Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells

Figures

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  Fig. 1. EPC colony-forming assays. A Colonies of primitive, dense, small, round EPCs showed higher proliferation potential and were composed of immature EPCs. Scale bar: 100 μm. B Colonies of definitive, large, spindle-type EPCs showed more vasculogenic properties and were composed of differentiating EPCs. Scale bar: 100 μm. C Effects of rivaroxaban on the numbers of total EPC colonies, small EPC colonies, and large EPC colonies. EPC: endothelial progenitor cell; DMSO, dimethyl sulfoxide. Bars are means ± standard deviations of nine samples

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  Fig. 2. Effects of rivaroxaban on adhesion and tube formation. A EPCs exposed to 0.1% DMSO (control) or rivaroxaban for 7 days were cultured for 6 h. The number of adhesive cells per low power field was counted. Bars are means ± standard deviations of five samples. B The adhesive cells were observed by phase-contrast microscopy. Scale bar: 100 μm. C EPCs exposed to 0.1% DMSO (control) or rivaroxaban for 7 days were cultured in Matrigel with human umbilical vein endothelial cells. The number of tubes per low power field was measured after 5 h. Bars are means ± standard deviations of five samples. D The tube formation observed by phase-contrast microscopy. Scale bar: 100 μm

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  Fig. 3. Flow cytometric analysis of the expression of endothelial cell markers on the surface of umbilical cord blood-derived CD34-positive cells. The effects of DMSO alone (control) and rivaroxaban on the percentages of cells positive for VEGFR-1, VEGFR-2, Tie-2, E-selectin, and CD31 were assessed. VEGFR: vascular endothelial growth factor receptor. Bars are means ± standard deviations of six or seven samples

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  Fig. 4. Western blot analysis of Akt/eNOS phosphorylation. A The effects of DMSO (control) and rivaroxaban on Akt phosphorylation levels were evaluated. p-Akt: phosphorylated Akt; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bars are means ± standard deviations of five samples. B The effects of DMSO (control) and rivaroxaban on eNOS phosphorylation levels were evaluated. p-eNOS: phosphorylated endothelial nitric oxide synthase. Bars are means ± standard deviations of 5 samples. The original images without crop and correction are shown in the Additional file 2: Figure S2

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  Fig. 5. Transfection with synthetic PAR-2 siRNA into EPCs. A: The effect of transfection with synthetic PAR-2 siRNA into EPCs were evaluated by westernblotting. Values are means ± standard deviations of five samples. B: Expression of PAR-2 in EPCs transfected with PAR-2 siRNA compared with that in cells transfected with control siRNA. PAR, protease-activated receptor; siRNA, small interfering RNA. The full-length gels of PAR-2 protein are shown in the Additional file 3: Figure S3 and Additional file 4: Figure S4

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  Fig. 6. The bioactivities of EPCs transfected with PAR-2 siRNA. A EPCs transfected with control or PAR-2 siRNA for 7 days were cultured for 6 h. The number of adhesive cells per low power field was counted. Bars are means ± standard deviations of five samples. B The adhesive cells were observed by phase-contrast microscopy. Scale bar: 100 μm. C EPCs transfected with control or PAR-2 siRNA for 7 days were cultured in Matrigel with human umbilical vein endothelial cells. The number of tubes per low power field was measured after 5 h. Bars are means ± standard deviations of five samples. D The tube formation observed by phase-contrast microscopy. Scale bar: 100 μm

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  Fig. 7. Surface protein expression of endothelial cell markers in PAR-2-deficient. After PAR-2 siRNA transfection, effects of PAR-2 knockdown on surface protein expression of VEGFR-1, VEGFR-2, Tie-2, E-selectin, and CD31 were evaluated. Bars are means ± standard deviations of five samples (seven samples for VEGFR-2)