Regulation of plasminogen activation by TGF-beta in cultured human retinal endothelial cells.
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.
Wileman
S M
SM
Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.
Booth
N A
NA
Moore
N
N
Redmill
B
B
Forrester
J V
JV
Knott
R M
RM
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
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