HYPOXIA-INDUCED EXOCYTOSIS OF ENDOTHELIAL-CELL WEIBEL-PALADE BODIES MECHANISM FOR RAPID NEUTROPHIL RECRUITMENT AFTER CARDIAC PRESERVATION

The period of hypoxia is an important priming event for the vascular d ysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postisch emic tissue, a process further amplified in an oxidant-rich milieu. Ex posure of human umbilical vein ECs to a hypoxic environment (pO(2) app roximate to 20 torr) stimulated release of von Willebrand factor (vWF) , stored in EC WP bodies, as well as increased expression of the WP bo dy-derived PMN adhesion molecule P-selectin at the EC surface. Increas ed binding of In-111-labeled PMNs to hypoxic EC monolayers (compared w ith normoxic controls) was blocked with a blocking antibody to P-selec tin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) w as sufficient to increase WP body exocytosis. To determine the relevan ce of these observations to hypothermic cardiac preservation, during w hich the pO(2) within the cardiac vasculature declines to similarly lo w levels, experiments were performed in a rodent (rat and mouse) cardi ac preservation/transplantation model. Immunodepletion of recipient PM Ns or administration of a blocking anti-P-selectin antibody before tra nsplantation resulted in reduced graft neutrophil infiltration and imp roved graft survival, compared with identically preserved hearts trans planted into control recipients. To establish the important role of en dothelial P-selectin expression on the donor vasculature, murine cardi ac transplants were performed using homezygous P-selectin deficient an d wild-type control donor hearts flushed free of blood/platelets befor e preservation/transplantation. P-selectin-null hearts transplanted in to wild-type recipients demonstrated a marked (13-fold) reduction in g raft neutrophil infiltration and increased graft survival compared wit h wild-type hearts transplanted into wild-type recipients. To determin e whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS ) was measured in 32 patients during open heart surgery. CS samples ob tained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac prese rvation, priming the vasculature to recruit PMNs rapidly during reperf usion.