S100A4 acts synergistically with VEGF on HUVEC migration, by increasing KDR expression and the production of active MMP-9.

<p>A) Dose-response of VEGF and S100A4. Cells were treated with EBM, S100A4, VEGF, or the combination of S100A4 plus VEGF for 24 h and migration was analyzed. B) Western-blot analysis of KDR expression after adding either S100A4 (0.3–30 µM) or VEGF (1–100 ng/mL) for 24 h. C) Levels of KDR expression and activation (phosphorylation) induced by VEGF (10 ng/mL), S100A4 (3 µM) or by the combination of the two proteins for 24 h of stimulation. All KDR signal intensities were normalized to α-tubulin. D) Time course (4 h and 8 h) of KDR expression and KDR phosphorylation upon incubation with S100A4 (3 µM) or VEGF (10 ng/mL) or the combination of the two proteins. E) Proteolytic activity of MMPs in HUVEC conditioned media. Cells were treated with S100A4 in EBM alone for 24 h. Active forms of MMP-9 induced by S100A4 are indicated by arrowheads. F) HUVEC were treated with VEGF (3 ng/mL), VEGF plus S100A4 (1 µM) or the combination of these proteins with the antibody 5C3 (0.25–4 µM) for 24 h. 5H4 was used as non-blocking antibody. G) 5C3 (1–2 µM) neutralized the production of active forms of MMP-9 induced by S100A4 (arrowhead). Each data point was normalized to the positive control of cells incubated with complete medium (left bar) that represents 100% migration. The control is the maximum of the possible migration (migration control). This migration is obtained by incubating HUVEC with basal medium containing 10% FCS plus hydrocortisone, brain bovine extract and hEGF. All the experimental points are referred to this control. Bars show the mean ± SEM. ns <i>p</i>>0.05, *<i>p</i><0.05, <i>**p<0.01,</i> ***<i>p</i><0.001.