Notch1-induced HUVEC morphological changes were enhanced by the VEGFR-1-specific ligand PlGF. HUVEC were transduced with Ad-LacZ or Ad-N1IC (MOI 40) and cultured on collagen type I gels in serum free medium with or without 50 ng/ml PlGF and evaluated for the formation of cellular extensions. Representative images from each cell culture are shown (10× magnification). (A-B) Ad-LacZ HUVEC remained a homogenous monolayer in the absence (NT) or presence of PlGF. (C) In the absence of PlGF, Ad-N1IC HUVEC undergo morphological differentiation characterized by one or two extensions per cell (black arrowheads). (D) In presence of PlGF, there is an increase number of Ad-N1IC HUVEC undergoing morphological changes and the number of extensions per cell (open arrowheads). (E) Quantification of total number of Ad-N1IC HUVEC with cellular extensions and cells with multiple (>3) processes with or without PlGF. Number of cells with cellular 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 cultures without PlGF. (F) RT-PCR of RNA isolated from Ad-LacZ and Ad-N1IC HUVEC for VEGF-A (22 cycles), PlGF (25 cycles) and GAPDH (22 cycles), as a control.

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.

Notch signaling induced cellular extensions and VEGFR-1 expression in HUVEC. HUVEC were transduced with either Ad-LacZ or Ad-N1IC (MOI 40) and seeded on collagen type I gel for five days. Representative images from each cell culture are shown (10× magnification). (A) Ad-LacZ HUVEC did not form extensions, (B) while Ad-N1IC HUVEC underwent morphological changes as seen by the sprouting of extensions into the underlying matrix. (C) Morphological differentiation of Ad-N1IC HUVEC was not affected by the addition of 1 μM PD166866 (FGFR inhibitor), or (D) 1 μM ZD1893 (EGFR inhibitor). (E) The addition of 0.5 μM SU5416 (a VEGFR inhibitor), suppressed the morphological differentiation of Ad-N1IC HUVEC. (F) Quantification of the effect of the different tyrosine kinase inhibitors on Notch-induced cellular extensions and cell number. (G) Quantification of the effect of increasing amounts of the VEGFR inhibitor (SU5416) on Notch-induced cellular extensions and cell number. For morphogenesis assays, HUVEC with sprouting extensions per 10× microscopy field were counted, for five separate fields. Data is representative of the mean plus or minus SEM of three separate experiments, relative to control. Cell number was determined as percent of control using colorimetric cell proliferation kit. (H) RT-PCR of RNA isolated from mock (X) or N1IC-expressing retrovirally transduced HUVEC lines for VEGFR-1 (30 cycles), VEGFR-2 (30 cycles) and β-actin (25 cycles) (I) Western blot of total cell lysate from Ad-LacZ or Ad-N1IC transduced HUVEC to detect VEGFR-1 protein expression. Detection of α-tubulin was used as a loading control.

Inhibition of Notch signaling decreased VEGFR-1 expression in neovessels. Chambers packed with VEGF-expressing KP1 pancreatic tumor cells (KP1/VEGF-A¹²¹) transduced with either Ad-GFP or Ad-Notch1 decoy were implanted under the dorsal skin of wild-type C57BL/6 mice. Four days after implantation, chambers were removed for immunohistochemistry of the overlying skin. Representative pictures are shown (20× magnification). (A-B) VEGFR-1 and VEGFR-2 expression in the smooth muscle cell layer of the skin overlying implants expressing Ad-GFP. (C) Expression of VEGFR-2 was detected in implants expressing the Notch1 decoy despite decreased vessel density (black arrowheads). (D) Expression of VEGFR-1 in implants expressing the Notch1 decoy was significantly reduced in the smooth muscle cell layer (open arrowheads). (E) Reduced VEGFR-1 expression relative to CD31 and VEGFR-2 was observed in skin overlying implants expressing the Notch1 decoy compared to those expressing Ad-GFP. Quantitative analysis of immunostaining intensity for each antibody, relative to Ad-GFP implants (set at 100%), was determined in five different areas of each sample. The average density ratio was determined by dividing the area of staining by the total area of the smooth muscle layer. Each group (Ad-GFP versus Ad-Notch1 decoy) consisted of 3-5 mice, and experiments were done in triplicate. Data represents the mean staining intensity in Notch1 decoy implants, expressed as percent of control staining, plus or minus SD. ⋆ P < 0.05. (F) Notch1 decoy expression in HUVEC reduced VEGF-induced VEGFR-1 expression. Adenovirus transduction of VEGF-A in HUVEC increased the expression of VEGFR-1 and VEGFR-2 as determined by RT-PCR (25 cycles) compared to control (Ad-LacZ) cells. When Ad-VEGF-A (VEGF) HUVEC were co-transduced with the Notch1 decoy, transcript levels of VEGFR-1 were reduced, while VEGFR-2 was unaffected. (G) Gamma secretase inhibitor (GSI) reduces VEGF-induced surface expression of VEGFR-1 in HUVEC. Stimulation of HUVEC with 50 ng/ml VEGF-A induced surface expression of VEGFR-1 (red) compared to control cells (black). Co-incubation with GSI (Compound E) inhibited the VEGF-A-induction of VEGFR-1 (green). 10,000 cells per experimental group were examined by FACs.