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

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 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.



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Figure 1  .

Immunohistochemical detection of u-PA, t-PA, and PAI-1 in human retinal endothelial cells. The detection of u-PA, t-PA, and PAI-1 was carried out using the APAAP method with HREC cultured in 5 mM glucose, 2.5% PDS in chamber slides. Positive staining was evident as pink coloration in the cytoplasm with u-PA (A), t-PA (B), and PAI-1 (C). A negative control is shown (D) (original magnification ×895).

Figure 2  .

Fibrin clot lysis by HREC. Standard clots, which were prepared with either no antibodies (♦) or antibodies to u-PA(-), t-PA(▪), and PAI-1(•), were overlaid on monolayers of HREC, pretreated in serum free containing medium supplemented with 5 mM glucose for 24 hours. Results are representative of three experiments using HREC from different donors. The presence of antibodies to t-PA significantly inhibited clot lysis.

Figure 3  .

Chromogenic detection of t-PA activity. Monolayers of HREC pretreated for 24 hours in serum free GMEM were incubated with plasminogen and S2251. Incorporation of anti-t-PA (▪) significantly reduced the generation of the colour while anti-u-PA (−) had no effect. A negative control using normal rabbit serum (⧫) is included for comparison.   

Figure 4  .

Effects of glucose on HREC fibrin clot lysis. Standard fibrin clots were prepared and overlaid on monolayers of HREC pretreated for 24 hours in serum free containing medium supplemented with 2 (•), 5(−), 15(*), or 25(▴) mM glucose. Results are representative of three experiments using HREC from different donors. The rate of clot lysis did not change when the cells were incubated in different glucose concentrations.   

Figure 5  .

TGF-β mediated increased expression of PAI-1 by HREC. HREC were stepped down overnight in serum free glucose free GMEM and then the medium was replaced with 5 or 15 mM glucose plus or minus TGF-β (1 ng/ml) for 24 hours. Results are the mean (SD) of six samples from one experiment and are representative of four experiments using HREC from different donors. Increased levels of PAI-1 antigen were evident when the cells were incubated with TGF-β as determined by a two tailed Student's t test (***p<0.001).

Figure 6  .

Effect of TGF-β on the level of PAI-1 mRNA. HREC were stepped down overnight in serum free glucose free GMEM. The medium was then replaced with serum free GMEM supplemented with 5 mM glucose plus or minus TGF-β (1 ng/ml). After 6 hours' incubation the cells were harvested and total RNA was extracted. Northern hybridisation indicated the presence of two PAI-1 specific mRNA bands (3.2 bp and 2.3 bp) and β-actin mRNA (A). The mRNA was quantified and is presented as the mean (SD) of the ratio of the respective PAI-1 transcripts with the level of β-actin mRNA with data from three separate experiments (B). The results were compared to a value of 100%—that is, the level of PAI-1 mRNA in the absence of TGF-β using a one tailed Student's t test (*p<0.05).

Figure 7  .

Effects of TGF-β treated HREC on fibrin clot lysis. HRECs, pretreated in serum free containing medium with either 5 mM glucose (♦) or 5 mM glucose plus TGF-β (1 ng/ml (▴) for 24 hours, were overlaid with fibrin. Antibody to PAI-1 was incorporated in the fibrin overlay of the TGF-β treated HREC (•). Results are representative of three experiments using different donors. The presence of TGF-β increased the rate of clot lysis and this effect was partially inhibited by the presence of antibody to PAI-1.