Platelet-derived serotonin links vascular disease and tissue fibrosis

Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage

Sirt1 regulates canonical TGF-β signalling to control fibroblast activation and tissue fibrosis

BACKGROUND Sirt1 is a member of the sirtuin family of proteins. Sirt1 is a class III histone deacetylase with important regulatory roles in transcription, cellular differentiation, proliferation and metabolism. As aberrant epigenetic modifications have been linked to the pathogenesis of systemic sclerosis (SSc), we aimed to investigate the role of Sirt1 in fibroblast activation. METHODS Sirt1 expression was analysed by real-time PCR, western blot and immunohistochemistry. Sirt1 signalling was modulated with the Sirt1 agonist resveratrol and by fibroblast-specific knockout. The role of Sirt1 was evaluated in bleomycin-induced skin fibrosis and in mice overexpressing a constitutively active transforming growth factor-β (TGF-β) receptor I (TBRIact). RESULTS The expression of Sirt1 was decreased in patients with SSc and in experimental fibrosis in a TGF-β-dependent manner. Activation of Sirt1 potentiated the profibrotic effects of TGF-β with increased Smad reporter activity, elevated transcription of TGF-β target genes and enhanced release of collagen. In contrast, knockdown of Sirt1 inhibited TGF-β/SMAD signalling and reduced release of collagen in fibroblasts. Consistently, mice with fibroblast-specific knockdown of Sirt1 were less susceptible to bleomycin- or TBRIact-induced fibrosis. CONCLUSIONS We identified Sirt1 as a crucial regulator of TGF-β/Smad signalling in SSc. Although Sirt1 is downregulated, this decrease is not sufficient to counterbalance the excessive activation of TGF-β signalling in SSc. However, augmentation of this endogenous regulatory mechanism, for example, by knockdown of Sirt1, can effectively inhibit TGF-β signalling and exerts potent antifibrotic effects. Sirt1 may thus be a key regulator of fibroblast activation in SSc

Inactivation of fatty acid amide hydrolase exacerbates experimental fibrosis by enhanced endocannabinoid-mediated activation of CB1

BACKGROUND: Selective targeting of the cannabinoid receptors CB1 and CB2 by synthetic compounds has revealed opposing roles of both receptors in fibrosis. OBJECTIVES: To characterise the role of endogenous cannabinoids (endocannabinoids) and their predominant receptor in fibrosis. METHODS: The levels of endocannabinoids in mice were modulated by pharmacological or genetic inactivation of the enzyme fatty acid amide hydrolase (FAAH). The predominant receptor for endocannabinoids was determined by selective inhibition of either CB1 or CB2. The extent of fibrosis upon challenge with bleomycin was determined by quantification of dermal thickness, hydroxyproline content and myofibroblast counts. RESULTS: The expression of FAAH is decreased in systemic sclerosis fibroblasts. FAAH-deficient mice with strongly increased levels of endocannabinoids were more sensitive to bleomycin. Consistently, pharmacological inhibition of FAAH significantly exacerbated bleomycin-induced fibrosis. Inhibition of CB1 completely abrogated the profibrotic effects of FAAH inactivation. In contrast, inhibition of CB2 only modestly enhanced fibrosis, indicating that CB1 is the predominant receptor for endocannabinoids in experimental fibrosis. CONCLUSIONS: Increased levels of endocannabinoids induced by inactivation of FAAH worsen experimental fibrosis via activation of CB1. These findings highlight the profibrotic effects of endocannabinoids and suggest that CB1 maybe a more promising candidate for targeted treatments in fibrotic diseases than CB2

Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis

BACKGROUND Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc). METHODS ATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation. RESULTS Transforming growth factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3. CONCLUSIONS We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc

Tribbles homologue 3 stimulates canonical TGF-β signalling to regulate fibroblast activation and tissue fibrosis

OBJECTIVES Tribbles homologue 3 (TRB3) is a pseudokinase that modifies the activation of various intracellular signalling pathways to control fundamental processes extending from mitosis and cell activation to apoptosis and modulation of gene expression. Here, we aimed to analyse the role of TRB3 in fibroblast activation in systemic sclerosis (SSc). METHODS The expression of TRB3 was quantified by quantitative PCR, western blot and immunohistochemistry. The role of TRB3 was analysed in cultured fibroblasts and in experimental fibrosis using small interfering RNA (siRNA)-mediated knockdown and overexpression of TRB3. RESULTS TRB3 expression was increased in fibroblasts of patients with SSc and in murine models of SSc in a transforming growth factor-β (TGF-β)/Smad-dependent manner. Overexpression of TRB3 stimulated canonical TGF-β signalling and induced an activated phenotype in resting fibroblasts. In contrast, knockdown of TRB3 reduced the profibrotic effects of TGF-β and decreased the collagen synthesis. Moreover, siRNA-mediated knockdown of TRB3 exerted potent antifibrotic effects and ameliorated bleomycin as well as constitutively active TGF-β receptor I-induced fibrosis with reduced dermal thickening, decreased hydroxyproline content and impaired myofibroblast differentiation. CONCLUSIONS The present study characterises TRB3 as a novel profibrotic mediator in SSc. TGF-β induces TRB3, which in turn activates canonical TGF-β/Smad signalling and stimulates the release of collagen, thereby inducing a positive feedback loop that may contribute to aberrant TGF-β signalling in SSc

Vitamin D receptor regulates TGF-β signalling in systemic sclerosis

BACKGROUND 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. METHODS 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)). RESULTS 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. CONCLUSIONS 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

