The Experts below are selected from a list of 22134 Experts worldwide ranked by ideXlab platform
Clara Dees - One of the best experts on this subject based on the ideXlab platform.
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tgf β induced epigenetic deregulation of socs3 facilitates stat3 signaling to promote Fibrosis
Journal of Clinical Investigation, 2020Co-Authors: Clara Dees, Andreas Ramming, Yun Zhang, Christina Bergmann, Sebastian Potter, Xiang Zhou, Markus Luber, Thomas Wohlfahrt, Emmanuel Karouzakis, Kolja GelseAbstract:Fibroblasts are key effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation may be initiated by external factors, prolonged activation can induce an "autonomous," self-maintaining profibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role in establishing this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, TGF-β induced the expression of DNA methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of STAT3 to promote fibroblast-to-myofibroblast transition, collagen release, and Fibrosis in vitro and in vivo. Reestablishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGF-β-dependent fibroblast activation, and ameliorated Experimental Fibrosis in murine models. These findings identify a pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of targeted therapies in fibrotic diseases.
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the histone demethylase jumonji domain containing protein 3 jmjd3 regulates fibroblast activation in systemic sclerosis
Annals of the Rheumatic Diseases, 2018Co-Authors: Christina Bergmann, Clara Dees, Yun Zhang, Sebastian Potter, Thomas Wohlfahrt, A Brandt, B Merlevede, L Hallenberger, Chihwei Chen, Tatiana MallanoAbstract:Objectives Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of Fibrosis. Methods JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced Fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. Results The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in Experimental Fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced Fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. Conclusion We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.
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stimulation of the soluble guanylate cyclase sgc inhibits Fibrosis by blocking non canonical tgfβ signalling
Annals of the Rheumatic Diseases, 2015Co-Authors: Christian Beyer, Alfiya Distler, Jingang Huang, Clara Dees, Pawel Zerr, Katrin Palumbozerr, Christoph Zenzmaier, Rossella Mancuso, Christiane Maier, Milena PachowskyAbstract:OBJECTIVES We have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor β (TGFβ) signalling that mediates the antifibrotic effects of the sGC. METHODS Human fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFβ. sGC knockout fibroblasts were isolated from sGCI(fl/fl) mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-β1 receptor. RESULTS sGC stimulation inhibited TGFβ-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFβ-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFβ target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFβ-driven dermal Fibrosis but did not change p-SMAD2 and 3 levels and TGFβ target gene expression, confirming that non-canonical TGFβ pathways mediate the antifibrotic sGC activity. CONCLUSIONS We elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFβ signalling and inhibits Experimental Fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of Fibrosis and vascular disease in systemic sclerosis.
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Stimulators of soluble guanylate cyclase (sGC) inhibit Experimental skin Fibrosis of different aetiologies
Annals of the rheumatic diseases, 2015Co-Authors: Clara Dees, Alfiya Distler, Katrin Palumbo-zerr, Christian Beyer, Oliver Distler, Georg Schett, Alina Soare, Yun Zhang, Peter Sandner, Jörg H W DistlerAbstract:Objectives Stimulators of the soluble guanylate cyclase (sGC) have recently been shown to inhibit transforming growth factor-β signalling. Here, we aimed to demonstrate that riociguat, the drug candidate for clinical trials in systemic sclerosis (SSc), is effective in Experimental Fibrosis and to compare its efficacy to that of phosphodiesterase V inhibitors that also increase the intracellular levels of cyclic guanosine monophosphate. Methods The antifibrotic effects of riociguat and sildenafil were compared in the tight-skin 1 model, in bleomycin-induced Fibrosis and in a model of sclerodermatous chronic graft-versus-host-disease (cGvHD). Doses of 0.1–3 mg/kg twice a day for riociguat and of 3–10 mg/kg twice a day for sildenafil were used. Result Riociguat dose-dependently reduced skin thickening, myofibroblast differentiation and accumulation of collagen with potent antifibrotic effects at 1 and 3 mg/kg. Riociguat also ameliorated Fibrosis of the gastrointestinal tract in the cGvHD model. The antifibrotic effects were associated with reduced phosphorylation of extracellular signal-regulated kinases. Sildenafil at doses of 3 and 10 mg/kg exerted mild antifibrotic effects that were significantly less pronounced compared with 1 and 3 mg/kg riociguat. Conclusions These data demonstrated potent antifibrotic effects of riociguat on Experimental skin and organ Fibrosis. These findings suggest a role for riociguat for the treatment of fibrotic diseases, especially for the treatment of SSc. A phase II study with riociguat in patients with SSc is currently starting.
