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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Objective

Activation of the coagulation cascade leading to generation of thrombin has been documented extensively in various forms of lung injury, including that associated with systemic sclerosis. We previously demonstrated that the direct thrombin inhibitor dabigatran inhibits thrombin-induced profibrotic signaling in lung fibroblasts. This study was undertaken to test whether dabigatran etexilate attenuates lung injury in a murine model of interstitial lung disease.

Methods

Lung injury was induced in female C57BL/6 mice by a single intratracheal instillation of bleomycin. Dabigatran etexilate was given as supplemented chow beginning on day 1 of bleomycin instillation (early treatment, study of antiinflammatory effect) or on day 8 following bleomycin instillation (late treatment, study of antifibrotic effect). Mice were killed 2 weeks or 3 weeks after bleomycin instillation, and lung tissue, bronchoalveolar lavage (BAL) fluid, and plasma were investigated.

Results

Both early treatment and late treatment with dabigatran etexilate attenuated the development of bleomycin-induced pulmonary fibrosis. Dabigatran etexilate significantly reduced thrombin activity and levels of transforming growth factor β1 in BAL fluid, while simultaneously reducing the number of inflammatory cells and protein concentrations. Histologically evident lung inflammation and fibrosis were significantly decreased in dabigatran etexilate–treated mice. Additionally, dabigatran etexilate reduced collagen, connective tissue growth factor, and α-smooth muscle actin expression in mice with bleomycin-induced lung fibrosis, whereas it had no effect on basal levels of these proteins.

Conclusion

Inhibition of thrombin using the oral direct thrombin inhibitor dabigatran etexilate has marked antiinflammatory and antifibrotic effects in a bleomycin model of pulmonary fibrosis. Our data provide preclinical information about the feasibility and efficacy of dabigatran etexilate as a new therapeutic approach for the treatment of interstitial lung disease.

In recent years there has been increasing evidence implicating involvement of the coagulation system in various fibrotic diseases, including idiopathic pulmonary fibrosis (IPF) and the interstitial lung fibrosis associated with systemic sclerosis (SSc) (1, 2). Activation of the coagulation cascade is one of earliest events following tissue injury, including lung injury (3). This complex and highly regulated system leads to the generation of insoluble, crosslinked fibrin to form plugs at the site of tissue injury. This process is critically dependent on the action of the serine protease thrombin (4).

In addition to its essential role in coagulation, thrombin has several important functions at the cellular level, both in normal health and in multiple disease processes (5). The majority of the cellular responses to thrombin are mediated via the G protein–coupled receptor protease-activated receptor 1 (PAR-1) (2–6). Previously, we demonstrated that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via PAR-1 and a protein kinase C–dependent pathway (7). Thrombin is mitogenic for lung fibroblasts (7–9) and enhances the proliferative effect of fibrinogen on fibroblasts (10). It is also a potent inducer of fibrogenic cytokines, such as transforming growth factor β (TGFβ) (11), connective tissue growth factor (CTGF) (12, 13), and platelet-derived growth factor AA (PDGF-AA) (9). In addition, thrombin increases expression of proinflammatory chemokines (14, 15) and extracellular matrix (ECM) proteins, such as collagen, fibronectin, and tenascin in various cells, including lung fibroblasts (16–18). Activation of these cells by thrombin is a likely mechanism for the development and progression of pulmonary fibrosis in general, and in particular SSc-associated interstitial lung disease (SSc-ILD), in which endothelial injury and activation of the coagulation cascade is widespread.

Activation of the coagulation cascade with generation of thrombin has also been shown to occur in an animal model of bleomycin-induced lung injury and fibrosis (1, 2, 19). Howell et al demonstrated in such a model that direct thrombin inhibition attenuates CTGF and lung collagen accumulation by lowering the profibrotic effects of thrombin (19). Additionally, increased thrombin activity and PAR-1 expression, similar to that which we have demonstrated in SSc-ILD (8, 9), have been observed in bleomycin-induced lung fibrosis (19, 20).

Dabigatran is a direct thrombin inhibitor that reversibly binds to the active site of thrombin, preventing the conversion of fibrinogen to fibrin (21). Recently we demonstrated that binding of dabigatran to thrombin prevents cleavage of the extracellular N-terminal domain of PAR-1 (22). In the absence of dabigatran, thrombin binds to PAR-1 and cleaves the peptide bond between residues Arg-41 and Ser-42, thereby unmasking a new amino terminus, SFLLRN, which then can bind to the second extracellular loop of PAR-1 and initiate receptor signaling (23). Dabigatran-bound thrombin is unable to cleave and activate PAR-1 (22). Further, we have shown that dabigatran inhibits thrombin-induced differentiation of normal lung fibroblasts to the myofibroblast phenotype and decreases CTGF, α-smooth muscle actin (α-SMA), and type I collagen in lung fibroblasts from patients with SSc (22).

In this study we investigated dabigatran etexilate, the oral prodrug of dabigatran. The prodrug does not have antithrombin activity; however, after oral administration, dabigatran etexilate is rapidly converted by ubiquitous esterases to the active moiety, dabigatran (21, 24). The present study was designed to determine whether the oral direct thrombin inhibitor dabigatran etexilate has preventive and/or therapeutic effects on bleomycin-induced pulmonary fibrosis in mice.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Animal model of fibrosis.

