Nitric oxide and microvessel permeability in vivo

项目总结 活性氧自由基(ROS)的增加被认为是高血压病的主要致病因素。 糖尿病患者血管功能障碍的发生发展。然而，ROS诱导的机制 微血管并发症及其与一氧化氮和活性氮物质的相互作用 糖尿病条件下的(RNS)仍然知之甚少。目前，ROS诱导的内皮型一氧化氮合酶 内皮型一氧化氮合酶解偶联和一氧化氮缺乏介导的血管功能障碍已在 培养内皮细胞和小动脉。关于ROS对eNOS活性的直接影响，我们知之甚少 小静脉的渗透性，这是溶质和液体交换的关键部位，也是炎症的主要部位。我们的 在完整的大鼠小静脉中进行的初步研究揭示了H_2O_2在eNOS激活、NO中的作用 H_2O_2介导的通透性的产生、过氧亚硝酸盐的形成及其细胞和分子机制 增加。我们的研究结果显示，糖尿病大鼠血浆过氧化氢含量升高，过氧化氢酶活性降低 H_2O_2介导的微血管通透性改变的机制可能类似于 糖尿病中ROS介导的微血管并发症。我们假设ROS不会减少NO产量， 而是导致小静脉中过量的NO产生和过氧亚硝酸盐的形成。NO衍生的过氧亚硝酸根 进一步激活eNOS，导致增加过氧亚硝酸盐的形成。这种自我促进的机制是 过氧化氢诱导的过氧亚硝酸盐介导的细胞损伤、内皮细胞内钙超载和微血管的关键 屏障功能障碍。这一假说将在三个具体目标上进行检验：1)研究细胞机制 H_2O_2诱导的NO生成和NO介导的微血管屏障功能障碍；2)探讨 NO衍生的过氧亚硝酸盐在过氧化氢诱导的微血管屏障功能障碍中的作用；以及3)研究细胞和 糖尿病患者ROS介导的微血管功能障碍的分子机制。设计性实验 微血管通透性与共焦和电子联合定量测量 单独灌流的微血管中的显微研究使ROS介导的信号变化成为可能 分子、酶活性和血管结构与血管屏障的变化直接相关 功能。新培育的Nrf2基因敲除大鼠将抗氧化剂防御基因修改为 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。从这项建议中得出的结果将提供新的信息，用于使用 动物、器官或血管床使用培养的内皮细胞进行研究，并提供更好的 了解糖尿病相关微血管并发症的发病机制并使其受益 靶向疗法的发展。

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.