title: Macitentan inhibits the transforming growth factor-β profibrotic action, blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts.
abstract: BACKGROUND
  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.
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. paola.cipriani@cc.univaq.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Cipriani
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. paola_dibenedetto@libero.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Di Benedetto
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. pierorus@hotmail.it.
authors.forename: Piero
authors.initials: P
authors.lastname: Ruscitti
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, University of L'Aquila, Coppito 2, 67100, L'Aquila, Italy. danielaverzella@tiscali.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Verzella
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, University of L'Aquila, Coppito 2, 67100, L'Aquila, Italy. faustafischietti@libero.it.
authors.forename: Mariafausta
authors.initials: M
authors.lastname: Fischietti
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, University of L'Aquila, Coppito 2, 67100, L'Aquila, Italy. francesca.zazzeroni@univaq.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Zazzeroni
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. vasiliki_liakouli@yahoo.it.
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Liakouli
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. francesco.carubbi@graduate.univaq.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Carubbi
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. berardicurtio@libero.it.
authors.forename: Onorina
authors.initials: O
authors.lastname: Berardicurti
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, University of L'Aquila, Coppito 2, 67100, L'Aquila, Italy. alesse@univaq.it.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Alesse
authors.affiliation: Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila, Delta 6 Building, Via dell'Ospedale, 67100, L'Aquila, Italy. roberto.giacomelli@cc.univaq.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Giacomelli
mesh_terms: D000199:Actins / Q000378:metabolism; D000328:Adult; D015153:Blotting, Western; D024042:Collagen Type I / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D020405:Dermis / Q000473:pathology; D065130:Endothelin A Receptor Antagonists / Q000494:pharmacology; D019332:Endothelin-1 / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D046912:Multiprotein Complexes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology*; D034622:RNA Interference; D044022:Receptor, Endothelin A / Q000235:genetics / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects; D055815:Young Adult
chemical_list: D000199:Actins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D065130:Endothelin A Receptor Antagonists; D019332:Endothelin-1; D046912:Multiprotein Complexes; D011743:Pyrimidines; D044022:Receptor, Endothelin A; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D013449:Sulfonamides; D053773:Transforming Growth Factor beta1; C533860:macitentan