Female C57BL/6 mice (6–8 weeks old; n = 160) were purchased from The Jackson Laboratory. All mice were maintained under pathogen-free conditions and were provided food and water ad libitum. Lung injury was induced when the mice were 8–10 weeks old (weighing 19–21 gm), by intratracheal instillation of bleomycin (0.045 units/mouse). On the same day as bleomycin treatment or 8 days following bleomycin treatment, dabigatran was administered with food, using chow supplemented with 10 mg/gm dabigatran etexilate or placebo (both from Boehringer Ingelheim). Two weeks or 3 weeks after bleomycin instillation, mice were killed by cervical dislocation under isoflurane anesthesia, and lungs, bronchoalveolar lavage (BAL) fluid, and plasma were collected. All experimental procedures were performed according to guidelines of the Institutional Animal Care and Use Committee of the Medical University of South Carolina.

BAL fluid collection and analysis.

BAL was performed when mice were killed, by instillation of saline (1 ml) using a tracheal cannula. The BAL fluid was centrifuged (500g for 10 minutes), and supernatants were stored at −80°C until assayed. The cell pellet was removed and subjected to total and differential cell counting as previously described (25). The protein concentration in BAL fluid was measured with a BCA protein assay kit (Pierce).

Thrombin assay.

The level of active thrombin in BAL fluid was determined by the spectrophotometric method. Aliquots of BAL fluid (100 μl) were mixed with 50 μl of buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, and 50 μl of the thrombin substrate Boc-Val-Pro-Arg-7-amido-4-methylcoumarin hydrochloride (Sigma) at 37°C. Absorbance at 405 nm was read on a spectrophotometer, and thrombin activity was determined by extrapolation from a thrombin standard curve.

Lung fixation and histologic examination.

Midline thoracotomy was performed after the mice were killed. The trachea was cannulated, and the lungs were fixed by instillation of 2% buffered formalin for 24 hours followed by perfusion with 70% ethanol for another 24 hours before routine processing and paraffin embedding. Multiple sections from each lung were stained with hematoxylin and eosin (H&E), or with Masson's trichrome for assessment of collagen and other ECM proteins. For the area analysis of fibrotic changes, a quantitative fibrotic scale (Ashcroft scale) was used (26). The severity of fibrotic changes in each lung section was recorded as the mean score from the observed microscopic fields. Ten fields within each lung section were observed at 20× magnification, and each field was assessed individually for severity and scored from 0 (normal) to 8 (total fibrosis). All histologic specimens were evaluated under blinded conditions. Each specimen was scored independently by 2 observers, and the mean of the scores was used. TGFβ immunohistochemistry was performed by the aminoethylcarbazole chromogen method (Invitrogen) using rabbit polyclonal anti-TGFβ1 antibody (Santa Cruz Biotechnology). CTGF and α-SMA immunohistochemistry was performed with a diaminobenzidine substrate kit (Vector) using anti-CTGF rabbit polyclonal antibodies (Santa Cruz Biotechnology) and anti–α-SMA rabbit polyclonal antibodies (Abcam).

Collagen, lung hydroxyproline, and TGFβ1 assays.

Collagen was assessed by Sircol assay in accordance with the instructions of the manufacturer (Accurate). The Sircol assay is a direct quantitative method for analysis of collagens based on the reagent Sirius red, which reacts specifically with the side chain groups of the basic amino acids present in collagen. Hydroxyproline levels in the lung were determined by colorimetric measurement of hydroxyproline in lung hydrolysates, reflecting total collagen in lung tissue, according to established procedures (27). Levels of TGFβ1 in BAL fluid samples (50 μl) were measured using the Quantikine Mouse/Rat TGFβ1 Immunoassay according to the instructions of the manufacturer (R&D Systems).

Immunoblotting.

CTGF was assayed as described previously (22). Smad3 was analyzed by Western blot using anti–phospho-Smad3 and anti-Smad3 rabbit monoclonal antibodies (Cell Signaling Technology), and α-SMA by Western blot using anti–α-SMA rabbit polyclonal antibody. The immunoblots were stripped and reblotted with anti–β-actin polyclonal antibody (Santa Cruz Biotechnology) as a loading control.

Assessment of dabigatran in mouse plasma and clotting tests.

Blood was collected by cardiac puncture when mice were killed. Plasma was prepared by centrifugation of citrated blood samples at 2,000g for 15 minutes. Plasma dabigatran levels were determined using validated liquid chromatography–tandem mass spectrometry methods described by Troconiz et al (28). Because the amount of plasma obtained in this study was too little to enable both performance of clotting tests and measurement of plasma dabigatran levels, plasma was obtained from separate mice (n = 7) as described above. An in vitro standard curve with increasing concentrations of dabigatran was used in order to correlate anticoagulant activity with plasma concentrations. Clotting tests and measurement of thrombin time (TT) and activated partial thromboplastin time (APTT) were performed with a CL4 coagulometer (Behnk Elektronik) as described by Palm et al (29). Mouse plasma was pooled, and each test was performed in duplicate. Increasing concentrations of dabigatran were preincubated with 500 μl plasma and then used in either test. Thrombin (3 IU stock; Siemens Healthcare) was used to initiate clotting for the TT measurement. The APTT reagent (Diagnostica Stago) was added to plasma, and CaCl2 (Dade Behring) was used to initiate clotting. The time until the plasma clotting was recorded in seconds.

