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Interstitial lung disease (ILD) is a serious complication and leading cause of mortality in patients with systemic sclerosis (SSc). In the current study we explored the role of LMCD1 (LIM and cysteine-rich domains-1) as a novel factor in the pathogenesis of SSc-ILD.Expression and effects of LMCD1 were studied lung tissue and fibroblasts from SSc-ILD patients and control subjects as well as in lung from animal models.LMCD1 is consistently elevated in lung tissue and in fibroblasts isolated from SSc-ILD patients as compared to controls. Additionally, LMCD1 is highly expressed in the lung in two different animal models of ILD: fibroblast specific protein (FSP)-driven constitutively active TGFβ receptor 1 (FSP-TβR1CA) transgenic and bleomycin-induced mouse models. In lung fibroblasts from SSc-ILD patients LMCD1 is an essential factor for TGFβ-induced generation of collagen type I, fibronectin, and smooth muscle alpha-actin (SMA). Depletion of LMCD1 by siRNA reduces the expression of ECM proteins and lowers transcriptional activity and expression of SMA, as well as decreases proliferation and contractile activity of SSc-ILD lung fibroblasts. In dense fibrotic areas of SSc-ILD lung LMCD1 co-localizes with SMA. In cultured scleroderma lung fibroblasts LMCD1 co-localizes and interacts with serum response factor (SRF) which mediates LMCD1-induced contractile activity of lung fibroblasts.Our study identifies LMCD1 as a profibrotic molecule contributing to the activation of myofibroblasts and the persistent fibroproliferation observed in SSc-ILD. Thus, LMCD1 may be a potential novel therapeutic target for patients with SSc-ILD. This article is protected by copyright. All rights reserved.

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human disease-related stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner.

OBJECTIVE To investigate the relationship between Krebs von den Lungen 6 (KL-6) and CCL18 levels and the severity and progression of systemic sclerosis (SSc)-related interstitial lung disease (ILD). METHODS Patients enrolled in the Scleroderma Lung Study II (cyclophosphamide [CYC] versus mycophenolate mofetil [MMF]) were included. Baseline and 12-month plasma samples were analyzed by enzyme-linked immunosorbent assay to assess CCL18 and KL-6 levels. The forced vital capacity (FVC) and the diffusing capacity for carbon monoxide (DLco) were measured every 3 months. Joint models were created to investigate the relationship between baseline CCL18 and KL-6 levels and the course of the FVC and DLco over 1 year according to treatment arm. RESULTS Baseline KL-6 and CCL18 levels each correlated with the extent of radiographic fibrosis. Levels of both CCL18 and KL-6 declined significantly at 1 year. In both treatment arms (n = 71 for CYC, n = 62 for MMF), a higher baseline KL-6 level predicted progression of ILD based on the course of FVC (P = 0.024 for CYC; P = 0.005 for MMF) and DLco (P < 0.001 for CYC; P = 0.004 for MMF) over 1 year. A higher baseline CCL18 level predicted progression of ILD based on the course of the FVC (P < 0.001 for CYC; P = 0.007 for MMF) and DLco (P = 0.001 for CYC; P < 0.001 for MMF) over 1 year, as well as mortality (P = 0.0008 for CYC arm only). CONCLUSION In a rigorously conducted clinical trial for SSc-related ILD, KL-6 and CCL18 levels correlated with ILD severity and declined with immunosuppression. Patients with higher baseline KL-6 and CCL18 levels were more likely to experience disease progression despite treatment. KL-6 and CCL18 levels could be used to identify patients with a progressive ILD phenotype who may benefit from a more aggressive initial treatment approach.

Objective Interstitial lung disease (ILD) is a frequent complication of systemic sclerosis (SSc) (scleroderma) and the leading cause of scleroderma-related deaths. There exists an unmet need for a new drug therapy for ILD-complicated SSc. Substantial evidence supports an important role for thrombin in the pathogenesis of SSc-associated ILD (hereafter SSc-ILD), and targeting thrombin with a direct thrombin inhibitor could prove to be a novel and effective treatment strategy. As a first step toward designing a clinical trial to test the efficacy of thrombin inhibition in SSc-ILD, we conducted this study to test the safety and tolerability of dabigatran in patients with SSc-ILD. Methods We performed a prospective, single-center, open-label treatment trial with the direct thrombin inhibitor, dabigatran, in patients with SSc-ILD. Any patient with a history of gastrointestinal hemorrhage or gastric antral vascular ectasia was excluded. Blood monitoring was performed monthly, and patient-reported outcomes, pulmonary function tests, and skin scores were obtained at baseline and at 3- and 6-month visits. Bronchoscopy with bronchoalveolar lavage (BAL) was performed at baseline and at 6 months for measurement of lung thrombin activity. Results Of 15 patients with SSc-ILD, 14 completed 6 months of treatment with dabigatran at 75 mg taken orally twice daily. Adverse events were uncommon and usually mild or unrelated to the study medication. No serious adverse event was observed. Dabigatran was well tolerated, and we observed no significant gastrointestinal, pulmonary, or other safety issues or intolerability. BAL fluid thrombin activity decreased or remained stable in 13 of 14 (92.8%) subjects. Conclusion Dabigatran appears to be safe and well tolerated in patients with SSc-ILD. A larger randomized controlled trial to test the efficacy of direct thrombin inhibition with dabigatran can be considered.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFβ1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O2 to hydrogen peroxide (H2O2), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-β-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of CD44V6 via an effect upon AP-1 activity. Further, CD44V6 is part of a positive-feedback loop with TGFβ1/TGFβRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic (CD44v6 shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.

