Figure 6.

Overexpression of NRP1 (neuropilin-1) cytoplasmic domain fragments in human umbilical vein endothelial cells (HUVECs) inhibits VEGF (vascular endothelial growth factor)-induced angiogenesis. A, Schematic diagram representing adenoviral NRP1 constructs comprising the cytoplasmic and transmembrane domains (Ad.NRP1Cyt-TM, residues 860–923), the cytoplasmic, transmembrane, and juxtamembrane regions (Ad.NRP1Cyt-JM, residues 797–923), and the cytoplasmic, transmembrane, juxtamembrane, and MAM domains (Ad.NRP1Cyt-MAM, residues 638–923). B, HUVECs transfected with adenoviruses overexpressing LacZ, wild-type NRP1 (Ad.NRP1WT), or the cytoplasmic domain species (Ad.NRP1Cyt-TM, Cyt-JM and Cyt-MAM, respectively) were used in a Transwell migration assay with (+, black symbols) and without (−, unfilled symbols) VEGF treatment (25 ng/mL, 4 h); the means±SEM of results from 3 independent assays are shown, P<0.05 vs Ad.NRP1WT plus VEGF. C, Aortic rings were incubated with the indicated adenoviruses in Opti-MEM overnight. The aortic ring assay was performed as detailed in the Materials and Methods with no treatment (−, unfilled symbols) or with VEGF-A₁₆₅ treatment (+, black symbols). Quantification of the number of branch points (left graph) and network area (right graph) are shown below the representative figures; **P<0.01, ***P<0.001 vs Ad.NRP1 plus VEGF, n=3 (each n includes aortic rings from 4 mice, to have sufficient sample to set up each condition using 6 replicate aortic rings). In this figure, values are presented as a scatterplot. Differences between samples were analyzed using 2-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.