The nuclear receptor constitutive androstane receptor/NR1I3 enhances the profibrotic effects of transforming growth factor β and contributes to the development of experimental dermal fibrosis

OBJECTIVE Nuclear receptors regulate cell growth, differentiation, and homeostasis. Selective nuclear receptors promote fibroblast activation, which leads to tissue fibrosis, the hallmark of systemic sclerosis (SSc). This study was undertaken to investigate the effects of constitutive androstane receptor (CAR)/NR1I3, an orphan nuclear receptor, on fibroblast activation and experimental dermal fibrosis. METHODS CAR expression was quantified by quantitative polymerase chain reaction, Western blotting, immunohistochemistry, and immunofluorescence. CAR expression was modulated by small molecules, small interfering RNA, forced overexpression, and site-directed mutagenesis. The effects of CAR activation were analyzed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor type I (TβRI-CA). RESULTS Up-regulation of CAR was detected in the skin and in dermal fibroblasts in SSc patients. Stimulation of healthy fibroblasts with TGFβ induced the expression of CAR messenger RNA and protein in a Smad-dependent manner. Pharmacologic activation or overexpression of CAR in healthy fibroblasts significantly increased the stimulatory effects of TGFβ on collagen synthesis and myofibroblast differentiation, and amplified the stimulatory effects of TGFβ on COL1A2 transcription activity. Treatment with CAR agonist increased the activation of canonical TGFβ signaling in murine models of SSc and exacerbated bleomycin-induced and TβRI-CA-induced fibrosis with increased dermal thickening, myofibroblast counts, and collagen accumulation. CONCLUSION Our findings indicate that CAR is up-regulated in SSc and regulates TGFβ signaling. Activation of CAR increases the profibrotic effects of TGFβ in cultured fibroblasts and in different preclinical models of SSc. Thus, inactivation of CAR might be a novel approach to target aberrant TGFβ signaling in SSc and in other fibrotic diseases

Inhibition of hedgehog signalling prevents experimental fibrosis and induces regression of established fibrosis

OBJECTIVES: Tissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach. METHODS: The activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice. RESULTS: Hedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis. CONCLUSIONS: Inhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials

Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis

OBJECTIVES: Epigenetic modifications such as DNA methylation and histone acetylation have been implicated in the pathogenesis of systemic sclerosis. However, histone methylation has not been investigated so far. We therefore aimed to evaluate the role of the trimethylation of histone H3 on lysine 27 (H3K27me3) on fibroblast activation and fibrosis. METHODS: H3K27me3 was inhibited by 3-deazaneplanocin A (DZNep) in cultured fibroblasts and in two murine models of dermal fibrosis. Fibrosis was analysed by assessment of the dermal thickening, determination of the hydroxyproline content and by quantification of the numbers of myofibroblasts. The expression of fos-related antigen 2 (fra-2) was assessed by real-time PCR, western blot and immunohistochemistry and modulated by siRNA. RESULTS: Inhibition of H3K27me3 stimulated the release of collagen in cultured fibroblasts in a time and dose-dependent manner. Treatment with DZNep exacerbated fibrosis induced by bleomycin or by overexpression of a constitutively active transforming growth factor β receptor type I. Moreover, treatment with DZNep alone was sufficient to induce fibrosis. Inhibition of H3K27me3 induced the expression of the profibrotic transcription factor fra-2 in vitro and in vivo. Knockdown of fra-2 completely prevented the profibrotic effects of DZNep. CONCLUSIONS: These data demonstrate a novel role of H3 Lys27 histone methylation in fibrosis. In contrast to other epigenetic modifications such as DNA methylation and histone acetylation, H3 Lys27 histone methylation acts as a negative regulator of fibroblast activation in vitro and in vivo by repressing the expression of fra-2

Interleukin-35 is upregulated in systemic sclerosis and its serum levels are associated with early disease

Objectives. IL-35 is a member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. IL-35 exerts immunomodulatory activities in experimental and human autoimmune inflammatory conditions. Our aim was to assess IL-35 expression in the skin and circulation of SSc patients and to characterize its potential association with SSc-related features. Methods. Expression of IL-35 in skin and dermal fibroblasts was quantified by quantitative PCR, immunohistochemistry and immunofluorescence. Serum levels of IL-35 (by ELISA), CRP (by turbidimetry), ANA (by immunofluorescence) and autoantibodies of the ENA complex (by immunoblot) were measured in 40 SSc patients. Serum IL-35 was determined in 40 age- and sex-matched healthy controls. Results. IL-35 expression was increased in SSc skin and dermal fibroblasts in a TGF-β-dependent manner. IL-35 induced an activated phenotype in resting fibroblasts and enhanced the release of collagen. IL-35 serum levels were increased in patients with SSc compared with healthy controls [median 83.9 (interquartile range 45.1–146.1) vs 36.2 (interquartile range 17.2–49.4) pg/ml, P < 0.0001]. Serum IL-35 was negatively correlated with disease duration (r = −0.4339, P = 0.0052). In line with this finding, serum IL-35 was increased in patients with an early SSc pattern on capillaroscopy assessment compared with those with active and late SSc patterns. Conclusion. The present study demonstrates overexpression of IL-35 in SSc skin, dermal fibroblasts and serum. TGF-β induces IL-35, which in turn activates resting fibroblasts and enhances the release of collagen, thereby contributing to aberrant TGF-β signalling in SSc. Increased serum IL-35 is associated with early, inflammatory stages of SSc