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

Molecular mechanisms of fibroblast activation in the pathogenesis of systemic sclerosis

In meiner Doktorarbeit wir untersuchten neue Mechanismen zur Hemmung der Fibroblastaktivierung und Therapie der Fibrose bei SSc Patienten. Im ersten Teil meiner Arbeit konnten wir zeigen, daß ROCK eine zentrale Rolle bei der Myofibroblastendifferenzierung spielt. Durch eine Hemmung von ROCK mit chemischen Inhibitoren oder siRNA ließ sich stark die Synthese von Kollagen und anderen extrazellulären Matrix-Proteinen in Fibroblasten auf der mRNA und Protein-Ebene signifikant reduzieren. Als zugrundeliegenden Mechanismus konnten wir eine verminderte Differenzierung von ruhenden Fibroblasten in aktivierte Myofibroblasten nachweisen. So stimulierte TGFβ über einen ROCK abhängigen Mechanismus die MAPK Erk. Eine Hemmung von ROCK unterdrückte die Aktivierung von Erk und verhinderte die TGFβ induzierte Myofibroblastdifferenzierung und Matrixsynthese. Aufgrund der potenten anti-fibrotischen Effekte von ROCK-Inhibitoren in pharmakologisch relevanten Konzentrationen, könnte die Hemmung von ROCK einer interessanten neuen therapeutischen Ansatz für die Behandlung der Fibrose bei SSc Patienten sein. Wir konnten bereits in vorherigen Projekten zeigen, daß die kombinierte Hemmung von c-abl and PDGF-Rezeptoren durch Imatinib eine starke anti-fibrotische Wirkung hat. Dasatinib und Nilotinib sind zwei neue niedermolekulare Inhibitoren von c-abl and PDGF-Rezeptoren, die erfolgreich bei Imatinib resistenten Patienten verwendet werden. Dasatinib und Nilotinib reduzierten in SSc Fibroblasten dosisabhängig die Synthese von Matrix-Proteinen. Im Maus Modell der Bleomycin-induzierten Hautfibrose konnten beide Substanzen der Entwicklung einer Hautfibrose vorbeugen. Dasatinib und Nilotinib reduzierten dosisabhängig die Hautdicke, die Zahl der Myofibroblasten und den Kollagen Gehalt. Dasatinib und Nilotinib könnten somit vielversprechende Medikamente für die Behandlung von SSc Patienten sein. Nachdem wir zeigen konnten, daß Hemmung von c-abl and PDGF-Rezeptoren dermale Fibrose in dem entzündlichen imitiertem frühe Phasen der SSc Modell verhindern, analysierten wir, ob Imatinib auch in einem entzündungsunabhängigen Tiermodell für spätere Stadien der SSc wirksam ist. Hierzu wurden Tsk-1 Mäuse mit Imatinib behandelt. Die Behandlung mit Imatinib verhinderte die Differenzierung von ruhenden Fibroblasten in Myofibroblasten und reduzierte das Ausmaß der Hautfibrose bei Tsk-1 Mäusen. Als nächstes untersuchten wir, ob die kombinierte Hemmung von c-abl und PDGF-Rezeptoren auch eine Regression bereits vorhandener fibrotischer Veränderungen induzieren kann, ob als neben Prävention auch eine Therapie schon etablierter Fibrose möglich ist. Tatsächlich verhinderte Imatinib nicht nur das weitere Fortschreiten der Fibrose in einem modifizierten Modell der Bleomycin induzierten etablierten Fibrose, sondern induzierte auch die Regression bereits vorbestehender fibrotischer Veränderungen mit Reduktion der Hautdicke unter den Ausgangswert. Diese Ergebnisse deuten darauf hin, dass die kombinierte Hemmung von c-abl und PDGF-Rezeptoren nicht nur in frühen, entzündlichen Krankheitsstadien, sondern auch in späteren, nicht entzündlichen Stadien der SSc wirksam sein könnte. Somit könnte Imatinib ein interessanter Kandidat für klinische Studien bei SSc Patienten mit einer langjährigen Krankheit und bereits bestehende Fibrose sein. Abschliessend konnten wir zeigen, daß der Cannabinoid-Rezeptor CB2 die Infiltration von Leukozyten und hierdurch sekundär die Aktiviertung von Fibroblasten im Modell der Belomycin-induzierten Fibrose hemmt. Mäuse, die kein CB2 exprimieren (CB2 -/-) zeigten eine stärkere fibrotische Reaktion nach Bleomycin-Injektionen und wiesen eine stärkere Hautfibrose auf als CB2 +/+ Mäuse. In injezierten Hautarealen von CB2 -/- Mäusen fanden sich signifikant mehr Leukozyten als bei CB2 +/+ Mäusen. Eine stärkere Hautfibrose und vermehrter Leukozyteninflux wurden auch bei Behandlung mit dem CB2-Antagonist AM-630 beobachtet. Im Gegensatz dazu hemmte der selektive CB2 Agonist JWH-133 die Infiltration von Leukozyten und die Entstehung einer Hautfibrose. Der Phänotyp der CB2 -/- Mäusen zu Bleomycin wurde durch die Transplantation von CB2 -/- Knochenmark in CB2 +/+ Mäuse imitiert. Im Gegensatz dazu zeigten CB2 -/- Mäuse, welche mit Knochenmark von CB2 +/+ Mäusen transplantiert wurden, nach Bleomycin-Injektion keine stärkere fibrotische Reaktion. Wir schlussfolgern daher, dass CB2 die Infiltration von Leukozyten in entzündlichen Stadien der experimentellen Fibrose hemmt, hierdurch die sekundäre, Leukozyten-abhängige Aktivierung von residenten Fibroblasten reduziert und dadurch der Entstehung einer Hautfibrose vorbeugt. Da selektive CB2-Agonisten für den klinischen Einsatz zur Verfügung stehen und gut verträglich sind, könnte CB2 ein interessantes Ziel für die Behandlung von frühen inflammatoischen