Background Recent studies demonstrated that the morphogenic pathways Notch, hedgehog (Hh) and Wnt are activated in systemic sclerosis and that inhibition of these morphogen pathways exerted potent anti-fibrotic effects in different pre-clinical models. As Notch and Wnt signaling are crucial for the proliferation especially of intestinal stem cells and Hh-ligands drive stem cell differentiation, interfering with morphogen pathways might be complicated by inhibitory effects on stem cell regeneration. Thus, high-dose therapies completely abrogating Notch, Hh and canonical Wnt signaling might be limited by adverse effects on stem cells
Objectives (I) To assess the anti-fibrotic effects of combined inhibition of Notch/Hh or Wnt/Hh-signaling and (II) to show the improved tolerability of the combination therapy with low doses of inhibitors in particular with respect to intestinal stem cells.
Methods Bleomycin was applied as described by . Mice were feed by gavages with the Notch-inhibitor DAPT (1.5 mg/kg/d), the Hh-inhibitor cyclopamine (5mg/kg/d)or injected i.p. with the Wnt-inhibitor ICG-001 (2 mg/kg/d). The applied dosages represent low-dose treatment. The combined treatment was administered accordingly. Skin thickness, myofibroblast counts and hydroxyproline contents of the skin were assessed as described by . Lgr5+ intestinal stem cells in sections of the colon- and the small intestine were detected by immunohistochemistry and counted in a blinded manner.
Results Low-dose single inhibition of all the three morphogen pathways, Notch, Hh and Wnt, efficiently reduced bleomycin-induced dermal fibrosis with reduced dermal thickness, decreased myofibroblast counts and reduced hydroxyproline content. Combined inhibition of the Notch and Hh-signaling or Notch and Wnt-signaling were superior to single low-dose therapy. In the mice treated with the combination of DAPT/cyclopamine or DAPT/ICG-001, dermal thickness was reduced up to 75% or 60%, numbers of myofibroblasts were decreased by 80% or 70% and the hydroxyproline content by 80% or 65% of control mice (bleomycin/vehicle), respectively.Of note, combination therapies were well tolerated and did not cause weight loss. Consistently, combined inhibition of either Notch/Hh- or of Wnt/Hh-signaling did not significantly reduce numbers of Lgr5+ positive stem cells in the colon and the small intestine compared to sham treated mice.
Conclusions Combined partial inhibition of morphogen-signaling with low doses of inhibition more potently prevents the development of bleomycin-induced skin fibrosis than single therapies. Combination therapies were well tolerated and did not deplete Lgr5+ intestinal stem cells. Combined targeting of different morphogenic pathways with low doses of inhibitors may not only reduce side-effects, but also increase the anti-fibrotic effects.
Dees C, Zerr P, Tomcik M, et al. Inhibition of Notch signaling prevents experimental fibrosis and induces regression of established fibrosis. Arthritis Rheum 2011;63:1396-1404.
Disclosure of Interest None Declared
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