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Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis
Annals of the rheumatic diseases, 2015Co-Authors: Tatjana Mallano, Jingang Huang, Clara Dees, Katrin Palumbo-zerr, Christian Beyer, Pawel Zerr, Andreas Ramming, Barbara Zeller, Oliver DistlerAbstract: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.
Christian Beyer - One of the best experts on this subject based on the ideXlab platform.
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stimulation of the soluble guanylate cyclase sgc inhibits Fibrosis by blocking non canonical tgfβ signalling
Annals of the Rheumatic Diseases, 2015Co-Authors: Christian Beyer, Alfiya Distler, Jingang Huang, Clara Dees, Pawel Zerr, Katrin Palumbozerr, Christoph Zenzmaier, Rossella Mancuso, Christiane Maier, Milena PachowskyAbstract:OBJECTIVES We have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor β (TGFβ) signalling that mediates the antifibrotic effects of the sGC. METHODS Human fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFβ. sGC knockout fibroblasts were isolated from sGCI(fl/fl) mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-β1 receptor. RESULTS sGC stimulation inhibited TGFβ-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFβ-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFβ target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFβ-driven dermal Fibrosis but did not change p-SMAD2 and 3 levels and TGFβ target gene expression, confirming that non-canonical TGFβ pathways mediate the antifibrotic sGC activity. CONCLUSIONS We elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFβ signalling and inhibits Experimental Fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of Fibrosis and vascular disease in systemic sclerosis.
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Stimulators of soluble guanylate cyclase (sGC) inhibit Experimental skin Fibrosis of different aetiologies
Annals of the rheumatic diseases, 2015Co-Authors: Clara Dees, Alfiya Distler, Katrin Palumbo-zerr, Christian Beyer, Oliver Distler, Georg Schett, Alina Soare, Yun Zhang, Peter Sandner, Jörg H W DistlerAbstract:Objectives Stimulators of the soluble guanylate cyclase (sGC) have recently been shown to inhibit transforming growth factor-β signalling. Here, we aimed to demonstrate that riociguat, the drug candidate for clinical trials in systemic sclerosis (SSc), is effective in Experimental Fibrosis and to compare its efficacy to that of phosphodiesterase V inhibitors that also increase the intracellular levels of cyclic guanosine monophosphate. Methods The antifibrotic effects of riociguat and sildenafil were compared in the tight-skin 1 model, in bleomycin-induced Fibrosis and in a model of sclerodermatous chronic graft-versus-host-disease (cGvHD). Doses of 0.1–3 mg/kg twice a day for riociguat and of 3–10 mg/kg twice a day for sildenafil were used. Result Riociguat dose-dependently reduced skin thickening, myofibroblast differentiation and accumulation of collagen with potent antifibrotic effects at 1 and 3 mg/kg. Riociguat also ameliorated Fibrosis of the gastrointestinal tract in the cGvHD model. The antifibrotic effects were associated with reduced phosphorylation of extracellular signal-regulated kinases. Sildenafil at doses of 3 and 10 mg/kg exerted mild antifibrotic effects that were significantly less pronounced compared with 1 and 3 mg/kg riociguat. Conclusions These data demonstrated potent antifibrotic effects of riociguat on Experimental skin and organ Fibrosis. These findings suggest a role for riociguat for the treatment of fibrotic diseases, especially for the treatment of SSc. A phase II study with riociguat in patients with SSc is currently starting.
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Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis
Annals of the rheumatic diseases, 2015Co-Authors: Tatjana Mallano, Jingang Huang, Clara Dees, Katrin Palumbo-zerr, Christian Beyer, Pawel Zerr, Andreas Ramming, Barbara Zeller, Oliver DistlerAbstract: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.
