The endothelial redox state is an important regulatory factor governing PMN extravasation. Using intravital microscopy to monitor postcapillary venules, we observed that oxidant exposure increased the number of rolling neutrophils (PMN), indicative of increased adhesion. Our in vitro data indicates that exposure of endothelial cells (EC) to subcytotoxic concentrations of H202 increased the transendothelial migration of PMN. To date, the mechanisms by which PMN adhesion and transmigration occurred in response to oxidants and regulated remain controversial and undefined. Further, the regulation of leukocyte transmigration through endothelial junctions is the least-understood step in the extravasation process. In this application, we propose to investigate the hypothesis that increased oxidative stress signals the regulation of adhesion molecules and junctional proteins of endothelial cells promoting PMN adhesion and transmigration. We will investigate the effects of acute and long-term exposure to H202 or that induced by hypoxia/reoxygenation on the expression of mRNA and protein, the organization and function of surface adhesion molecules (i.e., ICAM-1, P- and E-selectins) and whether they modulate PMN transmigration. Also, we propose to investigate whether oxidative stress alters the organization, expression, and function of junctional proteins (i.e., cadherins, occludin, and PECAM-1) and their importance in PMN transmigration. Preliminary data also showed that oxidant-stimulated EC released a bio-active factor(s) which promoted PMN transmigration and activated Ca2+ transients. The biochemical nature and function of the EC-derived factor(s) will be defined. The oxidant-responsive signalling mechanisms in EC (i.e., protein kinase C isoforms, tyrosine kinases, and the ras-MAPK cascade) will be identified and investigated for their functions in regulating adhesion molecules, junctional proteins, PMN adhesion and transmigration. To evaluate causal relationships between a signalling component and effector function, three methods of gene transfer (retrovirus vector, tetracycline-responsive plasmids, and adenovirus vectors) into EC will be used to selectively decrease endogenous gene expression or reduced kinase function with kinase- negative mutants of the oxidant-responsive protein kinase. The proposal will provide important information in the area of oxidative stress and endothelial dysfunction, which is the basis for a wide range of vascular diseases such as atherosclerosis, ischemia/reperfusion injury, diabetes, and the adult respiratory distress syndrome.
