Following ischemic injury of the myocardium, reperfusion is associated with a rapid influx of neutrophils (PMNs) that may cause additional myocardial damage. Adhesion between PMNs and vascular endothelial cells altered by ischemia/reperfusion appears to be essential for initial targeting of inflammatory cells, as well as for intravascular accumulation, transendothelial migration, and maximal release of free radicals and oxidants from PMNs. The goal of this Project is to elucidate the cellular mechanisms contributing to PMN-endothelial adhesion in post-ischemic myocardial inflammation. We have found, rising human aortic endothelial cell monolayers loaded with the fluorescent Ca2+ indicator indo-1, that initial attachment of a PMN causes a rapid large rise in endothelial cell Ca2+ concentration ([Ca2+]i), similar to the rise seen with agonists that increase adhesiveness of endothelial cells. The Ca2+ source and the intracellular signalling pathway responsible for the PMN-induced rise in {Ca2+]i will be determined, as will the importance of this rise in promoting subsequent PMN adhesion, expression of endothelial P-selectin and ICAM-1, and synthesis of the chemoattractant platelet activating factor (PAF). The in-vivo time course of expression of cell adhesion molecule protein and mRNA (for P-selectin, E-selectin, and ICAM-1) will be characterized in epicardial coronary arteries and veins, myocardial microvessels, and cardiac myocytes following ischemia/reperfusion. The role of oxygen radicals, endothelial derived nitric oxide, and PMNs in inducing the in-vivo expression of these adhesion molecules will be examined. Functional adhesion assays, immunohistology, immunoelectron microscopy, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR will be used to identify an localize the presence of new adhesion molecules or their genetic message within cells and in specific cell types. The ability of different """"""""anti-adhesion"""""""" therapies (treatments which prevent interaction between PMNs and endothelial cells) to limit myocardial infarct size when given before reperfusion will be determined. Treatments for which there is limited information (soluble selectin-IgG chimeras and small carbohydrate molecules to block selectin/ligand interactions) will be tested in the rat. Other treatments (including an anti-P-selectin antibody, and a PAF receptor antagonist) will be tested in dogs reperfused for 48 hr to provide more definitive information on infarct size, in combination with an assessment of global and regional left ventricular function. Additional studies will be performed with anti CD18 antibodies to determine the time limits after reperfusion within which anti-adhesion therapy is effective, and long term studies will be done to assess the safety of such therapy on infarct healing and susceptibility to infection. The proposed studies should provide important information about the role of PMN-endothelial adhesion in post-ischemic myocardial inflammation and should lead to the development of effective methods to inhibit the inflammatory response and related myocardial injury
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