Isolation of lung fibroblasts, collagen gel contraction assay, and α-SMA immunofluorescence studies.

Fibroblasts were isolated from the left lung of the mice, using procedures we have described previously (22). Lung fibroblasts were subjected to collagen gel contraction assay and to immunofluorescence analysis for α-SMA (22).

Statistical analysis.

Statistical analyses were performed by analysis of variance followed by post hoc testing or nonparametric test, as appropriate. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Bleomycin is a well-established agent for inducing pulmonary inflammation and fibrosis (30). In the present study, we compared the effects of oral administration of the direct thrombin inhibitor dabigatran etexilate on the day of bleomycin instillation (day 1) and the effects of administration on day 8 after bleomycin instillation in mice. It is recognized that drugs administered during the early injury phase predominantly act as antiinflammatory agents and should be considered as preventive treatment, whereas “true” antifibrotic agents might be effective irrespective of timing, particularly if administered during the “fibrotic” phase of the model (30). Administration of dabigatran etexilate beginning on day 1 and beginning on day 8 after bleomycin allowed us to distinguish between antiinflammatory and antifibrotic drug effects.

Histologic evaluation of lung inflammation and fibrosis.

In control mice that received saline alone or saline plus dabigatran etexilate, lung histology was characterized by alveolar structures composed of septa, vascular components, and connective tissue. Alveolar septa were thin, allowing maximum air to occupy the lung. Lung tissue isolated from bleomycin-treated mice exhibited extensive peribronchial and interstitial infiltration of inflammatory cells as seen on H&E staining, thickening of the alveolar walls, and multiple focal fibrotic lesions with excessive amounts of ECM protein shown by trichrome differential staining (Figure 1A). In contrast, significantly fewer cellular infiltrates, decreased thickness of alveolar septa, and reduced accumulation of ECM proteins were noted in dabigatran etexilate–treated mice. Importantly, beneficial effects of dabigatran etexilate on bleomycin-induced pulmonary fibrosis were observed not only in mice receiving dabigatran etexilate beginning on the same day as bleomycin, but also in mice that received dabigatran etexilate beginning on day 8 after bleomycin administration (Figure 1A).

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Figure 1. Effect of dabigatran etexilate on bleomycin-induced pulmonary fibrosis. A, Histologic findings of lung inflammation and fibrosis in a control mouse treated with saline plus placebo or saline plus dabigatran etexilate (designated 1), a mouse treated with bleomycin plus placebo (designated 2), a mouse treated with bleomycin plus dabigatran etexilate started on day 1 of bleomycin treatment (designated 3), and a mouse treated with bleomycin plus dabigatran etexilate started on day 8 of bleomycin treatment (designated 4). Representative results are shown (n = 10 mice per group). H&E = hematoxylin and eosin. Original magnification × 200. B, Quantitative evaluation of fibrotic changes (Ashcroft scores) (n = 10 mice per group). C, Lung collagen content determined by Sircol assay (n = 6 mice per group). D, Lung collagen content determined by hydroxyproline assay (n = 8 mice per group). Values in B–D are the mean ± SD. ∗ = P < 0.05 versus mice treated with bleomycin plus placebo.

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The overall level of fibrotic changes was assessed quantitatively based on the Ashcroft scoring system (26). The score in mice that received bleomycin and placebo was nearly 9-fold higher than that in control mice (mean ± SD 5.76 ± 1.64 versus 0.65 ± 0.7). The mean score in mice treated with dabigatran etexilate beginning on day 1 of bleomycin instillation was significantly reduced (2.8-fold; P < 0.05) when compared to that in the bleomycin plus placebo group, suggesting an antiinflammatory effect of dabigatran (Figure 1B). Interestingly, the score in mice that received dabigatran beginning on day 8 after bleomycin instillation was also significantly reduced (1.9-fold; P < 0.05) compared to the bleomycin plus placebo group, suggesting that, in addition to its antiinflammatory properties, dabigatran had a strong antifibrotic effect in bleomycin-induced pulmonary fibrosis.

Effect of dabigatran etexilate on collagen accumulation in bleomycin-induced pulmonary fibrosis.

The most profound development of fibrosis in the bleomycin model is observed by day 21; therefore, we determined the effect of dabigatran on collagen accumulation in the lungs 21 days after bleomycin treatment. To quantify collagen accumulation within the lungs we used 2 independent methods, the Sircol collagen assay and the hydroxyproline assay. We observed that collagen content in dabigatran etexilate–treated control mice was similar to that in saline-treated control mice (data not shown). Collagen content in bleomycin-treated mice was increased by 4.4-fold over control. Dabigatran etexilate significantly reduced collagen content, by 53.0% when administration began on day 1 and by 37.6% when administration began on day 8 (Figure 1C).

In the hydroxyproline assay, we observed that dabigatran etexilate did not affect basal levels of hydroxyproline (data not shown). However, dabigatran etexilate significantly reduced hydroxyproline levels in bleomycin-treated mice, by 63.9% and 38.9% when administered beginning on day 1 and on day 8, respectively (Figure 1D). From these results we conclude that dabigatran etexilate down-regulates lung collagen, consistent with an antifibrotic effect mediated by direct thrombin inhibition.

Effect of dabigatran etexilate on BAL fluid content.

