Endothelial Generation of Reactive Oxygen Species in Lung Ischemia

描述（由申请人提供）：该项目（目前是ARDS SCOR的一个组成部分）描述了活性氧（ROS）的产生与剪切应力（即机械转导）的急性改变相关的新范式，导致内皮细胞膜快速去极化，膜NADPH氧化酶的激活与ROS的产生，Ca2+内流和NO释放。相关事件包括细胞激酶（ERK1/2）的激活，转录因子（Nf-kappaB, AP-1）的激活和细胞增殖。细胞间释放Fe2+与ROS生成增加相关，导致关键细胞成分氧化，表现为脂质和蛋白质氧化。因此，这一机制可能与ARDS的病理生理有关。这个项目的一个重要发现是，缺血介导的ROS生成综合征可以在体外用内皮细胞复制，只要它们已经适应了流动。我们提出了四个具体的研究目标：1)内皮细胞去极化的机制，重点是KATP通道作为电位流量传感器；2)内皮生成ROS的途径，特别是NADPH氧化酶组分及其组装的作用；3)与钙内流相关的Ca2+通道，特别强调t型电压门控钙通道。Aim 4将使用蛋白质/DNA阵列技术来确定转录因子的激活，基因阵列技术来评估与细胞增殖和氧化剂/抗氧化平衡相关的基因的改变，以及流式细胞术来评估细胞增殖。将使用的实验模型包括从大鼠和小鼠身上分离的肺，以及从大鼠、小鼠和人类身上提取的肺微血管内皮细胞。这些研究将为内皮细胞ROS生成和随后的细胞信号传导的新机制提供更多的见解。这一机制在ARDS中具有潜在的重要性，因为它是与局灶性血管阻塞相关的ROS生成的来源。

DESCRIPTION (provided by applicant): This project (currently a component of the SCOR in ARDS) has described a novel paradigm for generation of reactive oxygen species (ROS) associated with acute alterations in shear stress (i.e., mechanotransduction) leading to rapid endothelial cell membrane depolarization, activation of membrane NADPH oxidase with ROS generation, Ca2+ influx, and NO release. Associated events include activation of cellular kinases (ERK1/2), activation of transcription factors (Nf-kappaB, AP-1) and cell proliferation. Intercellular release of Fe2+ associated with increased generation ROS results in oxidation of key cellular components manifested as lipid and protein oxidation. Thus, this mechanism can contribute to the pathophysiology of ARDS. A seminal finding of this project has been that the syndrome of ischemia-mediated ROS generation can be reproduced with endothelial cells in vitro provided they have been flow adapted. We propose 4 specific aims to investigate: 1) the mechanism for endothelial cell depolarization with emphasis on KATP channels as the potential flow sensor; 2) the pathway for ROS generation by the endothelium and in particular the role of NADPH oxidase components and their assembly; and 3) Ca2+ channels associated with calcium influx with special emphasis on the T-type voltage gated calcium channels. Aim 4 will use protein/DNA array technology to determine activation of transcription factors, gene array technology to evaluate alterations in genes associated with cell proliferation and oxidant/antioxidant balance, and flow cytometry to evaluate cell proliferation. The experimental models that will be used include isolated lungs from rats and mice and pulmonary microvascular endothelial cells from rats, mice and humans. These studies will provide additional insights into a novel mechanism for initiation of endothelial ROS generation and subsequent cell signaling. This mechanism is of potential importance in ARDS as a source of ROS generation in association with focal vascular obstruction.