Endothelial Generation of Reactive Oxygen Species in Lung Ischemia

描述(由申请人提供)：这个项目(目前是ARDS SCOR的一个组成部分)描述了一种新的范例，用于产生与剪切力(即机械转导)的急剧变化相关的活性氧物种(ROS)，导致内皮细胞膜快速去极化，激活膜NADPH氧化酶，产生ROS，钙内流，以及NO释放。相关事件包括细胞激活酶(ERK1/2)、转录因子(核因子-kappaB、AP-1)的激活和细胞增殖。随着ROS生成的增加，细胞内Fe2+的释放导致关键细胞成分的氧化，表现为脂质和蛋白质的氧化。因此，这一机制可能有助于ARDS的病理生理学研究。该项目的一个开创性发现是，只要内皮细胞进行了血流适应，就可以在体外复制缺血介导的ROS生成综合征。我们提出了4个具体目标来研究：1)内皮细胞去极化的机制，重点是KATP通道作为潜在的流量传感器；2)内皮细胞产生ROS的途径，特别是NADPH氧化酶成分及其组装的作用；以及3)与钙内流有关的钙通道，特别是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/抗氧化平衡相关的基因的变化，以及流式细胞术来评估细胞增殖。将使用的实验模型包括大鼠和小鼠的分离肺以及大鼠、小鼠和人类的肺微血管内皮细胞。这些研究将为启动内皮细胞ROS产生和随后的细胞信号转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产生的来源。

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.