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）的生物利用度降低通常是EC功能障碍的主要标志。然而，高血糖诱导一氧化氮生物利用度降低的分子机制仍然知之甚少。[我们假设糖尿病内皮细胞功能障碍/一氧化氮生物利用度降低是由活性氧（ROS）介导的，是一氧化氮和超氧化物（O2-）在内皮细胞水平上相互作用增加的结果。]增加的相互作用导致过氧亚硝酸盐（ONOO-）形成增加，一氧化氮合酶（eNOS）活性从NO生成转向O2生成，并导致EC损伤。可以通过减少ROS的形成和浓度来防止有害影响。设计了特定的目标来检验这些假设。Aim1。测定高血糖条件下EC中NO和O2的释放和细胞损伤。假设是：1)高糖通过增加ONOO-和O2-的形成而导致内皮功能长期障碍；2)O2-形成的减少是减轻内皮功能障碍的关键。我们将进行以下实验：1)确定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）和凋亡的短期和长期影响；2）确定增加NO或减少O2-形成是否能有效预防高糖的影响。Aim2。建立反应动力学输运计算模型，模拟Aim1实验，预测EC水平下NO， O2-和ONOO-的水平。假设是：1)由于NO和O2之间的高度相互作用，NO浓度降低，ONOO-增加，尽管在高葡萄糖条件下内皮细胞释放的NO在短时间内增加；2)当O2-形成或浓度降低时，NO浓度增加，ONOO-浓度降低。Aim3。建立一氧化氮、活性氧（O2-）和活性氮（RNS; ONOO-）在氧化应激过程中微循环运输的多尺度计算模型。假设是：i)内皮细胞功能障碍是超氧化物形成增加的结果；ii) ROS形成减少提高了NO的生物利用度；iii)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的形成。在EC水平，我们将模拟eNOS的调节以及NO和O2-的释放。在组织水平上，我们将模拟含有小动脉血管的组织体积，模拟NO、ROS和RNS的运输。这种实验与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疗法至关重要。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。高血糖诱导内皮细胞功能障碍的分子机制尚不清楚。该研究将采用综合计算和实验方法，从分子、细胞和组织水平评估高葡萄糖引起的氧化应激引起的内皮细胞功能障碍。氧化应激的综合实验测量和计算建模将提供一组最佳参数，这不仅将改善内皮细胞功能障碍/NO的生物利用度，而且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.