Systemic Sclerosis Skin Fibroblasts Research Articles

Alternative splicing and intron retention: Their profiles and roles in cutaneous fibrosis of systemic sclerosis

BackgroundAlternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). MethodsWe integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. FindingsAS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. ConclusionTotally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc.

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.

Upregulation of GPX4 drives ferroptosis resistance in scleroderma skin fibroblasts

The pathogenesis of systemic sclerosis (SSC) fibrosis involves the rapid proliferation of skin fibroblasts, and current anti-fibrotic treatments are limited. This study investigated the relationship between ferroptosis and SSC skin fibroblasts. We observed that erastin-induced ferroptosis was suppressed in SSC fibroblasts. RSL3, a direct inhibitor of Glutathione Peroxidase 4 (GPX4), significantly reduced the viability of the fibroblasts, and upregulation of GPX4 in the SSC fibroblasts contributed to ferroptosis resistance. Furthermore, we demonstrated that transferrin receptor 1 (TfR1) was a crucial transporter for iron deposition in the fibroblasts. Collectively, our results highlight that GPX4 inhibition could enhance the sensitivity to ferroptosis by SSC fibroblasts, which showed distinct characteristics of iron metabolism that were not observed in normal fibroblasts in this study. Taken together, these results suggest that targeting ferroptosis could be a therapeutic strategy for the treatment of SSC.

Xanthohumol attenuates collagen synthesis in scleroderma skin fibroblasts by ROS/Nrf2/TGFβ1/Smad3 pathway

Skin fibrosis, the most obvious clinical manifestation of systemic sclerosis (SSc), has a high unmet need for treatment. Xanthohumol (Xn) has been shown to have beneficial effects on fibrotic diseases, but its efficacy in SSc remains unreported. This study aims to elucidate the effects and mechanisms of Xn on collagen synthesis in SSc skin fibroblasts (SScF). We found increased collagen production in SScF cultured in vitro, accompanied by dysregulated levels of oxidative stress. Cell experiments showed that Xn inhibited cell proliferation and promoted apoptosis. In addition, Xn was shown for the first time to upregulate reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2)levels in SScF, and when combined with the ROS scavenger N-acetylcysteine (NAC), Nrf2 expression was decreased. Importantly, we demonstrated that Xn significantly attenuated collagen synthesis by blocking the fibrotic classical transforming growth factor beta 1 (TGFβ1)/Smad3 pathway, which interestingly was upregulated when combined with the Nrf2 inhibitor 385. Taken together, Xn suppressed the TGFβ1/Smad3 pathway to ameliorate collagen overproduction by promoting ROS-induced oxidative stress damage and activating Nrf2, suggesting that Xn administration may be an emerging therapeutic strategy for skin fibrosis in SSc.

Pharmacological inhibition of TAK1 prevents and induces regression of experimental organ fibrosis.

Multi-organ fibrosis in systemic sclerosis (SSc) accounts for substantial mortality and lacks effective therapies. Lying at the crossroad of transforming growth factor-β (TGF-β) and toll-like receptor (TLR) signaling, TGF-β-activated kinase 1 (TAK1) might have a pathogenic role in SSc. We therefore sought to evaluate the TAK1 signaling axis in patients with SSc, and investigate pharmacological TAK1 blockade using a novel drug-like selective TAK1 inhibitor, HS-276. Inhibiting TAK1 abrogated TGF-β1 stimulation of collagen synthesis and myofibroblasts differentiation in healthy skin fibroblasts, and ameliorated constitutive activation of SSc skin fibroblasts. Moreover, treatment with HS-276 prevented dermal and pulmonary fibrosis and reduced the expression of profibrotic mediators in bleomycin-treated mice. Importantly, initiating HS-276 treatment even after fibrosis was already established prevented its progression in affected organs. Together, these findings implicate TAK1 in the pathogenesis of SSc, and identify targeted TAK1 inhibition using a small molecule as a potential strategy for the treatment of SSc and other fibrotic diseases.

Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis.

