Evaluation neuer molekularer Ansätze für die Behandlung der Fibrose

The underlying pathology of systemic fibrotic diseases such as chronic graft-versus-host disease (cGvHD) and systemic sclerosis (SSc) still remains unknown. Fibrotic diseases are characterized by persistently activated fibroblasts that release excessive amounts of extracellular matrix proteins. The accumulation of extracellular matrix results in disruption of the physiological tissue architecture and subsequent dysfunction of affected organs. As molecular mechanisms underlying in fibrotic disorders have only partially been revealed, and molecular targeted therapies are not yet established for clinical use, the aim of this thesis was to investigate new potential molecular targets in the treatment of tissue fibrosis. In the first part of the thesis we examined the effects of the combined inhibition of the abelson kinase (c-Abl) and platelet-derived growth factor receptors (PDGFR) in experimental sclerodermatous cGvHD. Treatment with imatinib or nilotinib abolished the aberrant activation of c-Abl and PDGFR and protected against experimental cGvHD. Preventive therapy with imatinib or nilotinib inhibited the development of sclerodermatous cGvHD. Clinical features such as weight loss, alopecia and skin ulcers as well as histological features with dermal thickening and accumulation of collagen were significantly reduced in mice treated with imatinib or nilotinib, but not in prednisone treated mice. Of note, imatinib and nilotinib were also effective for the treatment experimental cGvHD, when it has already been clinically manifest. In the second part of the thesis we could demonstrate that hedgehog signaling was activated in human and murine cGvHD with increased expression of sonic hedgehog and accumulation of the transcription factors Gli-1 and Gli-2. Treatment with LDE223, a highly selective small molecule antagonist of the hedgehog co-receptor Smoothened (Smo), abrogated the activation of hedgehog signaling and protected against experimental cGvHD. Preventive therapy with LDE223 almost completely impeded the development of clinical and histological features of sclerodermatous cGvHD. Treatment with LDE223 was also effective, when initiated after the onset of clinical manifestations of cGvHD. Hedgehog signaling stimulated the release of collagen from cultured fibroblasts, but did not affect leukocyte influx in murine cGvHD suggesting direct, leukocyte-independent stimulatory effects on fibroblasts as the pathomechanism of hedgehog signaling in cGvHD. In the last part of the thesis we investigated the role of JunD, a member of the AP-1 family of transcription factors, as a downstream mediator of TGF-β signaling in SSc. We showed that JunD was overexpressed in SSc skin and in cultured fibroblasts in a TGF-β dependent manner. The expression of JunD co-localized with pSmad 3 in fibrotic skin and silencing of Smad 3 or Smad 4 by siRNA prevented the induction of JunD by TGF-β. JunD-/- fibroblasts were less responsive to TGF-β and released less collagen upon stimulation with TGF-β. Moreover, JunD-/- mice were protected from bleomycin induced fibrosis with reduced dermal thickening, decreased myofibroblast counts and lower collagen content of lesional skin. The findings of the present thesis might have translational implication and stimulate novel therapeutic approaches, because inhibitors of c-Abl and PDGFR and hedgehog signaling are already in clinical trials for other diseases and AP-1 inhibitors are currently in clinical trials.Die zugrunde liegende Pathologie systemisch fibrotischer Erkrankungen, wie der chronischen graft-versus-host Erkrankung (engl. cGvHD) und der systemischen Sklerose (SSc) ist bisweilen unbekannt. Charakteristisch für fibrotische Erkankungen sind die anhaltende Aktivierung von Fibroblasten und die exzessive Synthese von extrazellulärer Matrix. Die Anhäufung extrazellulärer Matrix führt zur Zerstörung der physiologischen Gewebearchitektur und zur Fehlfunktion der betroffenen Organe. Die zugrunde liegenden molekularen Mechanismen der fibrotischen Fehlfunktion konnten bisher nur teilweise identifiziert werden. Darüber hinaus sind gezielte molekulare Therapien für den klinischen Gebrauch bis dato noch nicht etabliert. Weshalb es die Zielsetzung dieser Dissertation war, neue potentielle Zielmoleküle für die Behandlung fibrotischer Erkrankungen zu identifizieren. Im ersten Teil dieser Arbeit untersuchten wir die Effekte der kombinierten Hemmung der abelson Kinase (c-Abl) und des platelet-derived growth factor receptors (PDGFR) in der experimentellen sklerodermiformen cGvHD. Die Behandlung mit Imatinib oder Nilotinib hob die abnorme Aktivierung von c-Abl und PDGFR auf, und bewahrte damit vor der experimentellen cGvHD. Die vorbeugende Therapie mit Imatinib oder Nilotinib hemmte die Entwicklung der sklerodermiformen cGvHD. Klinische Merkmale wie Gewichtsverlust, Alopezie und Hautulzerationen als auch histologische Ausprägungen wie Hautverdickung und Kollagenansammlung waren signifikant verringert in Mäusen, die mit Imatinib oder Nilotinib behandelt wurden, jedoch nicht in Prednison behandelten Mäusen. Von Bedeutung ist auch, dass Imatinib und Nilotinib auch effektiv in der Behandlung der experimentellen cGvHD waren, wenn sich diese bereits klinisch manifestiert hatte. Im zweiten Teil dieser Arbeit konnten wir zeigen, dass der Hedgehog Signalweg in der humanen und murinen cGvHD mit erhöhter Expression von Sonic Hedgehog (Shh) und der Akkumulation der Transkriptionsfaktoren Gli-1 und Gli-2 aktiviert ist. Die Behandlung mit LDE223, einem hoch selektiven niedermolekularen Antagonisten des Hedhehog Korezeptors Smoothened (Smo), hob die Aktivierung des Hedgehog Signalweges auf und bewahrte vor der experimentellen cGvHD. Mit der Präventivtherapie mittels LDE223 konnte eine annähernd vollständige Unterbindung der Entstehung von klinischen und histologischen Merkmalen der sklerodermiformen cGvHD erzielt werden. Die Behandlung mit LDE223 war ebenso erfolgreich, wenn gleich diese mit dem Einsetzen klinischer Manifestation der cGvHD eingeleitet wurde. Das Hedgehog-Signaling stimulierte die Freisetzung von Kollagen in Fibroblastenkulturen, hatte jedoch keinen Einfluss auf den Leukozyteneinstrom in muriner cGvHD. Dies führt zu der Annahme, dass das Hedgehog-Signaling in der cGvHD eine Leukozyten unabhängige, stimulierende Wirkung auf die Fibroblasten ausübt. Im letzten Teil dieser Arbeit wurde die Rolle von JunD, welches ein Mitglied der AP-1-Familie von Transkriptionsfaktoren ist, als nachgeschalteter Vermittler der TGF-β- Signalkaskade in der SSc untersucht. Wir zeigten, dass JunD sowohl in der SSc-Haut als auch in Fibroblastenkulturen in Abhängigkeit von TGF-β überexprimiert wurde. Die Expression von JunD kolokalisierte mit pSmad3 in fibrotischer Haut und das Silencing von Smad3 oder Smad4 mittels siRNA verhinderte die Induktion von JunD durch TGF-β. JunD-/- Fibroblasten wiesen eine verminderte TGF-β-Reaktion auf, und schütteten weniger Kollagen, im Zuge einer Stimulation mit TGF-β aus. Außerdem zeigten JunD-/- Mäuse eine schützende Wirkung vor der Bleomycin-induzierten Fibrose, welches sich durch eine reduzierte Hautverdickung, eine geringere Myofibroblastenzahl und eine niedrigere Kollageneinlagerungen in den Hautläsionen äußerte. Diese Ergebnisse haben direkte translationale Bedeutung, da sowohl kombinierte Inhibitoren von c-Abl und PDGFR als auch Smo-Antagonisten bereits zur Behandlung anderer Erkrankungen zugelassen sind und AP-1 Inhibitoren sich derzeit in fortgeschrittenen klinischen Studien befinde

