Dabigatran, a direct thrombin inhibitor, demonstrates antifibrotic effects on lung fibroblasts

Abstract

Objective: Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor 1 (PAR-1) and induces a myofibroblast phenotype in normal lung fibroblasts resembling the phenotype of scleroderma lung myofibroblasts. We undertook this study to investigate whether a selective direct thrombin inhibitor, dabigatran, interferes with signal transduction in human lung fibroblasts induced by thrombin and mediated via PAR-1.

Methods: Lung fibroblast proliferation was analyzed using the Quick Cell Proliferation Assay. Expression and organization of alpha-smooth muscle actin (alpha-SMA) was studied by immunofluorescence staining and immunoblotting. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Connective tissue growth factor (CTGF) and type I collagen expression was analyzed on Western blots.

Results: Dabigatran, at concentrations of 50-1,000 ng/ml, inhibited thrombin-induced cell proliferation, alpha-SMA expression and organization, and the production of collagen and CTGF in normal lung fibroblasts. Moreover, when treated with dabigatran (1 microg/ml), scleroderma lung myofibroblasts produced 6-fold less alpha-SMA, 3-fold less CTGF, and 2-fold less type I collagen compared with untreated cells.

Conclusion: Dabigatran restrains important profibrotic events in lung fibroblasts and warrants study as a potential antifibrotic drug for the treatment of fibrosing lung diseases such as scleroderma lung disease and idiopathic pulmonary fibrosis.

Figure 1. Effect of dabigatran on lung fibroblast proliferation

A, Dabigatran inhibits thrombin-induced cell proliferation in a dose-dependent manner. B, Dabigatran does not affect cell proliferation induced by the PAR-1 selective agonist PAR-1-AP. Serun-starved lung fibroblasts were incubated with or without thrombin (Thr), PAR-1-AP, and dabigatran (Dabi, 1- 1000ng/ml) in 96-well plates for 24 hr, and then subjected to Quick Cell Proliferation Assay. Data are presented as optical density (OD) determined at 450 nm. C, Time-dependant effects of thrombin and dabigatran on lung fibroblast proliferation. Normal and SSc lung fibroblasts in 1% FCS were incubated with thrombin (0.5U/ml) alone or with dabigatran (0.5μg/ml) for time indicated. At 24 hour intervals, cells were trypsinized and counted using a Coulter particle counter. Experiments were performed three times and mean values ± SD are presented. The asterisk represents statistically significant (p<0.05) differences between cells stimulated with thrombin versus cells stimulated with thrombin and dabigatran.

Figure 2. Dabigatran inhibits α–smooth muscle actin (α–SMA) expression in lung fibroblasts

A, cells were serum-deprived overnight and then treated with thrombin and/or dabigatran for 24 hr. Cell lysate proteins (40μg) were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti- α–SMA antibody. The immunoblots were then stripped and re-blotted with anti-β-actin antibody as a loading control. B, The images were scanned and analyzed with NIH Imaging software. Densitometric analysis of immunoblots from 3 independent experiments is presented. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone.

Figure 3. Effect of dabigatran on thrombin and PAR-1-AP-induced α–SMA expression and organization in lung fibroblasts

Cells were incubated with or without thrombin (0.5U/ml), PAR-1-AP (10μM), and dabigatran (1μg/ml) for 24 hr. A, Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti- α–SMA antibody followed by immunoblotting with anti-β-actin antibody as a loading control. B, Cells were labeled with anti-α–SMA mouse monoclonal antibody followed by anti-mouse antiserum tagged with Alexa Fluor 488 (green) and DAPI staining for nuclei (blue). Images were acquired with an Olympus IX71 fluorescence microscope, objective ×60/1.42. Image 1 represents control cells in serum-free medium, 2 represents cells treated with dabigatran, 3 represents cells treated with thrombin, 4 represents cells treated with PAR-1-AP, 5 represents cells treated with dabigatran and thrombin, and 6 represents cells treated with dabigatran and PAR-1-AP.

Figure 4. Effect of dabigatran on collagen gel contraction induced by thrombin and PAR-1-AP in lung fibroblasts

A, Dabigatran inhibits thrombin-induced collagen gel contraction in lung fibroblasts in a concentration-dependent manner. B, the degree of collagen gel contraction was determined as the difference between diameters of well itself and released gels. C, dabigatran does not affect PAR-1-AP-induced collagen gel contraction. The experiment was performed on floating gels seeded with lung fibroblasts stimulated with thrombin and/or dabigatran for 24h. Data are presented as mean values ± SD of three tests. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone.

Figure 5. Inhibition of floating and fixed collagen gel contraction by dabigatran in lung fibroblasts

Normal and SSc lung fibroblasts were subjected to collagen gel contraction assay as described in Materials and Methods. Cells were stimulated with thrombin (Thr, 0.5U/ml) and dabigatran (Dabi, 1μg/ml) for 48 h. A, left panel - floating gels; right panel – fixed gels. Data are presented as mean values ± SD of three experiments. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone. B, Collagen gels were digested with collagenase and analyzed by Western blot using anti-SM-α-actin and anti-β-actin antibodies. Note that thrombin promotes contraction and induces SM-α-actin in floating gels; dabigatran inhibits SM-α-actin and contraction in both floating and fixed collagen gels.

Figure 6. Effect of dabigatran on collagen type I, CTGF, and α–SMA in normal and scleroderma lung fibroblasts

A, Dabigatran inhibits thrombin- but not PAR-1-AP-induced collagen type I and CTGF. Serum-starved normal lung fibroblasts were incubated with or without thrombin (0.5U/ml), PAR-1-AP (10μM), and dabigatran (1μg/ml) for 48 hr. Cells were then collected with lysis buffer and analyzed by Western blot using anti-type I collagen antibody and anti-CTGF antibody. Anti-α-actin antibody was used as sample loading control. B, Dabigatran inhibits collagen type I, CTGF, and α–SMA expression in scleroderma lung fibroblasts. Confluent cultures of scleroderma lung fibroblasts were serum-starved for 24hr followed by incubation for 24, 48, 72 and 96hr with dabigatran (1μg/ml). C, Dabigatran inhibits α–SMA expression and organization in scleroderma lung fibroblasts. Scleroderma lung fibroblasts cultured on glass slides were incubated with dabigatran (1μg/ml) for 72 hr. Expression and organization of α–SMA were studied with an Olympus IX71 fluorescence microscope equiped with Olympus Slidebook 4.1 software. The experiments were repeated three times in three different cell lines and representative immunoblots and images are presented.