Regulation of Angiogenesis and Renin Expression in Rats

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，减少组织灌注量，限制药物治疗的效果。我们实验室和其他实验室的研究表明，肾素-血管紧张素系统在血管密度的增长和退化中都发挥了作用。因此，了解肾素的调控机制以及肾素调控与血管生成之间的关系至关重要。本计划项目3中提出的研究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，并导致低肾素Dahl盐敏感(SS)大鼠对生理刺激的异常血管生成反应。我们推测SS大鼠中的一个突变(S)是导致肾素基因调节受损和血管生成异常的原因。在三个特定的目标中，我们将识别序列变异，证明这些变异在体外影响肾素调节，并使用转基因方法，证明SS等位基因能够在体内消除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，以及捕获肾素远端候选区域的靶向同源品系开始，我们将确定它们发挥作用的变体和机制，控制肾素表达和血管生成反应。该项目将利用我们在高通量DNA测序、蛋白质组学以及一系列独特的动物和细胞模型方面的能力，并以上一个资助期中所做的发现为基础，证明：1)近交系SS既有高血压，又有能力增加血浆肾素活性的缺陷，从而导致血管生成反应受损；2)大鼠CHR 13的一个独特区域，位于近端肾素启动子下游1 Mb，负责这种SS中肾素活性和血管生成的损害。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模型，这些模型将为我们提供基因组工具来鉴定与肾素基因相互作用的突变，以削弱SS背景中肾素的产生。我们有能力从亚基因系中分离和研究原代微血管内皮细胞和肾小球旁细胞，以确定在我们的大鼠系中捕获的等位基因的功能。我们在脱氧核糖核酸结合蛋白的蛋白质组学分析方面拥有独特的优势，这将有助于确定与近端肾素启动子结合的蛋白质。最后，我们团队的联合优势带来了血管生成生理学、基因组学、体内基因操作以及成功完成拟议研究所需的所有相关技术方面的专业知识，例如，从识别基因(S)到在细胞培养和整个动物模型中使用新方法进行验证的生理图谱。 项目3是该计划的关键组成部分，因为它继续研究位于CHR 13不同区域的一组基因的复杂调节和相互作用(该计划的所有三个项目都关注CHR)，这些基因共同促进盐诱导的高血压、肾脏损伤和微循环的血管/血管生成。该项目与其他每个项目直接互动，是开发动物模型(核心B)、外科植入物(核心C)和管理(核心A)的所有核心的主要使用者。在项目3中，我们主要关注血管密度，这是高血压患者血管阻力和血流灌注的关键决定因素，以及肾素在其调节中的作用。

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 relationship 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 stimulation in the low renin Dahl salt- sensitive (SS) rat. We hypothesize that a mutation(s) in the SS rat is responsible for the impaired renin gene regulation 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 regulation 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 capabilities 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 mutation that interacts with the renin gene to impair renin production 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 validation 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.