R-Smad signaling-mediated VEGF expression coordinately regulates endothelial cell differentiation of rat mesenchymal stem cells.

A low-efficiency yield hinders the use of stem cells as a source of endothelial cells (ECs) for therapeutic vascularization, and the diversity of the transforming growth factor-β (TGF-β) superfamily has undermined understanding the effects of its potent vascularization-inducing. Herein, we studied the role of the TGF-β superfamily in EC differentiation of rat bone marrow mesenchymal stem cells (MSCs) induced by Smad2/3 and Smad1/5/8 signaling. MSCs that had been sorted by flow cytometry as CD31-negative were cultured for 14 days in medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as the control. The Smad2/3 pathway was activated by TGF-β1 and Smad1/5/8 by bone morphogenetic proteins (BMPs). In the early phase in the Smad2/3-activated group, there were 10% CD31-positive cells, which was significantly higher than in the control group. A low Smad1/5/8 phosphorylation level after BMP4 activation doubled the number of CD31-positive cells, while a higher phosphorylation level after BMP9 activation showed no effect. A Smad2/3 inhibitor initially blocked differentiation but later promoted it, while a Smad1/5/8 inhibitor reversed the induction observed with BMPs. Moreover, the positive effects of R-Smad on differentiation were weakened by the VEGF neutralizing antibody, and a Smad3 inhibitor decreased VEGF expression and blocked differentiation in both the early and late phases. In conclusion, differentiation of ECs from MSCs via Smad2/3 signaling is stage dependent. Activation, particularly by Smad3, significantly promotes differentiation at an early phase but later is suppressive. A low Smad1/5/8 phosphorylation level has a positive effect, and R-Smad effects are partly mediated by VEGF.