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

Heat shock protein 90 (Hsp90) inhibition targets canonical TGF-β signalling to prevent fibrosis

Objectives: Targeted therapies for systemic sclerosis (SSc) and other fibrotic diseases are not yet available. We evaluated the efficacy of heat shock protein 90 (Hsp90) inhibition as a novel approach to inhibition of aberrant transforming growth factor (TGF)-β signalling and for the treatment of fibrosis in preclinical models of SSc. Methods: Expression of Hsp90 was quantified by quantitative PCR, western blot and immunohistochemistry. The effects of Hsp90 inhibition were analysed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, in tight-skin (Tsk-1) mice and in mice overexpressing a constitutively active TGF-β receptor I (TβRI). Results: Expression of Hsp90β was increased in SSc skin and in murine models of SSc in a TGF-β-dependent manner. Inhibition of Hsp90 by 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) inhibited canonical TGF-β signalling and completely prevented the stimulatory effects of TGF-β on collagen synthesis and myofibroblast differentiation. Treatment with 17-DMAG decreased the activation of canonical TGF-β signalling in murine models of SSc and exerted potent antifibrotic effects in bleomycin-induced dermal fibrosis, in Tsk-1 mice and in mice overexpressing a constitutively active TβRI. Dermal thickness, number of myofibroblasts and hydroxyproline content were all significantly reduced on treatment with 17-DMAG. No toxic effects were observed with 17-DMAG at antifibrotic doses. Conclusions: Hsp90 is upregulated in SSc and is critical for TGF-β signalling. Pharmacological inhibition of Hsp90 effectively blocks the profibrotic effects of TGF-β in cultured fibroblasts and in different preclinical models of SSc. These results have translational implications, as several Hsp90 inhibitors are in clinical trials for other indications

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

The Wnt antagonists DKK1 and SFRP1 are downregulated by promoter hypermethylation in systemic sclerosis

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

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

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

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 (TBRICA). 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 TBRICA-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

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

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