The total cell count in BAL fluid on day 14 was markedly higher in the bleomycin-treated group as compared to saline-treated controls (Table 1). Dabigatran etexilate treatment starting on day 1 and starting on day 8 significantly reduced total cell counts in BAL fluid obtained from bleomycin plus placebo–treated mice (P < 0.01). Dabigatran etexilate alone did not affect BAL fluid cell counts (data not shown). In mice that received dabigatran etexilate starting on day 1, the percentage of macrophages compared to that in mice treated with bleomycin plus placebo was significantly increased, whereas the difference in mice receiving dabigatran etexilate starting on day 8 was not significant. The percentage of neutrophils in BAL fluid was significantly decreased in bleomycin plus dabigatran etexilate–treated mice, when dabigatran etexilate was started either on day 1 or on day 8, compared to that in bleomycin plus placebo–treated mice (P < 0.01). Total protein in BAL fluid was increased by 7.9-fold in bleomycin plus placebo–treated mice compared to controls, and this was significantly reduced by dabigatran treatment starting on either day 1 or day 8 (P < 0.01).

Table 1. Results of bronchoalveolar lavage fluid analysis*
ParameterTreatment group
ControlBleomycin/placeboBleomycin/dabigatran day 1Bleomycin/dabigatran day 8
  • *

    Bronchoalveolar lavage was performed on day 14 after bleomycin administration. In the bleomycin/dabigatran day 1 and bleomycin/dabigatran day 8 groups, dabigatran etexilate was administered starting on the day of bleomycin treatment and starting 8 days after bleomycin treatment, respectively. Values are the mean ± SD (n = 8 mice per treatment group).

  • P < 0.01 versus bleomycin/placebo-treated mice.

Total protein, mg/ml0.24 ± 0.091.89 ± 0.260.87 ± 0.410.89 ± 0.36
Total cell count, × 105/ml8.0 ± 3.541.9 ± 17.018.2 ± 7.521.0 ± 8.5
Macrophages, %97.8 ± 1.671.6 ± 8.384.8 ± 9.379.4 ± 9.0
Lymphocytes, %2.2 ± 1.410.3 ± 4.48.7 ± 3.79.1 ± 5.8
Neutrophils, %018.1 ± 5.66.5 ± 2.211.5 ± 5.4

On day 21 after bleomycin instillation there were notably fewer cells in BAL fluid from mice treated with bleomycin plus dabigatran etexilate compared to mice treated with bleomycin plus placebo. However, none of the differences in cell numbers among the studied groups reached statistical significance at that time point (data not shown).

Effect of dabigatran etexilate on thrombin activity in BAL fluid.

The level of active thrombin in BAL fluid from bleomycin plus placebo–treated mice was increased 35-fold compared to that in mice treated with saline plus placebo. Dabigatran etexilate treatment beginning on day 1 or on day 8 significantly reduced the level of active thrombin (by 74.1% in both groups) (Figure 2A). It also decreased basal levels of active thrombin in BAL fluid, from a mean ± SD of 1.32 ± 0.35 ng/ml to 0.61 ± 0.33 ng/ml. The average concentration of dabigatran in plasma obtained from mice fed dabigatran etexilate in chow was 342.1 ± 90.0 ng/ml (n = 21).

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Figure 2. A, Effect of dabigatran etexilate on levels of active thrombin in bronchoalveolar lavage (BAL) fluid from control mice treated with saline plus placebo or saline plus dabigatran etexilate (designated 1), with bleomycin plus placebo (designated 2), with bleomycin plus dabigatran etexilate started on day 1 of bleomycin treatment (designated 3), or with bleomycin plus dabigatran etexilate started on day 8 of bleomycin treatment (designated 4). Values are the mean ± SD (n = 10 mice per group). ∗ = P < 0.01 versus mice treated with bleomycin plus placebo. B, Effect of increasing concentrations of dabigatran on the thrombin time (TT) and activated partial thromboplastin time (APTT) in mouse plasma in vitro. Values are the mean of duplicate determinations. C, Effect of dabigatran etexilate on transforming growth factor β1 (TGFβ1) levels in BAL fluid. Values are the mean ± SD (n = 6 mice per group). ∗ = P < 0.01 versus mice treated with bleomycin plus placebo. D, Histologic analysis of the effect of dabigatran etexilate on TGFβ1 levels in lung tissue. Representative results are shown (n = 5 mice per group). Original magnification × 200.

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In vitro anticoagulation.

We observed a concentration-dependent increase in clotting time with increasing concentrations of dabigatran added in vitro (Figure 2B). The TT in control plasma was 7.5 seconds, and the APTT was 22.8 seconds. Mean plasma levels achieved in this study (342.1 ± 90.0 ng/ml) resulted in prolongation of the TT and APTT over baseline levels (∼10-fold and ∼2-fold, respectively).

Effect of dabigatran etexilate on TGFβ1 in BAL fluid and lung tissue.

We observed that TGFβ1 was up-regulated by 2.7-fold over control in mice treated with bleomycin plus placebo. Dabigatran etexilate significantly reduced TGFβ1 levels, from a mean ± SD of 54.9 ± 6.1 pg/ml in bleomycin plus placebo–treated mice to 29.9 ± 9.3 pg/ml and 31.1 ± 8.7 pg/ml when dabigatran was administered beginning on day 1 and beginning on day 8, respectively (P < 0.01) (Figure 2C). TGFβ1 expression in lung tissue was also assessed, by immunohistochemistry. In this analysis, TGFβ1 was not detectable in tissue from control mice, whereas it was strongly positive in fibrotic areas of tissue from mice treated with bleomycin plus placebo (Figure 2D). Dabigatran etexilate visibly reduced TGFβ1 accumulation when used as either early or late treatment.