Stadien der SSc sein.In my doctoral thesis, we evaluated novel strategies to prevent fibroblast activation and tissue fibrosis in SSc. First, we demonstrated that ROCK plays a central role for the differentiation of resting fibroblasts into metabolically active myofibroblasts and contributes to the activated phenotype of SSc fibroblasts. Inhibition of ROCK reduced the synthesis of the major extracellular matrix proteins on the mRNA and protein level. Inhibition of ROCK prevented myofibroblast differentiation. TGFβ activated Erk in a ROCK dependent manner by and Erk mediated in part the stimulatory effects of ROCK on myofibroblast differentiation. As inhibitors of ROCK potently prevented fibroblast activation and decreased the release of collagen at pharmacologically relevant concentrations, inhibition of ROCK might be an interesting novel therapeutic approach for the treatment of SSc. We have shown previously that the combined inhibition of c-abl and PDGF receptor by imatinib exerts potent anti-fibrotic effects. Dasatinib and nilotinib are two novel inhibitors of c-abl and PDGF receptor, which have recently been approved. Both inhibitors are more potent than imatinib, have been successfully used in CML patients resistant to imatinib and pocess a different spectrum of adverse effects. Thus, dasatinib and nilotinib might be interesting alternatives for patients that cannot tolerate imatinib. Dasatinib and nilotinib dose-dependently decreased the protein levels of extracellular matrix proteins in dermal fibroblasts from SSc patients. In a mouse model of bleomycin induced dermal fibrosis, a model for early inflammatory stages of SSc, dasatinib and nilotinib potently reduced the dermal thickening, the number of myofibroblasts and the collagen content of lesional skin at well tolerated doses. These data indicate that dasatinib and nilotinib potently inhibit the synthesis of extracellular matrix in vitro and in vivo. Thus, we provide the first evidence that dasatinib and nilotinib might be promising drugs for the treatment of patients with SSc. After demonstrating that the combined inhibition of c-abl and PDGF receptors by imatinib, dasatinib and nilotinib decreases the synthesis of extracellular matrix in cultured fibroblasts and completely prevents dermal fibrosis in inflammation driven models mimicking early stages of SSc, we next investigated whether imatinib can prevent dermal fibrosis in Tsk-1 mice, an inflammation-independent model mimicking later stages of SSc. Treatment with imatinib reduced the dermal and hypodermal thickening in Tsk-1 mice and prevented the differentiation of resting fibroblasts into myofibroblasts. Next, we investigated, whether the combined inhibition of c-abl and PDGF receptors can also induce regression of pre-established dermal fibrosis. Indeed, imatinib did not only stop further progression of fibrosis in a murine model of pre-established dermal fibrosis, but induced regression of pre-existing dermal fibrosis with reduction of the dermal thickness below pre-treatment levels. These results indicate that the combined inhibition of c-abl and PDGF receptors might also be effective for later, less inflammatory stages of SSc and for the treatment of established fibrosis. Thus, imatinib might an interesting candidate for clinical trials on patients with longstanding disease and pre-existing tissue fibrosis. Finally, we demonstrated that the cannabinoid receptor CB2 negatively regulates the influx of leukocytes and secondary fibroblast activation in experimental fibrosis. Mice lacking CB2 (CB2 -/-) were more sensitive to bleomycin induced dermal fibrosis than CB2 +/+ mice thickeining. Leukocytes counts were significantly higher in lesional skin of CB2 +/+ mice. Increased dermal fibrosis was also observed upon treatment with the CB2 antagonist AM-630. In contrast, the selective CB2 agonist JWH-133 reduced leukocyte infiltration and dermal thickening. The phenotype of CB2 -/- mice was mimiced by transplantation of CB2 -/- bone marrow into CB2 +/+ mice, whereas CB2 -/- mice transplanted with bone marrow from CB2 +/+ mice did not display an increased sensitivity to bleomycin induced fibrosis, indicating that the expression of CB2 on leukocytes, but not on mesenchymal cells is affecting the outcome of experimental fibrosis. Thus, CB2 limits leukocyte infiltration and tissue fibrosis in experimental dermal fibrosis. Since selective CB2 agonists are available and well tolerated, CB2 might be an interesting molecular target for the treatment of early inflammatory stages of SSc. In summary, we identified inhibition of ROCK, combined inhibition of c-abl and PDGF receptors and activation of CB2 as potential novel anti-fibrotic approaches for the treatment of SSc. Inhibition of ROCK, c-abl and PDGF receptors directly targets the activation of fibroblasts, whereas activation of CB2 exerts its anti-fibrotic effects indirectly by regulating leukocyte infiltration

