Systemic Sclerosis Dermal Fibroblasts Research Articles

P27 Chloride intracellular channel 4 is a global regulator of type 1 interferon signalling in systemic sclerosis skin

Abstract Introduction and aims Systemic sclerosis (SSc) is an autoimmune disease in which an immune-related injury induces fibrosis of the skin and can progress to affect the internal organs in the most serve cases. Aberrant type 1 interferon (IFN) signalling in the keratinocytes plays a major role in SSc disease progression. We have previously shown the chloride intracellular channel 4 (CLIC)4 is upregulated in SSc skin fibroblasts and plays an important role in SSc fibrosis by enhancing transforming growth factor-β signalling. In this study, we investigated the role of CLIC4 in SSc keratinocyte biology. Methods Skin biopsies from patients with SSc (N = 6) and healthy controls (HCs) (N = 6) were analysed by immunohistochemistry for the expression of CLIC4 and phosphorylated signal transducer and activator of transcription (STAT)1. The skin keratinocyte cell line HaCaTs was stimulated with a range of type 1 IFN signalling agonists including POLY I:C, POLY dA:dT, ODN 2216 and IFN-α. CLIC4 was inhibited with the chloride channel inhibitors NPPB and IAA-94 or small interfering RNA specific to CLIC4. Conditioned media from HC or SSc dermal fibroblasts was employed for indirect coculture of HaCaTs. Results Skin biopsies from SSc showed high levels of CLIC4 in SSc skin fibroblasts, keratinocytes and endothelial cells compared with HC. Indirect coculture of HaCaTs with SSc fibroblast media induced CLIC4 expression in the HaCaTs. The increased CLIC4 expression played an important role in type 1 IFN signalling in keratinocytes as inhibition of CLIC4 blocked Toll-like receptor (TLR)3, TLR9 and cGAS-mediated activation of the type 1 IFN signalling pathway transcription factor STAT1. Additionally, inhibition of CLIC4 prevented SSc fibroblast media from inducing a type 1 IFN response in keratinocytes. Conclusions The data presented in this study suggests that CLIC4 is a global regulator of the type 1 IFN signalling cascade in SSc epithelial cells.

EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis.

Erythropoietin-producing hepatocellular (Eph)/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models of skin fibrosis. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from patients with SSc and healthy controls, which was followed by in vivo analysis of fibroblast-specific Ephb2-deficient mice. Expression of EphB2 is up-regulated in SSc skin tissue and explanted SSc dermal fibroblasts compared with healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-β (TGF-β) cytokines up-regulate EphB2 in dermal fibroblasts via noncanonical TGF-β/mother against decapentaplegic signaling, and silencing EPHB2 in human dermal fibroblasts is sufficient to dampen TGF-β-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. Our data implicate TGF-β regulation of EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

Expression of collagen-related piRNA is dysregulated in cultured dermal fibroblasts derived from patients with scleroderma.

PIWI-interacting RNA (piRNA) is a class of recently discovered small non-coding RNAs. piRNAs derive from an initial transcript encompassing a piRNA cluster via a unique biosynthesis process, interact with PIWI proteins, bind to specific targets, and recruit chromatin modifiers to enable transcriptional repression. Abnormal expression of PIWI proteins and piRNAs has been reported in some human cancers, with participation of some PIWI/piRNAs complexes in tumorigenesis and association with cancer prognosis. Their expression in patients with systemic sclerosis (SSc) has not been widely elucidated. PIWI/piRNAs and their role in the pathogenesis of collagen accumulation in SSc was therefore investigated; no difference was found in the PIWIL1-4 levels between normal and cultured SSc dermal fibroblasts. Among piRNAs predicted to target SSc-related molecules, we first found significant piR-32364 up-regulation in SSc dermal fibroblasts, likely due to intrinsic TGF-β signaling. Forced piR-32364 overexpression in normal fibroblasts significantly reduced COL1A1 expression both at mRNA and protein levels, but not COL1A2. Thus, piR-32364 overexpression in SSc fibroblasts may be the negative feedback against collagen up-regulation, which could suggest the potential of piRNAs as a therapeutic target.

