Nitric Oxide-Superoxide Interactions in Endothelial Cell Dysfunction

描述(由申请人提供)：摘要内皮细胞(EC)功能障碍是许多糖尿病相关微血管和大血管并发症的常见致病框架。EC释放的一氧化氮(NO)的生物利用度降低是EC功能障碍的主要标志。然而，高血糖导致NO生物利用度降低的分子机制仍不清楚。[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，是内皮细胞水平上NO和超氧化物(O2-)相互作用增加的结果。相互作用的增加导致过亚硝酸根(ONOO-)的形成增加，一氧化氮合酶(ENOS)活性从NO产生转移到O2产生，并导致EC损伤。可以通过减少ROS的形成和浓度来预防这种有害影响。我们设计了特定的目标来检验这些假说。目的：1.测定高血糖条件下EC释放NO和O2-及细胞损伤情况。假说是：1)高糖通过增加ONOO-和O2-的生成而导致长时间的内皮功能障碍；2)减少O2-的生成是减轻内皮功能障碍的关键。我们将进行以下实验：i)检测高糖对人脐静脉内皮细胞(HUVECs)eNOS和NA(D)PH表达、NO和超氧化物释放、内皮细胞脂质过氧化(过氧亚硝酸盐形成的指标)和细胞凋亡的影响，以及ii)确定增加NO或减少O2-的生成是否对高糖的影响有效。AIM2.建立反应动力学-输运计算模型，模拟Aim1的实验，预测EC水平上的NO、O2-和ONOO-水平。假设：1)由于NO与O2的高度相互作用，即使在短期内高糖时内皮细胞释放的NO增加，但NO浓度降低，而ONOO-浓度增加；ii)当高糖中O2-的生成或浓度降低时，NO浓度增加，ONOO-浓度降低。Aim3.为氧化应激过程中微循环中的NO、ROS(O2-)和活性氮物种(RNS；ONOO-)的传输开发一个多尺度计算模型。假设：1)内皮细胞功能障碍是超氧化物生成增加的结果；2)ROS生成减少提高了NO的生物利用度；3)超氧化物歧化酶水平升高不仅降低了O2-水平，还增加了NO水平，并通过反馈机制减少了NO的生成。在EC水平上，我们将模拟eNOS的调节以及NO和O2-的释放。在组织水平上，我们将模拟包含小动脉血管的组织体积，并模拟NO、ROS和RNS的运输。]这种实验和计算相结合的方法对于我们理解EC功能障碍的分子机制和研究治疗EC功能障碍相关血管并发症的潜在治疗方法至关重要。公共卫生相关性：内皮细胞(EC)功能障碍是许多糖尿病相关微血管和大血管并发症的常见致病框架。高血糖引起血管内皮细胞功能障碍的分子机制尚不清楚。这项拟议的研究将使用综合的计算和实验方法，在分子、细胞和组织水平上评估高糖引起的氧化应激导致的内皮细胞功能障碍。氧化应激的综合实验测量和计算模型将提供一组最优参数，不仅将改善内皮细胞功能障碍/NO的生物利用度，还将指导我们开发糖尿病相关血管并发症的治疗方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.