DIABETES, TRANSPLANTATION AND VASCULAR ENDOTHELIUM

内皮细胞通过以下途径在调节血管张力方面发挥独特的作用 释放内皮源性舒张因子(EDRF)。EDRF中的缺陷 可能使糖尿病患者患高血压的风险更高， 动脉粥样硬化、冠状动脉血管痉挛和猝死。这 一项提案将检验糖尿病选择性地 解偶联对受体操作的内皮依赖的松弛 血管扩张剂(RO-EDV)，但不是受体非依赖性内皮 血管扩张剂(RI-EDV)。氧自由基的特殊作用 将对此缺陷中的(ODFR)进行调查。血管(大动脉、 股动脉和肠系膜动脉)来自链脲佐菌素糖尿病和 遗传糖尿病的BB大鼠将被用来与葡萄糖- 注射过的老鼠。RO-反应受损的选择性和特异性 EDV与RI-EDV和内皮非依赖性血管扩张剂将 审查(具体目标1)。体内干预(特定目标2) 使用外科手术(胰腺移植)或治疗性干预 (胰岛素、抗氧化剂、自由基清除剂、铁络合剂) 评估以测试内皮细胞的预防或逆转 功能障碍。EDRF受损的机制将被检查(具体 目标3)。生物测定技术将被用于内皮细胞- 对照或实验性供体节段的灌流受到挑战 释放EDRF并作用于探测器环的激动剂(没有 内皮细胞)。这将回答：糖尿病血管内皮细胞是否会释放 减少EDRF？；糖尿病血管平滑肌对 真实的EDRF和/或一氧化氮？；ODFR是否由糖尿病患者产生 内皮或糖尿病血管平滑肌受损或失活 EDRF活动？胰岛素对细胞内糖减少症的影响 或者EDRF的释放或动作也将受到测试。附加协议 会检测一种潜在的内皮来源的收缩因子或 CGMP的不同变化(采用放射免疫法)。超氧化物 歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶活性将 在血管中检查以评估清除自由基的能力。 由血管(有或没有内皮)产生ODFR将 用分光光度技术和最先进的技术进行检查 环隙谐振器的电子自旋共振 自旋捕获技术。细胞内钙信号转导 对RO-EDV和RI-EDV的响应将使用荧光进行评估 培养的牛主动脉内皮细胞的光谱(Fura-2) 受试者受模拟糖尿病环境的条件(特定目标 #4)。内皮细胞将与葡萄糖、脂肪 酸、酮体或糖尿病血清(包括极低密度 脂蛋白)检查细胞内钙离子的选择性解偶联 RO-EDV的信号转导。ODFR在这方面的作用 还将对去耦合进行评估。从这项研究中获得的数据将 帮助定义有缺陷的EDRF的机制并了解其作用 EDRF可能在高血压和其他血管的病因学中发挥作用 与糖尿病相关的并发症。它还将提供 胰腺移植疗效的实验证据 或治疗干预以防止或逆转内皮细胞 功能障碍。

Endothelial cells play a unique role in modulating vascular tone by releasing endothelium-derived relaxing factor (EDRF). Defects in EDRF may predispose the diabetic to a higher risk of hypertension, atherosclerosis, coronary artery vasospasm and sudden death. This proposal will examine the hypothesis that diabetes selectively uncouples relaxation to receptor-operated endothelium-dependent vasodilators (RO-EDV), but not receptor-independent endothelium vasodilators (RI-EDV). The specific role oxygen-derived free radicals (ODFR)in this defect will be investigated. Blood vessels (aorta, femoral and mesenteric artery) from streptozotocin-diabetic and genetically-diabetic BB rats will be used and compared to glucose- infused rats. Selectivity and specificity for impaired response to RO- EDV vs. RI-EDV and endothelium-independent vasodilators will be examined (Specific Aim #1). In vivo intervention (Specific Aim #2) using surgery (pancreatic transplantation) or therapeutic intervention (insulin, antioxidant, free radical scavengers, iron chelator) will be evaluated to test for prevention or reversal of endothelial dysfunction. The mechanism of impaired EDRF will be examined (Specific Aim #3). The bioassay technique will be utilized in which endothelial- perfusion of control or experimental donor segments are challenged with agonists which release EDRF and acts upon detector rings (without endothelium). This will answer: Does the diabetic endothelium release less EDRF?; Is the diabetic vascular smooth muscle less responsive to authentic EDRF and/or nitric oxide?; Do ODFR produced by the diabetic endothelium or diabetic vascular smooth muscle impair or inactivate EDRF activity? The influence of insulin on intracellular glucopenia or EDRF release or action will also be tested. Additional protocols will test for a potential endothelium-derived constricting factor or differential changes in cGMP (using radioimmunoassay). Superoxide dismutase, catalase and glutathione peroxidase activities will be examined in blood vessels to assess radical scavenging capacity. Production of ODFR by blood vessels (with or without endothelium) will be examined by spectrophotometric techniques and by state-of-the art electron spin resonance spectroscopy using loop-gap resonators with spin trapping techniques. Intracellular calcium signal transduction in response to RO-EDV and RI-EDV will be evaluated using fluorescence spectroscopy (Fura-2) in cultured bovine aortic endothelial cells subjects to conditions mimicking the diabetic environment (Specific Aim #4). Endothelial cells will be incubated with either glucose, fatty acids, ketone bodies or diabetic sera (including very low density lipoproteins) to examine selective uncoupling of intracellular calcium signal transduction in response to RO-EDV. The role of ODFR in this uncoupling will also be evaluated. Data obtained from this study will help define a mechanism for defective EDRF and understanding the role EDRF may play in the etiology of hypertension and other vascular complications associated with diabetes. It will also provide experimental evidence for the efficacy of pancreatic transplantation or therapeutic intervention to prevent or reverse endothelial dysfunction.