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 permeability and altered endothelial cell (EC) metabolism and function. Oxidants generated by activated inflammatory cells play and important pathogenic 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, xanthine plus xanthine oxidase, and H2O2 plus sodium orthovanadate (diperoxovanadate, DPV) will be employed as model oxidants to define the mechanisms which regulate oxidant-mediated 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 X/XO.
In Specific Aim #1, overexpression and characterization of PKCalpha and epsilon in immortalized ECs will be carried out.
In Specific Aim #2, the role of PKC and PKC isoenzymes in the regulation of oxidant-mediated barrier dysfunction will be characterized.
In Specific Aim #3, oxidant-mediated protein tyrosine phosphorylation modulated by tyrosine kinases and phosphatases will be examined as potential barrier regulatory mechanisms. Finally, in Specific Aim #4, physiologically relevant targets of the transcellular signals generated in response to oxidants will be investigated. Specifically, focal adhesion protein 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, as key feature in the pathophysiology of oxidant-mediated vascular disorders.
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