Angiogenic properties of Bcl-2 overexpressing DPSCs under hypoxia

Abstract

Objectives: The aim of this study was to investigate the angiogenic properties of Bcl-2 overexpressing dental pulp stem cells (DPSCs) under hypoxia in-vitro and in-vivo. Methods: Bcl2-overexpressing-DPSCs was established using target-specific lentiviral particles (GenTarget Inc, CA). Bcl2-overexpressing-DPSCs were cultured under hypoxia induced by 0.5mM CoCl2 up to 48 hours. Culture supernatants, total RNA and proteins were collected at different time points and used for ELISA, qPCR and western blotting assays, respectively. Expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF2) and hypoxia-inducible factor 1α (HIF-1α) was examined. In-vitro Matrigel assay was performed to assess the level of functionality of angiogenic factors secreted by Bcl2-overexpressing-DPSCs. In-vivo angiogenic potential of these cells was examined by Matrigel plug assay performed on severe-combined-immunodeficient mice. Briefly, cells mixed with Matrigel was injected subcutaneously, retrieved after 7 days and examined for histology and immunohistochemistry. DPSCs cultured under normoxia was used as the control group. Results: Both wild-type (WT) and Bcl2-overexpressing-DPSCs cultured under hypoxia showed increased levels of VEGF expression compared to that of cells cultured under normoxia. However, VEGF protein level in Bcl2-overexpressing-DPSCs cultured in hypoxia was threefold higher than that of WT-DPSCs, which was statistically significant (p<0.05). In contrast, no such increase was observed in relation to FGF2 expression in both Bcl2-overexpressing-DPSCs and WT-DPSCs. An increased expression of HIF-1α protein in Bcl2-overexpressing-DPSCs under hypoxia was observed, which also partially explained the mechanism behind enhanced VEGF levels. Accordingly, endothelial cells seeded under conditioned medium collected from Bcl2-overexpressing-DPSCs cultured under hypoxia formed an extensive vessel-like network on Matrigel. Conditioned medium from other cell groups did not support formation of such network. Histological assessment of in-vivo transplanted Matrigel plugs showed significantly higher vasculature in Bcl2-overexpressing-DPSC group compared to that of WT-DPSCs. Conclusions: Bcl-2 overexpression and hypoxia synergistically enhance angiogenic properties of DPSCs in-vitro and in-vivo.