BackgroundNintedanib is a multi-tyrosine kinase inhibitor that targets several pro-fibrotic pathways such as platelet derived growth factor receptor (PDGFR)-α and β, fibroblast growth factor receptor (FGFR)-1, 2, 3, vascular endothelial growth factor receptor (VEGFR)-1, 2, 3 and Src-family kinases Src. Nintedanib thus offers potential to simultaneously target multiple pro-fibrotic pathways in SSc. Nintedanib has been shown to slow disease progression in idiopathic pulmonary fibrosis in two replicate phase III clinical trials and has recently been approved by the FDA for the treatment of idiopathic pulmonary fibrosis (IPF).ObjectivesThe aim of this study was to analyze the anti-fibrotic effects of nintedanib in preclinical models of systemic sclerosis (SSc).MethodsThe effects of nintedanib on migration, proliferation, myofibroblast differentiation and release of extracellular matrix of dermal fibroblasts were analyzed by MTT- and scratch assays, stress fiber staining, qPCR and SirCol assays. The anti-fibrotic effects of nintedanib were evaluated in bleomycin-induced skin fibrosis, in murine sclerodermatous chronic graft-versus host disease and in tight-skin-1 mice at 30 mg/kg qd, 60 mg/kg qd and 50 mg/kg bid.ResultsTo assay the effects of nintedanib on migration and proliferation of fibroblasts, scratch assays were performed. Nintedanib delayed the PDGF- and TGFβ-induced closure of the scratch in a concentration-dependent manner. Nintedanib also inhibited proliferation of fibroblasts in MTT assays. Moreover, nintedanib dose-dependently reduced the mRNA levels of Col 1a1, Col 1a2 and fibronectin-1 as well as the release of collagen protein into the supernatant and decreased the basal levels of αSMA and of stress fibers in cultured fibroblasts from SSc patients in the absence of exogenous stimuli as well as upon incubation with TGFβ or PDGF. Nintedanib exerted potent anti-fibrotic effects in a variety of complementary mouse models. Treatment with nintedanib dose-dependently ameliorated dermal thickening, myofibroblast differentiation and collagen deposition in bleomycin-induced skin fibrosis, as a model of localized, inflammation-driven model of SSc, in a model of sclerodermatous chronic graft-versus host disease with systemic fibrotic changes as well as in tight-skin-1 mice resembling later stages of SSc with endogenous activation of fibroblasts. Maximal anti-fibrotic effects were observed with nintedanib in pharmacologically relevant doses of 50 mg/kg bid. Of note, treatment with nintedanib was effective in preventive as well as in therapeutic settings.ConclusionsNintedanib effectively inhibits the endogenous as well as cytokine-induced activation of SSc fibroblasts and that nintedanib exerts potent anti-fibrotic effects in complementary mouse models of SSc. The potent anti-fibrotic effects of nintedanib provide a scientific rational for clinical trials with nintedanib in SSc.Disclosure of InterestNone declared
BackgroundDipeptidyl-peptidase-4 (DPP4/CD26) has been recently identified as a marker for a special fibroblast lineage responsible for the tissue remodeling during physiological wound healing. Fibrotic disease may be considered as a consequence of persistent, exaggerated and uncontrolled tissue repair processes. Systemic sclerosis (SSc) is associated with the highest mortality among the connective tissue disorders and effective antifibrotic therapies are still lacking. DPP4 inhibitors are already used in treatment of diabetes.ObjectivesThe aim of the study was to characterize the DPP4 positive cells, investigate the expression of DPP4 in SSc skin and to evaluate the antifibrotic effect of DPP4 inhibitors in preclinical models of systemic sclerosis.MethodsMouse fibroblasts were isolated and DPP4 positive cells characterized by fluorescence activating cell sorting. Expression of DPP4/CD26 in human and murine skin was analyzed by immunofluorescence. DPP4 inhibitors were tested in two different concentrations administered orally (Sitagliptin 3mg/kg/d and 10mg/kg/d, Vildagliptin 1,5mg/kg/d and 15mg/kg/d) in bleomycin induced skin fibrosis and in sclerodermatous chronic graft-versus-host disease mouse model (cGvHD). The antifibrotic effect on skin was assessed by hydroxyproline assay, alpha smooth muscle cells quantification and measuring the dermal thickness. Inflammatory infiltrate was assessed by CD45 immunofluorescence staining.ResultsWe have demonstrated that DPP4/CD26 positive cells are a unique population of cells implicated in fibrosis. DPP4/CD26 positive cells were increased not only in experimental fibrosis, but also in skin biopsies from SSc patients as compared to healthy volunteers. Treatment with DPP4 inhibitor reduced dermal thickness in both mouse models (p<0.05). The differentiation of resting fibroblasts into myofibroblasts was also significantly decreased (p<0.05) in all treatment groups. Hydroxyproline content of the skin diminished by 40% in comparison with NaCl injected mice or syngeneic transplanted mice. Moreover, DDP4 inhibitors reduced the inflammatory infiltrate in a dose dependent manner in the bleomycin injected skin.ConclusionsDPP4/CD26 identifies a subpopulation of fibrosis-promoting fibroblasts that plays a key role in the pathogenesis of fibrosis in SSc. Moreover, inhibitors of DPP4 show a significant antifibrotic effect in several mouse models of SSc in well tolerated doses. These results may have direct clinical implications as DPP4 inhibitors are already in clinical use for diabetes.AcknowledgementAS received an EULAR Scientific Training Bursary (2014) and was an Articulum Fellow (2015).Disclosure of InterestNone declared
