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

PU.1 controls fibroblast polarization and tissue fibrosis

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs

Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation

Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Evaluation neuer molekularer Ansätze für die Behandlung der Systemischen Sklerose

Systemic sclerosis (SSc) is a chronic fibrotic autoimmune disease of unknown etiology that affects the skin and several internal organs. Besides vascular disease and cellular immune activation, the major histopathologic hallmark of SSc is an excessive accumulation of extracellular matrix proteins released from activated fibroblasts, which destroys the physiological tissue architecture and impairs the function of the involved organs. As current approaches to treat fibrosis are of limited efficacy and therapies targeting specifically the activation of fibroblasts are not yet available, this study aimed to investigate novel molecular targets which might be effective in the future treatment of tissue fibrosis. The first part of the study focused on the involvement of serotonin (5-hydroxytryptamine, 5-HT) signaling to the pathogenesis of SSc and revealed a link between vasculopathy, platelet activation and tissue fibrosis. Mice deficient for tryptophan hydroxylase-1 were protected from fibrosis and inhibition of platelet activation efficiently reduced tissue fibrosis in different animal models. Serotonin exerted potent pro-fibrotic effects on cultured fibroblasts by activating the 5-HT2B receptor. Furthermore, an upregulation of 5-HT2B in skin of SSc patients, particularly in activated myofibroblasts, was observed by immunohistochemical analyses. Activation of 5-HT2B induced transforming growth factor β (TGFβ) signaling with a time-dependent increase in nuclear levels of phospho-Smad3, the major downstream effector of TGFβ. Targeting 5-HT2B, either pharmacologically or genetically, not only prevented fibrosis in mouse models of SSc, but potently induced regression of pre-established fibrosis. The second part of the study evaluated the role of the Notch pathway to fibroblast activation and tissue fibrosis. Increased levels of activated Notch-1 were found in lesional skin of SSc patients and in cultured SSc fibroblasts. In addition, the ligand Jagged-1 (JAG1) was found to be overexpressed in T cells of inflammatory infiltrates. Activation of the Notch pathway by recombinant JAG1 in cultured dermal fibroblasts resulted in increased collagen mRNA and protein levels. Moreover, JAG1 induced myofibroblast differentiation with elevated expression of α-smooth-muscle-actin and formation of stress fibers. Consistent with the increased activation of Notch signaling in SSc fibroblasts, inhibition of the pathway by blockade of γ-secretase reduced collagen production only in SSc but not in healthy dermal fibroblasts. In vivo, Notch-1 antisense transgenic mice were protected from fibrosis and inhibition of γ-secretase potently reduced pre-established fibrosis. In conclusion, these findings might have translational implications. As inhibitors for 5-HT2B and γ-secretase are already in clinical use or in clinical trials for other diseases, they would also be available for clinical trials in SSc patients. Indeed, a fist proof-of-concept trial with the 5-HT2 inhibitor terguride in patients with SSc achieved promising anti-fibrotic effects.Systemische Sklerose (SSc) ist eine chronisch fibrosierende Autoimmunerkrankung mit unbekannter Ursache, welche die Haut und unterschiedliche innere Organe befallen kann. Neben einer Vaskulopathie und einer Aktivierung des Immunsystems ist das augenfälligste histopathologische Merkmal der SSc eine Akkumulation extrazellulärer Matrixproteine, welche die normale Gewebestruktur zerstört und die Funktion der betroffenen Organe beeinträchtigt. Da therapeutische Optionen zur Behandlung der Fibrose für den klinischen Gebrauch noch nicht zur Verfügung stehen, war es das Ziel dieser Studie, neue Zielmoleküle zu evaluieren, welche zukünftig eine effektive und gezielte Behandlung der Fibrose ermöglichen könnten. Der erste Teil der Studie befasst sich mit der Beteiligung des Serotonin (5-Hydroxytryptamin, 5-HT)- Signalweges an der Pathogenese der SSc und zeigte eine Verbindung zwischen vaskulären Veränderungen, Aktivierung von Thrombozyten und Fibrose. Tryptophanhydroxylase-1-defiziente Mäuse entwickelten keine Fibrose und die Inhibition der Thrombozytenaktivierung reduzierte deutlich die Entstehung von Fibrose in verschiedenen Tiermodellen. Serotonin zeigte starke pro-fibrotische Effekte auf kultivierte Fibroblasten, welche über den 5-HT2B Rezeptor vermittelt wurden. Desweiteren wurde mittels immunhistochemischer Färbung beobachtet, dass 5-HT2B in aktivierten Myofibroblasten in der Haut von SSc Patienten überexprimiert wird. Außerdem führte eine Aktivierung von 5-HT2B zu einer Induktion des transforming growth factor β (TGFβ)- Signalweges mit einer zeitabhängigen Akkumulation von phospho-Smad3, dem Hauptmediator von TGFβ, im Zellkern. Pharmakologische oder genetische Inhibition von 5-HT2B verhinderte nicht nur die Entstehung von Fibrose, sondern induzierte auch eine potente Regression etablierter Fibrose. Der zweite Teil der Studie evaluiert den Einfluss des Notch Signalweges auf die Aktivierung von Fibroblasten und die Entstehung von Fibrose. Eine verstärkte Aktivierung von Notch-1 konnte sowohl in der Haut von SSc Patienten als auch in kultivierten Fibroblasten gezeigt werden. Außerdem ist der Ligand Jagged-1 (Jag-1) in T-Zellen in Entzündungsinfiltraten überexprimiert. Aktivierung des Signalweges durch rekombinantes Jag-1 Protein führte zu einer erhöhten Kollagenproduktion und aktivierte die Differenzierung von Myofibroblasten mit verstärkter Expression von α-smooth-muscle actin und Bildung von Stressfasern. Passend zur erhöhten Aktivität von Notch-1 in SSc Fibroblasten reduzierte eine Blockade der γ-Sekretase die Kollagensynthese nur in SSc Fibroblasten, jedoch nicht in Fibroblasten gesunder Probanden. Im Tiermodell waren Notch-1 antisense transgene Mäuse vor Fibrose geschützt und Inhibition der Notch-Signalkaskade durch γ-Sekretase-Inhibitoren bewirkte eine starke Rückbildung etablierter Fibrose. Zusammenfassend könnten diese Ergebnisse zu einer verbesserten Therapie der SSc beitragen. Da Inhibitoren für 5-HT2B und γ-Sekretase bereits in klinischer Verwendung oder in klinischen Studien für andere Erkrankungen sind, stünden sie ebenso für klinische Studien mit SSc Patienten zur Verfügung. In der Tat konnten in einer ersten Machbarkeitsstudie (proof-of-concept trial) mit dem 5-HT2 Antagonisten Terguride in SSc Patienten vielversprechende anti-fibrotische Ergebnisse erzielt werden

