Nitric Oxide-Superoxide Interactions in Endothelial Cell Dysfunction

摘要 内皮细胞(EC)功能障碍是许多糖尿病相关微血管病变的常见致病框架 以及大血管并发症。EC释放的一氧化氮(NO)的生物利用度降低是主要原因 通常用于EC功能障碍的标志物。然而，高血糖诱导的分子机制 没有生物利用度的降低仍然知之甚少。[我们假设糖尿病内皮细胞 功能障碍/NO生物利用度降低是由活性氧(ROS)介导的，是以下因素的结果 内皮细胞水平上NO与超氧化物(O2-)的相互作用增强。互动增加的结果是 在较高的过氧亚硝酸盐(ONOO-)形成中，将一氧化氮合酶(ENOS)的活性从NO产生转移到 氧气的产生和EC的破坏。有害的影响可以通过减少ROS的形成和 集中精神。我们设计了特定的目标来检验这些假说。目的：1.确定EC释放的NO 以及高血糖条件下的O2-和细胞损伤。假设是：i)高糖导致内皮细胞 通过增加ONOO-和O2-的形成而导致的长期功能障碍和II)O2-形成的减少是关键 减少内皮功能障碍。我们将进行以下实验：i)确定HIGH的影响 葡萄糖对eNOS和NA(D)PH表达、NO和超氧化物歧化、内皮细胞脂质过氧化的影响 (过氧亚硝酸盐形成的指标)与人脐静脉内皮细胞(HUVECs)的凋亡 短时间段和长时间段，以及ii)确定是增加NO还是减少O2-的形成 有效预防高血糖的影响。AIM2.建立反应动力学-输运计算模型 模拟Aim1实验，预测EC水平的NO、O2-和ONOO-水平。假设是：i) 由于NO和O2之间的高度相互作用，NO浓度降低而ONOO-增加-即使 高糖短时间内内皮细胞释放的NO增加，II)NO浓度 当O2-形成或浓度降低时，ONOO-浓度增加，ONOO-浓度下降 葡萄糖。Aim3.建立NO、ROS(O2-)和活性氮物种的多尺度计算模型 (RNS；ONOO-)在氧化应激过程中的微循环中的运输。假设是：i) 内皮细胞功能障碍是更高的超氧化物形成的结果，II)ROS形成减少 提高NO的生物利用度和III)增加超氧化物歧化酶水平不仅降低O2-水平，而且 还通过反馈机制提高NO水平，减少NO生成。在欧共体层面，我们将 模拟内皮型一氧化氮合酶的调节以及一氧化氮和O2-的释放。在组织水平上，我们将模拟一定体积的组织 包含小动脉血管并模拟NO、ROS和RNS的运输。这两者结合在一起 实验和计算方法是理解EC分子机制的关键 并研究治疗EC功能障碍相关血管并发症的潜在治疗方法。项目叙事 内皮细胞(EC)功能障碍是许多糖尿病相关微血管病变的常见致病框架 以及大血管并发症。高血糖诱导内皮细胞损伤的分子机制 功能障碍仍然知之甚少。拟议的研究将使用综合计算和 评估高糖氧化应激所致内皮细胞功能障碍的实验方法 在分子、细胞和组织水平上。一体化的实验测量与计算 氧化应激的建模将提供一组最佳参数，不仅可以改善内皮细胞 细胞功能障碍/无生物利用度，但也将指导我们开发与糖尿病相关的疗法 血管并发症。

Abstract Endothelial cell (EC) dysfunction is a common pathogenic framework of many of the diabetes-related micro- and macro- vascular complications. Reduced bioavailability of EC-released nitric oxide (NO) is a primary marker generally used for EC dysfunction. However, the molecular mechanisms of hyperglycemia induced reduced NO bioavailability remain poorly understood. [We hypothesize that the diabetic endothelial cell dysfunction/reduced NO bioavailability is mediated by reactive oxygen species (ROS) and is a results of increased interaction of NO and superoxide (O2-) at the endothelial cell level. The increased interactions results in higher peroxynitrite (ONOO-) formation, shifting nitric oxide synthase (eNOS) activity from NO production to O2¿ production, and EC damage. The deleterious effects can be prevented by reducing ROS formation and concentration. Specific aims are designed to test these hypotheses. Aim1. Determine the EC release of NO and O2- and cell damage in hyperglycemic conditions. Hypotheses are: i) the high glucose causes endothelial dysfunction over long periods by increasing ONOO- and O2- formation and ii) reduction in O2- formation is key to reducing endothelial dysfunction. We will perform the following experiments: i) determining the effect of high glucose on eNOS and NA(D)PH expressions, NO and superoxide releases, endothelial cell lipid peroxidation (an indicator of peroxynitrite formation) and apoptosis in human umbilical vein endothelial cells (HUVECs) over short and long time-periods, and ii) determining whether increasing NO or decreasing O2- formation will be effective in preventing effects of high glucose. Aim2. Develop a reaction kinetic-transport computational model to simulate experiments of Aim1 and predict levels of NO, O2- and ONOO- at EC level. Hypotheses are: i) the NO concentration is reduced and ONOO- is increased due to high interaction between NO and O2- even though the NO release from endothelial cell increases in high glucose over short periods and ii) the NO concentration increases and ONOO- concentration decreases when O2- formation or concentration is reduced in high glucose. Aim3. Develop a multi-scale computational model for NO, ROS (O2-), and reactive nitrogen species (RNS; ONOO-) transport in the microcirculation underlying the process of oxidative stress. Hypotheses are: i) endothelial cell dysfunction is a results of higher superoxide formation, ii) a reduction in ROS formation enhances NO bioavailability and iii) increased superoxide dismutase levels not only reduces the O2- levels but also increases the NO levels, and reduces NO formation through feedback mechanism. At EC level, we will model the regulation of eNOS and the release of NO and O2-. At tissue level, we will model a volume of tissue containing an arteriolar blood vessel and simulate transport of NO, ROS and RNS.] This combined experimental & computational approach is critical in our understanding of molecular mechanism of EC dysfunction and examine the potential therapies to treat EC dysfunction related vascular complications. Project Narrative Endothelial cell (EC) dysfunction is a common pathogenic framework of many of the diabetes-related micro- and macro- vascular complications. The molecular mechanisms of hyperglycemia induced endothelial cell dysfunction remain poorly understood. The proposed research will use integrated computational and experimental approaches to assess endothelial cell dysfunction caused by oxidative stress due to high glucose at the molecular, cellular and tissue levels. The integrated experimental measurements and computational modeling of oxidative stress will provide an optimum set of parameters that will not only improve endothelial cell dysfunction/NO bioavailability but also will guide us in the development of therapies for diabetes related vascular complications.