Abstract 61: Endothelium-Targeted Deletion of MicroRNA-15a/16-1 Cluster Promotes Post-Stroke Angiogenesis and Improves Long-Term Neurological Functions

Introduction: Angiogenesis promotes functional recovery and is associated with longer survival time in stroke patients. Accumulating studies have shown important roles for microRNAs (miRs) in regulating angiogenesis. Previously, we have demonstrated that miR-15a/16-1 cluster in endothelium negatively regulates hindlimb ischemia-induced angiogenesis. Here we further investigate its functional significance and molecular mechanism on post-ischemic cerebral angiogenesis. Hypothesis: Genetic deletion of miR-15a/16-1 cluster in endothelium increases cerebral angiogenesis and improves long-term neurological functions after ischemic stroke. Methods: EC-selective miR-15a/16-1 conditional knockout mice (EC-miR-15a/16-1 cKO) and WT controls were subjected to 1h MCAO followed by 3-28d reperfusion. Neurobehavioral outcomes were determined by the foot fault, rotarod, adhesive tape removal and Morris water maze tests. Brain capillary density and functional vessels were examined by CD31 and tomato-lectin immunofluorescent staining. In vitro angiogenesis assays, including capillary tube formation, scratch assay and BrdU cell proliferation were applied to cultured mBMECs with lentiviral loss- or gain-of-miR-15a/16-1 function. Pro-angiogenic factors were determined by 3’-UTR luciferase reporter assay, ELISA and western blotting. Results: Brain capillary density and the number of functional vessels in the ischemic penumbra of EC-miR-15a/16-1 cKO mice are higher than those of WT controls. Accordingly, EC-miR-15a/16-1 cKO mice exhibit improved sensorimotor and cognitive outcomes. Moreover, loss- or gain-of-miR-15a/16-1 function in mBMECs significantly increases or reduces tube formation, cell migration and cell proliferation, respectively. Mechanistically, gain-of miR-15a/16-1 function decreases luciferase reporter activity of VEGF, and remarkably reduces VEGF expression in mBMECs. Conclusions: Our findings suggest endothelial miR-15a/16-1 cluster is a negative regulator for post-ischemic cerebral angiogenesis and long-term functional recovery. Clinical intervention of miR-15a/16-1 mediated angiogenesis may be helpful for the development of novel neurorestorative therapies after ischemic stroke.