Effect of dabigatran etexilate on Smad3 phosphorylation.

Most profibrotic effects of TGFβ are mediated through the Smad signaling pathway. Accordingly, we determined activation of Smad3 by measuring levels of phosphorylated Smad3 in lung tissue. Phosphorylated Smad3 was increased by 5.4-fold in mice treated with bleomycin plus placebo as compared to control mice (Figure 3). Dabigatran etexilate significantly reduced the levels of phosphorylated Smad3, by 8.1-fold and 6.4-fold when administered beginning on day 1 and beginning on day 8 respectively (P < 0.01). Basal levels of Smad3 were not affected by any treatment.

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Figure 3. Effect of dabigatran etexilate on connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and Smad3 expression in lung tissue. A, Immunoblots of CTGF, α-SMA, phosphorylated and basal Smad3, and β-actin (as loading control) from control mice treated with saline plus placebo or saline plus dabigatran etexilate (designated 1), mice treated with bleomycin plus placebo (designated 2), mice treated with bleomycin plus dabigatran etexilate started on day 1 of bleomycin treatment (designated 3), and mice treated with bleomycin plus dabigatran etexilate started on day 8 of bleomycin treatment (designated 4). Representative results are shown (n = 4 mice per group). B, Quantitative densitometric analysis of the levels of CTGF, α-SMA, and phosphorylated and basal Smad3 in lung tissue. Values are the mean ± SD. ∗ = P < 0.01 versus mice treated with bleomycin plus placebo.

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Effect of dabigatran etexilate on CTGF and α-SMA expression in bleomycin-induced pulmonary fibrosis.

We did not detect CTGF in lung tissue from control mice. However, CTGF was strongly expressed in lung tissue from mice treated with bleomycin (Figure 3). Dabigatran reduced CTGF expression by 10.5-fold and 9-fold when initiated on day 1 and day 8, respectively (Figure 3). Similarly, α-SMA was strongly up-regulated in lung tissue from bleomycin plus placebo–treated mice, whereas it was significantly reduced in lung tissue from both groups of bleomycin plus dabigatran etexilate–treated mice (2.7-fold and 2.5-fold when initiated on day 1 and on day 8, respectively) (Figure 3). We also measured CTGF and α-SMA expression by immunohistochemistry. These experiments showed that both proteins were strongly up-regulated in the lungs of bleomycin-treated mice, whereas there was no visible staining in control lung tissue, with the exception of some staining for α-SMA, which was restricted to smooth muscle cells located around blood vessels (Figure 4B). Dabigatran etexilate reduced the up-regulated expression of CTGF (Figure 4A) and α-SMA (Figure 4B) in lung tissue, when administered beginning on either day 1 or day 8 of bleomycin treatment.

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Figure 4. Immunohistochemical evaluation of the expression of connective tissue growth factor (A) and α-smooth muscle actin (B) in the lung tissue of control mice treated with saline plus placebo or saline plus dabigatran etexilate, with bleomycin (Bleo) plus placebo, with bleomycin plus dabigatran etexilate (Dabi) started on day 1 of bleomycin treatment, or with bleomycin plus dabigatran etexilate started on day 8 of bleomycin treatment. Representative results are shown (n = 5 mice per group). Original magnification × 200.

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Effect of dabigatran etexilate on α-SMA and collagen gel contraction in lung fibroblasts.

Fibroblasts were isolated from lung tissue of control mice, mice treated with bleomycin plus placebo, and mice treated with bleomycin plus dabigatran etexilate. Lung fibroblasts from bleomycin plus placebo–treated mice expressed high amounts of α-SMA stress fibers, while in control mice there were no visible α-SMA stress fibers (Figure 5A). Dabigatran etexilate strongly reduced α-SMA expression and organization when administered on day 1; the inhibition of stress fibers was observed to a lesser extent when dabigatran etexilate was administered on day 8. Lung fibroblasts isolated from bleomycin-treated mice contracted collagen gels to a significantly greater extent than was observed in controls, whereas cells isolated from dabigatran etexilate–treated mice exhibited less contractile activity (Figure 5B).

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Figure 5. A, Effect of dabigatran etexilate on α-smooth muscle actin expression/organization in fibroblasts from a control mouse treated with saline plus placebo or saline plus dabigatran etexilate (designated 1), a mouse treated with bleomycin plus placebo (designated 2), a mouse treated with bleomycin plus dabigatran etexilate started on day 1 of bleomycin treatment (designated 3), and a mouse treated with bleomycin plus dabigatran etexilate started on day 8 of bleomycin treatment (designated 4). Representative results are shown (n = 6 mice per group). Original magnification × 40. B, Effect of dabigatran etexilate on collagen gel contraction in mice from each treatment group. Values are the mean ± SD. ∗ = P < 0.01 versus mice treated with bleomycin plus placebo.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

Our recent in vitro studies demonstrated that the direct thrombin inhibitor dabigatran blocks thrombin-induced and PAR-1–mediated profibrotic signaling (22). Dabigatran inhibited thrombin-induced collagen and CTGF production in normal and SSc lung fibroblasts, blocked development of the myofibroblast phenotype from thrombin-activated normal lung fibroblasts, and reversed the myofibroblast phenotype expressed by lung fibroblasts from patients with SSc-ILD (22). These data suggest that dabigatran may serve as a novel and attractive therapeutic agent for pulmonary fibrosis. The present study was designed to determine whether dabigatran etexilate exhibits therapeutic and/or preventive effects on bleomycin-induced pulmonary fibrosis in mice. We demonstrate here for the first time that dabigatran etexilate attenuates bleomycin-induced pulmonary fibrosis by lowering thrombin activity to physiologic levels and by decreasing proinflammatory and profibrotic factors, supporting the idea that it has potential for use in the treatment of pulmonary fibrosis.

