In Vivo Mechanisms of Vascular Dysfunction in Obesity with Insulin Resistance

描述(由申请者提供)：这项资助计划旨在研究肥胖合并胰岛素抵抗的血管功能障碍的新的细胞和分子机制，胰岛素抵抗是代谢综合征的两个主要组成部分。具体地说，我们将研究钙依赖的蛋白水解酶在血管紧张素II的炎症信号中所起的作用，以及在体内血管紧张素Ⅱ受体阻断治疗的组织保护作用中所起的作用。我们的主要假设是，在肥胖相关的胰岛素抵抗状态下，血管紧张素Ⅱ信号的升高与中性粒细胞衍生的髓过氧化物酶(MPO)协同作用，通过激活血管内皮细胞中的钙蛋白酶而导致血管功能障碍。本文提出的综合研究结果可能揭示代谢综合征血管并发症的新机制。他们还将深入了解血管紧张素Ⅱ受体阻断治疗在这种疾病状态下的多效性作用。越来越多的证据表明，肾素-血管紧张素系统(RAS)的关键效应因子AngII诱导血管炎症，肥胖相关的胰岛素抵抗上调RAS和AngII信号。尽管进行了密集的研究，但血管紧张素转换酶炎症作用的细胞和分子机制仍基本不清楚，这限制了对日益增长的肥胖和胰岛素抵抗的美国人口的治疗干预。最近，我们的实验室发现在肥胖、胰岛素抵抗的实验啮齿动物中，有证据表明钙蛋白酶活性增加，内皮功能障碍和白细胞激活。其他人现在已经将钙蛋白酶系统与人类的新陈代谢综合征联系起来。本申请中提供的初步数据表明，钙蛋白酶在血管紧张素Ⅱ的炎症信号转导中发挥了作用，这引发了几个悬而未决的科学问题。对这些问题的回答最终将使我们能够开发新的、有效的治疗工具来对抗人类的血管疾病。为了实施这项研究，我们将使用Zucker肥胖大鼠，一种与肥胖相关的胰岛素抵抗的相关动物模型，并选择基因敲除小鼠技术。体内研究将阐明：(1)抑制Angii信号转导可阻止血管内皮细胞中Calain的激活和作用；(2)循环中的中性粒细胞在机械上对维持Angii反应中内皮细胞Calain的激活具有重要作用；(3)中性粒细胞衍生的MPO是Angii/Calain炎症信号级联反应的关键分子决定因素；(4)IKB/NFkB在Calain的内皮功能障碍中发挥机制作用。我们将利用以下生物化学和生理学技术：蛋白质印迹分析、免疫组织化学和免疫荧光、定量逆转录聚合酶链式反应、反义DNA技术、细胞和组织分离技术、活体显微镜、体内一氧化氮和超氧化物歧化的测量。我们预计，除了降低管理心血管并发症的总体经济成本外，我们的研究结果还将改善肥胖、胰岛素抵抗的美国人口的生活质量和预期。 公共卫生相关性：本申请中提出的研究旨在提供新的生物标记物和治疗策略，以对抗肥胖和胰岛素抵抗引起的血管并发症。根据美国心脏协会的数据，心血管疾病(CDV)仍然是美国的头号杀手。2006年心血管疾病的直接和间接费用估计为4031亿美元。我们已经确定了一条新的信号通路，Calain系统，它有助于解释a)血管紧张素II如何损害身体器官的血管系统，以及b)为什么阻断肾素-血管紧张素系统的药物保护心血管系统。我们预计，除了降低管理心血管疾病的总体经济成本外，我们的研究结果还将改善肥胖、胰岛素抵抗的美国人口的生活质量和预期。

DESCRIPTION (provided by applicant): This grant proposal is designed to study novel cellular and molecular mechanisms of vascular dysfunction in obesity with insulin resistance, two major components of the metabolic syndrome. Specifically, we will study the role that the calcium-dependent protease calpain plays in the inflammatory signaling of angiotensin II (AngII), and in the tissue protective action of AngII receptor blocking therapy in vivo. Our main hypothesis is that in states of obesity-associated insulin resistance, elevated AngII signaling synergizes with neutrophil-derived myeloperoxidase (MPO) to cause vascular dysfunction via activation of calpain in the vascular endothelium. The results of the integrative studies proposed here may uncover novel mechanisms of vascular complications in the metabolic syndrome. They will also provide an in depth understanding of the pleiotropic actions of AngII receptor blocking therapy in such disease state. Evidence has been accumulating in the literature to indicate that AngII, the key effector of the renin- angiotensin system (RAS), induces vascular inflammation, and that obesity-associated insulin resistance upregulates the RAS and AngII signaling. Despite intense research, the cellular and molecular mechanisms of the inflammatory action of AngII remain largely unknown, which limits therapeutic interventions in the ever-growing obese, insulin resistant U.S. population. Recently, our laboratory has found evidence of increased calpain activity with endothelial dysfunction and leukocyte activation in obese, insulin resistant laboratory rodents. Others have now linked the calpain system to the metabolic syndrome in humans. Preliminary data provided in the present application demonstrate a role for calpain in the inflammatory signaling of AngII, which gives rise to several, unresolved scientific questions. The answering of such questions will ultimately allow us to develop new, effective therapeutic tools to fight vascular disease in humans. To implement this research we will use the Zucker Obese rat, a relevant animal model of obesity-associated insulin resistance, and selected knockout mouse technology. In vivo studies will clarify whether (1) inhibition of AngII signaling prevents calpain activation and actions in the vascular endothelium; (2) circulating neutrophils are mechanistically important to sustain endothelial calpain activation in response to AngII; (3) neutrophil-derived MPO is a key molecular determinant of the AngII/calpain inflammatory signaling cascade; (4) IkB/NFkB plays a mechanistic role in the endothelial dysfunction of calpain. We will utilize the following biochemistry and physiology techniques: western blot analysis, immunohistochemistry and immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, antisense DNA technology, cells and tissue isolation techniques, intravital microscopy, in vivo measurements of nitric oxide and superoxide. We expect that the results of our research will improve the life quality and expectancy of the obese, insulin resistant U.S. population, in addition to lowering the overall economic cost of managing cardiovascular complications. PUBLIC HEALTH RELEVANCE: The research proposed in this application intends to provide new biological markers and therapeutic strategies to fight the vascular complications of obesity and insulin resistance. According to the American Heart Association, cardiovascular disease (CDV) remains the No. 1 killer in the United States. The estimated direct and indirect cost of CVD for 2006 is $403.1 billion. We have identified a novel signaling pathway, the calpain system, which helps explain a) how angiotensin II damage the vasculature of body organs, and b) why medications that block the renin-angiotensin system protect the cardiovascular system. We expect that the results of our research will improve life quality and expectancy of the obese, insulin resistant U.S. population, in addition to lowering the overall economic cost of managing cardiovascular disease.