Role of Calpain in Diabetic Endothelial Dysfunction

描述（由申请人提供）：心血管疾病占所有形式糖尿病患者发病率和死亡率的绝大部分。高血压被认为是糖尿病血管并发症的一个重要病因，是糖尿病血管并发症的初始触发因素。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，并且血管系统暴露于升高的环境葡萄糖会导致全身性内皮功能障碍，加速动脉粥样硬化过程，并损害微循环的重要功能。作为高血糖症的结果，糖尿病脉管系统经历异常炎症过程，其特征在于一氧化氮（NO）释放受损和内皮细胞增殖增加。钙蛋白酶是钙依赖性蛋白酶家族，近年来发现与心血管系统急性炎症性疾病有关。在初步的研究中，我们已经取得了新的观察，抑制钙蛋白酶活性，保持释放内皮NO和减轻炎症白细胞内皮细胞在高血糖症的微循环中的相互作用。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，这表明钙蛋白酶抑制剂在糖尿病中具有潜在的有益作用。因此，我们建议研究钙蛋白酶在急性和慢性高血糖时微循环炎症反应中的作用。我们将通过研究是否：（1）钙蛋白酶活性异常增加;（2）抑制钙蛋白酶活性防止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生理水平的丧失;（4）钙蛋白酶的激活通过上调NF-κ B活性增加促炎粘附分子的内皮细胞表面表达。我们将利用以下在体内和体外细胞生理学技术：活体显微镜，静态和流动条件下的微血管内皮细胞的培养，NO在体内和体外的测量，免疫组织化学，蛋白质印迹分析，和凝胶迁移分析。通过这些方法，本研究将阐明糖尿病微血管功能障碍的重要和新的机制。这些信息应该提供一个框架，为发展新的治疗策略，治疗危及生命的疾病，糖尿病。

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.