Thrombo-Inflammatory Role of CD39 In Vascular Stasis

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

? DESCRIPTION (provided by applicant): Limb immobilization after orthopedic surgery or prolonged travel can trigger deep vein thrombosis (DVT), which can result in embolization and death. This proposal models vascular flow in vitro and utilizes two surgical models of DVT to examine the relationship between vascular stasis, hypoxia, and a critical ectonucleotidase (CD39) which acts at the blood-vessel interface to maintain or restore homeostasis. CD39 is a membrane-spanning endothelial (and leukocyte) ectoenzyme, which catalyzes the phosphohydrolysis of extracellular ATP and ADP. By metabolically deleting these prothrombotic and proinflammatory danger signals released by injured and inflamed vessels, CD39 may prevent an explosive cascade of platelet aggregation and leukocyte recruitment in vessels made susceptible by flow impingment and/or the low oxygen tension environment characteristic of venous valve pockets. Using globally-deficient CD39 mice we have created through floxed CD39/EIIa Cre mouse intercrosses, our preliminary data shows for the first time that native CD39 acts to impede development of thrombus and inflammation in a murine model of DVT. Furthermore, CD39 antigen can be detected in mouse plasma and shed exosomes, where it may participate as a downstream thrombosis danger signal. The overall hypothesis to be tested is that CD39 is a critical endogenous suppressor of venous thrombosis/inflammation and that CD39-bearing extracellular vesicles may be shed to mark danger in DVT and protect downstream vascular segments. The Specific Aims of this project are: (1) To elucidate the molecular dynamics of endothelial CD39 expression under static or low-flow fluid shear. These experiments will use state-of-the art molecular techniques for ascertaining endothelial CD39 gene-promoter interactions driving response to laminar fluid shear or stasis; (2) To determine the effects of vascular stasis with or without systemic hypoxia on (i) local vein wall CD39 expression and activity; and (ii) release of CD39 into the circulation on leukocytes or CD39-bearing extracellular vesicles. Studies will leverage novel assays we have developed to quantify both CD39 antigen and nucleotidase activity of CD39-bearing exosomes shed into the circulation; (3) To ascertain the role for CD39 in in situ thrombus accretion and vascular wall inflammation under conditions of venous stasis or low flow, with or without systemic hypoxia. These experiments will use our unique strains of global- and cell lineage (macrophage, endothelial)-specific CD39 gene-deleted and - overexpressing mice, and test them in robust venous stasis and low-flow models of DVT. CD39 will be augmented (or reconstituted in knockout mice) via adoptively transferred leukocytes, or administration of recombinant soluble CD39 or immunopurified CD39-bearing exosomes. Studies should provide new insights into DVT pathogenesis and provide a window for understanding potential translation of our discoveries into new therapeutic approaches to mitigate the thromboinflammatory response to vascular stasis and hypoxemia.