Role of Calpain in Diabetic Endothelial Dysfunction

说明(申请人提供)：心血管疾病占所有形式糖尿病患者的发病率和死亡率的压倒性比例。高血糖被认为是糖尿病血管并发症的重要病因。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延长而增加，血管系统暴露在高血糖环境中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。由于高血糖，糖尿病血管经历了以一氧化氮(NO)释放受损和内皮粘附性增加为特征的异常炎症过程。钙蛋白酶是一类钙依赖的蛋白水解酶家族，最近被认为与心血管系统的急性炎症性疾病有关。在初步研究中，我们做了一个新的观察，即在高血糖时，抑制Calain活性可以保护内皮NO的释放，并减轻微循环中炎性白细胞与内皮细胞的相互作用。我们的数据有力地支持了钙蛋白酶在糖尿病血管疾病的病理生理学中的作用，这表明抑制钙蛋白酶在糖尿病中具有潜在的有益效果。因此，我们建议研究钙调蛋白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)抑制钙蛋白酶活性可防止微循环中的炎症事件；(3)钙蛋白酶活性增加下调eNOS酶，导致生理水平的一氧化氮丢失；(4)激活钙蛋白酶通过上调核因子-kB活性，增加内皮细胞表面促炎性黏附分子的表达，来检验以下假设：(1)钙蛋白酶活性异常增加；(2)抑制钙蛋白酶活性可防止微循环中的炎症事件发生；(3)钙酶活性增加可下调eNOS酶，导致生理水平的一氧化氮丧失；(4)钙酶的激活通过上调核因子-kB活性增加内皮细胞表面促炎黏附分子的表达。我们将利用以下体内和体外细胞生理学技术：活体显微镜、静态和流动条件下的微血管内皮细胞培养、体内和体外无测量、免疫组织化学、Western印迹分析和凝胶偏移分析。通过这些手段，本提案中的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。

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.