Under conditions of environmental or ischemic stress, the lungs exhibit a high degree of immunoreactivity and develop a procoagulant phenotype, leading to leukocyte recruitment and microvascular thrombosis. We hypothesized that endothelial cell (EC) expression of a transmembrane protein, CD39 (ectoapyrase), which metabolizes ATP and ADP in the releasate of activated cells, provides a critical mechanism by which ECs inhibit intravascular thrombosis (by inhibiting ADP-mediated platelet/platelet recruitment) and modulate leukocyte traffic(by reducing adhesion receptor expression). Mice null for the CD39 gene, created by deleting exons 4-6 containing the apyrase-conserved domains, exhibit a latent prothrombotic and leukoadhesive phenotype in the setting of unilateral lung ischemia/reperfusion, demonstrating impaired gas exchange and survival. Recombinant soluble CD39, lacking the transmembrane region but retaining apyrase activity, potently suppresses platelet aggregation and leukocyte adhesion to EC monolayers in vitro. Pilot data show that soluble CD39 """"""""reconstituted"""""""" the CD39 null mice to normalize their phenotype and confer functional rescue. Hypoxic CD39 -/-pulmonary ECs also showed increased adhesivity for monocytes and neutrophils, which was reversed by soluble CD39.
The Aims of this project are: (1) To establish the thromboregulatory role of CD39 in the lungs following ischemic, hypoxic, or inflammatory stress, focusing on identification of specific thrombotic or fibrinolytic paradigms which may be modulated by CD39; (2) To determine the leukoregulatory role of CD39 in the lungs following ischemic, hypoxic, or inflammatory stress, and in CD39 +/+ and CD39 -/- pulmonary microvascular ECs exposed to hypoxia or inflammatory mediators. Interactions between leukoadhesive and thrombotic mechanisms will also be studied; and (3) To identify the mechanisms(s) underlying EC CD39-mediated suppression of pulmonary leukosequestration following inflammatory, hypoxic, or ischemic stress, focusing on EC adhesion receptors or their cognate ligands on leukocytes. Overall, experiments will determine how endogenous CD39 modulates thrombosis and recruitment of specific leukocyte cell populations and identify the functional consequences of soluble CD39 administration in in vivo models of pulmonary ischemic, hypoxic, or inflammatory stress. The proposed studies should identify a new CD39-based mechanism of pulmonary vascular thromboregulation and leukoregulation which may lead to a novel therapeutic approach to treat pulmonary ischemia, sepsis, acute respiratory distress syndrome, or other inflammatory/thrombotic diatheses.
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