The etiology and reasons underlying the ethnic disparities in systemic sclerosis (SSc) remain unknown. African Americans are disproportionally affected by SSc and yet are underrepresented in research. The aim of this study was to comprehensively investigate the association of DNA methylation levels with SSc in dermal fibroblasts from patients of African ancestry. Reduced representation bisulfite sequencing (RRBS) was performed on primary dermal fibroblasts from 15 SSc patients and 15 controls of African ancestry, and over 3.8 million CpG sites were tested for differential methylation patterns between cases and controls. The dermal fibroblasts from African American patients exhibited widespread reduced DNA methylation. Differentially methylated CpG sites were most enriched in introns and intergenic regions while depleted in 5&prime, UTR, promoters, and CpG islands. Seventeen genes and eleven promoters showed significant differential methylation, mostly in non-coding RNA genes and pseudogenes. Gene set enrichment analysis (GSEA) and gene ontology (GO) analyses revealed an enrichment of pathways related to interferon signaling and mesenchymal differentiation. The hypomethylation of DLX5 and TMEM140 was accompanied by these genes&rsquo, overexpression in patients but underexpression for lncRNA MGC12916. These data show that differential methylation occurs in dermal fibroblasts from African American patients with SSc and identifies novel coding and non-coding genes.

Nintedanib is approved for the treatment of idiopathic pulmonary fibrosis (IPF) and was demonstrated to slow disease progression in patients with IPF by reducing decline in forced vital capacity by 50%. Recently, nintedanib has been reported to exert anti-fibrotic activity on systemic sclerosis (scleroderma, SSc) skin fibroblasts and to diminish skin and lung fibrosis in mouse models. The goal of the present study was to determine the effects of nintedanib on a cellular model of SSc-associated interstitial lung disease (ILD).Study was performed using lung fibroblasts (LF) isolated from five patients with SSc-ILD and from three control subjects.Nintedanib inhibited LF proliferation and migration in a concentration- and time-dependent manner. The proliferation rate of LF stimulated with PDGF in the presence of nintedanib was reduced 1.9-fold within 24 h as compared to cells stimulated with PDGF alone. Migration of SSc-ILD LF incubated with 100 nM nintedanib was reduced from 62.8±12.5% to 39.1±9.0% in the presence of PDGF and from 38.2±7.9% to 26.6±7.2% in serum-free medium. Nintedanib attenuated PDGF-induced Ca2+ efflux, reduced α-SMA promoter activity and α-SMA protein expression. Furthermore, nintedanib blocked PDGF-induced differentiation of normal LF to myofibroblasts, reduced production of collagen and fibronectin, and decreased contractility of SSc-ILD LF in both floating and fixed collagen gels.Our data demonstrate significant antifibrotic efficacy of nintedanib in SSc-ILD LF suggesting that nintedanib has the potential not only to prevent but also to reverse the increased activity of LF consequently attenuating excessive lung fibrosis observed in SSc-ILD.

Introduction: Activated lung fibroblasts (LF), or myofibroblasts (MF), from patients with systemic sclerosis (SSc) stay pro-fibrotic in culture. In collagen gel they can be used to explore fibrosis in SSc. Contraction of floating or fixed collagen gels resemble the initial phase of MF activation and the late phase of fibrotic scarring, respectively. Nintedanib (NTB) approved for the treatment of idiopathic pulmonary fibrosis was shown to have anti-fibrotic activity. Aim: To determine the effects of NTB on lung MF isolated from SSc patients with lung fibrosis. Materials and Methods: Study was performed using floating and fixed collagen gels populated with LF from 5 patients with SSc associated lung fibrosis and 3 control donors. Results: PDGF stimulates intracellular Ca2+ and activates smooth muscle α-actin (α-SMA) promoter in LF. PDGF does not interfere with actin polymerization and α-SMA organization, but increases α-SMA protein and collagen gel contraction. NTB attenuates PDGF-induced Ca2+ efflux, α-SMA promoter activity, α-SMA protein and collagen gel contraction in LF. NTB has no effects on basal Ca2+, α-SMA promoter or mRNA, but inhibits the baseline of α-SMA protein in SSc MF. Importantly, NTB reduces collagen gel contractions in SSc MF in both floating and fixed collagen gels. Conclusions: NTB blocks PDGF-induced differentiation of normal LF to MF and reduces contractility of SSc lung MF in both floating and fixed collagen gels suggesting that NTB may not only prevent but also reverse excessive matrix deposition that occurs in scleroderma-associated lung fibrosis.

