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

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

Autophagy regulates TNFα-mediated joint destruction in experimental arthritis

Objectives: Autophagy is a homeostatic process to recycle dispensable and damaged cell organelles. Dysregulation of autophagic pathways has recently been implicated in the pathogenesis of various diseases. Here, we investigated the role of autophagy during joint destruction in arthritis. Methods: Autophagy in osteoclasts was analysed in vitro and ex vivo by transmission electron microscopy, Western blotting and immunohistochemistry for Beclin1 and Atg7. Small molecule inhibitors, LysMCre-mediated knockout of Atg7 and lentiviral overexpression of Beclin1 were used to modulate autophagy in vitro and in vivo. Osteoclast differentiation markers were quantified by real-time PCR. The extent of bone and cartilage destruction was analysed in human tumour necrosis factor α transgenic (hTNFα tg) mice after adoptive transfer with myeloid specific Atg7-deficient bone marrow. Results: Autophagy was activated in osteoclasts of human rheumatoid arthritis (RA) showing increased expression of Beclin1 and Atg7. TNFα potently induced the expression of autophagy-related genes and activated autophagy in vitro and in vivo. Activation of autophagy by overexpression of Beclin1-induced osteoclastogenesis and enhanced the resorptive capacity of cultured osteoclasts, whereas pharmacologic or genetic inactivation of autophagy prevented osteoclast differentiation. Arthritic hTNFα tg mice transplanted with Atg7fl/fl×LysMCre+ bone marrow cells (BMC) showed reduced numbers of osteoclasts and were protected from TNFα-induced bone erosion, proteoglycan loss and chondrocyte death. Conclusions: These findings demonstrate that autophagy is activated in RA in a TNFα-dependent manner and regulates osteoclast differentiation and bone resorption. We thus provide evidence for a central role of autophagy in joint destruction in RA

Die Rolle des Matrixregulators Fra-1 in Arthritis and Leberfibrose

Herein we investigated the effect of matrix overproduction on bone and liver homeostasis. Our data show that overproduction of matrixproteins due to fra-1 overexpression have a dual effect since they 1) benefit new matrix synthesis in bones during inflammatory arthritis, but 2) induce liver fibrosis. We used two mouse models: the Fra-1 transgenic mouse, which shows increased bone synthesis and develops osteosclerosis and the hTNF transgenic mouse, which develops erosive arthritis and represents a mouse model of rheumatoid arthritis. In the first part of the study we could show that overexpression of the transcription factor fra-1 reduces bone and cartilage damage in the arthritic hTNFtg mouse. The Fra-1tg x hTNFtg mice show strong arthritic signs, similar to the hTNFtg mice. Arthritis development in Fra-1tg x hTNFtg mice was associated with strong inflammatory cell influx, but less bone erosions and decreased osteoclast number, whereas bone mass was strongly increased and osteosclerosis was evident. In addition, overexpression of fra-1 protected arthritic hTNFtg mice from cartilage destruction, which may be in part caused by increased chondrocytes proliferation due to fra-1 overexpression. In contrast to its benefical effects on bone metabolism, overexpression of fra-1 in liver tissue leads to development of liver fibrosis. Mice overexpressing fra-1 develop chronic hepatitis characterized by strong portal tract inflammation and increased bile duct number. Inflammatory infiltrates are composed of increased number of T cells, particularly of CD4+ cells, and decreased number of NK-, NKT- and B cells. Hepatitis development in Fra-1tg mice is accompanied by biliary fibrosis based on the upregulated expression of collagens and profibrogenic genes. Liver pathology is not directly triggered by the immune system as Fra-1tg x Rag2-/- mice showed no evident inflammation, but obvious fibrotic tissue. Fibrosis development in the Fra-1tg mice seems to be accomplished by activated biliary epithelial cells, which express the profibrotic proteins PDGF and TGFß1. In addition, biliary epithelial cells and inflammatory cells synthesize the Fra-1 protein in the Fra-1tg mice, but not in wildtype mice. These findings were replicated in human liver samples of advanced liver fibrosis. Our investigations show that overproduction of matrix proteins by overexpression of fra-1 reduces bone destruction during arthritis. However, this process should occur only locally since systemic overproduction of matrix proteins can lead to development of fibrosis in other organs such as the liver.Ziel dieser Doktorarbeit war es, den Effekt einer Matrixüberproduktion durch fra-1 Überexpression auf Knochen- und Leberhomoöstase zu untersuchen. Die erzielten Daten zeigten, dass eine erhöhte Matrixproduktion einen dualen Effekt hat, indem sie 1) die Knochenneubildung in entzündlichen Knochenerkrankungen wie der rheumatoiden Arthritis begünstigt, aber 2) die Bildung von fibrotischem Gewebe in der Leber induziert und damit Leberstruktur und –funktionen beeinträchtigt. Um den Fragestellungen nachgehen zu können wurden zwei Mausmodelle verwendet: die Fra-1 transgene Maus, welche vermehrt Knochen bildet und eine Osteosklerose entwickelt und die hTNF transgene Maus, welche eine erosive Arthritis entwickelt und ein murines Modell der rheumatoiden Arthritis darstellt. In dem ersten Teil der Studie konnte gezeigt werden, dass eine Überexpression des Transkriptionsfaktors fra-1 den Knochen- und Knorpelverlust in den arthritischen hTNFtg Mäusen vorbeugt. Dazu wurden Fra-1tg Mäuse mit den hTNFtg Mäusen gekreuzt und untersucht. Die Fra-1tg x hTNFtg Mäuse zeigten, wie die hTNFtg Mäuse, klinische Symptome der Arthritis. Des Weiteren wiesen die Fra-1tg x hTNFtg Mäuse auch histologisch eine synoviale Entzündung auf. Allerdings war der Abbau von subchondralem Knochen in den Fra-1tg x hTNFtg Mäusen reduziert und die Anzahl der Osteoklasten stark vermindert. Weiteres entwickelten die Fra-1tg x hTNFtg Mäuse Osteosklerose aufgrund erhöhter Knochensynthese. Zudem wiesen die Fra-1tg x hTNFtg Mäuse eine starke Reduktion des Proteoglykanverlustes und damit geringere Knorpelschädigung auf, was möglicherweise mit der erhöhten Proliferationsaktivität der fra-1tg Knorpelzellen erklärt werden kann. Die Überexpression von fra-1 führt auch in der Leber zur verstärkten Matrixneusynthese und damit einhergehend einer Leberfibrose. Die Fra-1 transgenen Mäuse wiesen eine erhöhte Anzahl an sklerosierenden intrahepatischen Gallengänge auf, begleitet von einer lokalen Entzündung, welche durch eine Mischung aus T-Zellen, überwiegend CD4+ T-Zellen und weniger B-, NK- und NKT-Zellen verursacht wurde. Im Verlauf der Erkrankung entwickelten die Fra-1tg Mäuse eine biliäre Fibrose, basierend auf einer Überexpression collagener und profibrogener Gene. Die pathologischen Veränderungen in der Leber waren nur teilweise eine Folge der Entzündungsaktivität, da Fra-1tg x Rag2-/- Mäuse kaum Entzündung, aber weiterhin eine Leberfibrose entwickelten. Die Entstehung von Leberfibrose in den Fra-1tg Mäusen scheint ein cholangiozyten-abhängiger Prozess zu sein, da fibrose-induzierende Moleküle wie PDGFs und TGFß1 in den Cholangiozyten von Fra-1tg Mäuse detektiert werden konnten. Zudem identifizierten wir die Cholangiozyten und inflammatorischen Zellen als Hauptsyntheseort des Fra-1 Proteins in der Leber der Fra-1tg Maus. Untersuchungen zur Fra-1 Produktion an humanen Proben von Patienten mit fortgeschrittener Leberfibrose unterstützten die experimentellen Ergebnisse. Unsere Untersuchungen zeigen, dass eine vermehrte Matrixproduktion durch fra-1 Überexpression die knöcherne Destruktion durch arthritische Vorgänge vermindern kann. Dieser Prozess sollte allerdings therapeutisch nur lokal stattfinden, da bei systemischer Anwendung auch in anderen Organen fibrotische Prozesse zu erwarten sind

