EphrinB2/EphB4 signaling regulates angiogenesis of dental pulp stem cells and endothelial cells

Abstract

Establishment of an instantaneous and adequate blood supply is critically important for dental pulp regeneration. However, the pulp cavity is enclosed by a rigid mineralized tissue shell and allows a single blood supply via a constricted apical opening, causing frequent ischemic necrosis. To overcome this challenge, stem cells with angiogenic potential, or in combination with endothelial cells (ECs), have been used to engineer pulp substitute in vitro, in order to accelerate in vivo vascular anastomosis upon transplantation. Dental pulp stem cells (DPSCs) and stem cells from exfoliated deciduous teeth (SHED) are promising cell sources for vascular tissue engineering. Previous evidence suggests that DPSCs and SHED could differentiate into functional endothelial-like cells under defined stimuli, but the efficiency is low. One reason may be the lack of in vivo microenvironment, such as the extracellular matrix (ECM) that provides various signaling molecules guiding cell differentiation. Therefore, in the 1st study, human umbilical vein endothelial cells (HUVECs) was decellularized and the ECM was extracted for endothelial induction. The results, for the first time, showed that the decellularized ECM significantly promoted adhesion, proliferation, as well as endothelial differentiation of SHED in vitro. The ligand EphrinB2 with its tyrosine kinase receptor EphB4 is one of the most essential ligand-receptor systems implicated in vascular development and remodeling. The 2nd and 3rd studies focus on investigating the synergistic effect of DPSCs and HUVECs in inducing angiogenesis and the specific role of EphrinB2/EphB4 signaling in regulating their interaction. To closely mimic in vivo angiogenic process, two three-dimensional (3-D) angiogenesis model–a fibrin gel microbead model and a Matrigel-dependent model were used for coculture of DPSCs and HUVECs. The results showed that the in vitro and in vivo vascular formation by HUVECs was greatly enhanced by coculture with DPSCs, which however was significantly suppressed by pharmacological inhibitors of EphB4. Knockdown of EphrinB2 and EphB4 expression in DPSCs reduced their tube forming capacity and delayed the coassembly of DPSCs with HUVECs into reticular structures. The EphrinB2/EphB4 signaling regulated angiogenesis in a phosphotyrosine-dependent manner and the Src family kinases are indispensably required for EphrinB2 reverse signaling-mediated angiogenesis. More importantly, the EphrinB2/EphB4 signaling showed a crosstalk with VEGF signaling. Stimulation of DPSCs with EphrinB2-Fc and EphB4-Fc enhanced the production of VEGF, and thus facilitating the vessel formation of HUVECs. In summary, the ECM derived from HUVECs could promote endothelial differentiation of SHED in vitro; and EphrinB2/EphB4 signaling may contribute to the in vitro and in vivo angiogenic functions of DPSCs and HUVECs. The current findings advance our understanding of the synergy function of DPSCs and ECs in enhancing angiogenesis as well as the underlying mechanisms. This present study has significant implications for developing strategies for promoting vascular tissue engineering in regenerative endodontics.