Regulation of plasminogen activation by TGF-β in cultured human retinal endothelial cells

摘要

BACKGROUND/AIMS—Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor β (TGF-β) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-β in retinal angiogenesis in the context of diabetes mellitus.
METHODS—Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-β (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay.
RESULTS—Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-β resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-β did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05).
CONCLUSIONS—These data demonstrate that TGF-β increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.