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the wnt antagonists dkk1 and sfrp1 are downregulated by promoter hypermethylation in systemic sclerosis
Annals of the Rheumatic Diseases, 2014Co-Authors: Clara Dees, Alfiya Distler, Neng-yu Lin, Christian Beyer, Oliver Distler, Pawel Zerr, Inga Schlottmann, Robin Funke, Katrin Palumbozerr, Georg SchettAbstract:Objectives Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists. Methods The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2′-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin Fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for β-catenin. Results The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated Experimental Fibrosis. Conclusions We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.
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op0016 tribbles homolog 3 mediates the stimulatory effects of tgf beta on fibroblast activation and dermal Fibrosis in systemic sclerosis
Annals of the Rheumatic Diseases, 2013Co-Authors: Alfiya Distler, Clara Dees, Pawel Zerr, Angelika Horn, Michal Tomcik, Jerome Avouac, Katrin Palumbozerr, A Khodzighorova, Christian BeyerAbstract:Background Tribbles Homolog 3 (TRB3), is a member of the family of pseudokinases called Tribbles which regulate activation of various intracellular signaling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. Objectives To investigate the role of TRB3 in the pathologic activation of fibroblasts in systemic sclerosis (SSc). Methods Activation of TRB3 was determined by real-time PCR, immunohistochemistry and immunofluorescence. Collagen synthesis was quantified by real-time PCR and SirCol collagen assay. A plasmid construct overexpressing TRB3 was designed. TRB3 expression was inhibited in cultured fibroblasts and murine models of Fibrosis via siRNA. The mouse models of dermal Fibrosis induced by bleomycin and attenuated adenovirus overexpressing a constitutively active TGF-β receptor I were used. Results Increased expression of TRB3 was detected in the upper layer of the dermis of SSc patients and colocalized with prolyl-4-hydroxylase, αSMA and phosphorylated Smad3 expression. The overexpression of TRB3 persisted in cultured SSc fibroblasts (4.1±0.4-fold increase, p Conclusions This is the first study on the key role of TRB3 in fibroblast activation and tissue Fibrosis in SSc. TRB3 is upregulated in SSc and in Experimental Fibrosis in a TGF-β dependent manner. TRB3 is essential for the pro-fibrotic effects of TGF-β and inhibition of TRB3 completely prevents the stimulatory effect of TGF-β. In addition, knockdown of TRB3 protected from Experimental dermal Fibrosis in different mouse models. Considering the potent anti-fibrotic effects in this study, TRB3 might be a promising candidate for molecular targeted therapies of SSc. Acknowledgements This study was performed with support of CMH Research Projects No 00000023728. Disclosure of Interest None Declared
Pawel Zerr - One of the best experts on this subject based on the ideXlab platform.
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stimulation of the soluble guanylate cyclase sgc inhibits Fibrosis by blocking non canonical tgfβ signalling
Annals of the Rheumatic Diseases, 2015Co-Authors: Christian Beyer, Alfiya Distler, Jingang Huang, Clara Dees, Pawel Zerr, Katrin Palumbozerr, Christoph Zenzmaier, Rossella Mancuso, Christiane Maier, Milena PachowskyAbstract:OBJECTIVES We have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor β (TGFβ) signalling that mediates the antifibrotic effects of the sGC. METHODS Human fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFβ. sGC knockout fibroblasts were isolated from sGCI(fl/fl) mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-β1 receptor. RESULTS sGC stimulation inhibited TGFβ-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFβ-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFβ target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFβ-driven dermal Fibrosis but did not change p-SMAD2 and 3 levels and TGFβ target gene expression, confirming that non-canonical TGFβ pathways mediate the antifibrotic sGC activity. CONCLUSIONS We elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFβ signalling and inhibits Experimental Fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of Fibrosis and vascular disease in systemic sclerosis.
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Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis
Annals of the rheumatic diseases, 2015Co-Authors: Tatjana Mallano, Jingang Huang, Clara Dees, Katrin Palumbo-zerr, Christian Beyer, Pawel Zerr, Andreas Ramming, Barbara Zeller, Oliver DistlerAbstract: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.