Background Aberrant Neutrophil Extracellular Trap (NET) formation contributes to the induction and propagation of inflammation and plays a key role in causing tissue damage in conditions like sepsis, SLE, RA and vasculitis. Citrullination of proteins is involved in the formation of NETs, autoimmunity, and the breaking of tolerance in NET-driven autoimmune diseases. In SLE and RA, neutrophils undergo enhanced NETosis, and NET components are observed in blood, inflamed tissues and joints. Objectives Our objective is to develop a novel first in class NET-inhibiting therapeutic anti-citrullinated protein antibody (tACPA) targeting citrullinated histones 2A and 4, for the treatment of human diseases in which aberrant NET formation add to the severity of the pathology with an initial focus on autoimmune diseases. Here, we demonstrate the utility of tACPA for different NET-based diseases beyond RA, including SLE, vasculitis, gout and idiopathic pulmonary fibrosis (IPF). Methods Previously, using two RA animal models, the therapeutic properties of tACPA have been demonstrated.Chirivi et al., 2013 In the current studies, neutrophils from RA and SLE donors, as well as biological NET-inducing stimuli, such as RA synovial fluid (SF), gout SF and activated platelets, have been used to demonstrate the NETosis-inhibiting properties of tACPA in different human disease contexts. We have further expanded tACPA’s therapeutic utility by testing it in a surrogate model for NET-mediated organ damage (sepsis) and IPF. Results NETosis in human RA and SLE neutrophils have been induced with a calcium ionophore and could be inhibited by tACPA treatment (40%–100% reduction). Similar results were obtained using RA and gout SF or activated platelets as NETosis inducers in combination with neutrophils from healthy donors. These observations have been confirmed with multiple NET readouts such as MPO activity, MPO/DNA ELISA, DNA quantification as well as imaging readouts. In addition, we demonstrated that in an LPS-induced sepsis model 30% of tACPA-treated mice survived (compared to 0% in placebo controls), showing protection against organ failure. In a bleomycin-induced IPF mouse model, tACPA protected mice from the development of lung fibrosis (compared to placebo controls). When determining neutrophil counts in bronchoalveolar lavage samples, we found that in tACPA-treated mice, neutrophil levels were normal, while levels in placebo-treated mice were elevated. Conclusions In a sepsis and IPF mouse model, tACPA prevented NET-mediated organ damage, providing evidence that tACPA could be a promising therapeutic strategy for diseases where NET-mediated endothelial toxicity causes organ damage like SLE, vasculitis and IPF. Central to our strategy for generating a preclinical data package supporting clinical testing, is to demonstrate that patient NETosis can be significantly inhibited ex vivo. We will present data that confirm that tACPA can block human SLE NETosis as well as human NETosis induced by activated platelets or gout SF. Disclosure of Interest R. Chirivi Shareholder of: Citryll BV, Employee of: ModiQuest BV, J. van Rosmalen Employee of: ModiQuest BV, K. Kambas: None declared, G. Schmets: None declared, H. Kalisvaart: None declared, G. Bogatkevich: None declared, T. Shaw: None declared, H. van Es Shareholder of: Citryll BV, J. Raats Shareholder of: Citryll BV

Contractility is a predominant feature of myofibroblasts and a key process in fibrotic tissue remodeling. We previously demonstrated that lung fibroblasts from scleroderma patients with interstitial lung disease express a robust contractile phenotype and contain large amounts of the IQ motif containing GTPase activating protein (IQGAP1). Here we show the contribution of IQGAP1 to two major features of lung myofibroblasts: α-smooth muscle actin (SMA) expression and collagen gel contraction. We found that IQGAP1 mediates SMA expression and contractile activity of scleroderma lung myofibroblasts as well as normal lung fibroblasts stimulated in vitro with thrombin, TGFβ1, or CTGF. We show that IQGAP1 co-immunoprecipitates with SMA and co-localizes with monomeric SMA, but not with the highly organized SMA in scleroderma lung myofibroblasts. In contrast, IQGAP1 binds directly to polymerized β- and γ-actin isoforms suggesting distinct mechanisms of α-SMA and β-/γ-actin polymerization in scleroderma lung myofibroblasts. We conclude that the primary role of IQGAP1 in promoting contractility of the lung parenchyma and contributing to restrictive lung disease is through the regulation of SMA expression and organization in lung fibroblasts.

Objective M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF) receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens. Experimental design An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for detection of M10 in biological fluids was developed using pharmaceutical grade synthetic M10 as a calibrator and commercially available anti-c-Met C12 antibody. Results M10 ELISA specifically detected in plasma M10, but not a scrambled peptide, following a single intraperitoneal administration of M10 (1mg/kg) to mice. The detection limit was 9.6 ng/ml, and the measuring limit was between 15 ng/ml and 200 ng/ml. The recovery limits of M10 were between 80% and 120%; intra-assay coefficient of variation was between 5.3% and 6.3%; inter-assay coefficient of variation was between 5.0% and 8.0% over the buffer concentration tested in the range from 15 ng /ml to 250 ng /ml. The peak of M10 concentration following a single intraperitoneal injection (1mg/kg) was achieved within 6 hours and declined to minimal levels by 48 hours. The experimentally obtained half-life for M10 was comparable to the theoretically predicted half-life for M10. Conclusions We have established a highly sensitive ELISA to detect the antifibrotic peptide M10 in plasma samples, which should prove to be a novel tool to study the pharmacokinetics and efficacy of M10 in the treatment of fibroproliferative disorders.

