Projects Cowley, Jr., Allen Wilson PROJECT 3 REGULATION OF ANGIOGENESIS AND RENIN EXPRESSION IN RATS Project Leader: Andrew S. Greene, Ph.D., Professor of Physiology Co-lnvestigators: Carol Moreno Quinn, M.D., Ph.D., Assistant Professor of Physiology Jozef Lazar, M.D., Ph.D., Associate Professor of Dermatology Mingyu Liang, Ph.D., Associate Professor of Physiology Howard J. Jacob, Ph.D., Professor of Physiology ABSTRACT In certain forms of hypertension and other diseases, vascular density is reduced and angiogenesis is impaired, increasing vascular resistance, reducing tissue perfusion, and limiting the efficacy of pharmacotherapy. Studies from our laboratory and others have implicated a role for the renin-angiotensin system in both the growth and regression of vessel density. Consequently, it is critical to understand the mechanisms by which renin is regulated and the relafionship between its regulation and angiogenesis. The studies proposed in Project 3 of this program represent a systematic effort to identify the defect that impacts renin gene expression and is responsible for the abnormal angiogenic response to physiological sfimulafion in the low renin Dahl salt- sensitive (SS) rat. We hypothesize that a mutafion(s) in the SS rat is responsible for the impaired renin gene regulafion and abnormal angiogenesis in this model. In three specific aims we will identify sequence variants, demonstrate that these variants impact renin regulation in vitro, and, using a transgenic approach, demonstrate that the SS allele is capable of eliminating normal renin regulafion and the angiogenic phenotype in vivo. Beginning with a series of congenic rat strains surrounding the renin gene on chromosome 13 (chr 13), as well as a targeted congenic strain that captures the candidate region distal to renin, we will identify the variants and mechanisms by which they act, controlling renin expression and the angiogenic response. This project will take advantage of our capabilifies in high-throughput DNA sequencing, proteomics, and a set of unique animal and cell models, and builds upon discoveries made in the previous funding period that demonstrate: 1) The inbred SS has both hypertension and a defect in the ability to increase plasma renin activity that results in an impaired angiogenic response and 2) a unique region of rat chr 13 located 1 Mb downstream from the proximal renin promoter is responsible for this impairment of renin activity and angiogenesis in the SS. Several aspects of this work make it unique in its approach. We have identified and produced a set of animal models that will provide us with the genomic tools for identifying the mutafion that interacts with the renin gene to impair renin producfion in the SS background. We have the ability to isolate and study primary microvascular endothelial and juxtaglomerular cells from the subcongenic lines in order to define the function ofthe alleles captured in our rat lines. We have unique strengths in proteomic analysis of DNA binding proteins that will help to determine proteins that bind to the proximal renin promoter Finally, the combined strength of our group brings expertise in the physiology of angiogenesis, genomics, in vivo gene manipulation and all of the associated techniques required for successful completion of the proposed studies i.e. from identifying gene(s) to physiological profiling with validafion in cell culture and in whole animal model using novel approaches. Project 3 is a critical component to this program because it continues the investigations of the complex regulation and interplay of a set of genes residing in different regions of chr 13 (the chr of interest in all three projects of this program) that collectively contribute to salt-induced hypertension, renal injury, and vascularity/angiogenesis of the microcirculation. This project interacts directly with each of the other projects and is a major user of all of the cores for the development of animal models (Core B), surgical implants (Core C), and administration (Core A). In Project 3, we are focused on vascular density, a critical determinant of vascular resistance and perfusion in hypertension and the role of renin in its regulation. 142 Program Director/Principal Investigator (Last, First, Middle): Cowley, Jr., Allen Wllson Project 3 DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM THROUGH DIRECT COSTS ONLY 07/01/11 06/30/12 List
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