Role of HO-1 in VEGF-induced angiogenesis in vitro. (A) HUVECs were cultured in M199 with 10% FBS for 24 hours prior to the addition of VEGF (25 ng/mL) and CoPP (12.5 and 25 μM) both for 72 hours. In test wells, ECs were preincubated with SnPP at the concentrations shown for 60 minutes prior to the addition of VEGF. ³H-thymidine (0.037 MBq/mL [1 μCi/mL]) was added 16 hours before the end of the assay and the plates were read on an automated plate reader. Bars show the mean + SD uptake of ³H-thymidine (n = 4). (B-F) Murine abdominal muscle was implanted in Matrigel and cultured in DMEM supplemented with 10% FBS and heparin (10 U/mL). VEGF (25 ng/mL) was added to the medium in the absence and presence of SnPP (20 μM). (B) After 9 days EC tube length was measured by image analysis. Data presented as mean + SD tube length (n = 3). Panels C-F show representative micrographs of the effect of SnPP on neovessels formed in the presence of VEGF. Panels C and E show VEGF alone and panels D and F show VEGF + SnPP. Original magnification: low power × 20 (C-D) and high power × 40 (E-F).