Figure 4.
ADAMs (a disintegrin and metalloproteinases) 9 and 10 mediate VEGF (vascular endothelial growth factor)-induced NRP1 (neuropilin-1) proteolytic cleavage. A, Human umbilical vein endothelial cells (HUVECs) were transfected with siRNAs as indicated, and after 72 h were then treated with 25 ng/mL VEGF-A for 60 min, and lysates were then immunoblotted as indicated. B, Quantification of the 10 kDa NRP1 cytoplasmic domain fragment in experiments in A in which HUVECs were treated without (C, unfilled symbols) or with VEGF-A (V, black symbols); VEGF significantly enhanced the generation of NRP1 cytoplasmic fragments in siScr-treated cells and this was blocked by siADAM9 (left graph) or siADAM10 (right graph); #P<0,05 vs siScr C, *P<0.05; **P<0.01; ***P<0.005 vs siScr plus VEGF (V; n≥3). C, HUVECs were either transfected with control (siScr) and ADAM10 specific siRNAs and were then either untreated (C, control, unfilled symbols)) or treated with 25 ng/mL VEGF (V, black symbols) for 60 min. Cell supernatant was then removed, concentrated, and immunoblotted NRP1 antibody specific for the extracellular domain with (AF3870) to detect sNRP1 (soluble NRP1). Quantification of results from 3 independent experiments is shown below; **P<0.01 for siADAM10 vs siScr. D, HUVECs were untreated (C, control, unfilled symbols), or were treated for 60 min with 25 ng/mL VEGF plus either DMSO (V, black symbols) or with 25 ng/mL VEGF plus an equal volume of GI254023X (V+G, black symbols). Quantification of results from 3 independent experiments is shown below; *P<0.05 for VEGF plus GI254023X (10 μmol/L) vs VEGF plus DMSO. In this figure, values are presented as a scatterplot. Differences between samples were analyzed using 1-way ANOVA with the Bonferroni correction for multiple pairwise comparisons after testing for normality and equal variance using the Shapiro-Wilk and Levene tests, respectively.