In Vivo Mechanisms of Vascular Dysfunction in Obesity with Insulin Resistance

描述（由申请人提供）：该资助提案旨在研究代谢综合征的两个主要组成部分胰岛素抵抗肥胖血管功能障碍的新细胞和分子机制。具体来说，我们将研究钙依赖性蛋白酶calpain在血管紧张素II （AngII）炎症信号传导中的作用，以及体内AngII受体阻断治疗的组织保护作用。我们的主要假设是，在肥胖相关的胰岛素抵抗状态下，升高的AngII信号与中性粒细胞衍生的髓过氧化物酶（MPO）协同作用，通过激活血管内皮中的钙蛋白酶导致血管功能障碍。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。他们也将为深入了解AngII受体阻断治疗在这种疾病状态下的多效性作用提供帮助。越来越多的文献证据表明，肾素-血管紧张素系统（RAS）的关键效应物AngII可诱导血管炎症，肥胖相关的胰岛素抵抗上调RAS和AngII信号。尽管进行了大量的研究，AngII的炎症作用的细胞和分子机制在很大程度上仍然未知，这限制了对日益增长的肥胖和胰岛素抵抗的美国人群的治疗干预。最近，我们的实验室在肥胖、胰岛素抵抗的实验室啮齿动物中发现了钙蛋白酶活性增加与内皮功能障碍和白细胞激活的证据。其他人现在将钙蛋白酶系统与人类的代谢综合征联系起来。本应用程序提供的初步数据表明，钙蛋白酶在AngII炎症信号传导中的作用，这引起了几个尚未解决的科学问题。这些问题的答案最终将使我们能够开发新的、有效的治疗工具来对抗人类血管疾病。为了实施这项研究，我们将使用Zucker肥胖大鼠，肥胖相关胰岛素抵抗的相关动物模型，并选择敲除小鼠技术。体内研究将阐明(1)抑制AngII信号传导是否会阻止钙蛋白酶在血管内皮中的激活和作用；(2)循环中性粒细胞在维持内皮细胞对AngII的激活中起着重要的机制作用；(3)中性粒细胞衍生的MPO是AngII/calpain炎症信号级联的关键分子决定因素；(4) IkB/NFkB在calpain内皮功能障碍中起机制作用。我们将利用以下生物化学和生理学技术：western blot分析，免疫组织化学和免疫荧光，定量逆转录-聚合酶链反应，反义DNA技术，细胞和组织分离技术，活体显微镜，一氧化氮和超氧化物的体内测量。我们期望我们的研究结果将改善肥胖，胰岛素抵抗的美国人口的生活质量和预期寿命，除了降低管理心血管并发症的总体经济成本。

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.