Semaphorin 4D Enhances Vascular Stabilization by Recruiting SHED Through Endothelial-Derived PDGF-BB

Abstract

Objectives: To investigate the role of soluble Semaphorin 4D (Sema4D) in recruiting stem cells from human exfoliated deciduous teeth (SHED) as mural cells to enhance vessel stabilization. Methods: To better mimic in-vivo neovessel formation stabilized by mural cell coverage, we adopted microfluidic chips that allow human umbilical vein endothelial cells (HUVECs) to form vessel networks first and recombinant Sema4D was added to examine its role in recruiting SHED as mural-like cells. Trans-well assay was performed to assess the direct and indirect chemotactic effects of Sema4D on SHED. The expression of mural cell markers was quantified by western blotting. Furthermore, plexin-B1(Sema4D receptor) was knocked down in HUVECs and introduced in trans-well assay and microfluidic assay to confirm its role in mediating Sema4D function. Additionally, the expression of PDGF-BB in Sema4D treated HUVECs was detected by ELISA and its function in recruitment of SHED was further investigated using PDGFR-β inhibitor. Results were statistically analyzed using Student’s t-test and 1-way ANOVA. Results: Sema4D significantly enhanced the vessel network formation and the percentage of SHED-covered vascular structures. Trans-well assay showed that Sema4D increased the migration of SHED through inducing the secretion of endothelial-derived factors. Furthermore, Sema4D-induced paracrine signaling transformed SHED into a more mature mural phenotype by increasing the expression of NG2, α-SMA, and SM22α. Compared to Sema4D-treated HUVEC conditioned-medium, the medium from Sema4D-treated plexin-B1KD-HUVECs significantly decreased the migration of SHED. Additionally, the microfluidic chip with plexin-B1KD-HUVECs showed significantly less total vessel length and SM22α+-SHED covered vessel structures. Sema4D-treated HUVECs expressed significantly higher levels of PDGF-BB compared to untreated HUVECs and there was no detectable expression of PDGF-BB in SHED. Accordingly, when PDGFR-β was blocked, the effects of Sema4D on SHED migration, vessel formation and stabilization by SM22α+ SHED were significantly reduced. Conclusions: Sema4D promotes vascular stabilization by recruiting SHED through endothelial-derived PDGF-BB.