The 12/15-lipoxygenase pathway counteracts fibroblast activation and experimental fibrosis

BACKGROUND: Idiopathic and inflammation-dependent fibrotic diseases such systemic sclerosis (SSc) impose a major burden on modern societies. Understanding endogenous mechanisms, which counteract fibrosis, may yield new therapeutic approaches. Lipoxins are highly potent lipid mediators, which have recently been found to be decreased in SSc. OBJECTIVES: To determine the potential role of 12/15-lipoxygenase (12/15-LO), the key enzyme for the synthesis of lipoxins, in fibrosis. METHODS: Two mouse models for experimental dermal fibrosis (bleomycin-induced dermal fibrosis and tight-skin 1 mouse model) together with bone marrow transfers were used in wildtype and 12/15-LO(-/-) mice to elucidate the role of this enzyme during dermal fibrosis. Primary dermal fibroblasts of wildtype and 12/15-LO(-/-) mice, and 12/15-LO-derived eicosanoids, were used to identify underlying molecular mechanisms RESULTS: In both models, 12/15-LO(-/-) mice exhibited a significant exacerbation of the fibrotic tissue response. Bone marrow transfer experiments disclosed a predominant role of mesenchymal cell-derived 12/15-LO in these antifibrotic effects. Indeed, 12/15-LO(-/-) fibroblasts showed an enhanced activation of the mitogen-activated protein-kinase pathway and an increased col 1a2 mRNA expression in response to stimulation with transforming growth factor β (TGFβ), whereas 12/15-LO-derived eicosanoids blocked these TGFβ-induced effects. CONCLUSIONS: These data indicate that 12/15-LO and its metabolites have a prominent antifibrotic role during dermal fibrosis. This opens new opportunities for therapeutic approaches in the treatment of fibrotic diseases

