Role of Calpain in Diabetic Endothelial Dysfunction

钙蛋白酶在糖尿病内皮功能障碍中的作用

基本信息

批准号：
6725622
负责人：
Rosario G Scalia
金额：
$ 32.16万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-30 至 2007-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Cardiovascular disease accounts for an overwhelming proportion of the morbidity and mortality suffered by patients with all forms of diabetes. Hyperglycemia is considered an important etiologic factor that serves as the initial trigger for diabetic vascular complications. Clinical evidence demonstrates that cardiovascular disease in diabetic patients increases as a function of the duration of hyperglycemia and that exposure of the vasculature to elevated ambient glucose causes generalized endothelial dysfunction, accelerates the atherosclerotic process, and impairs important functions of the microcirculation. As a consequence of hyperglycemia, the diabetic vasculature experiences abnormal inflammatory processes characterized by impaired release of nitric oxide (NO) and increased endothelial adhesiveness. Calpains are a family of calcium-dependent proteases, which have been recently implicated in acute inflammatory disorders of the cardiovascular system. In preliminary studies, we have made the novel observation that inhibition of calpain activity preserves release of endothelial NO and attenuates inflammatory leukocyte-endothelium interactions in the microcirculation during hyperglycemia. Our data strongly support a role for calpains in the pathophysiology of diabetic vascular disease, suggesting a potentially beneficial effect of calpain inhibition in diabetes. Therefore, we propose to study the role of calpains in the inflammatory response of the microcirculation during acute and chronic hyperglycemia. We will test the hypothesis that calpains cause vascular inflammation during hyperglycemia by studying whether: (1) calpain activity is abnormally increased; (2) inhibition of calpain activity prevents inflammatory events in the microcirculation; (3) increased calpain activity downregulates the eNOS enzyme leading to loss of physiologic levels of NO; (4) activation of calpains increases endothelial cell surface expression of pro-inflammatory adhesion molecules via upregulation of NF-kB activity. We will utilize the following in vivo and in vitro cell physiology techniques: intravital microscopy, culture of microvascular endothelial cells under static and flow conditions, NO measurements in vivo and in vitro, immunohistochemistry, Western blot analysis, and gel shift assays. By these means, the studies in this proposal will elucidate important and novel mechanisms underlying microvascular dysfunction in diabetes. This information should provide a framework for developing new therapeutic strategies for the treatment of the life-threatening disorder, diabetes mellitus.

描述（由申请人提供）：心血管疾病在所有形式的糖尿病患者的发病率和死亡率中占绝大多数。高血糖被认为是一个重要的病因，是糖尿病血管并发症的最初触发因素。临床证据表明，糖尿病患者的心血管疾病随着高血糖持续时间的增加而增加，脉管系统暴露于升高的环境葡萄糖会导致广泛的内皮功能障碍，加速动脉粥样硬化过程，并损害微循环的重要功能。由于高血糖，糖尿病血管系统会经历异常炎症过程，其特征是一氧化氮（NO）释放受损和内皮粘附性增加。钙蛋白酶是钙依赖性蛋白酶家族，最近发现其与心血管系统的急性炎症性疾病有关。在初步研究中，我们发现，抑制钙蛋白酶活性可以保留内皮NO的释放，并减弱高血糖期间微循环中炎症性白细胞-内皮细胞的相互作用。我们的数据强烈支持钙蛋白酶在糖尿病血管疾病的病理生理学中的作用，表明钙蛋白酶抑制对糖尿病具有潜在的有益作用。因此，我们建议研究钙蛋白酶在急慢性高血糖期间微循环炎症反应中的作用。我们将通过研究是否以下情况来检验钙蛋白酶在高血糖期间引起血管炎症的假设：（1）钙蛋白酶活性异常增加； (2)抑制钙蛋白酶活性可预防微循环中的炎症事件； (3) 钙蛋白酶活性增加，下调 eNOS 酶，导致 NO 生理水平下降； (4) 钙蛋白酶的激活通过上调 NF-kB 活性来增加内皮细胞表面促炎粘附分子的表达。我们将利用以下体内和体外细胞生理学技术：活体显微镜、静态和流动条件下微血管内皮细胞的培养、体内和体外 NO 测量、免疫组织化学、蛋白质印迹分析和凝胶位移测定。通过这些方式，本提案中的研究将阐明糖尿病微血管功能障碍的重要且新颖的机制。这些信息应该为开发治疗危及生命的疾病糖尿病的新治疗策略提供一个框架。