Thrombin is a multifunctional protease that promotes a wide range of cellular responses in addition to its central function in thrombosis and hemostasis (1–6). It mediates a variety of inflammatory and tissue repair responses associated with vascular injury (5). Additionally, thrombin stimulates secretion of mediators involved in the pathogenesis of fibrosis, such as TGFβ1 and PDGF (9, 11). TGFβ1 and PDGF are each elevated in BAL fluid of patients with IPF and SSc-ILD, as well as in BAL fluid in animal models of pulmonary fibrosis (9, 31–36). Utilizing a model of bleomycin-induced lung injury and fibrosis, we have demonstrated that dabigatran etexilate significantly decreases inflammatory cell numbers and protein content as well as levels of TGFβ1 in BAL fluid. It also significantly reduces CTGF and α-SMA expression in lung tissue of bleomycin-treated mice, suggesting that it exerts both antiinflammatory and antifibrotic effects.

There is compelling evidence that thrombin is an important mediator of interstitial lung disease including both IPF and SSc-ILD (1, 2, 5). We and others have demonstrated dramatically increased thrombin activity in BAL fluid from patients with SSc-ILD compared to normal subjects (9, 37). Elevated thrombin activity has also been observed in bleomycin-induced pulmonary fibrosis. Howell et al found that in bleomycin-induced lung fibrosis, the direct thrombin inhibitor UK-156406 attenuates collagen accumulation in the lung by lowering the profibrotic effects of thrombin and suppressing CTGF synthesis (19). Later, the same group demonstrated that mice lacking PAR-1 are significantly protected against bleomycin-induced lung fibrosis, with reductions in CCL2 and CTGF expression and TGFβ immunoreactivity (20). Recent studies by Thuillier et al (38) demonstrated that inhibition of thrombin reduced chronic kidney graft fibrosis and significantly improved survival rates in ischemia-reperfusion injury (38, 39).

Different models of pulmonary fibrosis have been developed over the years. Most mimic some but never all features of IPF and SSc-ILD, especially the progressive and irreversible condition (40). This may partially explain why some drugs effective in the treatment of bleomycin-induced pulmonary fibrosis may not demonstrate the same efficacy in pulmonary fibrosis in humans.

In this study, we utilized a single intratracheal administration of bleomycin, which is the most frequently used method for inducing pulmonary fibrosis in animal models. We studied both early and late treatment with dabigatran etexilate to distinguish antiinflammatory and antifibrotic effects of this drug. We observed that dabigatran etexilate markedly improved bleomycin-altered histopathology and reduced interstitial infiltration. It also reduced the thickness of alveolar septa and decreased the accumulation of ECM proteins. The collagen, CTGF, and α-SMA contents of the lung after bleomycin injury were significantly lower in dabigatran etexilate–treated mice than in placebo-treated mice. Importantly, we found that with both early treatment and late treatment, dabigatran etexilate was able to inhibit bleomycin-induced pulmonary fibrosis; however, the inhibition was more profound with early administration. The efficacy of early treatment with dabigatran etexilate was higher because it targeted the inflammatory stage of fibrosis, whereas late treatment was introduced at a stage when the disease was more established.

The role of inflammation in the pathogenesis of progressive pulmonary fibrosis remains a matter of controversy. Administration of bleomycin causes a severe acute inflammatory response followed by chronic inflammation and fibrosis. It has been repeatedly shown that the degree of inflammation in bleomycin-induced lung injury is associated with the intensity of fibrosis (40).

The concentration of dabigatran etexilate used in these experiments yielded plasma levels that are slightly higher than those achieved in patients treated with dabigatran etexilate for various thrombotic diseases (∼180 ng/ml peak levels achieved with 150 mg twice-daily dose) (41). The dose used in this study resulted in an ∼2-fold elevation of the APTT in mice and an ∼10-fold elevation of the TT. This trend is consistent with findings in human plasma, where it has also been shown that the TT is more sensitive to dabigatran than the APTT (41). Although it is not possible to directly relate findings regarding plasma levels and/or anticoagulation from mice to humans, the antifibrotic effects observed in our study were achieved with plasma levels and pharmacologic effects consistent with human dosing.

It is important to note that dabigatran etexilate in the concentrations used in this study significantly reduced, but did not completely inhibit, thrombin activity in BAL fluid. We did not observe any bleeding side effects during the study, suggesting that levels of dabigatran in mouse plasma were not sufficient to completely eliminate thrombin from the normal hemostatic process. However, dabigatran etexilate in the doses tested ameliorated lung fibrosis even after it was established, indicating that it could safely be administered in chronic forms of lung fibrosis, at least in mice.

