Coordination of Pathophysiologic Endothelial Cell Signaling by ROS

描述（由申请人提供）：内皮信号的扩增和传播可能依赖于局部旁分泌因子的释放。活性氧，包括来自活化炎症细胞的超氧化物，在炎症和缺血/再灌注条件下激活内皮细胞。随后，内皮损伤可能加剧炎症和缺血性器官损伤。因此，确定一种减少内皮损伤的方法是必要的，并可能导致治疗缺血性和炎症性疾病的新治疗方法的发展。该研究项目的长期目标是了解超氧化物介导的内皮损伤的分子机制。在本提案中，我们假设超氧化物是一种信号分子，促进内质网Ca2+信号传导并触发线粒体和核应激。指导假说是，超氧化物提高细胞内Ca2+，导致线粒体Ca2+超载，导致线粒体信号传导。细胞外超氧化物选择性诱导Ca2+依赖的去极化独立于其他氧化剂物种。此外，超氧诱发信号激活氧化还原敏感转录因子NF-:B。来自线粒体的细胞外超氧诱发信号的解偶联导致线粒体膜电位保存和内皮细胞存活。该项目的具体目的是研究（specific Aim 1）超氧化物如何在体外和体内触发上游Ca2+信号并选择性地激活肌醇1,4,5-三磷酸受体（InsP3R）；（Specific Aim 2）细胞外超氧化物介导的Ca2+信号在线粒体病理生理中的作用（线粒体氧化还原状态- nadh和谷胱甘肽水平，线粒体ROS生成和线粒体膜电位）；（Specific Aim 3）通过insp3r连接的ROS信号激活内皮炎症机制的核事件。提出的实验应提供以下信息：(A)在氧化应激过程中，InsP3Rs的参与及其在内皮细胞Ca2+信号传导中的选择性作用。(B)内皮功能障碍中线粒体功能障碍与炎症信号的关系。(C)这一结果将在病理生理条件下提供炎症和内皮细胞之间的新的信号联系，并导致新的治疗靶点的发展。肺血管内皮通过将血液信号转化为血管功能，在肺健康中起着关键作用。在脓毒性休克期间，一个重要但未被识别的信号是白细胞来源的超氧化物，它通过动员钙来刺激肺内皮细胞。了解内皮细胞中超氧化物介导的钙信号和线粒体核应激的机制对于阐明超氧化物在生理和病理过程中细胞存活和死亡中的基本作用至关重要。

DESCRIPTION (provided by applicant): Amplification and propagation of endothelial signaling is likely to be dependent upon the release of local paracrine factors. Reactive oxygen species, including superoxide derived from activated inflammatory cells, activate endothelial cells under inflammatory and ischemia/reperfusion conditions. Subsequently, endothelial damage may potentiate inflammation and ischemic organ injury. Determining a means to reduce endothelial damage is therefore warranted, and may lead to the development of novel therapeutic approaches to treat ischemic and inflammatory diseases. The long-term goal of the proposed research project is to understand the molecular mechanisms of superoxide- mediated endothelial injury. In this proposal, we postulate that superoxide is a signaling molecule that facilitates endoplasmic reticulum Ca2+ signaling and triggers mitochondrial and nuclear stress. The guiding hypothesis is that superoxide raises intracellular Ca2+ and leads to mitochondrial Ca2+ overload, resulting in mitochondrial signaling. Extracellular superoxide selectively induces Ca2+-dependent depolarization independent of other oxidant species. Further, superoxide-evoked signals activate the redox sensitive transcription factor NF-:B. The uncoupling of extracellular superoxide-evoked signal from the mitochondria results in mitochondrial membrane potential preservation and endothelial cell survival. The specific aims of this project are to examine (Specific Aim 1) how superoxide triggers upstream Ca2+ signaling and selectively activates inositol 1,4,5-trisphosphate receptors (InsP3R) both in vitro and in vivo; (Specific Aim 2) the role of extracellular superoxide-mediated Ca2+ signaling in mitochondrial pathophysiology (mitochondrial redox status-NADH and glutathione levels, mitochondrial ROS generation and mitochondrial membrane potential); (Specific Aim 3) the nuclear events that activate endothelial inflammatory mechanisms by InsP3R-linked ROS signaling. The proposed experiments should provide information on: (A) the involvement of InsP3Rs and their selective role in endothelial Ca2+ signaling during oxidative stress. (B) The relationship between mitochondrial dysfunction and inflammatory signaling in endothelial dysfunction. (C) This result will provide a novel signaling link between inflammatory and endothelial cells under pathophysiological conditions and lead to the development of novel therapeutic targets. PUBLIC HEALTH RELEVANCE The pulmonary vascular endothelium plays a key role in lung heath by translating blood signals into vascular function. During septic shock, an important and under-recognized signal is leukocyte-derived superoxide, which stimulates pulmonary endothelial cells by mobilizing calcium. Understanding the mechanisms of superoxide-mediated calcium signaling and mitochondria-nuclear stress in endothelial cells are crucial to elucidating the fundamental role of superoxide in both cell survival and death during physiological and pathological processes.