Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage.

Purpose Tissue fibrosis caused by pathological activation of fibroblasts with increased synthesis of extracellular matrix components is a major hallmark of systemic sclerosis (SSc). Notch signalling regulates tissue differentiation and pathologic activation of Notch signaling has been implicated in the pathogenesis of various malignancies. The aim of the present study was to investigate the role of Notch signaling in SSc and to evaluate the therapeutic potential of Notch inhibition for the treatment of fibrosis. Methods Activation of the Notch pathways was analyzed by staining for the Notch intracellular domain (NICD) and quantification of the mRNA levels hes-1. Notch signaling was inhibited in the mouse model of bleomycin induced dermal fibrosis and in tight-skin-1 mice by the γ-secretase inhibitor DAPT and by overexpression of a Notch-1 antisense construct. Results Notch signaling is activated in SSc in vivo with accumulation of the NICD and increased transcription of the target gene hes-1. Overexpression of a Notch antisense construct prevented bleomycin-induced fibrosis and hypodermal thickening in tight-skin 1 mice. Potent anti-fibrotic effects were also obtained by treatment with DAPT. In addition to prevention of fibrosis, targeting Notch signaling resulted in almost complete regression of pre-established experimental fibrosis. Conclusion We demonstrate that pharmacologic as well as genetic inhibition of Notch signaling exerts potent anti-fibrotic effects in different murine models of SSc. These findings might have direct translational implications because different inhibitors of the γ-secretase complex are available and yielded promising results in cancer trials.

No abstract

Objective. Fra-2 belongs to the activator protein 1 family of transcription factors. Mice transgenic for Fra-2 develop a systemic fibrotic disease with vascular manifestations similar to those of systemic sclerosis (SSc). The aim of the present study was to investigate whether Fra-2 plays a role in the pathogenesis of SSc and to identify the molecular mechanisms by which Fra-2 induces fibrosis.Methods. Dermal thickness and the number of myofibroblasts were determined in skin sections from Fra-2-transgenic and wild-type mice. The expression of Fra-2 in SSc patients and in animal models of SSc was analyzed by real-time polymerase chain reaction and immunohistochemistry. Fra-2, transforming growth factor ␤ (TGF␤), and ERK signaling in SSc fibroblasts were inhibited using small interfering RNA, neutralizing antibodies, and small-molecule inhibitors.Results. Fra-2-transgenic mice developed a skin fibrosis with increases in dermal thickness and increased myofibroblast differentiation starting at age 12 weeks. The expression of Fra-2 was up-regulated in SSc patients and in different mouse models of SSc. Stimulation with TGF␤ and platelet-derived growth factor (PDGF) significantly increased the expression of Fra-2 in SSc fibroblasts and induced DNA binding of Fra-2 in an ERK-dependent manner. Knockdown of Fra-2 potently reduced the stimulatory effects of TGF␤ and PDGF and decreased the release of collagen from SSc fibroblasts.Conclusion. We demonstrate that Fra-2 is overexpressed in SSc and acts as a novel downstream mediator of the profibrotic effects of TGF␤ and PDGF. Since transgenic overexpression of Fra-2 causes not only fibrosis but also vascular disease, Fra-2 might be an interesting novel candidate for molecular-targeted therapies for SSc.

OBJECTIVE: Systemic sclerosis-associated pulmonary arterial hypertension differs from idiopathic pulmonary arterial hypertension with respect to histopathology, treatment responses and survival. Medical progress on PAH is hampered by the lack of human biosamples and suitable animal models. In this study, the authors evaluated fos-related antigen 2 (Fra-2) transgenic mice as a novel model for systemic sclerosis-associated pulmonary arterial hypertension.METHODS: Lung sections of Fra-2 transgenic (n=12) and wild-type mice (n=6) were analysed at 16 weeks by histology using Dana Point criteria. Cellular and molecular key players were assessed by immunohistochemistry. To test the model's sensitivity to change over treatment, a subgroup of Fra-2 transgenic mice (n=6) was treated with the tyrosine kinase inhibitor nilotinib twice daily 37.5 mg orally from 8 weeks of age.RESULTS: Fra-2 transgenic mice developed severe vascular remodelling of pulmonary arteries and non-specific interstitial pneumonia-like interstitial lung disease resembling human systemic sclerosis-associated pulmonary hypertension. Histological features typical for systemic sclerosis-associated pulmonary arterial hypertension, such as intimal thickening with concentric laminar lesions, medial hypertrophy, perivascular inflammatory infiltrates, adventitial fibrosis, but not pulmonary occlusive venopathy were frequently detected. Platelet-derived growth factor signalling pathways were activated in pulmonary vessels of Fra-2 transgenic compared with wild-type mice. Since treatment with nilotinib strongly prevented the development of proliferative vasculopathy and lung fibrosis, the model proved to be sensitive to treatment.CONCLUSIONS: This study suggests that Fra-2 transgenic mice as an animal model of systemic sclerosis-associated pulmonary arterial hypertension display main characteristic features of the human disease. It therefore allows studying pathophysiological aspects and might serve as a preclinical model for interventional proof-of-concept studies. MethodsLung sections of Fra-2 transgenic (n=12) and wildtype mice (n=6) were analyzed at 16 weeks by histology using Dana Point criteria. Cellular and molecular key players were assessed by immunohistochemistry. To test the model's sensitivity to change over treatment, a subgroup of Fra-2 transgenic mice (n=6) was treated with the tyrosine kinase inhibitor nilotinib at 2x 37.5mg/d p. o. from 8 weeks of age. ResultsFra-2 transgenic mice developed severe vascular remodeling of pulmonary arteries and nonspecific interstitial pneumonia-like interstitial lung disease resembling human systemic sclerosis-associated pulmonary hypertension. Histological features typical for systemic sclerosis-associated pulmonary arterial hypertension such as intimal thickening with concentric laminar lesions, medial hypertrophy, perivascular inflammatory infiltrates, adventitial fibrosis, but not pulmonary occlusive venopathy were frequently detected. Platelet-derived growth factor signaling pathways were activate...

