Figure 7

Silencing of endogenous VEGF-A lacks an effect on mesenchymal stem cell differentiation into endothelial cells. Differentiating mesenchymal stem cells (MSCs) were transfected with small interfering RNA (siRNA) directed against vascular endothelial growth factor (VEGF-A) and then examined for endothelial cell (EC) marker expression. Treatment of MSCs with the cocktail of VEGF-A (2 ng/ml) plus angiotensin II (Ang II; 2 ng/ml) for 24 hours induced a synergistic increase in VEGF transcript as compared with VEGF-A alone or Ang II alone (A). VEGF-A mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (A). Standardization experiments demonstrated that 50 nM siRNA directed against VEGF completely abrogated VEGF protein expression (B). Fluorescence-activated cell sorting analysis revealed that there was no significant difference in EC marker expression between differentiating MSCs that received VEGF-A siRNA or scrambled control siRNA (C, D, E). Note that human umbilical vein endothelial cells (HUVECs) were excluded from the statistical analyses. *P <0.05 vs. naïve MSCs. ^# P <0.05 vs. VEGF-A (2 ng/ml)-treated MSCs or Ang II (2 ng/ml)-treated MSCs alone, n = 3.