Apoptosis of alveolar epithelial cells (AECs) and survival of lung fibroblasts are critical events in the pathogenesis of pulmonary fibrosis; however, mechanisms underlying the apoptosis of AECs and the resistance of lung fibroblasts to apoptosis remain obscure. Herein, we demonstrate that the fate of these two cell types depends on the expression of CCAAT enhancer–binding homologous protein (CHOP). We observed that thrombin, which is overexpressed in scleroderma (SSc; systemic sclerosis) and other interstitial lung diseases (ILDs), increases the expression of CHOP in primary AECs and in A549 cells via an Ets1-dependent pathway. In addition, thrombin activates caspase-3 in AECs and induces apoptosis of these cells in a CHOP-dependent manner. In contrast, thrombin decreases endoplasmic reticulum stress–induced CHOP in lung fibroblasts through Myc-dependent mechanisms and protects such cells from apoptosis. Furthermore, when lung fibroblasts are transfected with recombinant CHOP, they then undergo apoptosis, even in the presence of thrombin, suggesting that CHOP signaling pathways are downstream of thrombin. In accordance with the differential effects of thrombin on AECs and lung fibroblasts, we observed strong expression of CHOP in AECs in fibrotic lung tissue isolated from patients with SSc-associated ILD (SSc-ILD), but not in lung myofibroblasts nor in normal lung tissue. Expression of CHOP in SSc lung is accompanied by positive staining for the thrombin receptor, protease-activated receptor-1, and for terminal deoxynucleotidyl transferase dUTP nick end labeling, suggesting roles for both thrombin and CHOP in AEC apoptosis in SSc-ILD. We conclude that regulation of CHOP by thrombin directs AECs toward apoptosis while promoting survival of lung fibroblasts, ultimately contributing to the persistent fibroproliferation seen in SSc-ILD and other fibrosing lung diseases.

Author(s): Martyanov, Viktor; Kim, Grace-Hyun J; Hayes, Wendy; Du, Shuyan; Ganguly, Bishu J; Sy, Oumar; Lee, Sun Ku; Bogatkevich, Galina S; Schieven, Gary L; Schiopu, Elena; Marangoni, Roberta Goncalves; Goldin, Jonathan; Whitfield, Michael L; Varga, John | Abstract: BackgroundThere are no effective treatments or validated clinical response markers in systemic sclerosis (SSc). We assessed imaging biomarkers and performed gene expression profiling in a single-arm open-label clinical trial of tyrosine kinase inhibitor dasatinib in patients with SSc-associated interstitial lung disease (SSc-ILD).MethodsPrimary objectives were safety and pharmacokinetics. Secondary outcomes included clinical assessments, quantitative high-resolution computed tomography (HRCT) of the chest, serum biomarker assays and skin biopsy-based gene expression subset assignments. Clinical response was defined as decrease of g5 or g20% from baseline in the modified Rodnan Skin Score (MRSS). Pulmonary function was assessed at baseline and day 169.ResultsDasatinib was well-tolerated in 31 patients receiving drug for a median of nine months. No significant changes in clinical assessments or serum biomarkers were seen at six months. By quantitative HRCT, 65% of patients showed no progression of lung fibrosis, and 39% showed no progression of total ILD. Among 12 subjects with available baseline and post-treatment skin biopsies, three were improvers and nine were non-improvers. Improvers mapped to the fibroproliferative or normal-like subsets, while seven out of nine non-improvers were in the inflammatory subset (p = 0.0455). Improvers showed stability in forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLCO), while both measures showed a decline in non-improvers (p = 0.1289 and p = 0.0195, respectively). Inflammatory gene expression subset was associated with higher baseline HRCT score (p = 0.0556). Non-improvers showed significant increase in lung fibrosis (p = 0.0313).ConclusionsIn patients with SSc-ILD dasatinib treatment was associated with acceptable safety profile but no significant clinical efficacy. Patients in the inflammatory gene expression subset showed increase in skin fibrosis, decreasing pulmonary function and worsening lung fibrosis during the study. These findings suggest that target tissue-specific gene expression analyses can help match patients and therapeutic interventions in heterogeneous diseases such as SSc, and quantitative HRCT is useful for assessing clinical outcomes.Trial registrationClinicaltrials.gov NCT00764309.

Objectives Interstitial lung disease in systemic sclerosis (SSc-ILD) is often an irreversible and progressive fibrosing process that now is the leading cause of scleroderma-related deaths. In this review we present our current understanding of the role played by coagulation and particularly by thrombin in autoimmune-mediated tissue injury and fibrosis, mainly as it relates to SSc-ILD. Methods We used PubMed to search for articles published up to October 2010 for keywords referring to autoimmunity, coagulation, pulmonary fibrosis, and scleroderma. Results SSc-ILD is an autoimmune disease associated with lymphocyte activation and release of various cytokines and growth factors. The production of autoantibodies is a central feature in SSc. Activation of the coagulation cascade with release of thrombin is 1 of the earliest events following tissue injury. Thrombin contributes to autoimmune responses by activating of pathogenic Th2 lymphocyte profile in SSc. Thrombin also modulates tissue repair responses, stimulates transformation of epithelial cells, endothelial cells, and fibroblasts into myofibroblast phenotype, and induces secretion of several pro-immune and profibrotic factors, which serve as antigens for pathogenic autoantibodies production in SSc-ILD. Conclusions The identification of links between autoimmunity and coagulation would provide new insights into the pathogenesis of pulmonary fibrosis associated with autoimmune diseases and further acknowledge the importance of thrombin in the development of SSc-ILD.

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.

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.

