The goal of this project is to identify and determine the role of endogenous oxidases in initiating the endothelial and smooth muscle cell dysfunction that leads to pulmonary hypertension, dysregulated inflammation and tissue injury in acute lung injury (ALI). HYPOTHESIS: The overall hypothesis to be tested is that reactive oxygen intermediates (ROI) play a critical role in many types of ALI and that a vascular NAD(P)H oxidase analogous to the respiratory burst oxidase of phagocytes is highly-regulated and critically important for generation of ROI, oxygen sensing and iron uptake.
SPECIFIC AIMS : The first specific aim will characterize the NAD(P)H oxidase system of human endothelial and pulmonary artery smooth muscle cells and determine its contribution to the generation of ROI. The second specific aim will define the mechanisms by which cytokines or hypoxia direct the molecular and cellular regulation of vascular NAD(P)H oxidase. The third specific aim will determine the role of vascular NAD(P)H oxidase in pulmonary artery vasoconstriction, inflammation and increased vascular permeability in ALI. RESEARCH PLAN: Our strategy for the first specific aim is to define and fully characterize the structure of the components responsible for vascular NAD(P)H oxidase activity and pinpoint their subcellular location within the cell. We will also characterize the redox midpoint potential of vascular NAD(P)H oxidase and determine the role of individual oxidase components in the generation of ROI. Our strategy for the second specific aim is to delineate the effects of cytokines and hypoxia on the expression of vascular NAD(P)H oxidase components. In subsequent experiments, we will inclusively define elements in the promoters of the NAD(P)H oxidase components that account for transcriptional regulation. We will complete a functional analysis of the 5' regulatory region of the NAD(P)H oxidase components using step deletion constructs, followed by the definition of protein-DNA interactions via gel shift and DNase footprinting. Supershift and nuclear protein purification will be used to identify putative transacting factors. Our strategy for the third specific aim is to utilize animals and purified cells with genetic deficiencies of NAD(P)H oxidase components to directly establish the importance of the oxidase in clinically relevant models of lung injury (reperfusion injury and sepsis), hypoxic pulmonary hypertension, and iron uptake. SIGNIFICANCE: The work will provide a better understanding of the importance of vascular NAD(P)H oxidase in clinically relevant models of acute lung injury and in ARDS. The results will be of broad importance to determining the roles of vascular NAD(P)H oxidase in a host of other diseases and in normal physiology.
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