EphrinB2 Regulates DPSC Pericyte-like Functions via the Focal Adhesion Pathway

Mural cells, such as pericytes, integrate with endothelial cells (ECs) lining the capillaries, which are pivotal in vascular development and stabilization as well as in supporting EC function. Dental pulp stem cells (DPSCs) were recently revealed to have intimacy with pericytes in the dental pulp microenvironment and are regulated by EphB4/ephrinB2 signaling. However, how EphB4/ephrinB2 signaling regulates DPSC pericyte biology and their interactions with ECs remains unknown. In this study, single-cell RNA sequencing data analysis and immunofluorescence staining of healthy human dental pulp were used to demonstrate the roles of mesenchymal stem cells (MSCs) as pericytes and ephrinB phosphorylation between MSCs and ECs interaction. Bulk RNA-seq further showed transcriptomic similarity between DPSCs and human brain vascular pericytes. In vitro coculture of DPSCs and ECs further confirmed ephrinB2 activation at cell-cell contact sites. To investigate how ephrinB2 influenced DPSCs' pericyte function, <i>EFNB2</i> was either knocked down using small hairpin RNA or overexpressed via open reading frame (<i>ORF</i>) lentiviral transduction. Focal adhesion protein assessment was conducted, and a 3-dimensional (3D) fibrin beads assay was established to visualize the interaction between DPSCs and ECs in vitro. <i>EFNB2</i> knockdown in DPSCs significantly reduced cell proliferation, adhesion, and transwell migration but increased contractility. Conversely, <i>EFNB2</i> overexpression enhanced proliferation and adhesion but reduced migration and contractility. Interestingly, <i>EFNB2</i> overexpression significantly increased ECs' sprouting capability, improving pericyte coverage in 3D fibrin beads assays. These effects were mediated through the enhanced focal adhesion pathway involving Src, paxillin and FAK phosphorylation, and vinculin. Our findings demonstrate that ephrinB2 signaling regulates critical pericyte functions of DPSCs through the Src/FAK/paxillin signaling pathway, thus modulating their adhesion toward ECs. Targeting ephrinB2 signaling may therefore represent a promising strategy to enhance vascular formation and functional recovery in dental pulp tissue engineering.