Introduction: Systemic sclerosis (SSc) is a complex and not fully understood autoimmune disease associated with fibrosis of multiple organs. The main effector cells, the myofibroblasts, are collagen-producing cells derived from the activation of resting fibroblasts. This process is regulated by a complex repertoire of profibrotic cytokines, and among them transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) play a major role. In this paper we show that TGF-β and ET-1 receptors co-operate in myofibroblast activation, and macitentan, an ET-1 receptor antagonist binding ET-1 receptors, might interfere with both TGF-β and ET-1 pathways, preventing myofibroblast differentiation.
Methods: Fibroblasts isolated from healthy controls and SSc patients were treated with TGF-β and ET-1 and successively analyzed for alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression and for the Sma and Mad Related (SMAD) phosphorylation. We further tested the ability of macitentan to interfere with these process. Furthermore, we silenced ET-1 and endothelin-1 receptor A expression and evaluated the formation of an ET-1/TGF-β receptor complex by immunoprecitation assay.
Results: We showed myofibroblast activation in SSc fibroblasts assessing the expression of α-SMA and Col1A1, after stimulation with TGF-β and ET-1. Macitentan interfered with both ET-1- and TGF-β-induced fibroblast activation. To explain this unexpected inhibitory effect of macitentan on TGF-β activity, we silenced ET-1 expression on SSc fibroblasts and co-immunoprecipitated these two receptors, showing the formation of an ET-1/TGF-β receptor complex.
Conclusions: During SSc, ET-1 produced by activated endothelia contributes to myofibroblast activation using TGF-β machinery via an ET-1/TGF-β receptor complex. Macitentan interferes with the profibrotic action of TGF-β, blocking the ET-1 receptor portion of the ET-1/TGF-β receptor complex.