A Mesenchymal Stem cell line (B10) increases angiogenesis in a rat MCAO 1 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-group, vWF positive vessel and endoglin positive new vessels were increased in B10-8 transplanted MCAO groups in the lesion areas. The mRNA of angiogenesis factors 9 including placental growth factor and hypoxia inducible factor (HIF)-1α were 10 increased 3 days after MCAO in the core and IBZ areas of B10-transplanted group. 11 Angiopoetin1 mRNA was increased only in the IBZ. Western blotting results 12 showed that HIF-1α and vascular endothelial growth factor (VEGF) proteins levels 13 were increased in B10-transplanted group. Double immunofluorescence staining 14 results revealed that both HIF-1α and VEGF were expressed in 15 macrophage/microglia in the core area. In the IBZ, however, HIF-1α was 16 expressed both in astrocytes and macrophage/microglia, while VEGF was 17 expressed only in macrophage/microglia. Moreover, TGFβ protein levels were 18 found to be increased in B10-transplanted group in the core and IBZ regions. Cell 19 culture experiments using a human microglia cell line (HMO6) and B10 showed 20 that IL-1β induced VEGF mRNA expression in both cell types. IL-1β was found to 21 be highly expressed in B10 cells, and its co-culture with HMO6 further increased 22 that in B10. Co-culture increased VEGF mRNA in both B10 and HMO6. In the rat 1 brains, IL-1β was expressed in macrophage/microglia and transplanted-B10 cells 2 in the core. IL-1β positive cell number was increased slightly, but significantly in 3 B10-transplanted rats. To explore further, IL-1β expression was silenced in B10 4 cells by transfecting mRNA specific siRNA, and then transplanted in MCAO rats. 5 Immunostaining result showed that although total endoglin positive area was 6 decreased, vessel-like structure appeared as early as 3 days after MCAO when IL-7 1β-silenced B10 was transplanted. Thus our results demonstrated that B10 cells 8 increased angiogenesis in MCAO rat model, through the regulation of HIF-1α and 9 VEGF expression, where IL-1β might play a role.