EphrinB2/EphB4 signaling mediates HUVECs and DPSCs assembly into vascular-like structures

Abstract

Objectives: This study aimed to investigate the bidirectional EphrinB2/EphB4 signaling in mediating the interaction between human umbilical vein endothelial cells (HUVECs) and dental pulp stem cells (DPSCs) during postnatal angiogenesis. Methods: HUVECs, DPSCs were seeded alone or cocultured (at a ratio of 3:1) onto matrigel for in vitro angiogenesis and EphrinB2/EphB4 phosphorylation assay. The EphrinB2/EphB4 and VEGF/VEGFR-2 mRNA expression in HUVECs and DPSCs after coculture were analyzed through using Dynabead® CD31 to isolate HUVECs from DPSCs. EphrinB2 activity was then blocked with TNYL-RAW (EphB4 inhibitor) and SNEW (EphB2 inhibitor) peptides and the capillary-like structures formation within DPSCs-HUVECs coculture was evaluated. Additionally, HUVECs were coated onto Cytodex-3® microcarrier beads with or without pre-clustered EphrinB2-Fc or EphB4-Fc followed by seeding DPSCs or DPSCs pre-incubated with TNYL-RAW plus SNEW peptide onto fibrin gel to assess vessel-like structures formation after 10 days culture. Results: EphrinB2/EphB4 was time-dependently activated in matrigel-based DPSCs-HUVECs cocultures that the phosphorylation of EphrinB2 was detected at 1.5h and 2.5h when cord-like structures were actively formed, while undetectable after 5.5h culture. EphB4 was phosphorylated in DPSCs-HUVECs after 2.5h incubation and remained detectable at 5.5h time point. The mixture of TNYL-RAW and SNEW significantly reduced EphrinB2/EphB4 phosphorylation and blocked matrigel-supported vascular-like structure formation. For fibrin gel assay, there was more direct vessels sprouts with longer vessel segments formed in HUVECs cocultured with DPSCs than in DPSCs pre-incubated with inhibitory peptides, while the addition of EphrinB2-Fc and EphB4-Fc without DPSCs has no significant effects on the formation of vessel-like structures by HUVECs embedded within fibrin gel (p < 0.05). Conclusions: EphrinB2/EphB4 signaling plays a pivotal role in orchestrating HUVECs-DPSCs assembly into vascular-like structures on matrigel-supported culture.