Critical Role of LMCD1 in Promoting Profibrotic Characteristics of Lung Myofibroblasts in Experimental and Scleroderma-Associated 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 ß receptor type I (TGFßR1) 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 TGFß-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.

Differential DNA Methylation Landscape in Skin Fibroblasts from African Americans with Systemic Sclerosis.

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.

Antifibrotic efficacy of nintedanib in a cellular model of systemic sclerosis-associated interstitial lung disease.

OBJECTIVES: 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). METHODS: Study was performed using lung fibroblasts (LF) isolated from five patients with SSc-ILD and from three control subjects. RESULTS: 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. CONCLUSIONS: 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.

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.

Transforming growth factor ß1 (TGFß1)-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 TGFß1 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-ß-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 TGFß1/TGFßRI 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.

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 TGFß-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.

M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo.

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 TGFß-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFß-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.

Utilization of Glycosaminoglycans/Proteoglycans as Carriers for Targeted Therapy Delivery.

The outcome of patients with cancer has improved significantly in the past decade with the incorporation of drugs targeting cell surface adhesive receptors, receptor tyrosine kinases, and modulation of several molecules of extracellular matrices (ECMs), the complex composite of collagens, glycoproteins, proteoglycans, and glycosaminoglycans that dictates tissue architecture. Cancer tissue invasive processes progress by various oncogenic strategies, including interfering with ECM molecules and their interactions with invasive cells. In this review, we describe how the ECM components, proteoglycans and glycosaminoglycans, influence tumor cell signaling. In particular this review describes how the glycosaminoglycan hyaluronan (HA) and its major receptor CD44 impact invasive behavior of tumor cells, and provides useful insight when designing new therapeutic strategies in the treatment of cancer.

Roles of Proteoglycans and Glycosaminoglycans in Wound Healing and Fibrosis.

A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.

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.