title: Regulation of plasminogen activation by TGF-beta in cultured human retinal endothelial cells.
abstract: OBJECTIVE
  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 beta (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.
authors.affiliation: Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.
authors.forename: S M
authors.initials: SM
authors.lastname: Wileman
authors.forename: N A
authors.initials: NA
authors.lastname: Booth
authors.forename: N
authors.initials: N
authors.lastname: Moore
authors.forename: B
authors.initials: B
authors.lastname: Redmill
authors.forename: J V
authors.initials: JV
authors.lastname: Forrester
authors.forename: R M
authors.initials: RM
authors.lastname: Knott
mesh_terms: D018929:Cell Culture Techniques; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D010958:Plasminogen / Q000502:physiology; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism*; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D016212:Transforming Growth Factor beta; D005337:Fibrin; D010958:Plasminogen; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D005947:Glucose