EphrinB2/EphB4 signaling promote dental pulp stem cells-mediated angiogenesis

Abstract

Objectives: Dental pulp stem cells (DPSCs) reside in a perivascular niche and have shown great capacity to induce angiogenesis during dental pulp regeneration. The bidirectional EphrinB2 and EphB4 molecules represent an essential signaling system in vascular development and remodeling. Our present study was to explore whether and how EphrinB2/EphB4 signaling in DPSCs regulated their angiogenic function and supported endothelial cells (ECs) morphogenesis into functional blood vessels. Methods: We utilized in vitro Matrigel angiogenesis model to study the effects of 1) inhibition of EphrinB2/EphB4 signaling by TNYL-RAW (EphB4 inhibitor) and SNEW (EphB2 inhibitor) peptides, 2) EphrinB2 gene silencing by lentivirus-based short hairpin RNA (shRNA) system on angiogenic function of DPSCs and ECs. We used 3-D fibrin gel assay to interrogate the crosstalk between EphrinB2/EphB4 and VEGF/VEGFR2 pathways in modulating DPSCs-ECs interaction. ECs were coated onto Cytodex-3® microcarrier beads and embedded into fibrin gel. DPSCs pre-incubated with chimeric agonists (EphrinB2-Fc or EphB4-Fc) or inhibitor peptides were seeded atop for 2-week observation. Results: The results found endogenous EphrinB2/EphB4 signaling was required for vascular formation of DPSCs and ECs on Matrigel. Increased phosphorylation of EphrinB2 and EphB4 were associated with time-dependent vessel morphogenesis, while pharmacologic inhibition or knockdown of EphrinB2 dramatically abrogated vessel assembly. When stimulated by EphB4-Fc agonist, DPSCs demonstrated increased VEGF expression and secretion, with EphrinB2-specfic shRNA severely attenuated this increase. The elevated expression of VEGF was associated with increased phosphorylation of Erk and significantly blocked by MEK1/2 inhibitors U0126 and PD98059. Consistent with these observations, the addition of EphrinB2-Fc or EphB4-Fc significantly enhanced sprout formation by ECs cocultured with DPSCs atop fibrin gel. Conclusions: Collectively, these studies for the first time elucidate the contribution of EphrinB2/EphB4 signaling to DPSCs-ECs-mediated angiogenesis, suggest a novel link between EphrinB2/EphB4 and VEGF/VEGFR2 pathways, which may have implications for angiogenesis during dental pulp regeneration.