Project 1 characterizes the cellular and molecular mechanisms which mediate the acute inflammatory response and tissue injury ensuing upon reperfusion of the previously ischemic myocardium in a canine model. It takes advantage of a unique model which we have continually developed in which we are able to: 1) Cannulate the cardiac lymph duct so that we have access to cardiac extracellular fluid with a brief (1-2 minute) lag time which enables longitudinal temporal sampling during the ischemia reperfusion protocol. 2) By calibrating multiple tissue samples with respect to ischemic coronary blood flow and time of reperfusion we can correlate histologic, immunohistochemical, and molecular features and 3) Utilizing the above, we can exploit several independent strategies for measuring leukocyte trafficking as a function of the duration and extent of ischemia and duration of reperfusion. The experiments elucidate temporal relationships and pathological correlations by utilizing molecular and antibody probes to study induction of cytokines and adhesion molecules in various cell populations in vivo and in vitro. In addition, monoclonal antibodies and cell or molecular - specific inhibitors are utilized to evaluate the role of specific cellular mediator events. Each hypothesis is tested by several independent experimental strategies; the overall goals are highly concordant with those in projects 2 and 3. The four specific aims are designed to: 1) Characterize chemotactic signals mediating leukocyte integrin activation, 2) Examine the relationship between leukocyte influx, cell adhesion and cytokine induction during reperfusion, 3) Examine the induction of both tissue and soluble forms of ICAM-1 and VCAM-1 and 4) Compare the cell biology of inflammation during reperfusion after longer occlusion times with the mechanisms elucidated for a one hour occlusion.
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