Antifibrotic effect of apremilast in systemic sclerosis dermal fibroblasts and bleomycin-induced mouse model

Phosphodiesterase (PDE) 4 inhibitors have been reported to suppress the progression of dermal fibrosis in patients with systemic sclerosis (SSc); however, the precise mechanisms remain to be elucidated. Therefore, we conducted experiments focusing on the antifibrotic and anti-inflammatory effects of apremilast using dermal fibroblasts derived from patients with SSc and an SSc mouse model. Dermal fibroblasts derived from healthy controls and patients with SSc were incubated with apremilast in the presence or absence of 10 ng/ml transforming growth factor (TGF)-β1 for the measurement of intracellular cAMP levels and evaluation of mRNA and protein expression. A bleomycin-induced dermal fibrosis mouse model was used to evaluate the inhibitory effects of apremilast on the progression of dermal fibrosis. Intracellular cAMP levels were significantly reduced in dermal fibroblasts derived from patients with SSc compared with those derived from healthy controls. Apremilast reduced the mRNA expression of profibrotic markers and the protein expression of type I collagen and Cellular Communication Network Factor 2 (CCN2) in dermal fibroblasts. Additionally, apremilast inhibited the progression of dermal fibrosis in mice, partly by acting on T cells. These results suggest that apremilast may be a potential candidate for treating dermal fibrosis in SSc.

ATRA ameliorates fibrosis by suppressing the pro-fibrotic molecule Fra2/AP-1 in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to irreversible fibrosis of the skin and the internal organs. The etiology of SSc is complex, its pathophysiology is poorly understood, and clinical therapeutic options are restricted. Thus, research into medications and targets for treating fibrosis is essential and urgent. Fos-related antigen 2 (Fra2) is a transcription factor that is a member of the activator protein-1 family. Fra2 transgenic mice were shown to have spontaneous fibrosis. All-trans retinoic acid (ATRA) is a vitamin A intermediate metabolite and ligand for the retinoic acid receptor (RAR), which possesses anti-inflammatory and anti-proliferative properties. Recent research has demonstrated that ATRA also has an anti-fibrotic effect. However, the exact mechanism is not fully understood. Interestingly, we identified potential binding sites for the transcription factor RARα to the promoter region of the FRA2 gene through JASPAR and PROMO databases. In this study, the pro-fibrotic effect of Fra2 in SSc is confirmed. SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc animals exhibit increased levels of Fra2. Inhibition of Fra2 expression in SSc dermal fibroblasts with Fra2 siRNA markedly decreased collagen I expression. ATRA reduced the expressions of Fra2, collagen I, and α-smooth muscle actin(α-SMA) in SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc mice. In addition, chromatin immunoprecipitation and dual-luciferase assays demonstrated that retinoic acid receptor RARα binds to the FRA2 promoter and modulates its transcriptional activity. ATRA decreases collagen I expression both in vivo and in vitro via the reduction of Fra2 expression. This work establishes the rationale for expanding the use of ATRA in the treatment of SSc and indicates that Fra2 can be used as an anti-fibrotic target.

Amelioration of Fibrotic Remodeling of Human 3-Dimensional Full-Thickness Skin by Transglutamase 2 Inhibition.