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the wnt antagonists dkk1 and sfrp1 are downregulated by promoter hypermethylation in systemic sclerosis
Annals of the Rheumatic Diseases, 2014Co-Authors: Clara Dees, Alfiya Distler, Neng-yu Lin, Christian Beyer, Oliver Distler, Pawel Zerr, Inga Schlottmann, Robin Funke, Katrin Palumbozerr, Georg SchettAbstract:Objectives Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists. Methods The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2′-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin Fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for β-catenin. Results The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated Experimental Fibrosis. Conclusions We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.
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op0267 sirt1 regulates canonical tgf beta signaling to control fibroblast activation and tissue Fibrosis
Annals of the Rheumatic Diseases, 2014Co-Authors: Pawel Zerr, Alfiya Distler, Jingang Huang, Oliver Distler, Georg Schett, Katrin Palumbozerr, J DistlerAbstract:Background SIRT1 is a member of the sirtuin family (SIRT1-7) of proteins, homologs of the Sir2 in yeast. Sirtuins are characterized by a sirtuin core domain and grouped into four classes. SIRT1 is a class III histone deacetylase with important regulatory roles in transcription, cellular differentiation, proliferation and metabolism. Objectives Considering that various epigenetic modifications have recently been linked to the pathogenesis of SSc, we aimed to investigate the role of SIRT1 in fibroblast activation and tissue Fibrosis in SSc. Methods SIRT1 expression was analyzed by real-time PCR, Western Blot and immunohistochemistry in cultured human fibroblasts, in murine models of SSc and in skin samples from SSc patients. Collagen synthesis was quantified by real-time PCR, SirCol- and hydroxyproline assays. The effects of SIRT1 on canonical TGF-β signaling were evaluated by Smad reporter assays and analyses of TGF-β target genes. SIRT1 signaling was modulated by SIRT1 agonist resveratrol and by fibroblast-specific knockout. The role of SIRT1 was evaluated in bleomycin-induced skin Fibrosis in mice overexpressing a constitutively active TGF-β receptor I (TBR). Results SIRT1 expression was decreased in the skin of SSc patients. IHC demonstrated a pronounced decrease of SIRT1 in fibroblasts in SSc skin. SIRT1 mRNA and protein levels were also decreased in bleomycin or TBR-induced skin Fibrosis. TGF-β reduced mRNA and protein levels of SIRT1 in cultured fibroblasts. TBR overexpression reduced SIRT1 in murine skin, whereas treatment with the TBR inhibitor SD-208 prevented the downregulation of SIRT1 in Experimental Fibrosis. Activation of SIRT1 by treatment with resveratrol potentiated the stimulatory effects of TGF-β on cultured fibroblasts with more pronounced increases of Col1a1 mRNA and collagen protein. These effects were mediated by enhanced canonical TGF-β signaling as resveratrol induced SMAD-dependent transcription in reporter assays and increased the mRNA levels of CTGF of classical SMAD target genes. In contrast, knockdown of SIRT1 inhibited TGF-β/SMAD signaling and reduced release of collagen in fibroblasts. Consistent with a pro-fibrotic role of SIRT1, mice treated with resveratrol were more sensitive to bleomycin-induced dermal Fibrosis with more pronounced dermal thickening (74%, p=0.006), increased myofibroblast counts and higher hydroxyproline levels. Mice with fibroblast-specific SIRT1 knockdown were less susceptible to bleomycin induced Fibrosis. SIRT1 Inactivation also protected from Fibrosis induced by overexpression of TBR. Conclusions We identify SIRT1 as a crucial regulator of TGF-β/Smad signaling in SSc. Although SIRT1 is downregulated, this decrease is not sufficient to counter-balance the excessive activation of TGF-β signaling in SSc. However, augmentation of this endogenous regulatory mechanism, e.g. by knockdown of SIRT1 can effectively inhibit TGF-β signaling and exerts potent anti-fibrotic effects. Although further studies are required, these findings provide first evidence that SIRT1 may be an interesting target for pharmacological intervention. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2212
