A Mesenchymal stem cell line (B10) increases angiogenesis in a rat MCAO model

A human mesenchymal stem cell line (B10) transplantation has been shown to improve ischemia-induced neurological deficits in animal stroke models. To understand the underlying mechanism, we have investigated the effects of B10 transplantation on cerebral angiogenesis in a rat middle cerebral artery occlusion (MCAO) model. B10 cells were transplanted intravenously 24 h after MCAO. Immunofluorescence staining results showed that compared to PBS-groups, vWF positive vessel and endoglin positive new vessels were increased in B10-transplanted MCAO groups in the lesion areas. The mRNA of angiogenesis factors including placental growth factor and hypoxia inducible factor (HIF)-1 alpha were increased 3 days after MCAO in the core and IBZ areas of B10-transplanted group. Angiopoetin1 mRNA was increased only in the IBZ. Western blotting results showed that HIF-1 alpha and vascular endothelial growth factor (VEGF) proteins were increased in B10-transplanted group. Both HIF-1 alpha and VEGF were expressed in macrophage/microglia in the core area. In the IBZ, however, HIF-1 alpha was expressed both in astrocytes and macrophage/microglia, while VEGF was expressed only in macrophage/microglia. Moreover, TGF beta protein levels were found to be increased in B10-transplanted group in the core and IBZ regions. Cell culture experiments using a human microglia cell line (HMO6) and B10 showed that IL-1 beta induced VEGF mRNA expression in both cell types. IL-1 beta was found to be highly expressed in B10 cells, and its co-culture with HMO6 further increased that in B10. Co-culture increased VEGF mRNA in both B10 and HMO6. In the rat brains, IL-1 beta was expressed in macrophage/microglia and transplanted-B10 cells in the core. IL-1 beta positive cell number was increased slightly, but significantly in B10-transplanted rats. To explore further, IL-1 beta expression was silenced in B10 cells by transfecting mRNA specific siRNA, and then transplanted in MCAO rats. Immunostaining result showed that endoglin positive area was decreased in IL-1 beta-silenced B10 transplanted groups compared to nonsilenced-B10 transplanted groups. Interestingly, vessel-like structure appeared as early as 3 days after MCAO in IL-1 beta-silenced B10-transplanted group. Thus our results demonstrated that B10 cells increased angiogenesis in MCAO rat model, through the regulation of HIF-1 alpha and VEGF expression, where IL-1 beta might play a role.