Bcl-2 overexpression and hypoxia synergistically enhance VEGF secretion in DPSCs

Abstract

Objectives: Cells with higher cell survival and angiogenic properties are crucial for success in dental pulp regeneration approaches. Bcl-2 is known for its potent anti-apoptotic and pro-angiogenic properties. This study aimed to examine the pro-angiogenic properties of Bcl-2 overexpressing dental pulp stem cells (DPSCs). Methods: DPSCs were transduced with either Bcl-2-GFP or Null-GFP premade human target lentiviral particles (GenTarget Inc, San Diego, CA). Overexpression of Bcl-2 gene and protein was confirmed by quantitative-PCR and western blotting, respectively. Wild-type (WT) DPSCs and Bcl-2-overexpressing-DPSCs were cultured under normoxic and hypoxic (0.5mM CoCl2 in medium) conditions; and culture supernatants, total RNA and protein were collected at different time points. Expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) mRNA and protein was measured by quantitative-PCR and ELISA assays, respectively. Vessel morphogenesis and proliferation of endothelial cells were examined in the presence of VEGF, conditioned media from Bcl2-overexpressing-DPSCs and WT-DPSCs grown for 24 hours under hypoxia. Results: Quantitative-PCR and western blotting assays confirmed the overexpression of Bcl2 in transduced DPSCs compared to that of null-control and WT groups. Hypoxia increased secretion of VEGF in both WT-DPSCs and Bcl2-overexpressing-DPSCs compared to that of normoxia. Bcl2-overexpressing-DPSCs exhibited a threefold induction of VEGF secretion compared to the WT-DPSCs grown in hypoxia. The statistical analysis demonstrated a significant difference in VEGF secretion between control lines and bcl-2 transductants grown in hypoxia (p<0.05). In contrast, FGF2 secretion was reduced under hypoxia in both Bcl2-overexpressing and WT DPSCs. Quantitative-PCR results further confirmed the above findings. Cells seeded on matrigel in the presence of VEGF and conditioned media of Bcl2-overexpressing-DPSCs formed a network of capillary-like structures, which confirmed the functionality of secreted VEGF. Conclusions: Bcl-2 overexpression and hypoxia synergistically modulate vascular endothelial growth factor expression in DPSCs