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op0016 tribbles homolog 3 mediates the stimulatory effects of tgf beta on fibroblast activation and dermal Fibrosis in systemic sclerosis
Annals of the Rheumatic Diseases, 2013Co-Authors: Alfiya Distler, Clara Dees, Pawel Zerr, Angelika Horn, Michal Tomcik, Jerome Avouac, Katrin Palumbozerr, A Khodzighorova, Christian BeyerAbstract:Background Tribbles Homolog 3 (TRB3), is a member of the family of pseudokinases called Tribbles which regulate activation of various intracellular signaling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. Objectives To investigate the role of TRB3 in the pathologic activation of fibroblasts in systemic sclerosis (SSc). Methods Activation of TRB3 was determined by real-time PCR, immunohistochemistry and immunofluorescence. Collagen synthesis was quantified by real-time PCR and SirCol collagen assay. A plasmid construct overexpressing TRB3 was designed. TRB3 expression was inhibited in cultured fibroblasts and murine models of Fibrosis via siRNA. The mouse models of dermal Fibrosis induced by bleomycin and attenuated adenovirus overexpressing a constitutively active TGF-β receptor I were used. Results Increased expression of TRB3 was detected in the upper layer of the dermis of SSc patients and colocalized with prolyl-4-hydroxylase, αSMA and phosphorylated Smad3 expression. The overexpression of TRB3 persisted in cultured SSc fibroblasts (4.1±0.4-fold increase, p Conclusions This is the first study on the key role of TRB3 in fibroblast activation and tissue Fibrosis in SSc. TRB3 is upregulated in SSc and in Experimental Fibrosis in a TGF-β dependent manner. TRB3 is essential for the pro-fibrotic effects of TGF-β and inhibition of TRB3 completely prevents the stimulatory effect of TGF-β. In addition, knockdown of TRB3 protected from Experimental dermal Fibrosis in different mouse models. Considering the potent anti-fibrotic effects in this study, TRB3 might be a promising candidate for molecular targeted therapies of SSc. Acknowledgements This study was performed with support of CMH Research Projects No 00000023728. Disclosure of Interest None Declared
Oliver Distler - One of the best experts on this subject based on the ideXlab platform.
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Stimulators of soluble guanylate cyclase (sGC) inhibit Experimental skin Fibrosis of different aetiologies
Annals of the rheumatic diseases, 2015Co-Authors: Clara Dees, Alfiya Distler, Katrin Palumbo-zerr, Christian Beyer, Oliver Distler, Georg Schett, Alina Soare, Yun Zhang, Peter Sandner, Jörg H W DistlerAbstract:Objectives Stimulators of the soluble guanylate cyclase (sGC) have recently been shown to inhibit transforming growth factor-β signalling. Here, we aimed to demonstrate that riociguat, the drug candidate for clinical trials in systemic sclerosis (SSc), is effective in Experimental Fibrosis and to compare its efficacy to that of phosphodiesterase V inhibitors that also increase the intracellular levels of cyclic guanosine monophosphate. Methods The antifibrotic effects of riociguat and sildenafil were compared in the tight-skin 1 model, in bleomycin-induced Fibrosis and in a model of sclerodermatous chronic graft-versus-host-disease (cGvHD). Doses of 0.1–3 mg/kg twice a day for riociguat and of 3–10 mg/kg twice a day for sildenafil were used. Result Riociguat dose-dependently reduced skin thickening, myofibroblast differentiation and accumulation of collagen with potent antifibrotic effects at 1 and 3 mg/kg. Riociguat also ameliorated Fibrosis of the gastrointestinal tract in the cGvHD model. The antifibrotic effects were associated with reduced phosphorylation of extracellular signal-regulated kinases. Sildenafil at doses of 3 and 10 mg/kg exerted mild antifibrotic effects that were significantly less pronounced compared with 1 and 3 mg/kg riociguat. Conclusions These data demonstrated potent antifibrotic effects of riociguat on Experimental skin and organ Fibrosis. These findings suggest a role for riociguat for the treatment of fibrotic diseases, especially for the treatment of SSc. A phase II study with riociguat in patients with SSc is currently starting.
