Anna Ludwicka-Bradley

Surfactant Protein D and KL-6 as Serum Biomarkers of Interstitial Lung Disease in Patients with Scleroderma

Objective. To assess whether serum concentrations of surfactant protein D (SP-D) and Krebs von den Lungen-6 (KL-6), glycoproteins expressed by type II pneumocytes, correlate with the presence of “alveolitis” and measures of lung function in patients enrolled in the Scleroderma Lung Study (SLS). Methods. Serum obtained at baseline screening of patients with systemic sclerosis (SSc, scleroderma) in the SLS was assayed. “Alveolitis” was defined by either bronchoalveolar lavage or thoracic high-resolution computed tomography (HRCT) by SLS criteria. SP-D and KL-6 levels were measured by ELISA in 66 SSc patients (44 with “alveolitis,” 22 without “alveolitis”) and in 10 healthy controls. These were compared to clinical measures of lung disease and “alveolitis” in the SLS patients. Results. SP-D levels were 300 ± 214 ng/ml (mean ± SD) in the SSc patients compared to 40 ± 51 ng/ml in controls (p < 0.0001). KL-6 levels were 1225 ± 984 U/ml in the SSc patients and 333 ± 294 U/ml in controls (p < 0.0001). SSc patients with “alveolitis” had higher levels of both SP-D and KL-6 than those without “alveolitis.” The level of SP-D was 353 ± 219 ng/ml in patients with “alveolitis” and 161 ± 143 ng/ml without “alveolitis” (p = 0.0002). The level of KL-6 was 1458 ± 1070 U/ml in patients with “alveolitis” and 640 ± 487 U/ml without “alveolitis” (p = 0.0001). Receiver operator characteristic curve analysis demonstrated high sensitivity and specificity of both SP-D and KL-6 for the determination of “alveolitis.” KL-6 and SP-D were positively correlated with maximum fibrosis scores, but not with maximum ground-glass opacities, on HRCT. Conclusion. Serum levels of SP-D and KL-6 appear to be indicative of “alveolitis” in SSc patients as defined by the SLS, and are significantly higher than in SSc patients without “alveolitis.” Serum SP-D and KL-6 may serve as noninvasive serological means of assessing interstitial lung disease in patients with SSc.

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.

Depletion of protein kinase Cε in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression

OBJECTIVE To determine whether the extracellular matrix protein tenascin-C (TN-C) is overexpressed in lung fibroblasts from systemic sclerosis (SSc) patients, the molecular mechanisms regulating TN-C secretion in SSc and normal lung fibroblasts, and how these processes might contribute to lung fibrosis in SSc patients. METHODS TN-C secretion by SSc and normal fibroblasts was compared in vivo (in bronchoalveolar lavage [BAL] fluid) and in vitro (in culture medium). The ability of thrombin to induce TN-C was confirmed at both the protein and the messenger RNA (mRNA) level. The role of protein kinase Cepsilon (PKCepsilon) in the expression of TN-C was evaluated by determining the effects of thrombin on PKCepsilon levels and by directly manipulating PKCepsilon levels via the use of antisense oligonucleotides. RESULTS BAL fluid from SSc patients contained high levels of TN-C, whereas that from normal subjects contained little or no TN-C. In vitro, SSc lung fibroblasts expressed much higher amounts of TN-C than did normal lung fibroblasts. Consistent with the idea that thrombin is a physiologic inducer of TN-C, thrombin stimulated TN-C mRNA and protein expression in both SSc and normal lung fibroblasts by a mechanism that required proteolytic cleavage of the thrombin receptor. Surprisingly, thrombin treatment and antisense oligonucleotide-mediated depletion of PKCepsilon indicated that TN-C expression is regulated via opposite signaling mechanisms in SSc and normal cells. In SSc lung fibroblasts, thrombin decreased PKCepsilon levels, and the decreased PKCepsilon induced TN-C secretion; in normal fibroblasts, thrombin increased PKCepsilon levels, and the increased PKCepsilon induced TN-C secretion. Normal and SSc lung fibroblasts also differed in the subcellular localization of PKCepsilon, both before and after thrombin treatment. CONCLUSION These studies are the first to demonstrate that thrombin is a potent simulator of TN-C in lung fibroblasts and that PKCepsilon is a critical regulator of TN-C protein levels in these cells. Furthermore, our results indicate that both the regulation of PKCepsilon levels by thrombin and the regulation of TN-C levels by PKCepsilon are defective in SSc lung fibroblasts.

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.

Scleroderma Lung Fibroblasts

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.

Inhibitory Effects of the Dual Endothelin Receptor Blocker Bosentan on Thrombin-Activated Lung Fibroblasts Isolated from 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.