Figure 3.

VEGF-164 is the secreted factor that inhibits SMC apoptosis. (a) Effect of recombinant VEGF-164 on SMC apoptosis. Rat aortic SMCs were treated with 1, 2, 5, 10 or 20 ng/ml of VEGF for 12 h. The cells were then exposed to UV light for 5 min and apoptotic index was measured 6 h later as described in the Materials and Methods (*P<0.05 compared with solvent control; n=3). (b) Effect of VEGF neutralization on conditioned media-induced inhibition of SMC apoptosis. Rat aortic SMCs were infected with either AdGFP control or AdSmad3 followed by stimulation with solvent or TGF-β1 (5 ng/ml) for 48 h. Conditioned media were collected and incubated with the VEGF-neutralizing antibody (Nab) or goat IgG (10 μg/ml) for 1 h before application to naive vascular SMCs for 12 h. The cells were then exposed to UV light for 5 min and apoptotic index was measured 6 h later (*P<0.05; n=3). (c) Effect of VEGF siRNA on conditioned media-induced inhibition of SMC apoptosis. Rat aortic SMCs were infected with either AdGFP or AdSmad3 followed by stimulation with solvent or TGF-β1 (5 ng/ml) for 48 h in the presence of scrambled siRNA or VEGF siRNA. Conditioned media were collected and applied to naive vascular SMCs for 12 h. The cells were exposed to UV light for 5 min, fixed 6 h later and then subjected to TUNEL staining (see representative images) as described in the Materials and Methods, *P<0.05; n=3