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

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

Autophagy regulates TNFα-mediated joint destruction in experimental arthritis

OBJECTIVES: Autophagy is a homeostatic process to recycle dispensable and damaged cell organelles. Dysregulation of autophagic pathways has recently been implicated in the pathogenesis of various diseases. Here, we investigated the role of autophagy during joint destruction in arthritis. METHODS: Autophagy in osteoclasts was analysed in vitro and ex vivo by transmission electron microscopy, Western blotting and immunohistochemistry for Beclin1 and Atg7. Small molecule inhibitors, LysMCre-mediated knockout of Atg7 and lentiviral overexpression of Beclin1 were used to modulate autophagy in vitro and in vivo. Osteoclast differentiation markers were quantified by real-time PCR. The extent of bone and cartilage destruction was analysed in human tumour necrosis factor α transgenic (hTNFα tg) mice after adoptive transfer with myeloid specific Atg7-deficient bone marrow. RESULTS: Autophagy was activated in osteoclasts of human rheumatoid arthritis (RA) showing increased expression of Beclin1 and Atg7. TNFα potently induced the expression of autophagy-related genes and activated autophagy in vitro and in vivo. Activation of autophagy by overexpression of Beclin1-induced osteoclastogenesis and enhanced the resorptive capacity of cultured osteoclasts, whereas pharmacologic or genetic inactivation of autophagy prevented osteoclast differentiation. Arthritic hTNFα tg mice transplanted with Atg7(fl/fl)×LysMCre(+) bone marrow cells (BMC) showed reduced numbers of osteoclasts and were protected from TNFα-induced bone erosion, proteoglycan loss and chondrocyte death. CONCLUSIONS: These findings demonstrate that autophagy is activated in RA in a TNFα-dependent manner and regulates osteoclast differentiation and bone resorption. We thus provide evidence for a central role of autophagy in joint destruction in RA

Blockade of receptor activator of nuclear factor-\u3baB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus

Hepatic insulin resistance is a driving force in the pathogenesis of type 2 diabetes mellitus (T2DM) and is tightly coupled with excessive storage of fat and the ensuing inflammation within the liver. There is compelling evidence that activation of the transcription factor nuclear factor-\u3baB (NF-\u3baB) and downstream inflammatory signaling pathways systemically and in the liver are key events in the etiology of hepatic insulin resistance and \u3b2-cell dysfunction, although the molecular mechanisms involved are incompletely understood. We here test the hypothesis that receptor activator of NF-\u3baB ligand (RANKL), a prototypic activator of NF-\u3baB, contributes to this process using both an epidemiological and experimental approach. In the prospective population-based Bruneck Study, a high serum concentration of soluble RANKL emerged as a significant (P < 0.001) and independent risk predictor of T2DM manifestation. In close agreement, systemic or hepatic blockage of RANKL signaling in genetic and nutritional mouse models of T2DM resulted in a marked improvement of hepatic insulin sensitivity and amelioration or even normalization of plasma glucose concentrations and glucose tolerance. Overall, this study provides evidence for a role of RANKL signaling in the pathogenesis of T2DM. If so, translation to the clinic may be feasible given current pharmacological strategies to lower RANKL activity to treat osteoporosis