Tβ4 induces expression of PAI-1 in ECs. (A) Release of PAI-1 from HUVECs. These cells planted in 48-well microplates were starved overnight in medium containing 0.1% FBS and then stimulated for 20 hours with different concentrations of Tβ4 or Tβ10 (0-160 nM). Afterward, the supernatants were collected and assayed for PAI-1 antigen by ELISA using an Elisa-Imulyse PAI-1 (Biopool). The graph shows the average ± SD of duplicate measurements performed during 3 experiments. (B) Relative quantitative RT-PCR analysis of PAI-1 mRNA ± SD. In these experiments, EA.hy 926 cells were stimulated for 4 and 24 hours with different concentrations of Tβ4 (0-160 nM), then total cellular RNA was extracted. RT-PCR followed by staining with ethidium bromide was used to determine PAI-1 and t-PA mRNA levels. This experiment has been repeated 3 times with similar results. (C) Increase in PAI-1 mRNA but not in t-PA, VWF, and actin mRNAs on incubation of EA.hy 926 cells with different concentrations of Tβ4 for 4 and 24 hours. (D) When Tβ4 is used in the same concentration range as in the case of EA.hy 926 cells, it has no effect on the PAI-1 expression in human fibroblasts, PC3, and U937 cells.