Activation of TLR4 by its cognate damage-associated molecular patterns (DAMPs) elicits potent profibrotic effects and myofibroblast activation in systemic sclerosis (SSc), while genetic targeting of TLR4 or its DAMPs in mice accelerates fibrosis resolution. To prevent aberrant DAMP/TLR4 activity, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. These include radioprotective 105 kDa (RP105), a transmembrane TLR4 homolog that competitively inhibits DAMP recognition of TLR4, blocking TLR4 signaling in immune cells. The role of RP105 in TLR4-dependent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that levels of both TLR4 and its adaptor protein MD2 were elevated in SSc skin and significantly correlated with each other. Expression of RP105 was negatively associated with myofibroblast differentiation in SSc. Importantly, RP105-TLR4 association was reduced, whereas TLR4-TLR4 showed strong association in fibroblasts from patients with SSc, as evidenced by PLA assays. Moreover, RP105 adaptor MD1 expression was significantly reduced in SSc skin biopsies and explanted SSc skin fibroblasts. Exogenous RP105-MD1 abrogated, while loss of RP105 exaggerated, fibrotic cellular responses. Importantly, ablation of RP105 in mice was associated with augmented TLR4 signaling and aggravated skin fibrosis in complementary disease models. Thus, we believe RP105-MD1 to be a novel cell-intrinsic negative regulator of TLR4-MD2-driven sustained fibroblast activation, representing a critical regulatory network governing the fibrotic process. Impaired RP105 function in SSc might contribute to persistence of progression of the disease.

599 Selective inhibition Of fibro-inflammatory kinase TAK1: A potential therapeutic strategy to ameliorate systemic sclerosis

Fibrosis leads to failure of the skin, lungs, and other organs in systemic sclerosis (SSc); accounts for substantial morbidity and mortality; and lacks effective therapy. Recent findings implicated TGFβ-activated kinase 1 (TAK1), triggered by TGF-β and toll-like receptor signaling, in SSc pathogenesis. The objectives were to evaluate the activation of TAK1 signaling axis in SSc patients and to evaluate the antifibrotic ability of pharmacological TAK1 blockade in organ fibrosis. HS-276, a drug-like novel small molecule inhibitor of TAK1 was screened for its anti-fibrotic potential in cell cultures using foreskin, adult and SSc skin fibroblasts, and in bleomycin induced skin and lung fibrosis model.

Cytokine-mediated induction of human xylosyltransferase-I in systemic sclerosis skin fibroblasts

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-β1 (TGF-β1) and interleukin-1β (IL-1β). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-β signaling. Upon IL-1β treatment, SScF showed an increased mRNA expression level of XT-I and TGF-β receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1β in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-β1, we showed that the pro-fibrotic effect of IL-1β may be mediated by a miRNA-21/TGF-β receptor II axis, enhancing the autocrine TGF-β signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1β-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.

Bushen Yijing Decoction (BSYJ) exerts an anti-systemic sclerosis effect via regulating MicroRNA-26a /FLI1 axis

ABSTRACT Systemic sclerosis (SSc) refers to a group of autoimmune rheumatic diseases. Bushen Yijing decoction (BSYJ) is used for treating SSc. However, its underlying mechanism remains unknown. The present study aims to investigate potential roles of Friend leukemia integration factor 1 (FLI1) and microRNA in the beneficial effects of BSYJ on SSc. Primary skin fibroblasts were isolated from healthy individuals and SSc patients through tissue-explant technique and validated by immunocytochemistry. mRNA and microRNA levels were determined by quantitative RT-PCR. Protein expression was measured by western blotting. MiR-26a mimics or inhibitor were transfected to induce miR-26a overexpression or knockdown in vitro and in vivo, respectively. Histological changes of skin tissues from SSc mouse were evaluated by H&E and Masson trichrome staining. Results showed that FLI1 expression significantly decreased in primary skin fibroblasts of SSc patients. MiR-26a was predicted to target FLI1 untranslated region. Transfection of miR-26 mimics in SSc skin fibroblasts (SFB) leads to decrease in FLI1 expression and increase in collagen I gene expression and fibronectin accumulation. On the other hand, miR-26a knockdown increased FLI1 expression and decreased collagen I and fibronectin expression in SFB. In addition, BSYJ-containing rat serum suppressed miR-26a expression, while it elevated FLI1 expression and inhibited fibronectin and collagen I accumulation in SFB. In the mouse SSc model, BSYJ-containing serum inhibited dermal fibrosis by suppressing miR-26a expression and restoring FLI1 protein levels. Overall, our study demonstrates that BSYJ decoction exerts anti-dermal fibrosis in SSc patients via suppressing miR-26a level and thus to increase FLI1 expression in fibroblasts.

