This competitive renewal requests funding for a program that has been supported by NIH for the past 19 years. The program has demonstrated that ischemia in the lung leads to a reproducible response due to altered mechanotransduction and is characterized by endothelial cell membrane depolarization, activation of cell membrane associated NADPH oxidase (NOX2), generation of reactive oxygen species (ROS), activation of transcription factors (NF(B, AP-1, and others), and endothelial cell proliferation. We postulate that this sequence of events represents a signaling response leading to angiogenesis as a compensatory effort to restore blood flow. Intracellular release of Fe++ in the presence of increased ROS generation can result in oxidation of lipid and protein components leading to cell injury. Evidence of oxidative stress was increased lipid oxidation products and increased protein carbonyls. Thus, this mechanism results in lung signaling but can contribute to pulmonary pathophysiology. This project has made the seminal finding that the syndrome of ischemia-mediated ROS generation can be reproduced with endothelial cells in vitro provided that they have been flow adapted. We propose 4 specific aims to investigate: 1) the role of caveolae in sensing altered shear during ischemia; 2) the pathway for linkage between caveolae and endothelial cell depolarization; 3) the mechanism for ROS generation with ischemia and 4) the role of PI3Kinase/Akt in linking membrane depolarization with ROS production. The studies will utilize the isolated perfused mouse lung preparation, isolated mouse pulmonary microvascular endothelial cells that have been flow adapted in vitro and hind limb ischemia in vivo. These studies will provide additional insights into a novel mechanism for initiation of endothelial ROS generation and subsequent cell signaling. The proposed mechanism is of potential importance as a source of ROS leading to acute lung injury in association with focal vascular obstruction. PROJECT NARRATIVE: The pulmonary endothelium generates reactive oxygen species (ROS) when the blood flow through a vessel is interrupted due to various pathological conditions. Here we propose to identify the elements on the endothelium that lead to assembly of the reactive oxygen species generating machinery. ROS generation with stop of flow causes cell proliferation; thus understanding the mechanism by which these elements respond to stop of flow to produce ROS can help toward strategies to manipulate cell growth and proliferation in obstructed blood vessels. ? ? ?
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