TGF-β-induced epigenetic deregulation of SOCS3 facilitates STAT3 signaling to promote fibrosis

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

Translational engagement of LPA1_{1} receptor in skin fibrosis: from dermal fibroblasts of patients with scleroderma to Tsk1 mouse

BACKGROUND AND PURPOSE: Genetic deletion and pharmacological studies suggest a role for lysophosphatidic acid receptor-1 (LPA$_{1}$ receptor) in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA$_{1}$ receptor antagonist using dermal fibroblasts from patients and animal model of skin fibrosis. EXPERIMENTAL APPROACH: Dermal fibroblast and skin biopsies from SSs patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Therapeutic effect of SAR100842 was assessed in the tight skin mouse model-1 (Tsk1). KEY RESULTS: SAR100842 is equipotent against various LPA isoforms. SSc dermal fibroblasts and skin biopsies expressed high levels of LPA$_{1}$ receptor. The LPA functional response (Ca$^{2+}$ ) in SSc dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in SSc dermal and IPF lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from SSc dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice. CONCLUSION: The effects of SAR100842 on LPA -induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA$_{1}$ receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA$_{1}$ receptor antagonists in systemic sclerosis

Inactivation of tankyrases reduces experimental fibrosis by inhibiting canonical Wnt signalling

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

Inactivation of evenness interrupted (EVI) reduces experimental fibrosis by combined inhibition of canonical and non-canonical Wnt signalling

OBJECTIVES: Canonical as well as non-canonical Wnt signalling pathways have emerged as core pathways of fibrosis. Their profibrotic effects are mediated via distinct intracellular cascades independently of each other. Thus, inhibition of both pathways may have additive antifibrotic effects. Here, we knocked down evenness interrupted (EVI) to simultaneously target for the first time canonical and non-canonical Wnt signalling in experimental fibrosis. METHODS: The antifibrotic effects of siRNA-mediated knockdown of EVI were evaluated in the mouse models of bleomycin-induced skin fibrosis and in fibrosis induced by adenoviral overexpression of a constitutively active TGF-β receptor I (AdTBRI). RESULTS: Knockdown of EVI decreased the release of canonical and non-canonical Wnt ligands by fibroblasts and reduced the activation of canonical and non-canonical Wnt cascades in experimental fibrosis with decreased accumulation of β-catenin and phosphorylated JNK and cJun. Inactivation of EVI exerted potent antifibrotic effects and reduced dermal thickening, myofibroblast differentiation and accumulation of collagen in the mouse models of bleomycin-induced and AdTBR-induced fibrosis. CONCLUSIONS: Inhibition of Wnt secretion by knockdown of EVI inhibits canonical and non-canonical Wnt signalling and effectively reduces experimental fibrosis in different preclinical models. Inhibition of Wnt secretion may thus be an interesting approach for the treatment of fibrosis