Endothelial Generation of Reactive Oxygen Species in Lung Ischemia

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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.