The primary objective of this project is to determine the mechanisms whereby activated leukocytes induce injury to vascular endothelial cells (EC). The proposal will test the hypothesis that ICAM-1 and VCAM mediated adhesive interactions of leukocytes with endothelial cells and concomitant leukocyte production of H2O2 upregulate the activities of endothelial cell xanthine oxidase (XO) and nitric oxide synthase (NOS), with the attendant enhanced rates of endothelial cell production of - 02-,-NO, and ONOO- mediating cell injury. In vitro models of endothelial cell interactions with ethanol fixed leukocytes and antibodies to adhesion molecules will be used to determine whether ICAM- 1 mediated adhesion of neutrophils and monocytes or VCAM mediated adhesion of mononuclear leukocytes triggers upregulation of XO or NOS activity in aortic, arterial, and venular endothelial cells. Exogenous H2O2 or HOC1 or oxidant scavengers with intact leukocytes will be added to cultured endothelial cells to determine whether generation of reactive oxygen species by adherent leukocytes is required for upregulation of XO or NOS activity. Endothelial cells pre-loaded with calcium sensitive fluorescent dyes or intracellular calcium chelator will be used to determine whether upregulation of XO or NOS activity occurring in response to adhesion of activated leukocytes on the endothelial surface is mediated by increases in cytoplasmic Ca++. Generation of ONOO- within endothelial cells will be confirmed by HPLC analysis of nitrated tyrosine residues on cellular proteins or nitration of exogenous p-hydroxyphenyl acetate added to the endothelial cells prior to incubation with activated leukocytes. The role of 'NO, ONOO-, and -ON in mediating EC cytotoxicity will be determined by measuring release of 14C-adenine from EC incubated with leukocytes in the presence of NOS inhibitor, XO inhibitor, or iron chelator. These studies will elucidate how leukocytes cause injury to vascular endothelium during acute stroke, myocardial infarction, or vasculitis.
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