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Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis
Annals of the rheumatic diseases, 2015Co-Authors: Tatjana Mallano, Jingang Huang, Clara Dees, Katrin Palumbo-zerr, Christian Beyer, Pawel Zerr, Andreas Ramming, Barbara Zeller, Oliver DistlerAbstract: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.
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the wnt antagonists dkk1 and sfrp1 are downregulated by promoter hypermethylation in systemic sclerosis
Annals of the Rheumatic Diseases, 2014Co-Authors: Clara Dees, Alfiya Distler, Neng-yu Lin, Christian Beyer, Oliver Distler, Pawel Zerr, Inga Schlottmann, Robin Funke, Katrin Palumbozerr, Georg SchettAbstract:Objectives Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists. Methods The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2′-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin Fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for β-catenin. Results The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated Experimental Fibrosis. Conclusions We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.
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op0267 sirt1 regulates canonical tgf beta signaling to control fibroblast activation and tissue Fibrosis
Annals of the Rheumatic Diseases, 2014Co-Authors: Pawel Zerr, Alfiya Distler, Jingang Huang, Oliver Distler, Georg Schett, Katrin Palumbozerr, J DistlerAbstract:Background SIRT1 is a member of the sirtuin family (SIRT1-7) of proteins, homologs of the Sir2 in yeast. Sirtuins are characterized by a sirtuin core domain and grouped into four classes. SIRT1 is a class III histone deacetylase with important regulatory roles in transcription, cellular differentiation, proliferation and metabolism. Objectives Considering that various epigenetic modifications have recently been linked to the pathogenesis of SSc, we aimed to investigate the role of SIRT1 in fibroblast activation and tissue Fibrosis in SSc. Methods SIRT1 expression was analyzed by real-time PCR, Western Blot and immunohistochemistry in cultured human fibroblasts, in murine models of SSc and in skin samples from SSc patients. Collagen synthesis was quantified by real-time PCR, SirCol- and hydroxyproline assays. The effects of SIRT1 on canonical TGF-β signaling were evaluated by Smad reporter assays and analyses of TGF-β target genes. SIRT1 signaling was modulated by SIRT1 agonist resveratrol and by fibroblast-specific knockout. The role of SIRT1 was evaluated in bleomycin-induced skin Fibrosis in mice overexpressing a constitutively active TGF-β receptor I (TBR). Results SIRT1 expression was decreased in the skin of SSc patients. IHC demonstrated a pronounced decrease of SIRT1 in fibroblasts in SSc skin. SIRT1 mRNA and protein levels were also decreased in bleomycin or TBR-induced skin Fibrosis. TGF-β reduced mRNA and protein levels of SIRT1 in cultured fibroblasts. TBR overexpression reduced SIRT1 in murine skin, whereas treatment with the TBR inhibitor SD-208 prevented the downregulation of SIRT1 in Experimental Fibrosis. Activation of SIRT1 by treatment with resveratrol potentiated the stimulatory effects of TGF-β on cultured fibroblasts with more pronounced increases of Col1a1 mRNA and collagen protein. These effects were mediated by enhanced canonical TGF-β signaling as resveratrol induced SMAD-dependent transcription in reporter assays and increased the mRNA levels of CTGF of classical SMAD target genes. In contrast, knockdown of SIRT1 inhibited TGF-β/SMAD signaling and reduced release of collagen in fibroblasts. Consistent with a pro-fibrotic role of SIRT1, mice treated with resveratrol were more sensitive to bleomycin-induced dermal Fibrosis with more pronounced dermal thickening (74%, p=0.006), increased myofibroblast counts and higher hydroxyproline levels. Mice with fibroblast-specific SIRT1 knockdown were less susceptible to bleomycin induced Fibrosis. SIRT1 Inactivation also protected from Fibrosis induced by overexpression of TBR. Conclusions We identify SIRT1 as a crucial regulator of TGF-β/Smad signaling in SSc. Although SIRT1 is downregulated, this decrease is not sufficient to counter-balance the excessive activation of TGF-β signaling in SSc. However, augmentation of this endogenous regulatory mechanism, e.g. by knockdown of SIRT1 can effectively inhibit TGF-β signaling and exerts potent anti-fibrotic effects. Although further studies are required, these findings provide first evidence that SIRT1 may be an interesting target for pharmacological intervention. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2212