β-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis

OBJECTIVES: Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). β-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of β-catenin in fibroblasts for the development of SSc dermal fibrosis. METHODS: Nuclear accumulation of β-catenin in fibroblasts was assessed by triple staining for β-catenin, prolyl-4-hydroxylase-β and 4',6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of β-catenin in fibrosis. RESULTS: The auhors found significantly increased nuclear levels of β-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of β-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of β-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of β-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of β-catenin significantly reduced bleomycin-induced dermal fibrosis. CONCLUSIONS: The present study findings identify β-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of β-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of β-catenin/Wnt signaling have recently entered clinical trials

Levels of target activation predict antifibrotic responses to tyrosine kinase inhibitors

OBJECTIVES: To assess whether the discrepancy between the strong antifibrotic effects of tyrosine kinase inhibitors (TKIs) in animal models and the inconsistent results in clinical studies might be related to the activation levels of drug targets. METHODS: Skin sections of bleomycin, TSK1, Fra-2 transgenic mice, SSc patients and controls were analysed by histology and immunohistochemistry. Subgroups of mice were treated with the TKIs nilotinib or imatinib. Differences in the activation levels of the TKI targets p-PDGFRβ (platelet derived growth factor β) and p-c-abl were assessed. RESULTS: In bleomycin and TSK1 mice, expression of activated p-PDGFRβ (platelet derived growth factor receptor β) and p-c-abl was ubiquitous with strong upregulation compared with controls. Treatment with TKIs resulted in successful target inhibition and consequently reduced dermal fibrosis. In the Fra-2 model, the activation levels of p-PDGFRβ and p-c-abl were much lower than in the bleomycin and the TSK1 models. Accordingly, nilotinib did not prevent dermal fibrosis and target inhibition was unsuccessful. Notably, in skin biopsies of SSc patients, the mean activation levels of TKI targets were only moderate and in the majority of patients resembled those of the non-responsive Fra-2 model. CONCLUSIONS: Animal models for proof-of-concept studies should be selected based on a similar activation level and expression pattern of drug targets as in human SSc

Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis

BACKGROUND: Fibrosis and vascular disease are cardinal features of systemic sclerosis (SSc). Stimulators of soluble guanylate cyclase (sGC) are vasoactive drugs that are currently being evaluated in phase III clinical trials for pulmonary arterial hypertension. OBJECTIVE: To study the antifibrotic potency of sGC stimulators. METHODS: The effect of the sGC stimulator BAY 41-2272 on the release of collagen from dermal fibroblasts was examined. The antifibrotic effects of BAY 41-2272 on prevention and regression of fibrosis in bleomycin-induced dermal fibrosis and in Tsk-1 mice were also studied. Telemetric blood pressure studies in conscious mice were used to study potential hypotensive effects of sGC stimulation. RESULTS: sGC stimulation with BAY 41-2272 dose-dependently inhibited collagen release in dermal fibroblasts from patients with SSc and healthy individuals. Furthermore, BAY 41-2272 stopped the development of bleomycin-induced dermal fibrosis and skin fibrosis in Tsk-1 mice, preventing dermal and hypodermal thickening, reducing the numbers of myofibroblasts and reducing the hydroxyproline content. In addition, BAY 41-2272 was highly effective in the treatment of established fibrosis in the modified models of bleomycin-induced skin fibrosis and Tsk-1 mice. Treatment with sGC stimulators was well tolerated. Relevant antifibrotic doses of BAY 41-2272 did not affect systemic blood pressure and heart rate in mice. CONCLUSIONS: These findings demonstrate potent antifibrotic effects and good tolerability of sGC stimulators in various experimental models of SSc. Given their potential vasoactive properties, sGC stimulators may be promising candidates for the dual treatment of fibrosis and vascular disease in SSc

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