Pulmonary fibrosis as seen in both IPF and SSc-ILD is often a progressive and irreversible process that leads to respiratory failure and death (32, 42). There is an urgent need for new therapeutic approaches that would delay or reverse pulmonary fibrosis. Blocking of a single profibrotic factor has thus far not been proven to be an effective treatment method. A therapeutic strategy designed to act upstream of multiple fibrogenic pathways, e.g., altering procoagulant activity, might theoretically prove to be more efficacious. Indeed, anticoagulant therapy with warfarin or low molecular weight heparin in combination with prednisolone has been demonstrated to improve survival in patients with IPF (43). Dabigatran etexilate represents the first synthetic oral reversible direct inhibitor of thrombin with a very favorable biochemical and pharmacologic profile that translates into clinical efficacy and safety in patients with coagulation disorders (44). The current study provides important preclinical information about the feasibility and efficacy of dabigatran etexilate for the treatment of fibrotic diseases, including IPF and SSc-ILD, in which there is evidence of tissue injury with overexpression of thrombin. Future studies of thrombin inhibition for the treatment of SSc-ILD would need to demonstrate a positive risk/benefit ratio, taking into account potential risks such as gastrointestinal tract hemorrhage.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Bogatkevich had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Bogatkevich, Ludwicka-Bradley, van Ryn, Silver.

Acquisition of data. Bogatkevich, Akter, van Ryn.

Analysis and interpretation of data. Bogatkevich, Ludwicka-Bradley, Nietert, Akter, van Ryn, Silver.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

The manuscript was written entirely by the authors. Boehringer Ingelheim performed the measurement of plasma dabigatran levels and clotting tests and reviewed the manuscript, agreed to its submission, and approved the content.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES

