Aims: My overall objective is to understand mechanisms controlling lung endothelial permeability that may predispose to pulmonary edema. Previous work indicates ligation of the alpha(v)beta(3) integrin receptor on lung endothelium by the complement products SC5b-9 and multimeric vitronectin, increases lung endothelial permeability. Here, l will test the hypothesis that the underlying mechanism is attributable to alpha(v)beta(3) induced protein tyrosine phosphorylation of endothelial proteins that lead to further signalling events. The time course, concentration dependence and protein profile of tyrosine phosphorylation following alpha(v)beta(3) ligation with SC5b-9 and multimeric vitronectin, will be determined. The extent to which alpha(v)beta(3) induced tyrosine phosphorylation recruits a signalling pathway through phospholipase C-gamma (PLC), culminating in cytosolic calcium ([Ca2+]i) increases, in activation of phospholipase D (PLD) and protein kinase C (PKC), and in endothelial stress fiber formation will be determined. To understand endothelial regulation of the alpha(v)beta(3) integrin receptor, expression and distribution of the receptor will be determined by immunohistochemistry and by in situ hybridization. Procedures: Cultured monolayers of lung endothelial cells (EC) will be exposed to the alpha(v)beta(3) ligands, SC5b-9 and multimeric vitronectin. Subsequently, protein tyrosine phosphorylation will be determined by western blotting methods using antiphosphotyrosine. Tyrosine phosphorylated proteins will be identified by immunoprecipitation to determine whether signalling kinases such as FAK (focal adhesion kinase) or one of several actin associated proteins, are phosphorylated by alpha(v)beta(3) ligation. Endothelial [Ca2+]i will be quantified by the Fura-2 imaging method, and PLD and PKC by assay procedures. Rhodamine- phalloidin fluorescence of EC will be imaged to determine stress fiber formation. Anti-alpha(v)beta(3) will be ligated with a fluorescent antibody to determine alpha(v)beta(3) distribution in EC and intact lung capillaries. In situ hybridization for the alpha(v)beta(3) mRNA will conducted using the cDNA probes for alpha(v)beta(3). Significance: For the first time, this proposal addresses the mechanisms by which the alpha(v)beta(3) integrin of lung endothelium, initiates intracellular signalling. Since this integrin is implicated in increases of lung endothelial permeability attributable to complement activation products, the mechanisms by which alpha(v)beta(3) signals permeability changes must be understood. If preliminary data bear out, this research will prove for the first time that tyrosine phosphorylation is a critical mechanism in the regulation of lung endothelial permeability, particularly in complement activated states. No previous understanding of the role of tyrosine phosphorylation in endothelial permeability regulation exists. This proposed studies are therefore outstandingly novel.
