Figure 6

Activation of caspase-3 is abrogated by VEGF stimulation in HDMEC cultured on collagen I. HDMEC were seeded on either plastic or collagen I coated tissue culture dishes, and following removal of non-adherent cells by washing, were stimulated with either 50 ng/ml VEGF alone or in combination with 10, 50 or 250 ng/ml TSP-1 (panel A), 16, 80 or 400 ng/ml IP-10 (panel B), or 100, 500 or 2500 ng/ml recombinant endostatin (panel C) for 48 h. Cells were collected following collagenase treatment and counted. Cell pellets were lysed in appropriate volumes of lysis buffer to generate extracts containing equal number of cells per unit volume. A fluorometric based assay for caspase-3 activity was performed using Ac-DEVD-AMC as a substrate as described in the methods section. Bars are representative of the mean and standard error for triplicate samples. The experiment was performed three independent times with similar results. Statistical significance was determined using unpaired t-tests as compared to unstimulated samples or to VEGF stimulated samples for TSP-1, IP-10 or endostatin alone or in combination with VEGF respectively (* = p < 0.05).