Fibrotic tissues are characterized by excessive crosslinking between extracellular matrix (ECM) proteins, rendering them more resistant to degradation. Although increased crosslinking of ECM is thought to play an important role for progression of tissue fibrosis, enhanced ECM crosslinking has not yet been targeted therapeutically in systemic sclerosis (SSc). Here, we investigated the role of transglutaminase 2 (TG2), a central crosslinking enzyme, in the activation of SSc fibroblasts. We assessed TG2 expression and activity using TG2 staining, Western blotting, and TG2 activity assays. We inhibited TG2 in fibroblasts cultured under standard 2-dimensional conditions and in a 3-dimensional full-thickness equivalent skin model using monoclonal inhibitory anti-TG2 antibodies. TG2 expression was increased in the skin of patients with SSc compared with healthy controls, with levels particularly high in patients with SSc-associated interstitial lung disease. TG2 expression and TG2 activity were also increased in SSc dermal fibroblasts. Moreover, the levels of circulating TG2 in the plasma samples from SSc patients were increased versus samples from healthy controls. Anti-TG2 antibodies did not show consistent antifibrotic effects across different fibroblast cell lines under 2-dimensional culture conditions; however, anti-TG2 antibodies effectively reduced transforming growth factor β-induced dermal thickening, myofibroblast differentiation, and collagen accumulation in the 3-dimensional full-thickness model of human skin. We provide the first evidence, to our knowledge, that inhibition of TG2 might be a potential antifibrotic approach in SSc. Our findings have translational potential as anti-TG2 antibodies are currently evaluated in a phase II clinical trial in chronic allograft injury and would thus be available for clinical studies in SSc (ClinicalTrials.gov identifier: NCT04335578).

A Positive Loop Formed by SOX11 and Periostin Upregulates TGF-β Signals Leading to Skin Fibrosis

Systemic sclerosis (SSc) is a chronic, heterogeneous disease of connective tissue characterized by organ fibrosis together with vascular injury and autoimmunity. TGF-β plays a central role in generating fibrosis, including SSc. Periostin is a matricellular protein playing a key role in the generation of fibrosis by amplifying the TGF-β signals. SOX11 is a transcription factor playing several important roles in organ development in embryos. We have previously shown that SOX11 induces periostin expression. However, the roles of the interactions among the TGF-β signals, periostin, and SOX11 remain unknown in the pathogenesis of SSc. In this study, we found that most clones of dermal fibroblasts derived from patients with SSc showed constitutive, high expression of SOX11, which is significantly induced by TGF-β1. SOX11 forms a positive loop with periostin to activate the TGF-β signals in SSc dermal fibroblasts. Genetic deletion of Sox11 in Postn-expressing fibroblasts impairs dermal fibrosis by bleomycin. Moreover, using the DNA microarray method, we identified several fibrotic factors dependent on the TGF-β/SOX11/periostin pathway in SSc dermal fibroblasts. Our findings, taken together, show that a positive loop formed by SOX11 and periostin in fibroblasts upregulates the TGF-β signals, leading to skin fibrosis.

Esomeprazole alleviates fibrosis in systemic sclerosis by modulating AhR/Smad2/3 signaling

Systemic sclerosis (SSc) is a connective tissue disease with the involvement of complex signaling pathways, such as TGF-β/Smad2/3. SSc can lead to severe multiple organ fibrosis, but no effective therapy is currently available because of its unclear pathogenesis. Exploring new treatments is the focus of recent research on SSc. Recent studies have implied a potential antifibrotic role of esomeprazole (ESO), but with currently unidentified mechanisms. Signaling of AhR, a ligand-dependent transcription factor, has been described as a key controller of fibrosis, tumorigenesis, and immune balance. Recently, it has been reported that ESO may be an exogenous agonist of AhR signaling, while no previous study has revealed the effects of ESO on SSc and its underlying mechanisms. In this study, we demonstrate that ESO suppresses the migration of SSc dermal fibroblasts, downregulates profibrotic markers, including COLIA1, α-SMA CTGF and MMP1, and limits collagen production potentially via the activation of AhR signaling. More importantly, ESO could block Smad2/3 phosphorylation concurrently with the reduction in collagen via AhR signaling. Moreover, our results from the bleomycin (BLM)-induced SSc model in skin and lung shows that ESO ameliorates fibrosis in vivo, which in keeping with our in vitro results. We conclude that ESO is a potential therapeutic drug for SSc fibrosis.

