Journal List > Tuberc Respir Dis > v.70(5) > 1001619

Lee, Jeon, Yoo, Kim, Lee, Kwon, Son, Choi, and Na: The Effect of Tissue Plasminogen Activator on TGF-β1 Pre-Treated Human Mesothelial Cell Line

Abstract

Background

In an effort to find alternative therapeutic agents to prevent excessive fibrosis as a sequela to complicated parapneumonic effusion or empyema, we examined the effect of tissue plasminogen activator (tPA) as a fibrinolytic agent combined with talc or transforming growth factor (TGF)-β1 in a human pleural mesothelial cell line, MeT-5A.

Methods

MeT-5A cells were stimulated with various doses of talc, doxycycline or TGF-β1 for 24 h and then were treated with tPA for an additional 24 h. Cell viability was measured by MTT assay. The production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in the culture supernatants was measured by ELISA. Real-time PCR was carried out for measurement of type I collagen mRNA.

Results

MeT-5A cells treated with talc showed a dose-dependent increase in production of IL-8. Talc also increased production of type I collagen mRNA at low doses, but talc did not influence the induction of VEGF. Addition of tPA to talc-stimulated cells showed further increases in the production of IL-8, but tPA did not influence the production of VEGF or type I collagen mRNA. TGF-β1 increased the production of both VEGF and collagen type I mRNA, both of which were effectively inhibited by additional tPA treatment in MeT-5A cells.

Conclusion

TGF-β1 is a potent inducer of collagen synthesis without induction of IL-8 in MeT-5A cells. Addition of tPA after TGF-β1 stimulation inhibited further fibrosis by direct inhibition of collagen mRNA synthesis as well as by inhibition of VEGF production.

Figures and Tables

Figure 1
Effect of talc (A), doxycycline (B) or TGF-β1 (C) on cell viability of MeT-5A cells. Cells were treated with various concentrations of talc, doxycycline or TGF-β1, as indicated, for 24 hours. Cell viability was measured by MTT assay. *p< 0.01, p<0.001 compared with untreated group. MTT: magnetic tape transport.
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Figure 2
Effect of talc, doxycycline or TGF-β1 on production of IL-8 (A), VEGF (B) or type I collagen mRNA (C) from MeT-5A cells. Cells were treated with talc (200µg/ mL), doxycycline (5µg/mL) or TGF-β1 (5 ng/mL) for 24 hours, respectively. The level of IL-8 or VEGF in the culture supernatants were determined by ELISA kit. The level of type I collagen mRNA was determined by real time RT-PCR. *p<0.01, p<0.001, compared with untreated group. ELISA: enzyme-linked immunosorbent assay.
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Figure 3
Effect of tPA on MeT-5A cells. Cells were treated with various concentration of tPA, as indicated, for 24 hours. (A) Cell viability was measured by MTT assay. (B) The induction of cytokine mRNA or chemokine mRNA was determined by RT-PCR. The level of IL-8 (C) or VEGF (D) in culture supernatants were determined by ELISA. *p<0.01, compared with untreated group. MTT: magnetic tape transport; RT-PCR: reverse transcriptase polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay.
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Figure 4
Effect of additional treatment of tPA on IL-8 production from talc- or TGF-β1-treated MeT-5A cells. Cells were treated with various concentration of talc (A) or TGF-β1 (B), as indicated, for 24 hours. Then 50µg/mL of tPA were added to each cell and cultured for additional 24 hours. The level of IL-8 in culture supernatants were determined by ELISA kit. *p<0.05, p<0.01 compared with tPA-untreated group. tPA: tissue plasminogen activator; ELISA: enzyme- linked immunosorbent assay.
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Figure 5
Effect of additional treatment of tPA on VEGF production from talc- or TGF-β1-treated MeT-5A cells. Cells were treated with various concentration of talc (A) or TGF-β1 (B), as indicated, for 24 hours. Then 50µg/mL of tPA were added to each cell and cultured for additional 24 hours. The level of VEGF in culture supernatants were determined by ELISA kit. *p<0.01 compared with tPA-untreated group. tPA: tissue plasminogen activator; ELISA: enzyme-linked immunosorbent assay.
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Figure 6
Effect of additional treatment of tPA on type I collagen mRNA induction from talc- or TGF-β1-treated MeT-5A cells. Cells were treated with various concentration of talc (A) or TGF-β1 (B), as indicated, for 24 hours. Then 50µg/mL of tPA were added to each cell and cultured for additional 24 hours. The expression of type I collagen mRNA determined by real-time RT-PCR. *p<0.05 compared with tPA-untreated group. tPA: tissue plasminogen activator.
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Table 1
Primers used for RT-PCR or real-time RT-PCR
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Acknowledgements

This work was supported by Konyang University Myunggok Research Fund of 2007.

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