The primary objective of this project is to examine the mechanisms of endothelial activation leading to neutrophil (PMN) adherence in the setting of ischemia-reperfusion, focusing on the role of protein kinase C (PKC) in regulating endothelial P-selectin (CD62P) mobilization and synthesis. It has been shown that CD62P-mediated PMN-endothelial rolling and subsequent adherence and PMN-mediated endothelial injury play critical roles in many in vivo models of important surgical and trauma-related disease processes, including tissue and organ ischemia, shock, and burns. The mechanisms involved in the regulation of endothelial CD62P expression in the setting of ischemia-reperfusion, however, have not been examined in vivo. The central hypothesis of this proposal is that PKC activation plays a key role in the signal-transduction pathway leading to rapid endothelial surface expression of CD62P following stimulation, and that limited, reversible inhibition of this pathway can help define the mechanisms regulating CD62P and its role in PMN-mediated reperfusion injury, potentially leading to modulation of PMN-mediated injury. The effect of PKC activation and inhibition on stimulated endothelial CD62P expression will be examined in vitro in cultured human endothelial cells as well as intact rabbit vena cava endothelium, stimulated by direct PKC activation, pro-adhesive mediators of ischemia-reperfusion, and cellular anoxia and reoxygenation. The effect of PKC inhibition on PMN beta2- integrin expression will also be examined in vitro. The in vivo activation of PKC will then be examined in a rabbit model of isolated tissue ischemia-reperfusion. The effects of PKC modulation on CD62P expression, PMN accumulation, and tissue injury in this setting will be examined using trimethylsphingosine (TMS), a PKC inhibitor that rapidly and reversibly blocks PKC-mediated signal transduction with minimal toxicity. The effect of TMS-induced inhibition of CD62P expression on host defense will also be examined. PKC activation and subcellular translocation associated with ischemia-reperfusion will be examined both in vitro as well as in vivo. The hypothesis that increased CD62P expression occurs following reperfusion, not during ischemia will be tested by examining CD62P surface expression throughout the period of ischemia and reperfusion. Finally, this project will test the hypothesis that prolonged CD62P expression occurs following ischemia-reperfusion and involves de novo synthesis. in addition to initial translocation. This, too, will be tested in vivo, quantifying tissue CD62P mRNA by Northern blot analysis, throughout ischemia and reperfusion. These studies should yield new insights into the basic mechanisms of neutrophil-endothelial interactions in reperfusion injury and potentially suggest new therapeutic strategies to a wide range of important disease processes.