PDGF Promotes Dermal Fibroblast Activation via a Novel Mechanism Mediated by Signaling Through MCHR1.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs. To this day, no effective treatments to prevent the progression of fibrosis exist, and SSc patients have disabilities and reduced life expectancy. The need to better understand pathways that drive SSc and to find therapeutic targets is urgent. RNA sequencing data from SSc dermal fibroblasts suggested that melanin-concentrating hormone receptor 1 (MCHR1), one of the G protein-coupled receptors regulating emotion and energy metabolism, is abnormally deregulated in SSc. Platelet-derived growth factor (PDGF)-BB stimulation upregulated MCHR1 mRNA and protein levels in normal human dermal fibroblasts (NHDF), and MCHR1 silencing prevented the PDGF-BB-induced expression of the profibrotic factors transforming growth factor beta 1 (TGFβ1) and connective tissue growth factor (CTGF). PDGF-BB bound MCHR1 in membrane fractions of NHDF, and the binding was confirmed using surface plasmon resonance (SPR). MCHR1 inhibition blocked PDGF-BB modulation of intracellular cyclic adenosine monophosphate (cAMP). MCHR1 silencing in NHDF reduced PDGF-BB signaling. In summary, MCHR1 promoted the fibrotic response in NHDF through modulation of TGFβ1 and CTGF production, intracellular cAMP levels, and PDGF-BB-induced signaling pathways, suggesting that MCHR1 plays an important role in mediating the response to PDGF-BB and in the pathogenesis of SSc. Inhibition of MCHR1 should be considered as a novel therapeutic strategy in SSc-associated fibrosis.

The Contribution of LIGHT (TNFSF14) to the Development of Systemic Sclerosis by Modulating IL-6 and T Helper Type 1 Chemokine Expression in Dermal Fibroblasts

Systemic sclerosis (SSc) is an autoimmune and vascular disease resulting in multiple organ fibrosis, in which IL-6 and T helper (Th)2/Th17 cytokines serve as critical disease drivers. LIGHT is a proinflammatory cytokine promoting IL-6 production in lung fibroblasts and Th1 chemokine expression in dermal fibroblasts (DFs) stimulated with IFN-γ. In this study, we investigated the potential contribution of LIGHT to SSc development using clinical samples and animal models. In SSc-involved skin, LIGHT was upregulated in inflammatory cells, whereas herpesvirus entry mediator (HVEM), a receptor of LIGHT, was downregulated in DFs. Similar expression profiles of LIGHT and HVEM were reproduced in bleomycin-treated mice. Transcription factor FLI1 bound to the HVEM promoter, and FLI1 small interfering RNA suppressed HVEM expression in normal DFs. In SSc DFs, LIGHT significantly increased IL-6 production, whereas IFN-γ/LIGHT-dependent Th1 chemokine induction was decreased compared with that in normal DFs. Importantly, LIGHT small interfering RNA significantly attenuated bleomycin-induced skin fibrosis, and serum LIGHT levels were elevated in patients with diffuse cutaneous SSc and positively correlated with clinical parameters reflecting skin and pulmonary fibrosis. Taken together, these results suggest that altered response of DFs to LIGHT, namely increased IL-6 production and decreased Th1 chemokine expression, contributes to the development of skin fibrosis in SSc.

Oxidative Stress Induced by Reactive Oxygen Species (ROS) and NADPH Oxidase 4 (NOX4) in the Pathogenesis of the Fibrotic Process in Systemic Sclerosis: A Promising Therapeutic Target.

Numerous clinical and research investigations conducted during the last two decades have implicated excessive oxidative stress caused by high levels of reactive oxygen species (ROS) in the development of the severe and frequently progressive fibrotic process in Systemic Sclerosis (SSc). The role of excessive oxidative stress in SSc pathogenesis has been supported by the demonstration of increased levels of numerous biomarkers, indicative of cellular and molecular oxidative damage in serum, plasma, and other biological fluids from SSc patients, and by the demonstration of elevated production of ROS by various cell types involved in the SSc fibrotic process. However, the precise mechanisms mediating oxidative stress development in SSc and its pathogenetic effects have not been fully elucidated. The participation of the NADPH oxidase NOX4, has been suggested and experimentally supported by the demonstration that SSc dermal fibroblasts display constitutively increased NOX4 expression and that reduction or abrogation of NOX4 effects decreased ROS production and the expression of genes encoding fibrotic proteins. Furthermore, NOX4-stimulated ROS production may be involved in the development of certain endothelial and vascular abnormalities and may even participate in the generation of SSc-specific autoantibodies. Collectively, these observations suggest NOX4 as a novel therapeutic target for SSc.

