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2017 Cardiovascular Research Day Abstract Book

23 Substitution of Leu11

23 Substitution of Leu11 and Tyr12 in Mouse Angiotensinogen Does Not Affect Angiotensin IImediated Functions in Mice Chia-Hua Wu 1 • Congqing Wu 1 • Feiming Ye 1 • Deborah Howatt 1 • Anju Balakrishnan 1 • Jessica Moorleghen 1 • Craig Vander Kooi 2 • Alan Daugherty 3 • Hong Lu 1 1Saha Cardiovascular Research Center , University of Kentucky • 2 Molecular and Cellular Pharmacology, University of Kentucky • 3 Saha Cardiovascular Research Center, University of Kentucky Graduate Student Background and Objective: Angiotensinogen (AGT) is the unique precursor of angiotensin II (AngII), which is a critical contributor to atherogenesis. Renin cleavage of AGT exhibits species specificity. It has been determined in in vitro studies that Leu11-Tyr12 in mouse AGT and Val11- Ile12 in human AGT are essential for species-specific renin cleavage. In this study, we compared adeno-associated viral (AAV) vectors encoding human AGT or mouse AGT with Leu11Val and Tyr12Ile mutations to determine whether substitution of these residues regulated AngII-mediated functions. Methods and Results: Male hepatocyte-specific AGT deficient (hepAGT-/-) mice were injected intraperitoneally with AAV vector containing a null insert or encoding human AGT, while their wild type littermates (hepAGT+/+) were injected with AAV containing the null insert. All mice were in an LDLR -/- background. Two weeks after AAV injections, mice were fed a saturated fat-enriched diet for 12 weeks. Administration of AAV encoding human AGT led to high plasma human AGT concentrations, but had no effect on plasma renin concentrations and hypercholesterolemiainduced atherosclerosis. In a subsequent study, AAV encoding mutated mouse AGT with Leu11Val and Tyr12Ile were injected into hepAGT-/- mice. Mutated mouse AGT resulted in a significant increase of plasma AGT concentrations. Plasma renin concentrations in hepAGT-/- mice repopulated with mutated mouse AGT were decreased to a level comparable to their concentrations in hepAGT+/+ mice injected with a null AAV. AAV-driven expression of mutated mouse AGT also augmented atherosclerosis in hepAGT-/- mice. Conclusion: Human AGT does not repopulate AngII-mediated functions, whereas mutations of Leu11Val and Tyr12Ile in mouse AGT, to mimic the two amino acids in human AGT, does not affect AngII-mediated effects. 39

24 Vascular Inflammatory Regulation of Lipid Phosphate Phosphatase 3 Expression Patrick Van Hoose, PhD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1 1Cardiovascular Research Center, University of Kentucky Postdoc Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3, is a cell surface enzyme that regulates lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) availability and signaling. Genome wide association studies in humans identified heritable single nucleotide polymorphisms (SNPs) in the final intron of PLPP3 that independently predicted coronary artery disease (odds ratio, 1.17; P=3.81×10–19). PLPP3 is dynamically regulated during vascular inflammation and the risk allele reduces gene expression by disrupting binding of CCAAT enhancer binding protein beta (CEBPβ). However, other mechanisms may control dynamic regulation of PLPP3. The USC Genome Browser identifies potential target sequences for NFκB responsive elements in the PLPP3 promoter, including three potential RelA (p65) binding sites. Previous work has established that smooth muscle expression of Plpp3 attenuates experimental atherosclerosis and development of intimal hyperplasia.These observations led to the hypothesis that dynamic regulation of PLPP3 is a crucial step in controlling vascular inflammation and disease progression. Coronary human smooth muscle cells (caHSMCs) were treated for 72hrs with 1µM angiotensin II (ATII) in the presence or absence of 1 µM parthenolide. Ldlr-/- mice were randomized to receive either vehicle saline or 1000ng/kg/min angiotensin II via osmotic mini-pump for 7 days. Following 72hr exposure to angiotensin II (ATII) PLPP3 expression increases in caHSMCs and is accompanied by increases in p65 but not CEBPβ expression. Parthenolide, an inhibitor of IκBα degradation, blocks ATII induced PLPP3 expression. To determine whether Plpp3 is upregulated in response to ATII in vivo, Ldlr-/- mice were treated with ATII for 7 days. Plpp3 and p65 expression remained unchanged in the aortic arch, thoracic aorta, abdominal aorta and mesenteric arteries.These observations suggest potential novel regulation of PLPP3 expression governed by an ATII-NFκB pathway that could be important in the context of vascular inflammation and disease. 40

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