Antiinflammatory and antifibrotic effects of the oral direct thrombin inhibitor dabigatran etexilate in a murine model of interstitial lung disease

Abstract

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.

Figure 1

Effect of dabigatran etexilate on bleomycin-induced pulmonary fibrosis. A. Representative histological findings of lung inflammation and fibrosis. 1 – control (saline/placebo or saline/dabigatran), 2 - bleomycin/placebo, 3 – bleomycin/dabigatran etexilate (day 1), 4 – bleomycin/dabigatran etexilate (day 8), n = 40 (10 mice per group) B. Quantitative evaluation of fibrotic changes (Ashcroft scores). C. Collagen lung content determined by Sircol collagen assay, n = 24 (6 mice per group). D. Collagen lung content measured by hydroxyproline assay, n = 32 (8 mice per group). Values are the mean ± SD. * = P < 0.05 versus bleomycin/placebo-treated mice.

Figure 2

A. Effect of dabigatran etexilate on level of active thrombin in BALF. B. Effect of increasing concentrations of dabigatran on the thrombin time (TT) and activated partial thromboplastin time (aPTT) clotting times in mouse plasma in vitro. Data expressed as mean of duplicate determinations. C and D. Effect of dabigatran etexilate on TGF-β1 levels in BALF (C) and lung tissue (D). 1 – control (saline/placebo), 2 - bleomycin/placebo, 3 – bleomycin/dabigatran etexilate (day 1), 4 – bleomycin/dabigatran etexilate (day 8). Values are the mean ± SD. * = P < 0.01 versus bleomycin/placebo-treated mice.

Figure 3

Effect of dabigatran etexilate on Smad3, CTGF and α-SMA expression in lung tissue. Representative immunoblots of phosphorylated and basal Smad3, CTGF, α-SMA, and β-actin (as loading control) from control (1), bleomycin/placebo (2), bleomycin/dabigatran etexilate (day 1) (3), bleomycin/dabigatran etexilate (day 8) (4) are presented, n = 16 (4 mice per group).

Figure 4

Immunohistochemical evaluation for CTGF (A) and α-SMA (B) expression in lung tissue.

Figure 5

Effect of dabigatran etexilate on α-SMA expression/organization (A) and collagen gel contraction (B) in mouse lung fibroblasts. 1 – control (saline/placebo), 2 - bleomycin/placebo, 3 – bleomycin/dabigatran etexilate (day 1), 4 – bleomycin/dabigatran etexilate (day 8), n = 24 (6 mice per group). Values are the mean ± SD. * = P < 0.01 versus bleomycin/placebo-treated mice.