Regulation of Angiogenesis and Renin Expression in Rats

项目Cowley，Jr.，艾伦·威尔逊 项目3 大鼠血管生成与肾素表达的调节 项目负责人：Andrew S.格林博士，生理学教授 共同调查者：卡罗尔·莫雷诺·奎因，医学博士，哲学博士、生理学助理教授 约瑟夫·拉扎尔医学博士，哲学博士、皮肤科副教授 Mingyu Liang，Ph.D.，生理学副教授 霍华德J.雅各布博士，生理学教授 摘要 在某些形式的高血压和其他疾病中，血管密度降低并且血管生成受损， 增加血管阻力、减少组织灌注和限制药物治疗的功效。研究 来自我们实验室和其他人的研究表明，肾素-血管紧张素系统在生长和 血管密度的回归。因此，了解肾素与血管内皮细胞的相互作用机制是至关重要的。 调控及其调控与血管生成的关系。项目3中建议的研究 这个项目代表了一个系统的努力，以确定影响肾素基因表达的缺陷， 在低肾素Dahl盐中，负责对生理性收缩的异常血管生成反应- 敏感（SS）大鼠。我们假设SS大鼠中的一个突变与受损的肾素基因有关 调节和异常血管生成。在三个具体目标中，我们将鉴定序列变体， 证明这些变异体影响体外肾素调节，并使用转基因方法，证明 SS等位基因在体内能够消除正常的肾素调节和血管生成表型。 从13号染色体（chr 13）上的肾素基因周围的一系列同类大鼠品系开始， 作为捕获肾素远端候选区域的靶向同源菌株，我们将鉴定变体， 它们的作用机制，控制肾素表达和血管生成反应。该项目将采取 我们的优势在于高通量DNA测序、蛋白质组学和一套独特的动物和 细胞模型，并建立在上一个资助期的发现基础上，这些发现证明：1）近交系 SS既有高血压，又有增加血浆肾素活性的能力缺陷，这导致血浆肾素活性受损。 血管生成反应和2）一个独特的区域大鼠chr 13位于下游1 Mb的近端肾素 启动子负责这种损伤的肾素活性和血管生成的SS。 这项工作的几个方面使其在方法上独一无二。我们已经鉴定并制作了一组动物 这些模型将为我们提供基因组工具，用于识别与肾素基因相互作用的突变 在SS背景下损害肾素的产生。我们有能力分离和研究 微血管内皮细胞和肾小球细胞的亚同源系，以确定功能 在我们的老鼠品系中捕获的等位基因。我们在DNA结合蛋白的蛋白质组学分析方面具有独特的优势 这将有助于确定与近端肾素启动子结合的蛋白质。 我们的团队带来了血管生成生理学、基因组学、体内基因操作和所有 成功完成拟议研究所需的相关技术，即从确定 在细胞培养物和整个动物模型中使用新的基因进行生理学分析， 接近。 项目3是该计划的一个关键组成部分，因为它继续对该综合体进行调查 位于CHR 13不同区域的一组基因的调节和相互作用（所有三个区域中感兴趣的CHR 该计划的项目），共同导致盐引起的高血压，肾损伤， 微循环的血管分布/血管生成。这个项目与其他项目直接交互 并且是用于开发动物模型（Core B）、外科植入物（Core C）和管理（核心A）。在项目3中，我们专注于血管密度，这是一个关键的决定因素， 高血压的血管阻力和灌注以及肾素在其调节中的作用。 142 项目主任/主要研究者（最后一位，第一位，中间一位）：Cowley，Jr.，艾伦威尔森项目3 初始预算期的详细预算 仅直接费用11年7月1日12年6月30日 列表

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