Galectin-1 and Galectin-3 Expression in Lesional Skin of Patients With Systemic Sclerosis-Association With Disease Severity.

The aim of this study was to evaluate Gal-1 and Gal-3 expression in the lesional skin of patients with scleroderma regarding disease severity and organ involvement. A cross-sectional study was conducted on patients diagnosed as systemic sclerosis (SSc), after informed consent. Clinical and serological profiles were reviewed from medical records. Lesional skin biopsies were taken by losange incision from patients. Samples were analyzed by immunohistochemistry and compared with normal skin of a healthy patient. Parametric statistical analysis was done with Student t test and Pearson coefficient. Significance was established as p ≤ 0.05 with a 95% confidence interval. Biopsies of 10 patients and a healthy control (9 female, 1 male) were analyzed. The mean age was 54.5 years (18-74 years). Four of 10 patients had diffuse, 4 had limited scleroderma, 1 had overlap syndrome, and 1 had sclerodermiform graft-versus-host disease. The mean fibroblasts count per field was 13.2 in scleroderma versus 7.2 in normal skin. The mean expression of Gal-1 in scleroderma fibroblasts was 13% (0%-56%) and 47.5% for Gal-3 (6.5%-95.5%); in normal skin, the mean expression was 91% (90%-95%) for Gal-1 and 97% (89%-100%) for Gal-3. A higher Gal-3 expression in scleroderma (within its lower expression compared with normal skin) was associated with pulmonary artery hypertension (p = 0.004) and to a higher modified Rodnan's skin score (p = 0.0003). In a similar manner, anti-centromere antibodies were associated with a higher Gal-1 expression in SSc skin fibroblasts (p = 0.04). Gal-1 and Gal-3 had a lower expression in scleroderma lesional skin compared with a normal control. We found a significant correlation between a higher Gal-3 expression (within the lower ones compared with normal skin) in fibroblasts from SSc patients and severe disease (pulmonary hypertension and a higher modified Rodnan's skin score) compared with patients with lower expression of this protein. Similarly, the presence of anti-centromere antibodies was associated with a higher expression of Gal-1 within this group of patients.

Regulation of Fibroblast Apoptosis and Proliferation by MicroRNA-125b in Systemic Sclerosis.

To analyze the expression, regulation, and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc). MiR-125b expression was assessed by quantitative polymerase chain reaction (qPCR) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SSc patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR-125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase-Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real-time assay in dermal fibroblasts. Expression of miR-125b was significantly down-regulated in SSc skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35-0.69]; P < 0.001) and in SSc dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36-0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and α-smooth muscle actin (α-SMA) expression at the messenger RNA (mRNA) and protein levels. RNA-Seq identified BAK1, BMF, and BBC3 as potential targets of miR-125b. Quantitative PCR confirmed that knockdown of miR-125b up-regulated these genes (P < 0.01; n = 12). Bcl-2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level (P < 0.01; n = 10). Consequently, miR-125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR-125b overexpression decreased apoptosis. Our findings indicate that miR-125b is down-regulated in SSc skin and primary dermal fibroblasts. MiR-125b down-regulation increases apoptosis and decreases proliferation and α-SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option.

