Coordination of Pathophysiologic Endothelial Cell Signaling by ROS

描述（由申请人提供）：内皮信号的放大和传播可能依赖于局部旁分泌因子的释放。活性氧物质，包括来自活化的炎性细胞的超氧化物，在炎症和缺血/再灌注条件下活化内皮细胞。随后，内皮损伤可加重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，并且可能导致开发新的治疗方法来治疗缺血性和炎症性疾病。本研究的长期目标是了解超氧化物介导的内皮损伤的分子机制。在这个建议中，我们假设，超氧化物是一个信号分子，促进内质网钙信号和触发线粒体和核应激。指导性假设是超氧化物升高细胞内Ca 2+并导致线粒体Ca 2+过载，从而导致线粒体信号传导。细胞外超氧化物选择性地诱导依赖于Ca 2+的去极化，而不依赖于其他氧化剂。此外，超氧化物诱发的信号激活氧化还原敏感性转录因子NF-：B。细胞外超氧化物诱发信号与线粒体的解偶联导致线粒体膜电位保留和内皮细胞存活。该项目的具体目标是审查（具体目的1）超氧化物如何在体外和体内触发上游Ca 2+信号并选择性激活肌醇1，4，5-三磷酸受体（InsP 3R）;（特定目的2）细胞外超氧化物介导的Ca 2+信号在线粒体病理生理学中的作用（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）;（具体目标3）通过InsP 3R连接的ROS信号传导激活内皮炎症机制的核事件。所提出的实验应提供以下信息：（A）InsP 3R的参与及其在氧化应激期间内皮Ca 2+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细胞和内皮细胞之间的新型信号联系，并导致新型治疗靶点的开发。公共卫生相关性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关键作用。在感染性休克期间，一个重要的和未被认识到的信号是白细胞来源的超氧化物，其通过动员钙来刺激肺内皮细胞。了解超氧化物介导的钙信号和内皮细胞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.