Global gene expression analysis of systemic sclerosis myofibroblasts demonstrates a marked increase in the expression of multiple NBPF genes

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). Despite their importance to SSc pathogenesis, the specific transcriptome of SSc myofibroblasts has not been described. The purpose of this study was to identify transcriptome differences between SSc myofibroblasts and non-myofibroblastic cells. Alpha smooth muscle actin (α-SMA) expressing myofibroblasts and α-SMA negative cells were isolated employing laser capture microdissection from dermal cell cultures from four patients with diffuse SSc of recent onset. Total mRNA was extracted from both cell populations, amplified and analyzed employing microarrays. Results for specific genes were validated by Western blots and by immunohistochemistry. Transcriptome analysis revealed 97 differentially expressed transcripts in SSc myofibroblasts compared with non-myofibroblasts. Annotation clustering of the SSc myofibroblast-specific transcripts failed to show a TGF-β signature. The most represented transcripts corresponded to several different genes from the Neuroblastoma Breakpoint Family (NBPF) of genes. NBPF genes are highly expanded in humans but are not present in murine or rat genomes. In vitro studies employing cultured SSc dermal fibroblasts and immunohistochemistry of affected SSc skin confirmed increased NBPF expression in SSc. These results indicate that SSc myofibroblasts represent a unique cell lineage expressing a specific transcriptome that includes very high levels of transcripts corresponding to numerous NBPF genes. Elevated expression of NBPF genes in SSc myofibroblasts suggests that NBPF gene products may play a role in SSc pathogenesis and may represent a novel therapeutic target.

The Cell-Permeable Derivative of the Immunoregulatory Metabolite Itaconate, 4-Octyl Itaconate, Is Anti-Fibrotic in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-β1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-β1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.

MicroRNA-30c attenuates fibrosis progression and vascular dysfunction in systemic sclerosis model mice.

Systemic sclerosis (SSc) is characterized by peripheral circulatory disturbance and fibrosis in skin and visceral organs. We recently demonstrated that α2-antiplasmin (α2AP) is elevated in SSc dermal fibroblasts and SSc model mice, and is associated with fibrosis progression and vascular dysfunction. In the present study, we predicted that α2AP could be a target of microRNA-30c (miR-30c) using TargetScan online database, and investigated the effect of miR-30c on the pathogenesis of SSc using a bleomycin-induced SSc model mice. miR-30c attenuated α2AP expression, and prevented the pro-fibrotic changes (increased dermal thickness, collagen deposition, myofibroblast accmulation) and the vascular dysfunction (the reduction of vascular endothelial cells (ECs) and blood flow) in the skin of SSc model mice. Furthermore, miR-30c suppressed pulmonary fibrosis progression in the SSc model mice. miR-30c exerts the anti-fibrotic and anti-angiopathy effects on SSc model mice, and might provide a basis for clinical strategies for SSc.

