Nitric oxide and microvessel permeability in vivo

项目摘要 活性氧（ROS）的增加被认为是肝纤维化的主要致病因素。 糖尿病血管功能障碍的发展和进展。然而，ROS诱导的机制 微血管并发症和ROS与一氧化氮（NO）和活性氮的相互作用 (RNS)在糖尿病条件下仍然知之甚少。目前，ROS诱导的内皮NO合酶 eNOS解偶联和NO缺乏介导的血管功能障碍已在许多研究中得到广泛研究。 培养的内皮细胞和小动脉。关于ROS对eNOS活性的直接影响知之甚少 以及小静脉的渗透性，小静脉是溶质和液体交换的关键部位，也是炎症的主要部位。我们 在完整大鼠微静脉中进行的初步研究揭示了H2O2在eNOS激活、NO 过氧化氢的产生、过氧亚硝酸盐的形成以及H2O2介导的渗透性的细胞和分子机制 增大我们发现糖尿病大鼠血浆H2O2增加，过氧化氢酶活性降低，这表明 H2O2介导的微血管通透性变化的机制可能与 ROS介导的糖尿病微血管并发症我们假设ROS不会减少NO的产生， 而是引起小静脉中过量的NO产生和过氧亚硝酸盐形成。NO衍生的过氧亚硝酸盐 进一步激活eNOS，导致过氧亚硝酸盐形成增加。这种自我促进的机制是 H2O2诱导的过氧亚硝酸盐介导的细胞损伤、内皮细胞中的Ca2+超载和微血管的关键 屏障功能障碍本研究将从以下三个方面对这一假说进行检验：1）研究细胞机制 H2O2诱导的NO产生和NO介导的微血管屏障功能障碍; 2）研究H2O2诱导的NO产生和NO介导的微血管屏障功能障碍的作用。 NO衍生的过氧亚硝酸盐在H2O2诱导的微血管屏障功能障碍中的作用; 3）研究细胞和 ROS介导的糖尿病微血管功能障碍的分子机制设计的实验 结合微血管通透性的定量测量沿着与共聚焦和电子 在单独灌注的微血管中的显微镜研究使ROS介导的信号传导变化成为可能， 分子、酶活性和血管结构与血管屏障的变化直接相关 功能新开发的Nrf2基因敲除大鼠的加入， 这一建议将有利于ROS介导的微血管并发症的机制研究， 糖尿病从这一建议得出的结果将提供新的信息，桥梁研究使用整体 动物，器官或血管床与研究使用培养的内皮细胞，并提供了一个更好的 了解糖尿病相关微血管并发症的发病机制， 发展靶向治疗。

PROJECT SUMMARY Increased reactive oxygen species (ROS) have been considered to be the main pathogenic factors in the development and progression of vascular dysfunction in diabetes. However, the mechanisms of ROS-induced microvascular complications and the interplay of ROS with nitric oxide (NO) and reactive nitrogen species (RNS) under diabetic conditions remain poorly understood. Currently, ROS-induced endothelial NO synthase (eNOS) uncoupling and NO deficiency-mediated vascular dysfunction have been extensively studied in cultured endothelial cells and arterioles. Very little is known about the direct effect of ROS on eNOS activity and permeability in venules, a crucial site for solute and fluid exchange and a major site of inflammation. Our preliminary studies conducted in intact rat venules revealed the roles of H2O2 in eNOS activation, NO production, peroxynitrite formation, and cellular and molecular mechanisms of H2O2-mediated permeability increases. Our findings that diabetic rats have increased plasma H2O2 and decreased catalase activity suggest that the mechanisms of H2O2-mediated changes in microvascular permeability may resemble those involved in ROS-mediated microvessel complication in diabetes. We hypothesize that ROS do not reduce NO production, but rather cause excessive NO production and peroxynitrite formation in venules. The NO-derived peroxynitrite further activates eNOS, resulting in augmented peroxynitrite formation. This self-promoting mechanism is the key for H2O2-induced peroxynitrite-mediated cell injury, Ca2+ overload in endothelial cells, and microvascular barrier dysfunction. The hypothesis will be tested in three specific aims: 1) investigate the cellular mechanisms of H2O2-induced NO production and NO-mediated microvascular barrier dysfunction; 2) investigate the role of NO-derived peroxynitrite in H2O2-induced microvascular barrier dysfunction; and 3) investigate the cellular and molecular mechanisms of ROS-mediated microvascular dysfunction in diabetes. The designed experiments with combined quantitative measurements of microvessel permeability along with confocal and electron microscopic investigation in individually perfused microvessels enable ROS-mediated changes in signaling molecules, enzyme activities, and vascular structures to be directly linked with changes in vascular barrier function. The addition of newly developed Nrf2 knockout rats that genetically modify antioxidant defenses into the proposal will benefit the mechanistic investigations of ROS-mediated microvascular complications in diabetes. The results derived from this proposal will provide new information that bridges studies using whole animals, organs, or vascular beds with studies using cultured endothelial cells and provide a better understanding of the pathogenesis of diabetes-associated microvascular complication and benefit the development of targeted therapeutics.