EphrinB2/EphB4 and PDGF/PDGFRβ signalings coordinately regulate angiogenic functionof dental pulp stem cells

Abstract

Objectives: EphrinB2/EphB4 and PDGF/PDGFRβ are two signaling pathways that have been critically implicated in vascular development and remodeling, particularly in the contexts of pericytes recruitment for vessel maturation. Previous studies by our group as well as others have demonstrated dental pulp stem cells (DPSCs) exhibit pericyte-like properties with inherent angiogenic capacity to promote vascular formation by endothelial cells (ECs). In this study, we aimed to investigate the underlying molecular mechanisms that regulate the angiogenic function of DPSCs. Methods: PDGF/PDGFRβ and EphrinB2/EphB4 signaling were modulated pharmacologically or genetically by lentivirus-based shRNA systems. The tube formation capacity of DPSCs with/or human umbilical vein endothelial cells (HUVECs) were examined using an in vitro Matrigel assay together with confocal microscopy and Nikon’s NIS-Elements software for qualitative and quantitative analyses. To further test in vivo, DPSCs and/or HUVECs with the pharmacological inhibitors were encapsulated in Matrigel and implanted subcutaneously into severe combined immunodeficiency mice (SCIDM) for 7 days and 14 days observation. Results: We found the endogenous EphrinB2/EphB4 and PDGF/PDGFRβ signaling were intrinsically required for the capillary morphogenesis of DPSCs with/or HUVECs on 3-D extracellular matrix. The vessel formation was significantly diminished when EphrinB2/EphB4 or PDGF/PDGFRβ signaling were pharmacologically blocked (p < 0.05). Knockdown of EphrinB2 and EphB4 expressions in DPSCs inhibited their recruitment and association to the ECs-lined tubular networks. Evaluation of the Matrigel plugs retrieved from mice showed the microvessel density was significantly decreased in plug implants treated with EphB4 and PDGFRβ inhibitors (p < 0.05). More importantly, we identified PDGF/PDGFRβ signaling act as an upstream regulator of EphB4 signaling. PDGF up-regulated EphB4 expression of DPSCs, which was attenuated by treatment of PDGFRβ antagonist but rescued by EphB4-Fc agonist. Conclusions: Our results, for the first time, reveal a critical role of PDGF/PDGFRβ and EphrinB2/EphB4 signaling in coordinately regulate the angiogenic function of DPSCs in postnatal vascular formation and maturation.