DIABETES, TRANSPLANTATION AND VASCULAR ENDOTHELIUM

内皮细胞在调节血管张力方面发挥着独特的作用， 释放内皮源性舒张因子（EDRF）。 EDRF中的缺陷 可能使糖尿病患者更易患高血压， 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 舒张与受体操纵的内皮依赖性 血管舒张剂（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 （ODFR）在该缺陷中的作用将进行调查。 血管（主动脉， 股动脉和肠系膜动脉）， 将使用遗传性糖尿病BB大鼠并与葡萄糖- 输注的老鼠 对RO反应受损的选择性和特异性- EDV vs. RI-EDV和内皮非依赖性血管扩张剂将在 具体目标（1）。 体内干预（具体目标#2） 使用手术（胰腺移植）或治疗干预 （胰岛素，抗氧化剂，自由基清除剂，铁螯合剂）将是 评价以检测内皮细胞的预防或逆转 功能障碍 将检查EDRF受损的机制（具体 目标3）。 将使用生物测定技术，其中内皮- 对照或实验供体节段的灌注受到挑战， 释放EDRF并作用于检测环的激动剂（没有 内皮）。 这将回答：糖尿病内皮细胞是否释放 减少EDRF？ 糖尿病患者的血管平滑肌对 真正的EDRF和/或一氧化氮？ 糖尿病患者产生的ODFR 内皮或糖尿病血管平滑肌损伤或破坏 EDRF活动？ 胰岛素对细胞内葡萄糖减少的影响 或EDRF释放或行动也将进行测试。 附加议定书 将检测潜在的内皮源性收缩因子，或 cGMP的差异变化（使用放射免疫测定法）。 超氧 歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶的活性 在血管中检查以评估自由基清除能力。 血管（有或没有内皮）产生ODFR将 通过分光光度技术和最先进的 使用环隙共振器的电子自旋共振光谱学 自旋捕获技术。 细胞内钙信号转导 将使用荧光评价对RO-EDV和RI-EDV的响应 培养牛主动脉内皮细胞的Fura-2光谱 使受试者处于模拟糖尿病环境的条件下（具体目标 #4）。 将内皮细胞与葡萄糖、脂肪酸或其组合孵育。 酸、酮体或糖尿病血清（包括极低密度 脂蛋白）检测细胞内钙离子的选择性解偶联 响应RO-EDV的信号转导。 ODFR在其中的作用 还将评估解耦合。 本研究获得的数据将 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性疾病的病因学中起作用。 与糖尿病相关的并发症 它还将提供 胰腺移植有效性的实验证据 或治疗性干预，以防止或逆转内皮 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.