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 antifi brotic potency of sGC stimulators. Methods The effect of the sGC stimulator BAY 41-2272 on the release of collagen from dermal fi broblasts was examined. The antifi brotic effects of BAY 41-2272 on prevention and regression of fi brosis in bleomycin-induced dermal fi brosis 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 dosedependently inhibited collagen release in dermal fi broblasts from patients with SSc and healthy individuals. Furthermore, BAY 41-2272 stopped the development of bleomycin-induced dermal fi brosis and skin fi brosis in Tsk-1 mice, preventing dermal and hypodermal thickening, reducing the numbers of myofi broblasts and reducing the hydroxyproline content. In addition, BAY 41-2272 was highly effective in the treatment of established fi brosis in the modifi ed models of bleomycin-induced skin fi brosis and Tsk-1 mice. Treatment with sGC stimulators was well tolerated. Relevant antifi brotic doses of BAY 41-2272 did not affect systemic blood pressure and heart rate in mice.Conclusions These fi ndings demonstrate potent antifi brotic 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 fi brosis and vascular disease in SSc.

Objective. Cannabinoids are derivates of the marijuana component ⌬ 9 -tetrahydrocannabinol that exert their effects on mesenchymal cells and immune cells via CB1 and CB2 receptors. The aim of the present study was to evaluate the role of CB1 in systemic sclerosis.Methods. CB1-deficient (CB1 ؊/؊ ) mice and wildtype littermates (CB1 ؉/؉ mice) were injected with bleomycin. CB1 signaling was activated in vivo with the selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA). Bone marrow transplantation experiments were performed to investigate whether the phenotype of CB1 ؊/؊ mice was mediated by leukocytes or mesenchymal cells. The role of CB1 was also investigated in the TSK-1 mouse model.Results. CB1 ؊/؊ mice were protected from bleomycin-induced dermal fibrosis, with reduced dermal thickening, hydroxyproline content, and myofibroblast counts. Inactivation of CB1 decreased the number of infiltrating T cells and macrophages in lesional skin.In contrast, activation of CB1 with ACEA increased leukocyte infiltration and enhanced the fibrotic response to bleomycin. The phenotype of CB1 ؊/؊ mice was mimicked by transplantation of CB1 ؊/؊ mouse bone marrow into CB1 ؉/؉ mice, demonstrating that CB1 exerts its profibrotic effects indirectly by regulating leukocyte infiltration. Consistently, knockdown of CB1 did not prevent fibrosis in the inflammationindependent TSK-1 mouse model.Conclusion. We demonstrate that the cannabinoid receptor CB1 is crucial for leukocyte infiltration and secondary fibroblast activation and that inactivation of CB1 exerts potent antifibrotic effects in inflammationdriven models of fibrosis.

ObjectivesThe hallmark of systemic sclerosis (SSc) is the accumulation of extracellular matrix proteins by pathologically activated fibroblasts. This study analysed the antifibrotic effects of the selective c-Jun N-terminal kinase (JNK) inhibitor, CC-930, which recently entered first clinical trials as a novel antifibrotic approach.MethodsPhosphorylated c-Jun was detected by western blot and immunohistochemistry. The model of bleomycin-induced dermal fibrosis and the tight skin 1 (TSK1) mouse model were used to investigate the effects of CC-930 on the prevention of experimental fibrosis. The potential of CC-930 to induce regression of fibrosis was assessed in a modified model of established fibrosis.ResultsTransforming growth factor beta (TGFβ) and platelet-derived growth factor (PDGF) activate JNK and stimulate the phosphorylation of its downstream target c-Jun. Incubation with CC-930 prevented the phosphorylation of c-Jun and reduced the stimulatory levels of these cytokines on the release of collagen. Inhibition of JNK prevented dermal thickening, myofibroblast differentiation and the accumulation of collagen in a dose-dependent manner in mice challenged with bleomycin and in TSK1 mice. In addition to the prevention of fibrosis, treatment with pharmacologically relevant doses of CC-930 also induced regression of established experimental fibrosis.ConclusionsThese data identify JNK as a downstream mediator of the pro-fibrotic effects of of TGFβ and PDGF in SSc fibroblasts. Selective inhibition of JNK by CC-930 exerted potent antifibrotic effects in vitro and in different models in vivo. JNK might thus be a novel molecular target for the treatment of fibrosis in SSc.