Objective To compare the composition of cytokines in African American and Caucasian patients with systemic sclerosis (SSc; scleroderma) and in healthy individuals, particularly the expression and function of hepatocyte growth factor (HGF). Methods Bronchoalveolar lavage (BAL) fluid samples were analyzed using cytokine array techniques. HGF in plasma and cell culture medium samples was measured by enzyme-linked immunosorbent assay. Connective tissue growth factor (CTGF), type I collagen expression, and c-Met receptor phosphorylation were studied by immunoblotting. Results Overall greater expression of cytokines in BAL fluid from African American patients as compared with Caucasian patients was observed. Significant increases in HGF concentrations were detected in BAL fluid, plasma, and fibroblast culture medium from Caucasian SSc patients. In contrast, African American SSc patients did not demonstrate an increase in HGF. Recombinant HGF readily abolished CTGF expression and collagen accumulation in lung fibroblasts isolated from Caucasian SSc patients. Pretreatment of lung fibroblasts with neutralizing anti–c-Met antibody abolished the effects of HGF on CTGF expression and collagen accumulation, suggesting that the antifibrotic activity of HGF is mediated via c-Met receptor tyrosine kinase. Whereas recombinant HGF rapidly induced c-Met receptor phosphorylation in lung fibroblasts from Caucasian patients, c-Met receptor phosphorylation was significantly reduced in lung fibroblasts from African American subjects. Moreover, recombinant HGF failed to prevent CTGF expression and collagen accumulation in lung fibroblasts derived from African American subjects. Conclusion Ethnic differences exist in terms of antifibrotic HGF expression in lung fibroblasts derived from Caucasian and African American subjects. Reduced levels of HGF as well as a deficiency in c-Met receptor function appear to be present in African American patients with SSc. These findings may explain in part the greater disease severity and worse prognosis observed in African Americans with SSc.

Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

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) alpha-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)-epsilon-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM alpha-actin organization, collagen gel contraction, and PKC-epsilon-SM alpha-actin and PKC-epsilon-RhoA coimmunoprecipitation. However, it has no effect on PKC-epsilon activation or translocation of PKC-epsilon to the membrane. Overexpression of constitutively active PKC-epsilon and constitutively active RhoA induces collagen gel contraction or SM alpha-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-epsilon- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-epsilon-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-epsilon-SM alpha-actin coimmunoprecipitation, which leads to the PKC-epsilon-RhoA-SM alpha-actin ternary complex formation.

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-alpha actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-alpha actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-beta pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCepsilon abolishes SM-alpha actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCepsilon translocation also abolishes thrombin-induced collagen gel contraction, SM-alpha actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCepsilon is required for these effects. In normal lung fibroblasts PKCepsilon binds to SM-alpha actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-alpha actin may serve as a substrate for PKCepsilon in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.

Systemic sclerosis (scleroderma, SSc) is a heterogeneous autoimmune connective tissue disease of unknown etiology. Interstitial lung disease (ILD) is a frequent complication, and a significant contributor to morbidity and mortality among SSc patients. SSc-ILD most commonly occurs within 10 years of diagnosis, and may be seen in patients with either the limited or diffuse cutaneous subset of SSc. SSc-ILD is a multifaceted disease process in which different factors and pathways are involved. Aberrant function of a variety of lung cells, cytokines, growth factors, peptides, and bioactive proteins, in combination with genetic and epigenetic regulators, have crucial functions in the pathogenesis of this disease. Here we present our view on recent advances regarding the pathogenesis of SSc-ILD.

The hepatocyte growth factor (HGF) and the HGF receptor Met pathway are important in the pathogenesis of interstitial lung disease (ILD). Alternatively spliced isoforms of CD44 containing variable exon 6 (CD44v6) and its ligand hyaluronan (HA) alter cellular function in response to interaction between CD44v6 and HGF. TGF-β1 is the crucial cytokine that induces fibrotic action in ILD fibroblasts (ILDFbs). We have identified an autocrine TGF-β1 signaling that up-regulates both Met and CD44v6 mRNA and protein expression. Western blot analysis, flow cytometry, and immunostaining revealed that CD44v6 and Met colocalize in fibroblasts and in tissue sections from ILD patients and in lungs of bleomycin-treated mice. Interestingly, cell proliferation induced by TGF-β1 is mediated through Met and CD44v6. Further, cell proliferation mediated by TGF-β1/CD44v6 is ERK-dependent. In contrast, action of Met on ILDFb proliferation does not require ERK but does require p38(MAPK). ILDFbs were sorted into CD44v6(+)/Met(+) and CD44v6(-)/Met(+) subpopulations. HGF inhibited TGF-β1-stimulated collagen-1 and α-smooth muscle cell actin expression in both of these subpopulations by interfering with TGF-β1 signaling. HGF alone markedly stimulated CD44v6 expression, which in turn regulated collagen-1 synthesis. Our data with primary lung fibroblast cultures with respect to collagen-1, CD44v6, and Met expressions were supported by immunostaining of lung sections from bleomycin-treated mice and from ILD patients. These results define the relationships between CD44v6, Met, and autocrine TGF-β1 signaling and the potential modulating influence of HGF on TGF-β1-induced CD44v6-dependent fibroblast function in ILD fibrosis.

Copyright: © 2013 Bogatkevich GS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Welcome to the May 2013 issue of Rheumatology: Current Research. This issue of RCR covers a number of comprehensive reviews focused on bone, joints, autoimmune, and rheumatic diseases.

