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.
Clots which form in the deep veins of the leg or pelvis often break off and travel to the lungs, where they can cause vascular collapse and death. This proposal examines a natural mechanism by which an enzyme which degrades harmful molecules released from injured or dying cells can protect the body from deep vein clot formation. This enzyme lines the blood vessel surface and coats cells in the circulation, which can protect against clot formation, clot extension, or inflammation and reduce clot burden or recurrence. Experiments use a mouse model of deep vein clot formation using unique mice in which levels of the protective enzyme have been genetically altered, and also studies cells in culture to mimic blood flow in a vein. Ultimately, the goal is to develop means for boosting levels of the enzyme to protect against the devastating effects of deep vein clot formation.
| Knight, Jason S; Mazza, Levi F; Yalavarthi, Srilakshmi et al. (2018) Ectonucleotidase-Mediated Suppression of Lupus Autoimmunity and Vascular Dysfunction. Front Immunol 9:1322 |
| Pinsky, David J (2018) CD39 AS A CRITICAL ECTONUCLEOTIDASE DEFENSE AGAINST PATHOLOGICAL VASCULAR REMODELING. Trans Am Clin Climatol Assoc 129:132-139 |
| Baek, Amy E; Sutton, Nadia R; Petrovic-Djergovic, Danica et al. (2017) Ischemic Cerebroprotection Conferred by Myeloid Lineage-Restricted or Global CD39 Transgene Expression. Circulation 135:2389-2402 |
| Sutton, Nadia R; Hayasaki, Takanori; Hyman, Matthew C et al. (2017) Ectonucleotidase CD39-driven control of postinfarction myocardial repair and rupture. JCI Insight 2:e89504 |
| Meng, He; Yalavarthi, Srilakshmi; Kanthi, Yogendra et al. (2017) In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody-Mediated Venous Thrombosis. Arthritis Rheumatol 69:655-667 |
| Petrovic-Djergovic, Danica; Goonewardena, Sascha N; Pinsky, David J (2016) Inflammatory Disequilibrium in Stroke. Circ Res 119:142-58 |
| Visovatti, Scott H; Hyman, Matthew C; Goonewardena, Sascha N et al. (2016) Purinergic dysregulation in pulmonary hypertension. Am J Physiol Heart Circ Physiol 311:H286-98 |
| Kanthi, Yogendra; Hyman, Matthew C; Liao, Hui et al. (2015) Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis. J Clin Invest 125:3027-36 |
