Figure 3

Growth on collagen I impairs the ability of TSP-1 and IP-10, but not endostatin to induce apoptosis of HDMEC. HDMEC were seeded onto plastic or collagen I coated tissue culture dishes, monolayers were washed to remove nonadherent cells, and subsequently adherent cells 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 60 h. Non-adherent and adherent cells were then collected, washed twice with PBS, and permeabilized in 70% ethanol for a minimum of 24 h prior to staining with propidium iodide. The percentage of apoptotic cells was determined following identification of the subG1 population of cells using FACS analysis. The adherent and nonadherent populations of unstimulated cells were included as a control for the basal level of apoptosis of HDMEC (unstimulated-all) and an adherent-only population of unstimulated cells was included to demonstrate the surviving fraction of untreated HDMEC (unstim-adherent). Cells treated with 50 nM of actinomycin D were included as a positive control for apoptosis. All samples were normalized to the levels of apoptosis observed following stimulation with 50 ng/ml VEGF alone. Bars are representative of the mean and standard error of duplicate dishes from three independent experiments (n = 6). Statistical significance was determined following analysis using unpaired t-tests as compared to VEGF (* = p < 0.05).