Few effective treatments exist for ischemic stroke. Interruption of the brain's blood supply triggers innate immune mechanisms at the brain/blood vessel interface which exacerbate the primary injury caused by lack of nutritive flow. When cells are injured or die, extravasation of intracellular nucleotides sets off a series of primordial dangr signals which drive inflammation and microvascular coagulation in the ischemic brain. These danger signals are suppressed locally by the vascular ectonucleotidases CD39 and CD73, which sequentially phosphohydrolyze ATP, ADP, and AMP to generate adenosine, dissipating proinflammatory and procoagulant signaling in platelets and leukocytes. New data indicate that extracellular microvesicles (microparticles and exosomes) express CD39 which can be enzymatically active, and shed in stroke. Mice we have engineered to lack CD39, or mice lacking CD73, exhibit larger infarcts following middle cerebral artery occlusion, and recombinant / soluble nucleotidases can reduce the ischemic damage. These studies identified leukocyte surface CD39 as a key auto-suppressor of leukocyte influx into ischemic tissue. New data suggest that after stroke, CD73 modulates innate immune effector mechanisms including costimulatory molecule expression on microglia (CD40-CD40L and CD80/86), chemokine secretion, and expression of the leukocyte chemorepulsion/neuronal guidance factor netrin-1 and its receptor, UNC5B. Studies will test the hypothesis that metabolic deletion of extracellular purinergic nucleotides and generation of adenosine by the vascular ectoenzymes CD39 and CD73 suppress innate immune amplification in stroke, tilt the balance towards leukocyte emigration from the ischemic brain, and quash platelet activation so as to limit ischemic cerebrovascular injury. Using unique reagents we have developed, including globally- and conditionally-CD39 deficient (and overexpressing) mice, and CD73 deficient mice, experiments will in a photothrombotic model of stroke: (1) determine the role of extracellular nucleotide dissipation by CD39 and CD73 and adenosine signaling; (2) elucidate the mechanism(s) by which ectonucleotidases on specific cell types (endothelial cells, macrophages, and microglia) drive microvascular coagulation, inflammation, and immune activation; (3) characterize ectonucleotidase-bearing extracellular microvesicles (microparticles and exosomes) shed in ischemic stroke, and determine if they retain biological activity to signal danger or protect against it. Functionally active, ectonucleotidase-bearing extracellular microvesicles will be administered into the cerebral ventricle to ascertain whether they protect in stroke, and if so, how they modulate local coagulation or innate immune effector mechanisms. Understanding key vascular homeostatic enzymes (ectonucleotidases) can open a new opportunity for therapy based on nucleotide dissipation, nucleoside generation, or purinergic receptor signaling to improve outcomes in ischemic stroke.
Stroke, which has very few treatment options, remains the third leading cause of death and the leading cause of disability in the United States. This proposal examines a natural mechanism by which blood vessels in the brain can protect against damage in stroke by expressing a family of enzymes which degrade harmful molecules released from injured or dying cells. Experiments use a mouse model of stroke and unique mice in which levels of the protective enzymes have been genetically altered. Ultimately, the goal is to develop means for boosting levels of these enzymes to protect the brain from damage in stroke.
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