Butyrate Improves Skin/Lung Fibrosis and Intestinal Dysbiosis in Bleomycin-Induced Mouse Models.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Despite several studies on SSc treatments, effective treatments for SSc are still lacking. Since evidence suggests an association between intestinal microbiota and SSc, we focused on butyrate, which has beneficial effects in autoimmune diseases as a bacterial metabolite. Here, we investigated the therapeutic potential of sodium butyrate (SB) using a bleomycin-induced fibrosis mouse model of SSc and human dermal fibroblasts (HDFs). SB attenuated bleomycin-induced dermal and lung fibrosis in mice. SB influenced fecal microbiota composition (phyla Actinobacteria and Bacteroidetes, genera Bifidobacterium and Ruminococcus_g2). SB controlled macrophage differentiation in mesenteric lymph nodes, spleen, and bronchoalveolar lavage cells of mice with bleomycin-induced skin fibrosis. Profibrotic and proinflammatory gene expression was suppressed by SB administration in skin. Furthermore, SB inhibited transforming growth factor β1-responsive proinflammatory expression with increased acetylation of histone 3 in HDFs. Subcutaneous SB application had antifibrogenic effects on the skin. Butyrate ameliorated skin and lung fibrosis by improving anti-inflammatory activity in a mouse model of SSc. Butyrate may exhibit indirect and direct anti-fibrogenic action on fibroblasts by regulating macrophage differentiation and inhibition of histone deacetylase 3. These findings suggest butyrate as an SSc treatment.

Long non-coding RNA HOTAIR induces GLI2 expression through Notch signalling in systemic sclerosis dermal fibroblasts

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation.MethodsPatient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2).ResultsOverexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR.ConclusionsOur data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.

The Phosphodiesterase Type 5 Inhibitor Sildenafil Improves DNA Stability and Redox Homeostasis in Systemic Sclerosis Fibroblasts Exposed to Reactive Oxygen Species.

Systemic sclerosis (SSc) is a multi-system connective tissue disease characterized by the increased deposition of extracellular matrix proteins such as collagen and fibronectin. Although the pathogenesis is not completely understood, a number of studies suggest that free radicals could be the major contributors to the disease. Indeed, different studies demonstrated how oxidative stress could contribute to the fibrotic process activation at the level of the skin and visceral organs. Emerging evidences highlight the beneficial effects of sildenafil, a phosphodiesterase type 5 inhibitor (PDE5i), which protects different cell lines from the cell damage induced by reactive oxygen species (ROS). These data make sildenafil a good candidate for therapeutic treatment aimed to protect biological macromolecules against oxidative damage, thus preserving cell viability. The purpose of this study was to evaluate the sensitivity of SSc dermal fibroblasts to an oxidative insult and the ability for sildenafil to prevent/reduce the DNA damage due to ROS action. Additionally, we evaluated the capacity for sildenafil to influence redox homeostasis and cytotoxicity, as well as cell proliferation and cell cycle progression. We demonstrated that SSc fibroblasts have an increased sensitivity to a pro-oxidant environment in comparison to healthy controls. The sildenafil treatment reduced ROS-induced DNA damage, counteracted the negative effects of ROS on cell viability and proliferation, and promoted the activity of specific enzymes involved in redox homeostasis maintenance. To our knowledge, in this report, we demonstrate, for the first time, that sildenafil administration prevents ROS-induced instability in human dermal fibroblasts isolated by SSc patients. These results expand the use of PDE5i as therapeutic agents in SSc by indicating a protective role in tissue damage induced by oxidative insult.

Translational engagement of lysophosphatidic acid receptor 1 in skin fibrosis: from dermal fibroblasts of patients with scleroderma to tight skin 1 mouse.

Background and PurposeGenetic deletion and pharmacological studies suggest a role for lysophosphatidic acid (LPA1) receptor in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA1 receptor antagonist using dermal fibroblasts from patients and an animal model of skin fibrosis.Experimental ApproachDermal fibroblast and skin biopsies from systemic sclerosis patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Pharmacolgical effect of SAR100842 was assessed in the tight skin 1 (Tsk1) mouse model.Key ResultsSAR100842 is equipotent against various LPA isoforms. Dermal fibroblasts and skin biopsies from patients with systemic sclerosis expressed high levels of LPA1 receptor. The LPA functional response (Ca2+) in systemic sclerosis dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in systemic sclerosis dermal and idiopathic pulmonary fibrosis lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from systemic sclerosis dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice.Conclusion and ImplicationsThe effects of SAR100842 on LPA‐induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA1 receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA1 receptor antagonists in systemic sclerosis.

