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2017 Cardiovascular Research Day Abstract Book

33 Promoter Enhancer

33 Promoter Enhancer Interactions Regulating PLPP3 Gene Expression Guogen Mao, PhD 1 • Shaojing Ye, PhD 1 • Susan S. Smyth, MD, PhD 2 • Andrew J. Morris, PhD 2 1Gill Heart and Vascular Institute, University of Kentucky • 2 Gill Heart and Vascular Institute, Veterans Affairs Medical Center,, University of Kentucky Staff Common heritable intronic variants within the final fifth intron of the PLPP3 gene associate with significant inter individual differences in human coronary artery disease risk. PLPP3 is one of three closely related genes encoding lipid phosphate phosphatases 1-3 which are integral membrane enzymes that dephosphorylate lipid phosphate mono esters and some lipid polyphosphates. PLPP3 expression in vascular endothelial cells is required for mouse development and while individuals with heterozygosity for loss of function PLPP3 alleles have been identified homozygotes have not so PLPP3 is likely also essential for human development. Because the three PLPP genes have overlapping expression patterns and encode enzymes with very similar activities it is reasonable to hypothesize that differences in regulation of expression may underlie these unique functions of PLPP3 gene in atherosclerosis and development. We used informatic approaches to identify regulatory elements within the PLPP3 promoter and coronary artery disease risk haplotype block. We then cloned the relevant sequences into reporter constructs and used transfection assays and site directed mutagenesis to examine sequence and allele specific effects on promoter and enhancer activity and on how these interact to control gene expression. The ~1.5 KB PLPP3 promoter comprises a ~0.5 KB enhancer indicated by H3K4ms1/H3K27Ac markers and a ~1KB core promoter containing three NF-kB (p50/RelA and p42/RelB) binding sites. Chromatin immunoprecipitation and reporter assays indicate that all three of these are functional regulators of basal activity of the PLPP3 promoter with very strong upregulation of promoter activity observed in transfection assays with these transcription factors. Similar informatic approaches identified cFOS and C/EBP target sequences in accessible chromatin within the coronary artery disease risk associated haplotype block in the final intron of the PLPP3 gene. These sequences are conserved in mice and humans and interact with their cognate transcription factors in immunoprecipitation and gel shift assays and regulate expression of both generic promoters and the PLPP3 promoter in reporter gene/transfection assays. The cFOS and C/EBP target sequences exhibit allelic variation in humans with coronary artery disease risk variants decreasing transcription factor binding and transcriptional enhancer activity. Taken together these results identify an NF-kB driven pathway that likely explains the widely observed strong upregulation of PLPP3 expression during inflammation and atherosclerosis and suggest a mechanism by which coronary artery disease risk associated variants of intronic enhancer sequences could decrease PLPP3 expression and reduce the normally protective functions of this gene observed in experimental models of atherosclerosis. 49

34 Endothelial Mineralocorticoid Receptor and Neutrophils Mediate Aldo plus Salt-Induced Abdominal Aortic Aneurysm Shu Liu 1 • Yu Zhong 1 • Zhenheng Guo 2 • Ming Gong 1 1Physiology, University of Kentucky • 2 Pharmacology and Nutritional Sciences, University of Kentucky Staff Abstract Objective—We recently reported that administration of mice with aldosterone (Aldo) or deoxycorticosterone acetate (DOCA) plus salt induces AAA via mineralocorticoid receptor (MR). The current study defines the specific roles of endothelial and myeloid cell MR in DOCA-salt induced AAA. Approach and Results—A tamoxifen inducible endothelial cell (EC)-specific MR knockout mouse model (iECMRKO) and a myeloid cell-specific MR knockout mouse model (MyMRKO) were developed. The iECMRKO mice, but not the MyMRKO mice were protected from Aldo- or DOCA-saltinduced AAA. Mechanistically, EC-specific MR deletion had little effect on Aldo-salt-induced salt retention, hypertension, and renal fibrosis, but largely suppressed aortic elastin degradation, matrix metalloproteinase-2 (MMP-2) and MMP-9 upregulation, macrophage and neutrophil infiltration. Surprisingly, neutrophils, but not macrophages, were observed in the aorta 1 week after Aldo-salt administration in control mice, but not in iECMRKO mice. Treatment of C57BL/6 mice with an anti-PMN antibody selectively suppressed Aldo-salt-induced circulating Ly6G-postive neutrophils, but not CD4-postive leukocytes, and protected mice from Aldo-salt-induced AAA. In cell cultures, Aldo-induced endothelial adhesion molecules (E-selectin, P-selectin, and ICAM-1, but not VCAM-1) and proinflammatory cytokine (IL-6 and MCP-1) mRNA expressions were abolished in MR-deficient ECs. Importantly, Aldo-salt-induced ICAM-1 but not VCAM-1 protein upregulation was abolished in aortas from iECMRKO mice. Conclusions—Endothelial MR, but not myeloid cell MR, plays an important role in Aldo-salt-induced AAA. Moreover, ICAM-1, but not VCAM-1; and neutrophils, but not macrophages, mediate the early processes of Aldo-salt-induced and endothelial MR-mediated AAA development. 50

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