS quintile (Odds Ratio 1.33, 95% confidence interval 1.08-1.63, p<0.01), an observation that was replicated in three further German GWAS. In VSMC, oxLDL led to significant reduction of ADAMTS7 expression (-40%, p<0.01) whereas it was not altered by sChol. Silencing of OLR1 led to a partial recovery of reduced ADAMTS7 expression (+16%, p<0.05). Conclusions: Our in vitro and genetic data provide evidence for an interaction of the CAD risk gene ADAMTS7 with LDL-C. Interestingly, relevance of the ADAMTS7 risk allele increased at low LDL-C. Hence, inhibition of ADAMTS-7 might be particularly beneficial for individuals

with low LDL-C. Additionally, the ADAMTS7-attributed risk might be underestimated if LDL-cholesterol is not taken into account.

SAG018. MATERNAL DIETARY ALA PREVENTS ATHEROGENIC LIPID DISARRAY IN THE OFFSPRING VIA FETAL GENE PROGRAMMING Alicia Leikin-Frenkel1, Aviv Shaish2, Dror Harats2. 1 Bert Strassburger Lipid Center,Sheba,Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 2 Bert Strassburger Lipid Center,Sheba,Tel Hashomer, Tel Aviv, Israel Aim: The origin of atherosclerosis is still unknown and its treatment is an ongoing challenge. The atherogenic linked lipid disarray seems to begin in utero. Maternal dietary Saturated Fatty Acids (SFA) promote detrimental modifications of lipid metabolism in the offspring whereas Alpha Linolenic Acid (ALA) prevents them. The aim of this work is to study the metabolic and molecular pathways associated with ALA preventive effects Methods: Rodent plasma and tissue lipids, desaturase enzymes and genes were studied in the offspring of C57Bl6/J and LDLr -/- mice receiving dietary fatty acid treatment during pregnancy. Results: Maternal dietary ALA prevented increased plasma and lipoproteins triglycerides and cholesterol levels in 6 month old offspring following a 2 month high fat diet. ALA decreased monounsaturated and n-6 polyunsaturated fatty acids. The activities of Stearoyl Coenzyme A (SCD1) and d 6 desaturases were decreased by up to 30% and 10%, respectively, in maternal ALA offspring while they were increased by up to 35% and 30%, respectively, in maternal SFA offspring. FADS2 and SCD1 gene expression was down-regulated by maternal dietary ALA compared to SFA. Genes related to thermogenesis Pparcg1-a,Plin 5 and Lpin 1 were up-regulated by ALA compared to SFA. Conclusions: ALA and SFA in maternal diet differentially affect atherogenic linked lipid metabolic and gene expression patterns in the offspring. ALA normalizes fatty acid’s profile by lowering SCD1 and FADS2 activities. ALA in utero prevents offspring lipid metabolic disarray by programming down-regulation of desaturases expression and up-regulation of thermogenesis processes.

SAG019. TARGETING LIPID METABOLISM-RELATED GENES BY IN VIVO INHIBITION OF MIR-486 AND MIR-92A LOWERS PLASMA CHOLESTEROL LEVELS IN HYPERLIPIDEMIC HAMSTERS Natalia Simionescu, Mihaela G. Carnuta, Elena V. Fuior, Camelia S. Stancu, Mina Raileanu, Madalina D. Dulceanu, Emanuel Dragan, Anca V. Sima, Loredan S. Niculescu. Institute of Cellular Biology and Pathology N. Simionescu, Bucharest, Romania Aim: To identify and validate the lipid metabolism-related target genes of miR-486 and miR-92a, and to evaluate in vivo inhibitors for miR-486 and miR-92a as a potential treatment for atherogenic dyslipidemia. Methods: Target genes of miR-486 and miR-92a were identified by miRWalk analysis, validated by 3’UTR sequences’ cloning in pmirGLO Dual-Luciferase vector and confirmed in HepG2 hepatocytes and THP-1 macrophages using miR-486 and miR-92a precursors. Fat diet-induced hyperlipidemic hamsters (HLH) were subcutaneously injected with LNA-inhibitors for either miR-486 or miR-92a. Plasma cholesterol, miR486 and miR-92a levels, and liver expression of target genes were assessed. Results: Bioinformatics analysis, 3’UTR molecular cloning and precursor’s transfection proved that SOAT2 and SREBF1 are targeted by miR-486, while ABCG4 and NPC1 are modulated by miR-92a. In HLH, after 10 weeks of fat diet, plasma levels of miR-486 and miR-92a increased 2-fold and, respectively, 1.8-fold compared to controls (standard diet). In HLH liver, SOAT2 gene expression was reduced 4.5-fold, SREBF1 9-fold, ABCG4 4fold and NPC1 2.5-fold versus controls. After 2 weeks of LNA-inhibitors