Decrease in matrix metalloproteinase‑3 activity in systemic sclerosis fibroblasts causes α2‑antiplasmin and extracellular matrix deposition, and contributes to fibrosis development.

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune origin characterized by fibrosis of the skin and visceral organs, and peripheral circulatory disturbance. α2‑antiplasmin (α2AP) is the major circulating inhibitor of plasmin and is a key regulator of fibrinolysis. It has been demonstrated that the expression of α2AP is elevated in dermal fibroblasts obtained from patients with SSc patients. It has also been determined that α2AP is associated with the development and progression of fibrosis in SSc. The present study assessed the relationship between α2AP and matrix metalloproteinase‑3 (MMP‑3), an extracellular matrix (ECM)‑degrading enzyme. Serum levels of α2AP and MMP‑3 were measured in healthy controls and patients with SSc using ELISA. No significant differences were determined between these two groups. α2AP, MMP‑3 and tissue inhibitor of metalloproteinase‑1 (TIMP‑1) expression was subsequently evaluated in normal and SSc fibroblasts via western blotting. The results revealed that α2AP expression increased in SSc dermal fibroblasts, while the ratio of MMP‑3/TIMP‑1 decreased. Additionally, incubation of recombinant α2AP with MMP‑3 caused α2AP degradation. The mixture of recombinant α2AP with MMP‑3 was subsequently added to normal fibroblasts prior to western blotting. The results revealed decreased α‑smooth muscle actin (α‑SMA; a marker of the myofibroblast phenotype) and typeI collagen expression. The stimulation of SSc fibroblasts with MMP‑3 decreased protein levels of α2AP, α‑SMA and typeI collagen, thus reversing the pro‑fibrotic phenotype of SSc fibroblasts. SSc fibroblast transfection with microRNA‑29a resulted in a decreased TIMP‑1 expression, but also decreased the protein expression of α2AP. The results indicated that MMP‑3 attenuated fibrosis progression by degrading α2AP and ECM, and might therefore contribute to a novel therapeutic approach for SSc treatment.

Systemic Sclerosis Dermal Fibroblasts Induce Cutaneous Fibrosis Through Lysyl Oxidase-like 4: New Evidence From Three-Dimensional Skin-like Tissues.

Systemic sclerosis (SSc) is a clinically heterogeneous disease characterized by increased collagen accumulation and skin stiffness. Our previous work has demonstrated that transforming growth factor β (TGFβ) induces extracellular matrix (ECM) modifications through lysyl oxidase-like 4 (LOXL-4), a collagen crosslinking enzyme, in bioengineered human skin equivalents (HSEs) and self-assembled stromal tissues (SAS). We undertook this study to investigate cutaneous fibrosis and the role of LOXL-4 in SSc pathogenesis using HSEs and SAS. SSc-derived dermal fibroblasts (SScDFs; n = 8) and normal dermal fibroblasts (NDFs; n = 6) were incorporated into HSEs and SAS. These 3-dimensional skin-like microenvironments were used to study the effects of dysregulated LOXL-4 on ECM remodeling, fibroblast activation, and response to TGFβ stimulation. SScDF-containing SAS showed increased stromal thickness, collagen deposition, and interleukin-6 secretion compared to NDF-containing SAS (P < 0.05). In HSE, SScDFs altered collagen as seen by a more mature and aligned fibrillar structure (P < 0.05). With SScDFs, enhanced stromal rigidity with increased collagen crosslinking (P < 0.05), up-regulation of LOXL4 expression (P < 0.01), and innate immune signaling genes were observed in both tissue models. Conversely, knockdown of LOXL4 suppressed rigidity, contraction, and α-smooth muscle actin expression in SScDFs in HSE, and TGFβ-induced ECM aggregation and collagen crosslinking in SAS. A limitation to the development of effective therapeutics in SSc is the lack of in vitro human model systems that replicate human skin. Our findings demonstrate that SAS and HSE can serve as complementary in vitro skin-like models for investigation of the mechanisms and mediators that drive fibrosis in SSc and implicate a pivotal role for LOXL-4 in SSc pathogenesis.