Purpose of review Racial disparities appear to exist in the susceptibility and severity of systemic sclerosis (SSc, scleroderma) and are responsible for a greater health burden in blacks as compared with whites. Disparities in socioeconomic status and access to healthcare do not sufficiently explain the observed differences in prevalence and mortality. It is important to determine whether there might be a biologic basis for the racial disparities observed in SSc. Recent findings We present data to suggest that the increased susceptibility and severity of SSc in blacks may result in part from an imbalance of profibrotic and antifibrotic factors. Racial differences in the expression of transforming growth factor-β1 (TGF-β1) and caveolin-1, as well as differences in the expression of hepatocyte growth factor and PPAR-γ, have been demonstrated in blacks with SSc, as well as in normal black individuals. A genetic predisposition to fibrosis may account for much of the racial disparities between black and white patients with SSc. Summary A better understanding of the biologic basis for the racial disparities observed in SSc may lead to improved therapies, along with the recognition that different therapies may need to be adapted for different groups of patients.

Activation of the coagulation cascade leading to generation of thrombin has been extensively documented in various forms of lung injury including systemic sclerosisassociated interstitial lung disease (SSc-ILD). The molecular mechanisms underlying the pathogenesis and progression of lung fibrosis in SSc-ILD and in idiopathic pulmonary fibrosis (IPF) are not entirely clear. The conceptual process of fibrogenesis involves tissue injury and activation of the coagulation cascade, the release of various fibrogenic factors, and the induction of myofibroblasts culminating in enhanced extracellular matrix deposition. Cells with a myofibroblast phenotype appear in the early stages of fibrosis and are characterized by an increased proliferative capacity and abundant expression of ┙-SMA, collagens and other extracellular matrix proteins (Hinz et al., 2007). Myofibroblasts can be cultured from bronchoalveolar lavage (BAL) fluid of SSc-ILD patients, and thrombin activity is also significantly greater in BAL fluid from SSc-ILD patients compared with healthy controls (Ludwicka et al., 1992; Ohba et al., 1994). Thrombin differentiates lung fibroblasts to a myofibroblast phenotype, increases lung fibroblast proliferation (Bogatkevich et al., 2001), and enhances the proliferative effect of fibrinogen on fibroblasts (Gray et al., 1993). Thrombin is also a potent inducer of fibrogenic cytokines, such as transforming growth factor-┚ (TGF-┚) (Bachhuber et al., 1997), connective tissue growth factor (CTGF) (Chambers et al., 2000; Bogatkevich et al., 2006), platelet-derived growth factor-AA (PDGF-AA) (Ohba et al., 1994), chemokines (Mercer et al., 2007), and ECM proteins such as collagen, fibronectin, and tenascin in various cells, including lung fibroblasts (Tourkina et al., 2001; Chambers et al., 1998; Armstrong et al., 1996). Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin and prevents the cleavage of Arg-Gly bonds of fibrinogen needed for the formation of fibrin. Recently, we have demonstrated that binding of dabigatran to thrombin prevents cleavage of the extracellular N-terminal domain of the protease-activated receptor 1 (PAR-1), which is responsible for most profibrotic events induced by thrombin (Bogatkevich et al., 2009). In

My research experience in the field of scleroderma began in year 2000 when I was recruited as a postdoctoral fellow to the laboratory of Dr. Richard M. Silver. Since that time my research interests has focused on understanding the cellular and molecular mechanisms of pulmonary fibrosis in scleroderma patients.

Background: Endothelin-1 and thrombin are elevated during lung injury and repair, as seen in scleroderma and other interstitial lung diseases, and each plays important roles in remodeling epithelium, blood vessels and connective tissue. Both factors promote lung myofibroblast differentiation, the hallmark of pulmonary fibrosis. This study was undertaken to investigate whether bosentan, the dual, specific and competitive inhibitor of endothelin, interferes with thrombin signaling in scleroderma lung fibroblasts. Methods: Endothelin-1 secretion was measured by enzyme-linked immunosorbent assay. Lung fibroblast proliferation was studied by DNA Synthesis and Quick Cell Proliferation assays. Expression of α-smooth muscle actin (α-SMA) was analyzed on Western blots. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Lung fibroblast migration was studied by wound-healing “scratch” assay. Results: We show that thrombin significantly induces endothelin-1 expression in human lung fibroblasts. Bosentan significantly decreases α-SMA and collagen gel contraction of human lung fibroblasts stimulated by thrombin, suggesting that thrombin-induced differentiation of fibroblasts to the myofibroblast phenotype is at least in part regulated by endothelin-1. Bosentan decreases thrombin-induced migration of normal and scleroderma lung fibroblasts and inhibits innately increased migration of scleroderma lung fibroblasts suggesting that endogenous endothelin may be in part responsible for enhanced migration of lung fibroblasts in pulmonary fibrosis. We also report that bosentan inhibits thrombin-induced thymidine incorporation and decreases lung fibroblast proliferation. Conclusions: Fibrogenic effects of thrombin in scleroderma lung fibroblasts are mediated in part by endothelin-1. The dual endothelin receptor blocker bosentan restrains profibrotic effects of endogenous and thrombin-induced endothelin-1 in scleroderma lung fibroblasts.

We present novel data demonstrating that the expression of PPARγis reduced in lung fibroblasts from black SSc-ILD patients as compared to white patients. Activating PPARγwith the agonist rosiglitazone increased the expression of MMP-1 and inhibited collagen type I in lung fibroblasts isolated from white, but not black, SSc-ILD patients. Blocking the c-Met receptor abolishes rosiglitazone's effects on collagen and MMP-1 in lung fibroblasts isolated from white SSc-ILD patients, while augmenting the expression of the c-Met receptor in fibroblasts from black SSc-ILD patients replicates the effects of rosiglitazone seen in whites. We conclude that PPARγagonists warrant consideration as potential antifibrotic drugs in patients with SSc-ILD. Differential therapeutic effects might be anticipated especially relative to racial differences and the functional expression of the c-Met receptor.

