Galina S. Bogatkevich

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

OBJECTIVE Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor 1 (PAR-1) and induces a myofibroblast phenotype in normal lung fibroblasts resembling the phenotype of scleroderma lung myofibroblasts. We undertook this study to investigate whether a selective direct thrombin inhibitor, dabigatran, interferes with signal transduction in human lung fibroblasts induced by thrombin and mediated via PAR-1. METHODS Lung fibroblast proliferation was analyzed using the Quick Cell Proliferation Assay. Expression and organization of alpha-smooth muscle actin (alpha-SMA) was studied by immunofluorescence staining and immunoblotting. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Connective tissue growth factor (CTGF) and type I collagen expression was analyzed on Western blots. RESULTS Dabigatran, at concentrations of 50-1,000 ng/ml, inhibited thrombin-induced cell proliferation, alpha-SMA expression and organization, and the production of collagen and CTGF in normal lung fibroblasts. Moreover, when treated with dabigatran (1 microg/ml), scleroderma lung myofibroblasts produced 6-fold less alpha-SMA, 3-fold less CTGF, and 2-fold less type I collagen compared with untreated cells. CONCLUSION Dabigatran restrains important profibrotic events in lung fibroblasts and warrants study as a potential antifibrotic drug for the treatment of fibrosing lung diseases such as scleroderma lung disease and idiopathic pulmonary fibrosis.

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

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.

Proteomic analysis of CTGF-activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration

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-alpha (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.

Racial differences between blacks and whites with systemic sclerosis.

Purpose of reviewRacial 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 findingsWe 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-&bgr;1 (TGF-&bgr;1) and caveolin-1, as well as differences in the expression of hepatocyte growth factor and PPAR-&ggr;, 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. SummaryA 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.

Overexpression of c-Met and CD44v6 Receptors Contributes to Autocrine TGF-β1 Signaling in Interstitial Lung Disease

Background: CD44v6 and c-Met contribute to TGF-β1 signaling in interstitial lung disease (ILD). Results: CD44v6/TGF-β1 signaling regulates activation of ILD fibroblasts, whereas the HGF/Met pathway down-regulates the activation. Conclusion: Overexpression of HGF in ILD fibroblasts sustains the TGF-β1-regulated CD44v6 expression that promotes collagen synthesis. Physiological concentrations of HGF are insufficient to influence its antifibrotic effect in these cells. Significance: These results should provide CD44v6 as new drug target to treat 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 p38MAPK. 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.

The PPARγ Agonist Rosiglitazone Is Antifibrotic for Scleroderma Lung Fibroblasts: Mechanisms of Action and Differential Racial Effects

We present novel data demonstrating that the expression of PPARγ is reduced in lung fibroblasts from black SSc-ILD patients as compared to white patients. Activating PPARγ with 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 rosiglitazones 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 PPARγ agonists 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.

Coagulation and Autoimmunity in Scleroderma Interstitial Lung Disease

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.

Thrombin Increases Lung Fibroblast Survival while Promoting Alveolar Epithelial Cell Apoptosis via the Endoplasmic Reticulum Stress Marker, CCAAT Enhancer–Binding Homologous Protein

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.

Novel lung imaging biomarkers and skin gene expression subsetting in dasatinib treatment of systemic sclerosis-associated interstitial lung disease

BACKGROUND There 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). METHODS Primary 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 >5 or >20% from baseline in the modified Rodnan Skin Score (MRSS). Pulmonary function was assessed at baseline and day 169. RESULTS Dasatinib 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). CONCLUSIONS In 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 REGISTRATION Clinicaltrials.gov NCT00764309.

Transforming growth factor β1 (TGFβ1)-induced CD44V6-NOX4 signaling in pathogenesis of idiopathic pulmonary fibrosis.

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.