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.