ACUTE LUNG INJURY--ROLE OF VASCULAR NAD(P)H OXIDASE

目的：本项目的目标是识别和确定 内源性氧化酶在启动内皮和平滑肌 细胞功能障碍导致肺动脉高压，失调 急性肺损伤（ALI）中炎症和组织损伤。 假设：待检验的总体假设是活性氧 中间体（ROI）在许多类型的ALI中起着关键作用， 血管NAD（P）H氧化酶类似于呼吸爆发氧化酶， 吞噬细胞是高度调节的，对于产生 ROI、氧感测和铁摄取。 具体目标：第一个具体目标将是NAD（P）H的特征 人内皮和肺动脉平滑肌氧化酶系统 细胞，并确定其对ROI生成的贡献。第二 具体的目标将定义细胞因子或缺氧的机制， 指导血管NAD（P）H氧化酶的分子和细胞调节。 第三个具体目标将确定血管NAD（P）H氧化酶的作用， 在肺动脉血管收缩、炎症和血管增加中， ALI的通透性。 研究人员：我们的第一个具体目标的战略是定义和 充分表征负责以下方面的组件的结构： 血管NAD（P）H氧化酶活性，并确定其亚细胞定位 在细胞内。我们还将表征的氧化还原中点电位 血管NAD（P）H氧化酶和确定单个氧化酶的作用 在ROI的生成过程中。 第二个具体目标的策略是描述 细胞因子和缺氧对血管NAD（P）H氧化酶表达的影响 件.在随后的实验中，我们将包含定义元素 在NAD（P）H氧化酶组分的启动子中， 转录调控我们将完成一个功能分析， NAD（P）H氧化酶的5'调控区组分采用分步缺失 构建体，然后通过凝胶定义蛋白质-DNA相互作用 移位和DNA酶足迹法。超位移与核蛋白纯化 将被用来确定假定的交易因素。 我们的第三个具体目标的战略是利用动物和纯化 NAD（P）H氧化酶成分遗传缺陷的细胞直接 确定氧化酶在临床相关模型中的重要性， 肺损伤（再灌注损伤和脓毒症），缺氧性肺损伤 高血压和铁吸收。 意义：这项工作将提供一个更好的理解 血管NAD（P）H氧化酶在临床相关模型中的重要性 急性肺损伤和ARDS。结果将具有广泛的重要性， 确定血管NAD（P）H氧化酶在许多其他疾病中的作用 疾病和正常生理。

OBJECTIVE: The goal of this project is to identify and determine the role of endogenous oxidases in initiating the endothelial and smooth muscle cell dysfunction that leads to pulmonary hypertension, dysregulated inflammation and tissue injury in acute lung injury (ALI). HYPOTHESIS: The overall hypothesis to be tested is that reactive oxygen intermediates (ROI) play a critical role in many types of ALI and that a vascular NAD(P)H oxidase analogous to the respiratory burst oxidase of phagocytes is highly-regulated and critically important for generation of ROI, oxygen sensing and iron uptake. SPECIFIC AIMS: The first specific aim will characterize the NAD(P)H oxidase system of human endothelial and pulmonary artery smooth muscle cells and determine its contribution to the generation of ROI. The second specific aim will define the mechanisms by which cytokines or hypoxia direct the molecular and cellular regulation of vascular NAD(P)H oxidase. The third specific aim will determine the role of vascular NAD(P)H oxidase in pulmonary artery vasoconstriction, inflammation and increased vascular permeability in ALI. RESEARCH PLAN: Our strategy for the first specific aim is to define and fully characterize the structure of the components responsible for vascular NAD(P)H oxidase activity and pinpoint their subcellular location within the cell. We will also characterize the redox midpoint potential of vascular NAD(P)H oxidase and determine the role of individual oxidase components in the generation of ROI. Our strategy for the second specific aim is to delineate the effects of cytokines and hypoxia on the expression of vascular NAD(P)H oxidase components. In subsequent experiments, we will inclusively define elements in the promoters of the NAD(P)H oxidase components that account for transcriptional regulation. We will complete a functional analysis of the 5' regulatory region of the NAD(P)H oxidase components using step deletion constructs, followed by the definition of protein-DNA interactions via gel shift and DNase footprinting. Supershift and nuclear protein purification will be used to identify putative transacting factors. Our strategy for the third specific aim is to utilize animals and purified cells with genetic deficiencies of NAD(P)H oxidase components to directly establish the importance of the oxidase in clinically relevant models of lung injury (reperfusion injury and sepsis), hypoxic pulmonary hypertension, and iron uptake. SIGNIFICANCE: The work will provide a better understanding of the importance of vascular NAD(P)H oxidase in clinically relevant models of acute lung injury and in ARDS. The results will be of broad importance to determining the roles of vascular NAD(P)H oxidase in a host of other diseases and in normal physiology.