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

S M Wileman, N A Booth, N Moore, B Redmill, J V Forrester, R M Knott

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Abstract

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 (TGF-beta) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-beta 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-beta (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-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 transcript 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 decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

Original languageEnglish
Pages (from-to)417-422
Number of pages6
JournalBritish Journal of Ophthalmology
Volume84
Publication statusPublished - 2000

Keywords

  • PROLIFERATIVE DIABETIC-RETINOPATHY
  • GROWTH-FACTOR-BETA
  • MESSENGER-RNA
  • TRANSFORMING GROWTH-FACTOR-BETA-1
  • MONOCLONAL-ANTIBODIES
  • GENE-EXPRESSION
  • INHIBITOR PAI-1
  • HIGH GLUCOSE
  • FACTOR-ALPHA
  • IGF-I

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