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. 2020 Aug 26;21(17):6159.
doi: 10.3390/ijms21176159.

Bcl-2 Overexpression and Hypoxia Synergistically Enhance Angiogenic Properties of Dental Pulp Stem Cells

Waruna L Dissanayaka  1 , Yuanyuan Han  1 , Lili Zhang  1 , Ting Zou  2 , Chengfei Zhang  2
Affiliations

Bcl-2 Overexpression and Hypoxia Synergistically Enhance Angiogenic Properties of Dental Pulp Stem Cells

Waruna L Dissanayaka et al. Int J Mol Sci. .

Abstract

Post-implantation cell survival and angio-/vasculogenesis are critical for the success of cell-based regenerative strategies. The current study aimed to overexpress B-cell lymphoma 2 (Bcl-2) gene in dental pulp stem cells (DPSCs) and examine the anti-apoptotic and angio-/vasculogenic effects both in-vitro and in-vivo. DPSCs were transduced with Bcl-2-green fluorescent protein (GFP) lentiviral particles and examined for cell proliferation and apoptosis. The cells were cultured under normoxic or hypoxic (0.5 mM CoCl2) conditions and examined for the expression of angiogenic factors and effects on endothelial cell proliferation, migration and vessel morphogenesis. Cells with or without hypoxic preconditioning were used in in-vivo Matrigel plug assay to study the post-implantation cell survival and angio-/vasculogenesis. Bcl-2-overexpressing-DPSCs showed significantly lower apoptosis than that of null-GFP-DPSCs under serum-free conditions. Under hypoxia, Bcl-2-overexpressing-DPSCs expressed significantly higher levels of vascular endothelial growth factor compared to that under normoxia and null-GFP-DPSCs. Consequently, Bcl-2-overexpressing-DPSCs significantly enhanced endothelial cell proliferation, migration and vascular tube formation on Matrigel. Immunohistological assessment of in-vivo transplanted Matrigel plugs showed significantly higher cell survival and vasculature in hypoxic preconditioned Bcl-2-overexpressing-DPSC group compared to null-GFP-DPSC group. In conclusion, Bcl-2 overexpression and hypoxic-preconditioning could be synergistically used to enhance post-implantation cell survival and angio-/vasculogenic properties of DPSCs.

Keywords: Bcl-2; angiogenesis; dental pulp stem cells; gene modification; post-implantation cell survival; tissue regeneration; vascularization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
B-cell lymphoma 2 (Bcl-2) overexpression in dental pulp stem cells (DPSCs). (A) Western blot analysis and (B) real-time polymerase chain reaction of Bcl-2 protein and mRNA expression in Bcl-2- and green fluorescent protein (GFP)-transfected, and parental DPSCs. β-actin – endogenous control *** p < 0.001 versus the corresponding controls.
Figure 2
Figure 2
Characterization of Bcl-2-DPSCs. (A) Cell proliferation rates of Bcl-2-DPSCs and GFP-DPSCs as shown by Cell Counting Kit-8 (CCK-8) assay. Except at day 4, no significant difference was observed between the two groups. (B) Apoptosis in cultures of Bcl-2-DPSCs and GFP-DPSCs in the presence and absence of serum. Bcl-2-DPSCs demonstrated significantly lower apoptotic levels compared to GFP-DPSCs at all the time points under both conditions. Under serum starvation, the difference was markedly increased. (C) Caspase-3 activity in Bcl-2-DPSCs and GFP-DPSCs in the presence and absence of serum. Caspase-3 levels were significantly lower in Bcl-2-DPSCs in serum-free condition compared to that of GFP-DPSCs. * p < 0.05, ** p < 0.01versus the corresponding controls.
Figure 3
Figure 3
Expression of angiogenic factors in Bcl-2-DPSCs. Secretory (A) vascular endothelial growth factor (VEGF) and (B) fibroblast growth factor-2 (FGF2) levels at 6, 12, 24, and 48 h in Bcl-2-DPSCs and GFP-DPSCs under normoxia and hypoxia as detected by enzyme-linked immunosorbent assay (ELISA). (C) VEGF mRNA expression in Bcl-2-DPSCs and GFP-DPSCs under normoxia and hypoxia at 12, 24, and 48 h. (D) VEGF and hypoxia inducible factor (HIF)-1α protein levels at 24 h in Bcl-2-DPSCs and GFP-DPSCs under normoxia and hypoxia. β-actin – endogenous control * p < 0.05, ** p < 0.01, *** p < 0.001 versus the corresponding controls.
Figure 4
Figure 4
Paracrine effects of Bcl-2-DPSCs on human umbilical vein endothelial cell (HUVEC) proliferation and migration. (A) Cell proliferation rates as shown by CCK-8 assay and (B) Transwell migration assay of HUVECs cultured under conditioned media of Bcl-2-DPSCs and GFP-DPSCs collected under normoxia and hypoxia. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the corresponding controls.
Figure 5
Figure 5
(A) Brightfield images of Matrigel tube formation assay. HUVECs cultured in conditioned media (CM) from Bcl-2-DPSCs under hypoxia formed a vascular network similar to that cultured in endothelial cell medium (ECM). (B) Quantification of length and number of vascular tubes formed by HUVECs. * p < 0.05, ** p < 0.01, versus the corresponding controls.
Figure 6
Figure 6
In vivo Matrigel plug assay: (A) Representative microscopic images of hematoxylin and eosin (H&E) (20× and 50× magnification) and immunohistochemistry for human CD31 (H CD31; 20× and 50× magnification) and mouse CD31 (M CD31; 20× and 50× magnification) of Matrigel plugs at 7 days of implantation. Broken black lines—interface between the mouse tissue and the Matrigel plug. Blue arrows—perfused blood vessels. Red arrows—human CD31 positive non-perfused lumens. Yellow arrows—mouse CD31 positive perfused blood vessels. (B) Representative microscopic images of immunohistochemistry for DNA damage (20× and 50× magnification) and quantified percentage cells with DNA damage. Black arrows—DNA damage positive cells. *** p < 0.001 versus the corresponding controls.
Figure 7
Figure 7
Role of HIF-1α in VEGF expression in Bcl-2-DPSCs under hypoxia. (A) Expression of protein levels of VEGF and HIF-1α in GFP-DPSCs and Bcl-2-DPSCs under normoxia (N), hypoxia (H), and YC-1 treatment as shown by western blotting. (B) Relative mRNA expression levels of VEGF in GFP-DPSCs and Bcl-2-DPSCs under normoxia (N), hypoxia (H), and YC-1 treatment as shown by qRT-PCR assay. β-actin – endogenous control * p < 0.05, ** p < 0.01 *** p < 0.001 versus the corresponding controls.
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