Regulation of plasminogen activation by TGF-beta in cultured human retinalendothelial cells

Background/aims-Regulation of plasmin mediated extracellular matrix degrada tion by vascular endothelial cells is important in the development of angio genesis. The aim was to determine whether transforming growth factor (TGF-b eta) affected the regulation of components of the plasminogen system by hum an retinal endothelial cells, in order to define more clearly the role of T GF-beta in retinal angiogenesis in the context of diabetes mellitus. Methods-Human retinal endothelial cells (HREC) were isolated from donor eye s and used between passages 4-8. The cells were cultured in medium suppleme nted with 2, 5, 15, or 25 mM glucose, plus or minus TGF-beta (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminog en 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 mRN A 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, a nd trace amounts of u-PA. Cell surface plasminogen activation observed by l ysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-P A. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-beta resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-beta did not occur uniformly, the 2.3 kb mRNA trans cript was preferentially increased in comparison with the 3.2 kb mRNA (p<0. 05). Conclusions-These data demonstrate that TGF-beta increases PAI-1 and decrea ses cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.