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.

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 α‐smooth muscle actin (α‐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, α‐SMA expression and organization, and the production of collagen and CTGF in normal lung fibroblasts. Moreover, when treated with dabigatran (1 μg/ml), scleroderma lung myofibroblasts produced 6‐fold less α‐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.

Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts that resembles the phenotype of scleroderma lung fibroblasts. We now demonstrate that PAR-1 expression is dramatically increased in lung tissue from scleroderma patients, where it is associated with inflammatory and fibroproliferative foci. We also observe that thrombin induces resistance to apoptosis in normal lung fibroblasts, and this process is regulated by protein kinase C (PKC)-ε but not by PKC-α. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-ε significantly inhibits Fas ligand-induced apoptosis in lung fibroblasts, whereas scleroderma lung fibroblasts are resistant to apoptosis de novo. Thrombin translocates p21Cip1/WAF1, a signaling molecule downstream of PKC, from the nucleus to cytoplasm in normal lung fibroblasts mimicking the localization of p21Cip1/WAF1 in scleroderma lung fibroblasts. Overexpression of c-a PKC-α or PKC-ε results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-α or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-α, but not PKC-ε, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G₁ phase regulatory protein cyclin D1, suggesting that PKC-α, MAPK, and cyclin D1 mediate lung fibroblast proliferation. These data demonstrate that two distinct PKC isoforms mediate thrombin-induced resistance to apoptosis and proliferation and suggest that p21Cip1/WAF1 promotes both phenomena.

Activated fibroblasts, or myofibroblasts, are crucial players in tissue remodeling, wound healing, and various fibrotic disorders, including interstitial lung fibrosis associated with scleroderma. Here we characterize the signaling pathways in normal lung fibroblasts exposed to thrombin as they acquire two of the main features of myofibroblasts: smooth muscle (SM) α-actin organization and collagen gel contraction. Our results show that the small G protein Rho is involved in lung myofibroblast differentiation. Thrombin induces Rho-³⁵S-labeled guanosine 5′- O-(3-thiotriphosphate) binding in a dose-dependent manner. It potently stimulates Rho activity in vivo and initiates protein kinase C (PKC)-ϵ-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM α-actin organization, collagen gel contraction, and PKC-ϵ-SM α-actin and PKC-ϵ-RhoA coimmunoprecipitation. However, it has no effect on PKC-ϵ activation or translocation of PKC-ϵ to the membrane. Overexpression of constitutively active PKC-ϵ and constitutively active RhoA induces collagen gel contraction or SM α-actin organization, whereas, individually, they do not perform these functions. We therefore conclude that the contractile activity of myofibroblasts induced by thrombin is mediated via PKC-ϵ- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-ϵ-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-ϵ-SM α-actin coimmunoprecipitation, which leads to the PKC-ϵ-RhoA-SM α-actin ternary complex formation.

Connective tissue growth factor (CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF-induced proteins, such as pro-α (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF-responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF-induced proteins, IQ motif containing GTPase activating protein (IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF-induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF-induced cell migration and did not interfere with CTGF-induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and propose that CTGF-induced migration of lung fibroblasts to the damaged tissue is mediated via IQGAP1 and MAPK signaling pathways.