The cellular dysfunction that occurs when ischemic skeletal muscle is reperfused is becoming increasingly recognized as one form of acute inflammation in which both chemotactic mediators are released and in which activated neutrophils play a key role. Recent studies from our laboratory indicate that neutrophil adherence to vascular endothelium is required to produce microvascular dysfunction in postischemic skeletal muscle. In vitro evidence indicates that the establishment of adhesive interactions between granulocytes and vascular endothelium is a very complex process that involves a highly coordinated and dynamic interplay among several different adhesion molecules expressed both by the endothelium (eg, ICAM-1 and ELAM-1) and the granulocyte (eg, CD11/CD18 and LAM-1). The overall goal of the projects outlined in this application is to determine the role of those molecular determinants of neutrophil/endothelial cell adhesion in the genesis of microvascular dysfunction induced by ischemia/reperfusion (I/R) in skeletal muscle. These studies will utilize evaluation of vascular permeability and the no-reflow phenomenon as indices of microvascular dysfunction and intravital microscopic techniques to monitor neutrophil/endothelial cell interactions in vivo. In addition, the effect of I/R on cell surface expression of ICAM-1 and ELAM-1 will be evaluated. Immunofluorescence flow cytometry will be used to quantitate changes in surface expression of the leukocyte adhesion molecules, CD11a/CD18,CD11b/CD18, and LAM-1, on neutrophils obtained after I/R relative to control (no ischemia). We propose to determine: (1) the role of the leukocyte adherence molecules CD11a/CD18(LFA-1)CD11b/CD18(Mac-1), and LAM-1 (MEL-14 antigen or LEC- CAM-1) in the increased microvascular permeability and neutrophil infiltration induced by I/R; (2) whether administration of monoclonal antibodies to the endothelial cell adhesive molecules ICAM-1 and ELAM-1 will attenuate postischemic neutrophil infiltration and increased microvascular permeability; (3) the role of neutrophil/endothelial cell adherence reactions in the genesis of the no-reflow phenomenon in postischemic skeletal muscle; and (4) the molecular basis for ischemia/reperfusion-induced leukocyte adherence and emigration in skeletal muscle. These studies should improve our understanding of the role of neutrophil adhesion in the pathogenesis of I/R-induced microvascular dysfunction in skeletal muscle.
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