The primary objective of this project is to characterize the time course, severity and source of endothelial cell Injury following trauma or ischemia + reperfusion of the coronary and splanchnic vasculatures. Characterization will take the form of physiological, humoral and morphological assessment of endothelial integrity. The physiological assessment will focus on EDRF-dependent and EDRF-independent vasodilator profiles; the humoral evaluation will be related to determining eicosanoid production (e.g., prostacyclin release); and the morphological assessment will concentrate on transmission and scanning electron microscopy of endothelial cells. The three experimental settings of potential endothelial injury to be studied are (a) myocardial ischemia + reperfusion, (b) splanchnic ischemia + reperfusion, and (c) traumatic shock. In vitro findings (e.g., vascular ring preparations, isolated perfused organs) will be correlated with in vivo studies performed in the intact animal. Particular attention will be paid to the precise time course of endothelial injury following reperfusion in all models. Moreover, studies will be conducted to determine the role of oxygen derived free radicals (e.g., superoxide, hydroxyl ions) using appropriate free radical scavengers, as well as the role of other mediators of endothelial injury including cytokines (e.g., TNFalpha) and adhesive proteins and analysis of subsequent lipid peroxidation resulting from the action of oxygen derived free radicals. Appropriate blocking agents including human superoxide dismutase (h-SOD) against superoxide ions (e.g., N-2-mercaptoproprionyl glycine), against hydroxyl ions, Transforming Growth Factor (TGF-beta) against cytokines especially TNFalpha and against the expression of integrins, the monoclonal antibody (RI5.7) directed against the common subunit (CD/18) of the LFA-1 heterodimer family of adhesive proteins, acting against neutrophil and endothelial adhesive proteins. Additionally, we will study the interaction of neutrophils with the endothelium by studying perfused hearts and isolated coronary and mesenteric artery rings in the presence and absence of neutrophils and by measuring myeloperoxidase, a specific neutrophil marker in intact animals subjected to ischemia and reperfusion. These findings should provide valuable basic information on the pathophysiology of myocardial ischemia, bowel ischemia, and traumatic shock, and should open up new therapeutic approaches to these potentially lethal circulatory disorders.
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