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. 2011 May;6(3):238–250. doi: 10.1016/j.scr.2011.02.001

Figure 6.

Figure 6

VEGF-A inhibited HBMSC differentiation to vSMCs but promoted EC differentiation. To determine whether secreted VEGF-A inhibited vSMC marker expression in HBMSCs cultured at high density, the effects of blocking VEGF-A signaling was determined. (A,B) HBMSCs cultured at high density for 24 h were (A) exposed to 1 μg/ml VEGF neutralisation antibody (VEGF-I) (lane 2) or (B) transfected with VEGF-A siRNA (lane 2), and then SM-MHC-1 expression was determined by immunoblot analysis. Untreated HBMSCs (Cont) or scrambled (Scr) siRNA-transfected HBMSCs (lane 1) were used as respective controls. Pixel density was normalised to β-actin and plotted as a bar graph. * P < 0.05 compared to controls. Data are representative of two independent experiments for each analysis. (C,D) HBMSCs cultured at high density for 14 days were exposed to 1 μg/ml VEGF-I (lane 2) or 0.5 μM VEGFR tyrosine kinase inhibitor (VEGFR-I) (C, lane 3, and D, lane 5), and then (C) VEGFR1 and (D) VE-cadherin expression was determined by immunoblot analysis. Untreated HBMSCs were used as controls (Cont) (C, lane 1, and D, lanes 1 and 4). Pixel density was normalised to β-actin and plotted as a bar graph. * P < 0.05 compared to untreated HBMSCs. A representative of two independent experiments is shown in each case. (E) HBMSCs cultured at high density for 14 days were exposed to 50 ng/ml recombinant VEGF-A, and then VEGFR transcript expression was determined by semiquantitative RT-PCR analysis. Lanes 1–2, GAPDH in untreated (control) HBMSCs. VEGFRs 1–3 in untreated (control) HBMSCs, lanes 3–5, or following exposure to recombinant VEGF-A, lanes 6–8, respectively. Two different primer pairs for VEGFRs 1–3 gave similar results. Red boxes denote unstimulated cells; blue boxes denote VEGF-A-stimulated cells. (F) Immunoblot analysis of VEGFR1 protein level in untreated HBMSCs (lane 1), or following exposure to recombinant VEGF-A for 14 days (lane 2). * P < 0.05 compared to untreated HBMSCs cultured after plating at high cell density (HBMSC-H). In each case, fresh medium supplemented with VEGF-A or inhibitors was added to cells every 2 days.