The adhesion of leukocytes and platelets to endothelial cells is an early manifestation of the microvascular dysfunction that is elicited by ischemia and reperfusion (I/R). While leukocytes are generally recognized as key mediators of the microvascular dysfunction and tissue injury associated with I/R, relatively little is known about the role of platelets in this pathologic process. The work outlined in this project will focus on defining the mechanism that underlies the recruitment of adherent platelets in postischemic intestinal venules and will determine if I/R-induced leukocyte recruitment in postcapillary venules is dependent on platelet-endothelial cell (P/E) adhesion. Intravital fluorescence microscopy and the dual radiolabeled monoclonal antibody technique will be used to test our central hypothesis that ischemia/reperfusion promotes the adhesion of platelets to venular endothelium by altering the balance between nitric oxide and superoxide production, and that the resultant platelet-endothelial cell adhesion modulates the recruitment of leukocytes via the expression of P-selectin on platelets that are adherent to the vessel wall. This I/R-induced adhesion of platelets in intestinal venules is more pronounced when venular shear rate is reduced in the postischemic period.
Five specific aims are proposed: 1) to define the influence of varying durations of ischemia, followed by reperfusion, on the adhesion of platelets and leukocytes in intestinal postcapillary venules, the expression of P-selectin, and the expression/activity of nitric oxide- and superoxide-producing enzymes, 2) to define the dependence of P/E adhesion on venular shear rate and to determine the adhesion glycoproteins that mediate the shear rate-dependent P/E adhesion, 3) to determine if I/R-induced leukocyte recruitment in postcapillary venules is dependent on platelet-endothelial cell adhesion & P-selectin expression on platelets and/or endothelial cells, 4) to define the contribution of nitric oxide to I/R-induced P/E adhesion and microvascular dysfunction, and 5) to define the contribution of superoxide in mediating I/R-induced P/E adhesion and microvascular dysfunction. Mutant mouse models and specific pharmacological agents will enable us to dissect the contributions and sources of both superoxide and nitric oxide in mediating the early and late recruitment of both platelets and leukocytes into the postischemic microvasculature.
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