Antifibrotic effects of 2-carba cyclic phosphatidic acid (2ccPA) in systemic sclerosis: contribution to the novel treatment

BackgroundCyclic phosphatidic acid (cPA) has an inhibitory effect on the autotaxin (ATX)/lysophosphatidic acid (LPA) axis, which has been implicated to play an important role in the progression of fibrosis in systemic sclerosis (SSc). The purpose of this study is to assess the antifibrotic activity of cPA for the treatment of SSc using SSc skin fibroblasts and an animal model of bleomycin-induced skin fibrosis.MethodsWe used a chemically stable derivative of cPA (2ccPA). First, we investigated the effect of 2ccPA on extracellular matrix (ECM) expression in skin fibroblasts. Next, the effect of 2ccPA on the intracellular cAMP levels was determined to investigate the mechanisms of the antifibrotic activity of 2ccPA. Finally, we administered 2ccPA to bleomycin-induced SSc model mice to evaluate whether 2ccPA prevented the progression of skin fibrosis.Results2ccPA decreased ECM expression in SSc skin fibroblasts and TGF-β1-treated healthy skin fibroblasts without LPA stimulation. 2ccPA increased the intracellular cAMP levels in skin fibroblasts, suggesting that the antifibrotic effect of 2ccPA was the consequence of the increase in the intracellular cAMP levels. Administration of 2ccPA also ameliorated the progression of bleomycin-induced skin fibrosis in mice.ConclusionsOur data indicated that 2ccPA had inhibitory effects on the progression of skin fibrosis by abrogating ECM production from activated skin fibroblasts. These cells were repressed, at least in part, by increased intracellular cAMP levels. 2ccPA may be able to be used to treat fibrotic lesions in SSc.

Integration of microRNA and mRNA expression profiles in the skin of systemic sclerosis patients

MicroRNAs (miRNAs) play important roles in the fibrosis of systemic sclerosis (SSc). However, the underlying miRNA-mRNA regulatory network is not fully understood. A systemic investigation of the role of miRNAs would be very valuable for increasing our knowledge of the pathogenesis of SSc. Here, we combined miRNA and mRNA expression profiles and bioinformatics analyses and then performed validation experiments. we identified 21 miRNAs and 2698 mRNAs that were differentially expressed in SSc. Among these, 17 miRNAs and their 33 target mRNAs (55 miRNA-mRNA pairs) were involved in Toll-like receptor, transforming growth factor β and Wnt signalling pathways. Validation experiments revealed that miR-146b, miR-130b, miR-21, miR-31 and miR-34a levels were higher whereas miR-145 levels were lower in SSc skin tissues and fibroblasts, normal fibroblasts and endothelial cells that were stimulated with SSc serum. ACVR2B, FZD2, FZD5 and SOX2 levels were increased in SSc skin fibroblasts, normal fibroblasts and endothelial cells that were stimulated with SSc serum. We did not identify any negative correlations among these miRNA-mRNA pairs. miR-21 was specifically expressed at higher levels in SSc serum. Six miRNAs and 4 mRNAs appear to play important roles in the pathogenesis of SSc are worth investigating in future functional studies.

Sildenafil attenuates the fibrotic phenotype of skin fibroblasts in patients with systemic sclerosis

Systemic sclerosis (SSc) is a multi-organ fibrotic disease that affects the skin and various internal organs. Therapeutic strategies for tissue fibrosis have not been established; however, aberrantly activated fibroblasts in affected lesions are key targets for modulating fibrosis. Recently, increased intracellular cyclic GMP (cGMP) levels were demonstrated to improve fibrosis levels in various diseases. The purpose of this study was to assess the anti-fibrotic properties of cGMP in cultured fibroblasts from patients with SSc. The phosphodiesterase (PDE) 5 inhibitor sildenafil increased the intracellular cGMP levels in skin fibroblasts in a dose-dependent manner. Sildenafil treatment also significantly decreased the expression of several pro-fibrotic factors that were upregulated by TGF-β1 treatment in SSc skin fibroblasts. These inhibitory effects occurred via non-canonical TGF-β signaling. Our findings revealed that sildenafil might be a novel strategy to treat tissue fibrosis and vasculopathy in SSc.

