ANG II: Microvascular rarefaction and angiogenesis

在过去五年中，该项目的研究表明，血管紧张素II（ANGII）是一种重要的生长因子，它是维持骨骼肌和大脑中阻力血管正常功能所必需的，并且它与盐敏感性高血压有重要关系。我们最近已经确定ANGII在成人骨骼肌中的血管生成中起关键作用，并且已经建立了大鼠后肢肌肉组织的慢性电刺激模型，该模型产生稳健且可再现的血管生成。在这个模型中， 高盐饮食或ANGII形成的抑制阻断了“受刺激的血管生成”，但其发生的机制或相互作用的途径尚未建立。在项目5中，我们假设，ANGII，这是本地产生的骨骼肌血管中的电刺激，与一氧化氮，和20-HETE相互作用，改变抗血管生成（稳态）和血管生成机制之间的平衡，导致微血管密度的增加。在这个项目中，我们将利用几种基因操纵的大鼠品系，这将有助于我们更好地剖析ANGII和钠摄入在调节中的作用。 和维持微循环。Dahl S大鼠（SS/Mcw）是一种盐敏感性高血压模型，在高盐和低盐饮食中都具有低肾素水平。该大鼠不经历响应于本申请中使用的电刺激方案的血管生成。染色体13（含有肾素基因的染色体）已从BN/Mcw大鼠品系置换到亲本SS/Mcw大鼠的等基因背景中的同源大鼠模型（SS. BN 13）与SS/Mcw品系具有98%的同一性，但其血压正常，并且具有 恢复了肾素和血管生成反应，使其成为本项目研究的理想选择。目的1将使用这些动物来确定局部产生的ANGII在由电刺激诱导的血管生成中的重要性，使用我们的高度特异性和灵敏度的提取和测定程序来测量组织中的ANGII。近交系大鼠的独特品系将使我们能够将ANGII的潜在致病作用与许可作用分开。目标2将利用一系列良好表征的抑制剂和测定系统的可用性来研究 体内骨骼肌血管生成中的肾素-血管紧张素系统、一氧化氮和20-HETE产生之间的相互作用。在独特的骨骼肌、微血管、内皮细胞培养模型中进行的其他研究将确认体内获得的结果。目标3集中在高盐饮食抑制血管生成的机制，并将测试两个具体的假设，关于生产ANGII及其受体在骨骼肌的调节。在这些研究中，将使用我们的近交系大鼠分离血压和盐摄入量的作用，其中血压和血管生成的盐敏感性并不总是相关的。 ANGII受体亚型的调节将使用我们的用于手解剖和分离微血管的技术，然后通过定量真实的时间RT-PCR和蛋白质印迹技术来研究。总之，这些研究将确定肾素-血管紧张素系统在骨骼肌血管生成调节中的作用，并可能提供对血管生成抑制情况下抑制RAS的临床益处的了解。 适得其反

Studies in this program over the last five years have shown that angiotensin II (ANGII) is an important growth factor, that it is required for the maintenance of normal function of resistance vessels in the skeletal muscle and brain, and that it is importantly involved in salt sensitive hypertension. We have recently established that ANGII plays a key role in angiogenesis in adult skeletal muscle and have established a model of chronic electrical stimulation of the hind limb musculature in rats that produces a robust and reproducible angiogenesis. In this model, high salt diet or inhibition of ANGII formation blocks "stimulated angiogenesis", but the mechanism by which this occurs, or the pathways that are interacting, have not been established. In Project 5, we hypothesize that ANGII, which is produced locally in blood vessels of skeletal muscle in response to electrical stimulation, interacts with nitric oxide, and 20-HETE, to alter the balance between antiangiogenic (homeostatic) and angiogenic mechanisms resulting in an increase in microvessel density. In this project we will take advantage of several genetically manipulated rat strains that will help us to better dissect out the roles of ANGII and sodium intake in the regulation and maintenance of the microcirculation. The Dahl S rat (SS/Mcw) is a model of salt sensitive hypertension with low renin levels on both high and low salt diets. This rat does not undergo angiogenesis in response to the electrical stimulation protocol used in this application. The consomic rat model (SS.BN13) in which chromosome 13 (the chromosome containing the renin gene) has been substituted from the BN/Mcw rat strain into the isogenic background of the parental SS/Mcw rat is 98% identical to the SS/Mcw strain, yet it is normotensive and has restored renin and angiogenic responses making it ideal for the studies in this project. Aim 1 will use these animals to determine the importance of locally produced ANGII in angiogenesis induced by electrical stimulation using our highly specific and sensitive extraction and assay procedures for the measurement of ANGII in tissue. The unique strains of inbred rats will allow us to separate a potential causative from a permissive role for ANGII. Aim 2 will take advantage of the availability of a series of well-characterized inhibitors and assay systems to investigate the interactions between the renin-angiotensin system, nitric oxide, and 20-HETE production in skeletal muscle angiogenesis in vivo. Additional studies in a unique skeletal muscle, microvascular, endothelial cell culture model will allow confirmation of results obtained in vivo. Aim 3 focuses on mechanisms involved in the inhibition of angiogenesis by high salt diet and will test two specific hypotheses regarding the regulation of production ANGII and its receptors in skeletal muscle. In these studies the role of blood pressure and salt intake will be separated using our strains of inbred rats in which salt sensitivity of blood pressure and angiogenesis are not always related. Regulation of ANGII receptor subtypes will be studied using our techniques for hand dissection and isolation of microvessels followed by quantitative real time RT-PCR and Western blotting techniques. Taken together, these studies will define the role of the renin-angiotensin system in the regulation of angiogenesis in skeletal muscle and may provide insight into the clinical benefit of inhibition of the RAS in situations where angiogenesis is counterproductive.