Increased generation of reactive oxygen species (ROS) in the vasculature has been linked to inflammation, vascular leakiness, atherosclerosis and other cardiovascular disorders. In the vascular endothelium, NADPH oxidase has been identified as a major source of ROS, which regulates signaling pathways involved in endothelial cell growth, migration, cytoskeletal organization and barrier function. The endothelial NADPH oxidase, similar to phagocytic oxidase, consists of cytosolic (p47phox, p67phox and Rac1) and membranebound (gp91phox and p22phox) components. Recently, several homologues of Nox 2 (gp91phox) namely Nox1, Nox3, Nox4 and Nox5 have been identified. We and others have found that Nox4 mRNA is expressed at much higher levels, compared to Nox2, in the endothelial cells from different vascular beds;however, the role of Nox4 in ROS production and endothelial cell functions is not well understood. In preliminary experiments, we have identified that Nox4 is not only involved in hyperoxia- and TNF-alpha-induced superoxide/ROS production but also in endothelial cell signal transduction regulating motility and capillary tube formation. The overall hypothesis of this proposal is that "Nox4 plays a key role in regulating endothelial signaling pathways involved in superoxide/ROS production, migration and capillary tube formation". This hypothesis will be tested in primary human lung endothelial cells and in genetically modified Nox4 and Nox2 (gp91phox) mice using hyperoxia- and TNF-alpha as modulators of NADPH oxidase. Specific Aim 1: To characterize expression of Nox 4 in HPAECs and mouse lung ECs and determine its role in ROS production;Specific Aim 2: To investigate molecular mechanisms of increased expression and activation of Nox4 by hyperoxia in human lung endothelial cells;Specific Aim 3: To characterize signaling pathways that regulate Nox4 dependent endothelial cell migration and capillary tube formation in response to hyperoxia;Specific Aim 4: To investigate the role of Nox4 in ROS generation and vascular leakiness in an in vivo murine model of lung injury. These experiments will provide novel and new insights into the role of Nox4, as a component of NADPH oxidase, in regulating endothelial cell ROS production and function that may lead to development of therapeutic strategies minimizing lung injury.