FIGURE 4.

The stimulation of tube formation by Fc rNRP-1 depends on VEGFR-2 kinase activity, but not on VEGF-A in HDMEC. A, HDMECs were placed in a collagen gel and stimulated with Fc rNRP-1 (5 nm) in the presence and absence of the inhibitor of the VEGFR-2 tyrosine kinase, ZM323881. B, angiogenic effect of Fc rNRP-1 (5 nm) on HDMEC cells grown 7 days prior to serum starvation in medium supplemented with VEGF-A₁₆₅ or VEGF-A₁₂₁ (white bar indicates 100 μm). C, tubes from A and B were photographed and analyzed as described under “Experimental Procedures.” Significance of the effect of addition of the ZM323881 inhibitor to the Fc rNRP-1 variant and the effect of the Fc rNRP-1 in the two tested pools of cells versus their respective controls were determined by Student's t test. Single asterisk (*) indicates that the p value is <0.001. D, Western blot of cells from a parallel experiment to B conducted on a collagen layer, where the top panel presents effect of stimulation with VEGF-A₁₆₅ (10 ng/ml) and recombinant NRP-1 proteins (10 nm Fc rNRP-1 and 20 nm shNRP-1) on cells grown 7 days prior to serum starvation in medium supplemented with VEGF-A₁₂₁, and panel E presents an experiment with cells grown in standard conditions in the medium supplemented with VEGF-A₁₆₅. The band intensities were analyzed as described under “Experimental Procedures,” with the exception, that all band intensities were normalized to their respective actin bands.