The structural and functional integrity of the vascular endothelium is critical to normal lung function and vessel wall homeostasis. Injury to the endothelium results in increased vascular permeability and altered endothelial cell (EC) metabolism and function. Oxidants generated by activated inflammatory cells play an important pathogenetic role in EC barrier dysfunction and non-cardiogenic pulmonary edema. Although the mechanisms of oxidant-induced barrier dysfunction are not completely defined, oxidants function as bioregulators of EC signal transduction pathways. This proposal postulates that oxidant-mediated alterations of intracellular signals and effectors play a critical role in the pathophysiology of barrier dysfunction. To investigate this hypothesis, hydrogen peroxide (H2O2), xanthine plus xanthine oxidase, and H2O2 plus sodium ortho-vanadate (diperoxovanadate, DPV) will be employed as model oxidants to define the mechanisms which regulate oxidant-induced barrier dysfunction. Based upon our preliminary data, we will test the hypothesis that """"""""oxidant-mediated protein kinase and phosphatase activities regulate EC barrier function via modulation of effector targets"""""""". ECs derived from several vascular sites will be examined following treatment with physiologically relevant doses of oxidant stress generated by H2O2 or xanthine plus xanthine oxidase. In S.A.#1, the role of protein kinase C (PKC) and PKC isoenzymes in the regulation of oxidant-mediated barrier dysfunction, gap formation, force development and transmonolayer resistance will be characterized. In S.A.#2, oxidant-mediated protein tyrosine phosphorylation modulated by tyrosine kinases and phosphatase will be examined as potential barrier regulatory mechanisms. In S.A.#3, the role of nitric oxide (NO) in the regulation of basal and oxidant- induced barrier dysfunction will be defined. Finally, in S.A.#4, physiologically relevant targets of the transcellular signals generated in response to oxidants will be investigated. Specifically, local adhesion kinase and myosin light chain kinase will be investigated as potential targets for PKC and tyrosine kinases. These studies will provide a better understanding of the molecular mechanisms regulating oxidant-mediated barrier dysfunction, a key feature.
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