Angiostatin induces endothelial cell apoptosis without affecting cell cycle progression. (A) Simultaneous detection of cell proliferation and apoptosis by BrdU labeling and annexin V staining. Low-density cultures of BME cells grown in 5% serum were treated either with vehicle alone (A and C) or with 5 μg/mL rhAst (B and D) for 72 hours. Apoptotic cells were detected by incubation with FITC-labeled annexin V (A and B, arrows). In the same field, proliferating cells were identified with a MoAb against BrdU (C and D, arrows). Original magnification × 80. (B) Lack of effect of angiostatin on endothelial cell S-phase entry. Low-density cultures of BME cells grown in medium containing 5% serum were treated with increasing amounts of angiostatin for 72 hours. The cells were pulsed for 5 hours with BrdU, and uptake was analyzed immunohistochemically as described in Materials and Methods. The percentage of positive nuclei was estimated by fluorescence microscopy. Results are shown as the mean of triplicate samples; error bars depict SEM.