Hepatopulmonary syndrome is a pulmonary vascular complication of cirrhosis in which intrapulmonary vasodilatation (IPV) results in hypoxemia. Endothelin-1 (ET-1), produced by proliferating cholangiocytes, has been identified as a mediator of IPV in an animal model of HPS, but the pathophysiology of IPV in humans has not been defined.The purpose of this study was to assess whether cirrhosis with IPV, which often leads to HPS, is associated with increased hepatic venous ET-1 blood levels.We performed a prospective cohort pilot study of 40 patients with liver disease undergoing transjugular liver biopsy from November 1, 2008 to September 1, 2009. Patients were categorized according to absence (-) or presence (+) of IPV as determined by bubble-contrasted echocardiography. Hepatic venous blood was assayed for ET-1 by ELISA. The percent volume of cholangiocytes in the liver biopsy specimen was determined by morphometric analysis, as a measure of bile duct proliferation.Nine subjects were excluded, due to absence of cirrhosis (6) and patent foramen ovale (3). Of the remaining 31 subjects, IPV was present in 18 (58%). Median hepatic venous ET-1 was higher with IPV+ than IPV- at levels of 9.1 pg/mL (range 7.5-11.7) versus 2.1 pg/mL (1.3-5.6), respectively (P = 0.004). ET-1 levels correlated positively with cholangiocyte percent volume (r = 0.72, P0.001) but not with measures of liver dysfunction (bilirubin, INR, MELD score, or hepatic venous pressure gradient).In human cirrhosis, increased hepatic venous ET-1 is associated with IPV and increased hepatic cholangiocyte volume.

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.

RATIONALE: Activation of the coagulation cascade and generation of thrombin has been extensively documented in pulmonary fibrosis, both in acute and chronic lung injury, and in animal models of lung injury and fibrosis. The oral direct thrombin inhibitor (DTI), dabigatran etexilate, modulates the coagulation cascade and inhibits thrombin-induced profibrotic signaling in lung fibroblasts. This study tested whether thrombin inhibition by dabigatran etexilate attenuates bleomycin-induced pulmonary fibrosis in a murine model of lung injury.

Objective To study the mechanisms by which hepatocyte growth factor (HGF) down-regulates collagen and connective tissue growth factor (CTGF) in scleroderma (systemic sclerosis [SSc]) lung fibroblasts. Methods CTGF, type I collagen, and IκBα expression, together with MAPK phosphorylation, were studied by immunoblotting of lung fibroblasts derived from white SSc patients. Matrix metalloproteinase 1 (MMP-1) expression in cell culture medium samples was measured by enzyme-linked immunosorbent assay, MMP-1 activity was studied using an MMP-1 assay, and NF-κB DNA binding activity was determined using a transcription factor assay. Results In lung fibroblasts from white SSc patients, HGF activated MAPK (ERK-1/2) signaling pathways and MMP-1, while it inhibited NF-κB and significantly down-regulated CTGF and collagen in a time- and dose-dependent manner. Small interfering RNA (siRNA)–mediated depletion of Grb2 expression disrupted c-Met receptor downstream signaling, which resulted in diminished HGF-induced ERK-1/2 phosphorylation and the recovery of HGF-inhibited expression of MMP-1, NF-κB, collagen, and CTGF. The MAPK inhibitor, U0126, blocked MMP-1 activity and restored HGF-inhibited collagen and CTGF accumulation. Inhibition of MMP activity by MMP inhibitor GM1489 and inhibition of MMP-1 expression by siRNA did not prevent HGF-induced ERK-1/2 phosphorylation and NF-κB activity, but significantly restored HGF-inhibited collagen and CTGF accumulation. NF-κB inhibitor BAY 11-7082 did not interfere with MAPK phosphorylation or MMP-1 expression and activation, but significantly inhibited NF-κB DNA binding activity and acted synergistically with HGF to completely diminish the expression of CTGF. Conclusion In lung fibroblasts from white SSc patients, HGF down-regulates the accumulation of CTGF via MAPK/MMP-1 and NF-κB signaling pathways, whereas collagen down-regulation is mediated mainly by a MAPK/MMP-1–dependent pathway.

The role of myofibroblasts in various fibrotic disorders is currently well established. These smooth-muscle-like fibroblasts promote deposition of ECM proteins and contractility of lung parenchyma. The present studies were performed to characterize the contractile activity of SSc lung fibroblasts. Previously, we demonstrated that the early stages of interstitial lung disease of SSc are characterized by a prominence of cells prossessing a myofibroblast phenotype. A major feature of such myofibroblasts is contractility, explained by an over-expression of α-smooth muscle actin. Here, we demonstrate for the first time that the contractility of SSc lung fibroblasts dependends on expression of CTGF as well, and that the VWC domain is primarily responsible for the contractile activity of CTGF in human lung fibroblasts. Future studies are required to identify the mechanisms by which CTGF stimulates collagen gel contraction.