MicroRNA-130b regulates scleroderma fibrosis by targeting peroxisome proliferator-activated receptor γ

Objectives. To investigate the role of microRNA-130b (miR-130b) in systemic sclerosis (SSc) skin fibrosis and its regulatory effect on peroxisome proliferator-activated receptor γ (PPARγ).Methods. miR-130b was identified from microarray analyses in our previous studies. The expression of miR-130b, PPARγ, and fibrosis-related genes were determined by real-time PCR analysis. PPARγ protein levels were detected by immunohistochemistry and Western blot. Cells were transfected with microRNA mimics/inhibitor/scramble of miR-130b using Lipofectamine. Luciferase reporter gene assays were used to identify the direct target of miR-130b. Transforming growth factor β (TGF-β) was used for stimulation.Results. The expression of miR-130b was significantly upregulated and level of PPARγ was decreased in the dermis of the SSc skin biopsy samples and fibroblasts. Similar to human SSc, the same expression patterns of miR-130b, PPARγ, and fibrosis-related genes were observed in the bleomycin-induced skin fibrosis model; TGF-β induced the expression of miR-130b and fibrosis-related genes expression, but downregulated the expression of PPARγ. Overexpression of miR-130b in normal or SSc skin fibroblasts significantly decreased, and accordingly, knockdown of miR-130b increased the levels of PPARγ and fibrosis-related genes. In the reporter gene assay, cotransfection with miR-130b mimics significantly decreased the relative luciferase activity, which suggested a direct regulation of PPARγ by miR-130b.Conclusions. These studies demonstrated that miR-130b played important profibrotic roles in SSc fibrosis, and enhanced TGF-β signaling through negative regulation of PPARγ expression. MiR-130b may be a potential therapeutic target in SSc fibrosis.

Effects of macitentan and its active metabolite on cultured human systemic sclerosis and control skin fibroblasts.

To investigate the effects of the endothelin 1 (ET-1) receptor antagonists (ETRA) macitentan, its active metabolite ACT-132577, and bosentan on myofibroblast activation and extracellular matrix production induced by ET-1 in cultured systemic sclerosis (SSc) and control skin fibroblasts. Fibroblasts were obtained from skin biopsies of 6 patients with SSc and 5 healthy subjects. Some cultured cells were untreated or treated with macitentan, ACT-132577, or bosentan alone (10 μM). Other cultured cells were treated with ET-1 alone (100 nM) or with ETRA, and after 1 h, also with ET-1. After 48 h of treatment, myofibroblast activation was investigated to evaluate the α-smooth muscle actin (α-SMA) expression by immunofluorescence; type I collagen (COL-1) and fibronectin (FN) were investigated by immunocytochemistry, Western blotting, and quantitative real-time PCR (qRT-PCR). Statistical analysis was performed by the nonparametric Mann-Whitney U test. In cultured SSc skin fibroblasts, only the treatment with macitentan significantly reduced the basal level of α-SMA expression (p = 0.03 vs untreated cells). Macitentan also significantly reduced the basal level of COL-1 synthesis, similarly to bosentan (p < 0.05 vs untreated cells). Macitentan or ACT-132577 antagonized the ability of ET-1 to further induce α-SMA expression (p = 0.03), COL-1, and FN synthesis (p = 0.03, p = 0.005); bosentan showed similar effects. These results obtained by immunofluorescence and immunocytochemistry were confirmed by Western blotting and qRT-PCR. The downregulatory effects exerted by ETRA were observed also in cultured human control skin fibroblasts. Macitentan and ACT-132577 seem to downregulate in vitro the profibrotic myofibroblast phenotype induced by ET-1 in cultured human SSc skin fibroblasts.

