MiR-150and Shh Regulate Angiogenesis After Ischemia In Rat

Objective:to explore the effect of miR-150on the angiogenesis and explored the underlying molecular basis after ischemic stroke in rat.Methods:SD rats were subjected ischemic stroke by the middle cerebral artery occlusion (MCAO) for1,3and7days respectively, and then the expression of miR-150in the brain and in the serum were detected by RT-PCR. The primary rat brain microvascular endothelial cells (BMEVCs) were cultured and were subjected oxygen glucose deprivation (OGD) for2,4, and6hours, then the expression of miR-150in the brain and in the serum were also detected by RT-PCR. The up-or down-regulation of miR-150were obtained by miR-150mimic or miR-150inhibitor transfection; the tube formation, proliferation and migration of BMEVCs were respectively detected by matrigel assay, by MTT assay and quantified by anti-Ki-67positive immunofluorescence and by scratch test. SD rat subjected MCAO were tansfected with miR-150inhibitor by daily intracerebroventricular injection, and the density of microvascules and blood flow in the peri-infarction regions were calculated by FITC-labeled dextran. The mRNA and protein expression of c-myb and VEGF in BMEVCs of miR-150inhibitor or negative inhibitor transfected were detecte by RT-PCR and western blotting. The down-regulation of c-myb or VEGF was achieved by lentiviral transfection or special antibody, and the role of c-myb and VEGF on miR-150mediated angiogensis were detected.Results:We found that:1) the expression of miR-150in the brain and serum were down-regulated after1-,3-,7d-ischemia, and the expression of miR-150in BMEVCs was also down-regulated after2-,4-,6h-OGD.2) The tube formation, proliferation and migration of BMEVCs were decrease by miR-150mime trasfection, while the tube formation, proliferation and migration of BMEVCs were increase by miR-150inhibitor trasfection. The density of microvascules and blood flow in the peri-infarction regions were increased with miR-150inhibitor transfection by intracerebroventricular injection after MCAO.3) The mRNA and protein expression of c-myb and VEGF in BMEVCs were up-regulated by miR-150transfection. The increase of tube formation, proliferation, and migration of BMEVCs were reversed by the down-regulation of c-myb or VEGF.Conclusions:the expression of miR-150is down-regulated after ischemia. MiR-150could mediate angiogensis by modulating c-myb and VEGF after ischemia in rat. MiR-150could be a potential target of the treatment for cerebral ischemia. Objective:to investigate the role of astrocytes in the angiogenesis of brain microvascular endothelial cells after oxygen glucose deprivation and to explore the underlying molecular basis.Methods:SD rat brain microvascular endothelial cells (BMVECs) and astrocytes were primary cultured. Oxygen glucose deprivation (OGD) was used as the ischemia model in vitro. The proliferation, migration and tube formation in BMECs were detected by anti-ki-67immunofluorescence, scratch test and matrigel assay respectively. The concentration of Shh in the medium was detected by Elisa. Cyclopamine (a Shh antagonist) was added to exam the role of Shh in angiogenesis. The expression mRNA and protein of RhoA and ROCK were detected by RT-PCR and west blotting. Lentiviral transfection of RhoA and ROCK inhibitor, Y27632were used respectively to exam the effect of RhoA and ROCK on angiogenesis.Results:We found that1) the treatment of oxygen-glucose deprivation increase astrocytic secretion of Shh; co-cultured astrocytes with BMVECs increase the tube formation, proliferation and migration after OGD; the increase was reversed by Cyclopamine.2) Co-cultured astrocytes with BMVECs increase the mRNA and protein expression of RhoA and ROCK, and the increase was reversed by Cyclopamine.3) The increase of the tube formation, proliferation and migration after OGD were respectively reversed by Lentiviral transfection of RhoA and ROCK inhibitor, Y27632.Conclusions:astrocytes may promote the angiogenesis in BMVECs after OGD by the secretion of Shh.