IADR Abstract Archives
Semaphorin 4D Enhances Vascular Stabilization by Recruiting SHED
Objectives: To investigate the role of soluble Semaphorin 4D (Sema4D) in mediating the interactions between human umbilical vein endothelial cells (HUVECs) and stem cells from human exfoliated deciduous teeth (SHED) during vasculogenesis.
Methods: Recombinant Sema4D was commercially purchased and used to treat SHED and HUVEC co-cultures in in-vitro Matrigel and microfluidic assays in order to examine its role in vascular stabilization. Trans-well (8µm-pore) assay was performed to assess the direct and indirect chemotactic effects of Sema4D on SHED. Furthermore, the expression of plexin-B1 (Sema4D receptor) was quantified by PCR and western blots. Additionally, plexin-B1 was knocked down in HUVECs and introduced in trans-well assay to confirm its role in mediating Sema4D function. The expression of mural cell markers was further quantified by western blots to investigate the effect of Sema4D on differentiation of SHED into pericytes. Results were statistically analyzed using Student’s t-test and 1-way ANOVA.
Results: According to in-vitro Matrigel and microfluidic assay results, addition of Sema4D significantly enhanced the vessel-like network formed by HUVECs and the number of SHED co-localized with vascular structures. Trans-well assay showed that Sema4D indirectly increased the migration of SHED by inducing the secretion of endothelial derived factors. Western blotting results confirmed that the plexin-B1 expression was significantly higher in HUVECs compared to that of SHED. Accordingly, the plexin-B1 knockdown in HUVECs significantly blocked the effects of Sema4D on the migration of SHED in trans-well assay. Furthermore, when SHED was cultured under Sema4D-treated conditioned medium from HUVECs, they were transformed into a more mature phenotype of mural cells as shown by significantly higher expression of mural cell markers, NG2, α-SMA, and SM22α compared with that of the control group at 72h. Similarly, microfluidic assay demonstrated that the addition of Sema4D significantly enhanced the co-localization of SM22α+ SHED with HUVEC vasculature confirming enhanced mural cell function.
Conclusions: Sema4D promotes vascular stabilization by recruiting SHED through endothelial derived factors.
Methods: Recombinant Sema4D was commercially purchased and used to treat SHED and HUVEC co-cultures in in-vitro Matrigel and microfluidic assays in order to examine its role in vascular stabilization. Trans-well (8µm-pore) assay was performed to assess the direct and indirect chemotactic effects of Sema4D on SHED. Furthermore, the expression of plexin-B1 (Sema4D receptor) was quantified by PCR and western blots. Additionally, plexin-B1 was knocked down in HUVECs and introduced in trans-well assay to confirm its role in mediating Sema4D function. The expression of mural cell markers was further quantified by western blots to investigate the effect of Sema4D on differentiation of SHED into pericytes. Results were statistically analyzed using Student’s t-test and 1-way ANOVA.
Results: According to in-vitro Matrigel and microfluidic assay results, addition of Sema4D significantly enhanced the vessel-like network formed by HUVECs and the number of SHED co-localized with vascular structures. Trans-well assay showed that Sema4D indirectly increased the migration of SHED by inducing the secretion of endothelial derived factors. Western blotting results confirmed that the plexin-B1 expression was significantly higher in HUVECs compared to that of SHED. Accordingly, the plexin-B1 knockdown in HUVECs significantly blocked the effects of Sema4D on the migration of SHED in trans-well assay. Furthermore, when SHED was cultured under Sema4D-treated conditioned medium from HUVECs, they were transformed into a more mature phenotype of mural cells as shown by significantly higher expression of mural cell markers, NG2, α-SMA, and SM22α compared with that of the control group at 72h. Similarly, microfluidic assay demonstrated that the addition of Sema4D significantly enhanced the co-localization of SM22α+ SHED with HUVEC vasculature confirming enhanced mural cell function.
Conclusions: Sema4D promotes vascular stabilization by recruiting SHED through endothelial derived factors.