Altered MCM Protein Levels and Autophagic Flux in Aged and Systemic Sclerosis Dermal Fibroblasts

Aging is a common risk factor of many disorders. With age, the level of insoluble extracellular matrix increases leading to increased stiffness of a number of tissues. Matrix accumulation can also be observed in fibrotic disorders, such as systemic sclerosis (SSc). Although the intrinsic aging process in skin is phenotypically distinct from SSc, here we demonstrate similar behavior of aged and SSc skin fibroblasts in culture. We have used quantitative proteomics to characterize the phenotype of dermal fibroblasts from healthy subjects of various ages and from patients with SSc. Our results demonstrate that proteins involved in DNA and RNA processing decrease with age and in SSc, while those involved in mitochondrial and other metabolic processes behave the opposite. Specifically, mini-chromosome maintenance (MCM) helicase proteins are less abundant with age and SSc, and they exhibit an altered subcellular distribution. We observed that lower levels of MCM7 correlate with reduced cell proliferation, lower autophagic capacity and higher intracellular protein expression phenotypes of aged and SSc cells. Additionally, we show that SSc fibroblasts exhibit higher levels of senescence than their healthy counterparts, suggesting further similarities between the fibrotic disorder and the aging process. Hence, at the molecular level, SSc fibroblasts exhibit intrinsic characteristics of fibroblasts from aged skin.

Vitamin D receptor regulates TGF-β signalling in systemic sclerosis

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. Its ligand, 1,25-(OH)2D, is a metabolically active hormone derived from vitamin D3. The levels of vitamin D3 are decreased in patients with systemic sclerosis (SSc). Here, we aimed to analyse the role of VDR signalling in fibrosis. VDR expression was analysed in SSc skin, experimental fibrosis and human fibroblasts. VDR signalling was modulated by siRNA and with the selective agonist paricalcitol. The effects of VDR on Smad signalling were analysed by reporter assays, target gene analyses and coimmunoprecipitation. The effects of paricalcitol were evaluated in the models of bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active transforming growth factor-β (TGF-β) receptor I (TBRI(CA)). VDR expression was decreased in fibroblasts of SSc patients and murine models of SSc in a TGF-β-dependent manner. Knockdown of VDR enhanced the sensitivity of fibroblasts towards TGF-β. In contrast, activation of VDR by paricalcitol reduced the stimulatory effects of TGF-β on fibroblasts and inhibited collagen release and myofibroblast differentiation. Paricalcitol stimulated the formation of complexes between VDR and phosphorylated Smad3 in fibroblasts to inhibit Smad-dependent transcription. Preventive and therapeutic treatment with paricalcitol exerted potent antifibrotic effects and ameliorated bleomycin- as well as TBRI(CA)-induced fibrosis. We characterise VDR as a negative regulator of TGF-β/Smad signalling. Impaired VDR signalling with reduced expression of VDR and decreased levels of its ligand may thus contribute to hyperactive TGF-β signalling and aberrant fibroblast activation in SSc.

Proinflammatory effects of diesel exhaust nanoparticles on scleroderma skin cells.

Autoimmune diseases are complex disorders of unknown etiology thought to result from interactions between genetic and environmental factors. We aimed to verify whether environmental pollution from diesel engine exhaust nanoparticulate (DEP) of actually operating vehicles could play a role in the development of a rare immune-mediated disease, systemic sclerosis (SSc), in which the pathogenetic role of environment has been highlighted. The effects of carbon-based nanoparticulate collected at the exhaust of newer (Euro 5) and older (Euro 4) diesel engines on SSc skin keratinocytes and fibroblasts were evaluated in vitro by assessing the mRNA expression of inflammatory cytokines (IL-1α, IL-6, IL-8, and TNF-α) and fibroblast chemical mediators (metalloproteases 2, 3, 7, 9, and 12; collagen types I and III; VEGF). DEP was shown to stimulate cytokine gene expression at a higher extent in SSc keratinocytes versus normal cells. Moreover, the mRNA gene expression of all MMPs, collagen types, and VEGF genes was significantly higher in untreated SSc fibroblasts versus controls. Euro 5 particle exposure increased the mRNA expression of MMP-2, -7, and -9 in SSc fibroblasts in a dose dependent manner and only at the highest concentration in normal cells. We suggest that environmental DEP could trigger the development of SSc acting on genetically hyperreactive cell systems.

Endothelin-1 mediates downstream profibrotic effects by transforming growth factor-beta 1 in systemic sclerosis skin fibroblasts

Endothelin-1 mediates downstream profibrotic effects by transforming growth factor-beta 1 in systemic sclerosis skin fibroblasts