The authors would like to thank Dr. Karl Wagner for producing the dabigatran-supplemented chow and C. Beth Singleton and Isaac J. Van Duys for excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. Acknowledgements
  9. REFERENCES
  • 1
    Chambers RC. Procoagulant signaling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention? Br J Pharmacol 2008; 153 Suppl 1: S36778.
  • 2
    Howell DC, Laurent GJ, Chambers RC. Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis. Biochem Soc Trans 2002; 30: 2116.
  • 3
    Green D. Coagulation cascade. Hemodial Int 2006; 10 Suppl 2: S24.
  • 4
    Mann KG. Thrombin formation. Chest 2003; 124: 410S.
  • 5
    Ludwicka-Bradley A, Bogatkevich G, Silver RM. Thrombin- mediated cellular dysfunction in pulmonary fibrosis associated with systemic sclerosis (scleroderma). Clin Exp Rheumatol 2004; 22 Suppl 33: S3846.
  • 6
    Coughlin SR. Thrombin signaling and protease-activated receptors. Nature 2000; 407: 25864.
  • 7
    Bogatkevich GS, Tourkina E, Silver RM, Ludwicka-Bradley A. Thrombin differentiates normal lung fibroblast to a myofibroblast phenotype via proteolytically activated receptor-1 and protein kinase C-dependent pathway. J Biol Chem 2001; 276: 4518492.
  • 8
    Bogatkevich GS, Gustilo E, Oates J, Feghali-Bostwick C, Harley RA, Silver RM, et al. Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. Am J Physiol Lung Cell Mol Physiol 2005; 288: L190201.
  • 9
    Ohba T, McDonald JK, Silver RM, Strange C, LeRoy EC, Ludwicka A. Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of the platelet-derived growth factor α-receptor. Am J Respir Cell Mol Biol 1994; 10: 40512.
  • 10
    Gray AJ, Bishop JE, Reeves JT, Laurent GJ. Aα and Bβ chains of fibrinogen stimulate proliferation of human fibroblasts. J Cell Sci 1993; 104: 40913.
  • 11
    Bachhuber BG, Sarembock IJ, Gimple LW, Owens GK. α-thrombin induces transforming growth factor-β1 mRNA and protein in cultured vascular smooth muscle cells via a proteolytically activated receptor. J Vasc Res 1997; 34: 418.
  • 12
    Chambers RC, Leoni P, Blanc-Brude OP, Wembridge DE, Laurent GJ. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1. J Biol Chem 2000; 275: 3558491.
  • 13
    Bogatkevich GS, Ludwicka-Bradley A, Paul J. Nietert, Silver RM. Scleroderma lung fibroblasts: contractility and connective tissue growth factor. In GabbianiG, ChaponnierC, DesmouliereA, editors. Myofibroblasts. Georgetown (TX): Landes Bioscience; 2006. p 2531.
  • 14
    Mercer PF, Deng X, Chambers RC. Signaling pathways involved in proteinase-activated receptor1-induced proinflammatory and profibrotic mediator release following lung injury. Ann N Y Acad Sci 2007; 1096: 868.
  • 15
    Ludwicka-Bradley A, Tourkina E, Suzuki S, Tyson E, Bonner M, Fenton JW II, et al. Thrombin upregulates interleukin-8 in lung fibroblasts via cleavage of proteolytically activated receptor-I and protein kinase C-γ activation. Am J Respir Cell Mol Biol 2000; 22: 23543.
  • 16
    Tourkina E, Hoffman S, Fenton JW II, Lipsitz S, Silver RM, Ludwicka-Bradley A. Depletion of protein kinase Cϵ in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression. Arthritis Rheum 2001; 44: 137081.
  • 17
    Chambers RC, Dabbagh K, McAnulty RJ, Gray AJ, Blanc-Brude OP, Laurent GJ. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1. Biochem J 1998; 333: 1217.
  • 18
    Armstrong MT, Fenton JW II, Andersen TT, Armstrong PB. Thrombin stimulation of matrix fibronectin. J Cell Physiol 1996; 166: 11220.
  • 19
    Howell DC, Goldsack NR, Marshall RP, McAnulty RJ, Starke R, Purdy G, et al. Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis. Am J Pathol 2001; 159: 138395.
  • 20
    Howell DC, Johns RH, Lasky JA, Shan B, Scotton CJ, Laurent GJ, et al. Absence of proteinase-activated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis. Am J Pathol 2005; 166: 135365.
  • 21
    Sorbera LA, Bozzo J, Castaner J. Dabigatran/dabigatran etexilate. Drugs Future 2005; 30: 87785.
  • 22
    Bogatkevich GS, Ludwicka-Bradley A, Silver RM. Dabigatran, a direct thrombin inhibitor, demonstrates antifibrotic effects on lung fibroblasts. Arthritis Rheum 2009; 60: 345564.
  • 23
    Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev 2001; 53: 24582.
  • 24
    Wienen W, Stassen JM, Priepke H, Ries UJ, Hauel N. In-vitro profile and ex-vivo anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate. Thromb Haemost 2007; 98: 15562.
  • 25
    Silver RM, Metcalf JF, Stanley JH, LeRoy EC. Interstitial lung disease in scleroderma: analysis by bronchoalveolar lavage. Arthritis Rheum 1984; 27: 125462.
  • 26
    Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988; 41: 46770.
  • 27
    Laurent GJ, Cockerill P, McAnulty RJ, Hasting JR. A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples. Anal Biochem 1981; 113: 30112.
  • 28
    Troconiz IF, Tillmann C, Liesenfeld KH, Schaefer HG, Stangier J. Population pharmacokinetic analysis of the new oral thrombin inhibitor dabigatran etexilate (BIBR 1048) in patients undergoing primary elective hip replacement surgery. J Clin Pharmacol 2007; 47: 37182.
  • 29
    Palm M, Frankenberg L, Johansson M, Jalkesten E. Evaluation of coagulation tests in mouse plasma. Comp Haematol Int 1997; 7: 2436.
  • 30
    Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int J Biochem Cell Biol 2008; 40: 36282.
  • 31
    Ludwicka A, Ohba T, Trojanowska M, Yamakage A, Strange C, Smith EA, et al. Elevated levels of platelet derived growth factor and transforming growth factor-β1 in bronchoalveolar lavage fluid from patients with scleroderma. J Rheumatol 1995; 10: 187683.
  • 32
    Wilson MS, Wynn TA. Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol 2009; 2: 10321.
  • 33
    Scala E, Pallotta S, Frezzolini A, Abeni D, Barbieri C, Sampogna F, et al. Cytokine and chemokine levels in systemic sclerosis: relationship with cutaneous and internal organ involvement. Clin Exp Immunol 2004; 138: 5406.
  • 34
    Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev 2004; 15: 25573.
  • 35
    Maeda A, Ishioka S, Taooka Y, Hiyama K, Yamakido M. Expression of transforming growth factor-β1 and tumour necrosis factor-α in bronchoalveolar lavage cells in murine pulmonary fibrosis after intraperitoneal administration of bleomycin. Respirology 1999; 4: 35963.
  • 36
    Tani K, Ogushi F, Takahashi H, Kawano T, Endo T, Sone S. Thrombin stimulates platelet-derived growth factor release by alveolar macrophages in rats—significance in bleomycin-induced pulmonary fibrosis. J Med Invest 1997; 44: 5965.
  • 37
    Hernandez-Rodriguez NA, Cambrey AD, Harrison NK, Chambers RC, Gray AJ, Southcott AM, et al. Role of thrombin in pulmonary fibrosis. Lancet 1995; 346: 10713.
  • 38
    Thuillier R, Favreau F, Celhay O, Macchi L, Milin S, Hauet T. Thrombin inhibition during kidney ischemia-reperfusion reduces chronic graft inflammation and tubular atrophy. Transplantation 2010; 90: 61221.
  • 39
    Favreau F, Thuillier R, Cau J, Milin S, Manguy E, Mauco G, et al. Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model. Am J Transplant 2010; 10: 309.
  • 40
    Moore BB, Hogaboam CM. Murine models of pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2008; 294: L15260.
  • 41
    Van Ryn J, Stangier J, Haertter S, Liesenfeld KH, Wienen W, Feuring M, et al. Dabigatran etexilate—a novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost 2010; 103: 111627.
  • 42
    Kim R, Meyer KC. Therapies for interstitial lung disease: past, present and future. Ther Adv Respir Dis 2008; 2: 31938.
  • 43
    Kubo H, Nakayama K, Yanai M, Suzuki T, Yamaya M, Watanabe M, et al. Anticoagulant therapy for idiopathic pulmonary fibrosis. Chest 2005; 128: 147582.
  • 44
    Eisert WG, Hauel N, Stangier J, Wienen W, Clemens A, van Ryn J. Dabigatran: an oral novel potent reversible nonpeptide inhibitor of thrombin. Arterioscler Thromb Vasc Biol 2010; 30: 18859.