Regulation of Angiogenesis and Renin Expression in Rats

小考利项目，艾伦·威尔逊 项目3 大鼠血管生成和肾素表达的调控 项目负责人：安德鲁·S·格林博士，生理学教授 共同研究人员：卡罗尔·莫雷诺·奎恩，医学博士，生理学助理教授 Jozef Lazar，医学博士，皮肤科副教授 梁明宇，博士，生理学副教授 霍华德·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，血管生成受损， 增加血管阻力，减少组织灌注，限制药物治疗的效果。研究 来自我们实验室和其他人的研究表明，肾素-血管紧张素系统在肾素-血管紧张素系统的生长和生长中都发挥了作用 血管密度减退。因此，了解肾素的作用机制是至关重要的。 调控及其与血管生成的关系。项目3建议进行的研究 这一计划代表了一项系统性的努力，以确定影响肾素基因表达的缺陷，并 低肾素Dahl盐对生理性Sfimulafion异常血管生成反应的责任 敏感(SS)大鼠。我们假设SS大鼠中的一个突变(S)是肾素基因受损的原因 在该模型中调节和异常血管生成。在三个具体目标中，我们将识别序列变体， 证明这些变体在体外影响肾素调节，并使用转基因方法证明 SS等位基因能够消除体内正常的肾素调节和血管生成表型。 从围绕13号染色体肾素基因(Chr 13)的一系列同源大鼠品系开始 作为一种捕获肾素远端候选区域的靶向同源菌株，我们将鉴定变异体和 它们的作用机制，控制肾素的表达和血管生成反应。这项工程将需要 我们在高通量DNA测序、蛋白质组学和一套独特的动物和 细胞模型，并建立在前一个资助期的发现基础上，这些发现证明：1)近亲交配 SS既有高血压，又有提高血浆肾素活性的能力缺陷，从而导致 血管生成反应和2)位于肾素近端下游1Mb的大鼠CHR 13的独特区域 启动子是SS肾素活性和血管生成受损的原因。 这项工作的几个方面使其方法独一无二。我们已经鉴定并生产了一套动物 这些模型将为我们提供识别与肾素基因相互作用的突变子的基因组工具 在SS背景下损害肾素的产生。我们有能力隔离和研究初级 来自亚基因系的微血管内皮细胞和肾小球旁细胞以确定其功能 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 这将有助于确定与肾素近端启动子结合的蛋白质，最终，结合强度 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，即通过确定 基因(S)在细胞培养和全动物模型中的应用 接近了。 项目3是该方案的关键组成部分，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(这三个区域都是CHR 该计划的项目)，共同导致盐源性高血压、肾损伤和 微循环的血管形成/血管生成。此项目与其他每个项目直接交互 并且是开发动物模型(核心B)、外科植入物(核心)的所有核心的主要使用者 C)和管理(核心A)。在项目3中，我们重点关注血管密度，这是 高血压患者的血管阻力和血流灌注以及肾素在其调节中的作用。 一百四十二 项目总监/首席调查员(最后、第一、中间)：小Cowley，Allen Wllson Project 3 初始预算期间的详细预算，从 直接成本仅为07/01/11 06/30/12 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