Figure 1.

Detachment-induced apoptosis in HUVECs is Fas/Fas-L dependent. HUVECs were detached and either kept in suspension or on polyhema-coated dishes in complete growth medium (EGM medium containing 2% FCS, 10 ng/ml hEGF, and 1 μg/ml hydrocortisone, supplemented with 12 μg/ml of bovine brain extract, 2 mM l-glutamine, 50 μg/ml streptomycin, and 50 U/ml penicillin) for the indicated time periods, and cell death was determined by (A) trypan blue exclusion or (B) DNA fragmentation analysis. Identical results were obtained under the two experimental conditions; results shown are for cells kept in suspension, and these experimental conditions were used in all subsequent experiments involving detachment-induced cell death. (C) HUVECs were kept adherent or in suspension for 12 h in the presence or absence of 5 μg/ml of inhibitory anti–Fas-L antibodies (NOK-2), inhibitory anti-Fas antibodies (ZB4), or with control antibodies (IgG). Apoptosis was determined by DNA fragmentation analysis. In A and B, results are shown for representative experiments independently carried out three times. In C, bars indicate SD in a representative experiment done in triplicate.