Tanjina Akter

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.

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.

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.

D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts.

Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated “M10,” with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFβ-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients.

Inhibition of Thrombin as a Novel Strategy in the Treatment of Scleroderma-Associated Interstitial Lung Disease

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

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10

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.

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.