BackgroundHedgehog signalling plays a key-role in the pathogenesis of fibrosis in SSc. Besides the canonical hedgehog pathway with activation of Gli transcription factors via ligand-binding to the cell surface receptors, Gli can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways).ObjectivesThe aim of the present study was to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct Gli-inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.MethodsThe Gli-inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the smo-inhibitor vismodegib was used to selectively target canonical hedgehog signalling. The effects of inhibition of Gli2 on TGF-β signalling were analysed in cultured fibroblasts, in the mouse model of bleomycin-induced pulmonary fibrosis and in a model of fibrosis induced by overexpression of a constitutively active TGF-b receptor I (TBRI) in vivo.ResultsTGF-β upregulated the expression of Gli2 in cultured fibroblasts and in murine skin by direct transcriptional regulation. TGF-β as well as its downstream mediator phosphorylated Smad3 were co-expressed with Gli-2 in the skin of SSc patients. Consistent a novel role of Gli2 as a downstream mediator of TGF-β in fibroblasts, inhibition of Gli2 by GANT-61 ameliorated the stimulatory effects of TGF-β on fibroblasts in vitro and in vivo. Treatment with GANT-61 downregulated the mRNA levels of prototypical TGF-b targeted genes, whereas inhibition of canonical hedgehog signalling had no effects. Moreover, GANT-61 reduced the mRNA levels of col1a2 and the release of collagen protein. GANT-61 also blocked TGF-b-induced myofibroblast differentiation and decreased the levels of a-smooth muscle actin and the formation of stress fibres. Moreover, GANT-61 ameliorated experimental fibrosis of the skin and lungs more efficiently as compared to vismodegib. In the model of TBRI-induced fibrosis, mice treated with GANT-61 showed reduced dermal thickening, lower myofibroblast counts and decreased the hydroxyproline content. In contrast, vismodegib had no anti-fibrotic effect in this model. GANT-61 also exerted potent anti-fibrotic effects in the model of bleomycin-induced pulmonary fibrosis and induced regression of pre-established fibrosis. In both models, GANT-61 did not only reduce levels of the hedgehog target genes, but also the levels of the TGF-b target genes PAI-1, CTGF and Smad7, thus confirming inhibition of TGF-b signalling upon targeting of Gli2.ConclusionsWe characterize Gli2 as a novel intracellular mediator of the pro-fibrotic effects of TGF-b. Pharmacologic inhibition of Gli2 targets canonical as well as non-canonical hedgehog signalling and ameliorates the pro-fibrotic effects of TGF-b. The potent anti-fibrotic effects of Gli2 inhibitors on cultured fibroblasts, dermal and pulmonary fibrosis and availability of Gli2 inhibitors for clinical use encourage additional studies to further explore the ...
Background Casein kinase-2 (CK2)is a ubiquitous, highly conserved serine/threonine kinase. CK2 presents as a tetramer composed of 2 catalytic subunits (α orα’) and 2β regulatory subunits, whichareessential for cell viability. Meanwhile, JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. Recently, we have shown thattargeting of JAK2 might be an interesting molecular approach for the treatment of systemic sclerosis (SSc). However, the role of CK2 in SSc and the functional relationship between CK2 and JAK-STAT signaling in SSc have not been established. Objectives The purpose of the study was to characterize whether CK2 contributes to the pathologic activation of fibroblasts in patients with SSc, to evaluate the anti-fibrotic potential of CK2 inhibition for the treatment of SSc, and to investigate the CK2-JAK2-STAT3 signaling interactions in fibrosis. Methods Activation of CK2, JAK2, and STAT3 in human skin and in experimental fibrosis were determined by immunohistochemical analysis. CK2 signaling was inhibited by the selective CK2 inhibitor 4, 5, 6, 7-Tetrabromobenzotriazole (TBB). The mouse models of bleomycin-induced and TGF-β receptor I (TBR)-induced dermal fibrosis were used to evaluate the anti-fibrotic potential of specific CK2 inhibition in vivo. Results Increased expression of CK2 was detected by immunohistochemistry in skin sections of SSc patients, particularly in fibroblasts. Inhibition of CK2 by TBB in cultured fibroblasts completely abrogated the stimulatory effects of TGFβ on collagen release (p<0.05). After TBB treatment, stress fiber formation and α-smooth muscle actin (α-SMA) expression in TGFβ-stimulated fibroblasts were significantly reduced by 97% (p=0.0064) and 69% (p=0.0280). Besides reduced fibroblast activation, western blot analyses showed almost complete normalization of phosphorylated JAK2 (pJAK2) levels in the cytoplasm and of phosphorylated STAT3 (pSTAT3) levels in the nucleus of TGFβ-treated fibroblasts upon pre-incubation with TBB (p=0.0004 and p=0.0214). In addition, treatment with TBB effectively prevented bleomycin-induced fibrosis in mice with decreased dermal thickness by up to 70% (p<0.0001) and efficient reductions in myofibroblast counts by up to 68% (p=0.0002). TBR-induced fibrosis in mice was strongly ameliorated by TBB with efficient reductions of dermal thickening by 75% (p<0.0001). Myofibroblast counts and hydroxyproline content also decreased by 59% and 40% (p<0.0001 and p=0.0193), respectively. In both murine models, we observed reduced pJAK2 and pSTAT3 expression as analyzed by immunohistochemistry. Conclusions We demonstrate that CK2 is activated in SSc and prove that inhibition of CK2 reduces canonical TGF-β signaling and prevents experimental fibrosis in different preclinical models. Considering the potent anti-fibrotic effects of CK2 inhibition, our study might have direct translational implications. These data provide first evidence that targeting CK2 may be a novel therapeutic approach for fibrotic dise...
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