Time course of su-PAR accumulation in hMVEC supernatant.
(A) The hMVECs were stimulated with or without the combination of bFGF (10 ng/mL) and TNF-α (10 ng/mL) in the presence of vehicle (0.1% DMSO) or BB94 (10 μg/mL) for 8, 24, 48, and 72 hours. At the indicated times, supernatant was collected and total su-PAR was determined by ELISA. The data of one representative time course experiment are expressed as mean accumulative su-PAR ± SEM of triplicate wells (in nanograms per well). (B) The hMVECs were stimulated with or without the combination of bFGF (10 ng/mL) and TNF-α (10 ng/mL), or PMA (10−8 M) in the presence of vehicle (0.1% DMSO) or BB94 (10 μg/mL) for 72 hours. Supernatant was collected, and total su-PAR was determined by ELISA. Data of 5 independent experiments (mean ± SEM) are shown and expressed as nanograms per milliliter of su-PAR accumulation. (C) The hMVECs were stimulated with or without the combination of bFGF (10 ng/ml) and TNF-α (10 ng/mL) in the presence of hirudin (5 U/mL), Trasylol (100 U/ml), pepstatin A (100 μM), leupeptin (100 μM), or BB94 (10 μg/ml) for 9 days. At days 3, 6, and 9, the supernatants were collected, the cells refreshed with the same stimulators, and the su-PAR determined by ELISA. The data are expressed as mean ± SEM of triplicate wells (in nanograms per well) and are representative of 2 independent experiments.
