This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. 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 beta2-integrin-mediated PMN- endothelial 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 burn. Beta2-integrin blockade, however, increases susceptibility to infection, limiting that approach as a therapeutic strategy. Blocking leukocyte rolling, which precedes integrin-mediated adherence, by blocking endothelial CD62P does not increase the risk of infection, yet is equally effective in attenuating injury. The mechanisms involved in endothelial P-selectin (CD62P) signal transduction in the setting of ischemia- reperfusion 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 mechanism regulating CD62P and its role in PMN-mediated reperfusion injury, potentially leading to modulation of PMN-mediated injury. The effect of PKC inhibition on stimulated endothelial CD62P expression will first be examined in ex vivo rabbit vena cava endothelium using immunohistochemical techniques. The in vivo effects of PKC modulation on CD62P expression, PMN accumulation, and tissue injury will then be examined in a rabbit model of isolated tissue ischemia-reperfusion, using trimethylsphingosine (TMS), a PKC inhibitor with a less than 5 minute half-life that rapidly and reversibly blocks PKC-mediated signal transduction with minimal toxicity. This will potentially allow """"""""selective"""""""" in vivo examination of """"""""early"""""""" PKC-mediated signal transduction processes (as with CD62P) vs late. The effect of TMS-induced inhibition of CD62P expression on host defense will also be examined. 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. It is hoped that these studies will 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.