To the Editor: We read with interest the article regarding warfarin in idiopathic pulmonary fibrosis by Noth and colleagues (1). Despite an overall negative result, the value of the data collected is very high because it leads to a very important conclusion: namely, that the general inhibition of the coagulation cascade is not a good strategy for the treatment of pulmonary fibrosis. Our laboratory has investigated the profibrotic effects of thrombin for more than a decade. Based on our work, we have come to a very similar conclusion with important implications for potential therapy for idiopathic pulmonary fibrosis (IPF) and other fibrosing lung diseases, which is that there is no need to inhibit the coagulation cascade to inhibit the profibrotic effects of thrombin. Activation of thrombin is one of the earliest events after tissue injury. Thrombin promotes differentiation of fibroblasts to a myofibroblast phenotype, increases fibroblast proliferation, and enhances the proliferative effect of fibrinogen on fibroblasts. Thrombin is also a potent inducer of fibrogenic cytokines, including transforming growth factor-β, connective tissue growth factor, platelet-derived growth factor (PDGF), as well as various chemokines and extracellular matrix proteins like collagen, fibronectin, and tenascin (reviewed in Reference 2). There is compelling evidence that thrombin is an important mediator of interstitial lung diseases, including both IPF and scleroderma-associated interstitial lung disease (SSc-ILD). Our laboratory and others have demonstrated dramatically increased levels of thrombin in bronchoalveolar lavage fluid (BALF) from patients with SSc-ILD and other fibrosing lung diseases (3, 4). BALF samples from normal subjects have a low level of thrombin activity, while BALF samples from patients with SSc-ILD express up to 100-fold higher thrombin activity. Elevated thrombin activity is also observed in the bleomycin rodent model of pulmonary fibrosis. In this setting, highly concentrated thrombin binds to the protease-activated receptor 1 and initiates profibrotic signaling. We recently demonstrated that inhibition of thrombin using dabigatran etexilate has marked antiinflammatory and antifibrotic effects in the bleomycin pulmonary fibrosis model (5). Importantly, the antifibrotic effects of dabigatran etexilate were achieved without inhibition of the coagulation cascade. Thrombin activity in BALF was significantly reduced in bleomycin-treated mice receiving dabigatran etexilate as compared with bleomycin-treated mice receiving placebo, but not significantly different compared with saline-treated control mice receiving placebo. We have not observed any hemorrhagic side effects during our studies of dabigatran etexilate in this rodent model of ILD, suggesting that levels of dabigatran in mouse plasma are not sufficient to perturb the normal hemostatic process, yet were sufficient to ameliorate thrombin-induced and protease-activated receptor 1–mediated lung inflammation and fibrosis. The study published by Noth and coworkers was not designed to address the molecular mechanisms involved in the respiratory worsening (exacerbation or progression) associated with warfarin treatment (1). One obvious explanation for the respiratory worsening and higher mortality rate of warfarin-treated patients with IPF is inhibition of vitamin K–dependent synthesis of protein C, known to already be at a very low level in patients with IPF. Activated protein C plays a protective role in lung tissue repair and remodeling. Yasui and colleagues demonstrated that intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis (6). In contrast to warfarin, dabigatran etexilate does not interfere with protein C synthesis; therefore, the protective effect of protein C on lung tissue should remain intact even when dabigatran etexilate is administered. We suggest that thrombin inhibition as a strategy to treat fibrosing lung diseases needs to be explored and not abandoned based on studies employing warfarin as an anticoagulant, particularly since lung fibrosis may be ameliorated with doses of a thrombin inhibitor not sufficient to cause significant perturbation of hemostasis.

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)-epsilon but not by PKC-alpha. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-epsilon 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-alpha or PKC-epsilon results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-alpha or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-alpha, but not PKC-epsilon, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G(1) phase regulatory protein cyclin D1, suggesting that PKC-alpha, 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.

Gbetagamma binds directly to the third intracellular (i3) loop subdomain of the M(3)-muscarinic receptor (MR). In this report, we identified the Gbetagamma binding motif and G-protein-coupled receptor kinase (GRK2) phosphorylation sites in the M(3)-MR i3 loop via a strategy of deletional and site-directed mutagenesis. The Gbetagamma binding domain was localized to Cys(289)-His(330) within the M(3)-MR-Arg(252)-Gln(490) i3 loop, and the binding properties (affinity, influence of ionic strength) of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain were similar to those observed for the entire i3 loop. Site-directed mutagenesis of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain indicated that Phe(312), Phe(314), and a negatively charged region (Glu(324)-Asp(329)) were required for interaction with Gbetagamma. Generation of the full-length M(3)-MR-Arg(252)-Gln(490) i3 peptides containing the F312A mutation were also deficient in Gbetagamma binding and exhibited a reduced capacity for phosphorylation by GRK2. A similar, parallel strategy resulted in identification of major residues ((331)SSS(333) and (348)SASS(351)) phosphorylated by GRK2, which were just downstream of the Gbetagamma binding motif. Full-length M(3)-MR constructs lacking the 42-amino acid Gbetagamma binding domain (Cys(289)-His(330)) or containing the F312A mutation exhibited ligand recognition properties similar to wild type receptor and also effectively mediated agonist-induced increases in intracellular calcium following receptor expression in Chinese hamster ovary and/or COS 7 cells. However, the M(3)-MRDeltaCys(289)-His(330) and M(3)-MR(F312A) constructs were deficient in agonist-induced sequestration, indicating a key role for the Gbetagamma-M(3)-MR i3 loop interaction in receptor regulation and signal processing.