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Inactivation of tankyrases reduces Experimental Fibrosis by inhibiting canonical Wnt signalling
Annals of the Rheumatic Diseases, 2012Co-Authors: Alfiya Distler, Lisa Deloch, Jingang Huang, Clara Dees, Neng-yu Lin, Katrin Palumbo-zerr, Christian Beyer, Alexander Weidemann, Oliver Distler, Georg SchettAbstract:OBJECTIVES: Canonical Wnt signalling has recently emerged as a key mediator of fibroblast activation and tissue Fibrosis in systemic sclerosis. Here, we investigated tankyrases as novel molecular targets for inhibition of canonical Wnt signalling in fibrotic diseases. METHODS: The antifibrotic effects of the tankyrase inhibitor XAV-939 or of siRNA-mediated knockdown of tankyrases were evaluated in the mouse models of bleomycin-induced dermal Fibrosis and in Experimental Fibrosis induced by adenoviral overexpression of a constitutively active TGF-β receptor I (Ad-TBRI). RESULTS: Inactivation of tankyrases prevented the activation of canonical Wnt signalling in Experimental Fibrosis and reduced the nuclear accumulation of β-catenin and the mRNA levels of the target gene c-myc. Treatment with XAV-939 or siRNA-mediated knockdown of tankyrases in the skin effectively reduced bleomycin-induced dermal thickening, differentiation of resting fibroblasts into myofibroblasts and accumulation of collagen. Potent antifibrotic effects were also observed in Ad-TBRI driven skin Fibrosis. Inhibition of tankyrases was not limited by local or systemic toxicity. CONCLUSIONS: Inactivation of tankyrases effectively abrogated the activation of canonical Wnt signalling and demonstrated potent antifibrotic effects in well-tolerated doses. Thus, tankyrases might be candidates for targeted therapies in fibrotic diseases.
Frank A. Schildberg - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii type 1 receptor mediated janus kinase 2 activation induces liver Fibrosis
Hepatology, 2014Co-Authors: Martin Granzow, Benita Kowallick, Irela Gretchen Reza Mazar, Jan Gortzen, Robert Schierwagen, A Vogt, Sebastian Huss, Markus Linhart, S. Klein, Frank A. SchildbergAbstract:Activation of the renin angiotensin system resulting in stimulation of angiotensin-II (AngII) type I receptor (AT1R) is an important factor in the development of liver Fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver Fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real-time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental Fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human-derived LX2 cells. JAK2 expression and activity were increased in Experimental rodent and human liver Fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild-type animals led to activation of HSCs and Fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects were prevented in AT1R−/− mice. Pharmacological inhibition of JAK2 attenuated liver Fibrosis in rodent Fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. Conclusion: Our study substantiates the important cell-intrinsic role of JAK2 in HSCs for development of liver Fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver Fibrosis. (Hepatology 2014;60:334–348)
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angiotensin ii type 1 receptor mediated janus kinase 2 activation induces liver Fibrosis
Hepatology, 2014Co-Authors: Martin Granzow, Benita Kowallick, Irela Gretchen Reza Mazar, Jan Gortzen, Robert Schierwagen, A Vogt, Sebastian Huss, Markus Linhart, S. Klein, Frank A. SchildbergAbstract:Activation of the renin angiotensin system resulting in stimulation of angiotensin-II (AngII) type I receptor (AT1R) is an important factor in the development of liver Fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver Fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real-time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental Fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human-derived LX2 cells. JAK2 expression and activity were increased in Experimental rodent and human liver Fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild-type animals led to activation of HSCs and Fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects were prevented in AT1R−/− mice. Pharmacological inhibition of JAK2 attenuated liver Fibrosis in rodent Fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. Conclusion: Our study substantiates the important cell-intrinsic role of JAK2 in HSCs for development of liver Fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver Fibrosis. (Hepatology 2014;60:334–348)
