Galina S. Bogatkevich

Introduction: Activation of the coagulation cascade leading to generation of thrombin is well documented in various forms of lung injury including systemic sclerosis-associated interstitial lung disease (SSc-ILD). We previously demonstrated that the direct thrombin inhibitor dabigatran inhibits thrombin-induced profibrotic signaling in lung fibroblasts isolated from scleroderma patients. The objective of this study was to characterize and compare lung fibroblasts from an SSc-ILD patient at baseline and after dabigatran treatment to ascertain this therapy’s differential effects on fibrogenic gene expression. Materials and methods: Lung fibroblasts isolated by bronchoalveolar lavage (BAL) from a SSc-ILD patient before and after receiving dabigatran (Pradaxa ^® ) 75 mg twice daily for 6 months (ClinicalTrials.gov Identifier NCT02426229) were analyzed by RNA sequencing, real-time quantitative reverse transcription polymerase chain reaction (qRT)-PCR, and immunofluorescent staining. Results: Thrombin inhibition for six-months by oral dabigatran resulted in significantly decreased expression of 708 lung fibroblast genes as compared to basal levels before dabigatran treatment. Using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, we determined that thrombin-inhibition by dabigatran primarily affected extracellular matrix (ECM) and ECM-related genes. Fibrosis-associated genes, including smooth muscle alpha-actin (SMA, ACTA2), tenascin C, collagen 1 alpha 1 (COL1A1), collagen 3 alpha1 (COL3A1), collagen 8 alpha 2 (COL8A2), collagen 10 alpha 1 (COL10A1), collagen 5 alpha 1 (COL5A1), fibronectin 1, connective tissue growth factor (CTGF), and procollagen-lysine-2-oxoglutarate-5-dioxygenase-2 (PLOD2) were all significantly down regulated following thrombin inhibition by dabigatran treatment. Real-time qRT-PCR and immunofluorescent staining confirmed significant downregulation of the selected genes at the mRNA and protein levels. Conclusion: Inhibition of thrombin in this SSc-ILD patient treated with low-dose dabigatran etexilate downregulated profibrotic proteins in lung fibroblasts, providing further support for the use of thrombin inhibitors as a therapeutic approach for the treatment of patients with SSc-ILD.

We previously found IQ motif containing GTPase activating protein (IQGAP1) to be consistently elevated in lung fibroblasts (LF) isolated from patients with scleroderma (systemic sclerosis, SSc)-associated interstitial lung disease (ILD) and reported that IQGAP1 contributed to SSc by regulating expression and organization of α-smooth muscle actin (SMA) in LF. The aim of this study was to compare the development of ILD in the presence and absence of IQGAP1. Pulmonary fibrosis was induced in IQGAP1 knockout (KO) and wild-type (WT) mice by a single-intratracheal instillation of bleomycin. Two and three weeks later, mice were euthanized and investigated. We observed that the IQGAP1 KO mouse was characterized by a reduced rate of actin polymerization with reduced accumulation of actin in the lung compared to the WT mouse. After exposure to bleomycin, the IQGAP1 KO mouse demonstrated decreased contractile activity of LF, reduced expression of SMA, TGFβ, and collagen, and lowered overall fibrosis scores compared to the WT mouse. The numbers of inflammatory cells and expression of pro-inflammatory cytokines in lung tissue were not significantly different between IQGAP1 KO and WT mice. We conclude that IQGAP1 plays an important role in the development of lung fibrosis induced by bleomycin, and the absence of IQGAP1 reduces the contractile activity of lung fibroblast and bleomycin-induced pulmonary fibrosis. Thus, IQGAP1 may be a potential target for novel anti-fibrotic therapies for lung fibrosis.

Objective: Interstitial lung disease (ILD) is a serious complication and leading cause of mortality in patients with systemic sclerosis (SSc). In this study, we explored the role of LIM and cysteine‐rich domains protein 1 (LMCD1) as a novel factor in the pathogenesis of SSc‐related ILD (SSc‐ILD).Methods: The expression and effects of LMCD1 were studied in lung tissue samples and fibroblasts from SSc‐ILD patients and control subjects as well as in lung tissue samples from animal models.Results: LMCD1 was consistently elevated in lung tissue samples and in fibroblasts isolated from SSc‐ILD patients as compared to controls. Additionally, LMCD1 was found to be highly expressed in the lung in the fibroblast‐specific protein (FSP)–driven, constitutively active transforming growth factor β receptor type I (TGFβR1) transgenic mouse model of ILD and the bleomycin‐induced mouse model of ILD. In lung fibroblasts from SSc‐ILD patients, LMCD1 is an essential factor for the TGFβ‐induced generation of type I collagen, fibronectin, and α‐smooth muscle actin (α‐SMA). Depletion of LMCD1 by small interfering RNA reduced the expression of extracellular matrix proteins and lowered transcriptional activity and expression of α‐SMA, as well as decreased the proliferation and contractile activity of SSc‐ILD lung fibroblasts. In dense fibrotic areas of affected lung tissue, lung LMCD1 colocalized with α‐SMA. In cultured scleroderma lung fibroblasts, LMCD1 colocalized and interacted with serum response factor which mediates LMCD1‐induced contractile activity of lung fibroblasts.Conclusion: 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.

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′ 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’ 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.

Objective: The etiology and reasons underlying the ethnic disparities in systemic sclerosis (SSc) remain unknown. African Americans are disproportionally affected by SSc, yet 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. Methods: Reduced representation bisulfite sequencing (RRBS) was performed on primary cultured 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. Gene set enrichment (GSEA) and gene ontology (GO) analyses were computed to elucidate the underlying biological processes. Quantitative PCR (qPCR) was performed to assess correlations between DNA methylation changes and gene expression levels of top candidate genes. Results: Skin 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’ UTR, promoters, and CpG islands. Seventeen genes and eleven promoters showed significant differential methylation, mostly in non-coding RNA genes and pseudogenes. GSEA and GO enrichment analysis revealed enrichment of immune, metabolism, cell development, and cell signaling pathways, including those related to interferon signaling and mesenchymal differentiation. The hypomethylation of DLX5 and TMEM140 was accompanied by these genes’ overexpression, while for the IncRNA MGC12916 , it was accompanied by its under-expression in patients. Conclusion: 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.

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. 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. 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. 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.

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.