Figure 3

Notch-induced sprouting in HUVEC did not depend on VEGFR-2 expression. (A, B) Ad-N1IC HUVEC were transfected with 200 pmol of control, VEGFR-1 or VEGFR-2 siRNA and cultured on collagen gels. (A, upper panels) RT-PCR of Ad-N1IC HUVEC transfected with control (CT), or VEGFR-1 siRNA for VEGFR-1 and VEGFR-2 (25 cycles each) and (lower panels) western blot with an antibody against VEGFR-1 demonstrated decreased transcript and protein levels of VEGFR-1 in cells transfected with VEGFR-1 siRNA relative. VEGFR-2 transcripts were unchanged by VEGFR-1 siRNA. GAPDH (25 cycles) and α-tubulin were used as controls for the RT-PCR and western blot, respectively. (B, upper panels) RT-PCR of Ad-N1IC HUVEC transfected with control (CT) or VEGFR-2 siRNA for VEGFR-1, VEGFR-2 and GAPDH (25 cycles each) and (lower panels) western blot with an antibody against VEGFR-2. VEGFR-2 siRNA suppressed VEGFR-2 transcripts and protein, but did not alter VEGFR-1 transcripts. (C-E) Three days after transfection with control, VEGFR-1 or VEGFR-2 siRNAs, Ad-N1IC HUVEC were evaluated for morphological changes (compare cell extensions, black arrowheads). Representative images are shown (10× magnification). (C) N1IC-induced cellular extensions was unaffected by control siRNA. (D) VEGFR-2 siRNA resulted in a modest decrease in Notch-induced morphological changes. (E) VEGFR-1 siRNA suppressed the morphological differentiation of Ad-N1IC HUVEC. (F) Quantification of the effect of either 100 or 200 pmol VEGFR-1 siRNA on Notch-induced HUVEC undergoing morphological changes. (G, H) Ad-VEGF-A (VEGF) HUVEC were transfected with 200 pmol of control (CT) or VEGFR-2 siRNA and cultured on collagen gels for three days. (G) Ad-VEGF HUVEC underwent morphological changes with control siRNA. (H) VEGFR-2 siRNA suppressed Ad-VEGF HUVEC morphological changes. (I) Quantification of the effect of 200 pmol VEGFR-2 siRNA on VEGF-A or Notch-induced morphological changes. VEGFR-2 siRNA only modestly affected Ad-N1IC HUVEC morphogenesis, while it strongly suppressed Ad-VEGF HUVEC morphogenesis. (F, I) Cells with extensions were counted per 10× field, for five separate fields. Data is representative of the mean plus or minus SD of three separate experiments. * P < 0.01 compared with control.