FIG. 9.

VEGFR2 is upregulated in response to dRP in an NF-κB-dependent manner and its activity is necessary for dRP-dependent angiogenesis. (A) qPCR analysis of VEGFR2 expression on HUVECs treated with or without 200 μM dRP for 4 h. The 2^−ΔΔCt analysis method was used to analyze the data with GAPDH used as normalizer. Statistical significance of the difference was tested using a nonparametric Mann–Whitney test (mean ± SEM, n = 4, *p < 0.05). (B) HUVECs were treated with increasing concentration of dRP (2 μM to 1 mM) for 6 h. Alternatively, HUVECs were incubated with (C) 100 nM QNZ or (D) 10 μM Nox2ds-tat for 1 h and then stimulated with 200 μM dRP for 6 h. Cell lysates were immunoblotted for VEGFR2 and β-actin. Data are representative of four independent experiments. (E) Effects of VEGFR2 inhibitors pazopanib and mAB3572 on dRP-induced tube formation. HUVECs with or without 200 μM dRP were tested in the presence of 10 μg/ml pazopanib and 50 ng/ml mAB3572 antibody. Representative pictures (i) and quantification (ii). (F) Effects of the NF-κB inhibitor QNZ (100 nM) on dRP-induced tube formation. Representative pictures (i) and quantification (ii). Bar graphs represent quantification of tube number per optical field performed using ImageJ software with Angiogenesis Analyzer plug-in and compared by one-way ANOVA with Bonferroni post-test (*p < 0.05, **p < 0.01, n = 6). Bar: (E, F) 300 μm. qPCR, real-time quantitative polymerase chain reaction; VEGFR2, VEGF receptor 2. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars