Paola Cipriani1, Paola Di Benedetto2, Piero Ruscitti2, Daria Capece2, Francesca Zazzeroni2, Vasiliki Liakouli2, Ilenia Pantano2, Onorina Berardicurti2, Francesco Carubbi2, Gianluca Pecetti2, Stefano Turricchia2, Edoardo Alesse2, Marc Iglarz2, Roberto Giacomelli2. 1. From the Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, and the Department of Applied Clinical Sciences and Biotechnology, General Phatology Unit, University of L'Aquila, L'Aquila; Medical and Scientific Direction, Actelion Pharmaceuticals Italy, Imola, Italy; Drug Discovery Department, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland.P. Cipriani, MD, PhD; P. Di Benedetto, PhD; P. Ruscitti, MD, Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila; D. Capece, PhD; F. Zazzeroni, PhD, Department of Applied Clinical Sciences and Biotechnology, General Phatology Unit, University of L'Aquila; V. Liakouli, MD, PhD; I. Pantano, MD; O. Berardicurti, MD; F. Carubbi, MD, Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila; G. Pecetti, MD; S. Turricchia, MD, Medical and Scientific Direction, Actelion Pharmaceuticals Italy; E. Alesse, MD, PhD, Department of Applied Clinical Sciences and Biotechnology, General Pathology Unit, University of L'Aquila; M. Iglarz, PhD, Drug Discovery Department, Actelion Pharmaceuticals Ltd.; R. Giacomelli, MD, PhD, Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila. paola.cipriani@cc.univaq.it. 2. From the Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, and the Department of Applied Clinical Sciences and Biotechnology, General Phatology Unit, University of L'Aquila, L'Aquila; Medical and Scientific Direction, Actelion Pharmaceuticals Italy, Imola, Italy; Drug Discovery Department, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland.P. Cipriani, MD, PhD; P. Di Benedetto, PhD; P. Ruscitti, MD, Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila; D. Capece, PhD; F. Zazzeroni, PhD, Department of Applied Clinical Sciences and Biotechnology, General Phatology Unit, University of L'Aquila; V. Liakouli, MD, PhD; I. Pantano, MD; O. Berardicurti, MD; F. Carubbi, MD, Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila; G. Pecetti, MD; S. Turricchia, MD, Medical and Scientific Direction, Actelion Pharmaceuticals Italy; E. Alesse, MD, PhD, Department of Applied Clinical Sciences and Biotechnology, General Pathology Unit, University of L'Aquila; M. Iglarz, PhD, Drug Discovery Department, Actelion Pharmaceuticals Ltd.; R. Giacomelli, MD, PhD, Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L'Aquila.
Abstract
OBJECTIVE: High endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) levels may induce in healthy endothelial cells (EC) an endothelial-to-mesenchymal transition (EndMT). The same cytokines are associated with fibrosis development in systemic sclerosis (SSc). Although EndMT has not been definitively shown in SSc, this process, potentially induced by a stimulatory loop involving these 2 cytokines, overexpressed in this disease might contribute to fibroblast accumulation in affected tissues. Macitentan (MAC), an ET-1 receptor antagonist interfering with this loop, might prevent EndMT and fibroblast accumulation. METHODS: EC, isolated from healthy controls (HC) and patients with SSc, were treated with ET-1 and TGF-β and successively analyzed for gene and protein expressions of endothelial and mesenchymal markers, and for Sma- and Mad-related (SMAD) phosphorylation. Further, in the supernatants, we evaluated ET-1 and TGF-β production by ELISA assay. In each assay we evaluated the ability of MAC to inhibit both the TGF-β and ET-1 effects. RESULTS: We showed that both TGF-β and ET-1 treatments induced an activation of the EndMT process in SSc-EC as reported in HC cells. The ELISA assays showed a mutual TGF-β and ET-1 induction in both SSc-EC and HC-EC. A statistically significant increase of SMAD phosphorylation after treatment was observed in SSc-EC. In each assay, MAC inhibited both TGF-β and ET-1 effects. CONCLUSION: Our work is the first demonstration in literature that SSc-EC, under the synergistic effect of TGF-β and ET-1, may transdifferentiate toward myofibroblasts, thus contributing to fibroblast accumulation. MAC, interfering with this process in vitro, may offer a new potential therapeutic strategy against fibrosis.
OBJECTIVE: High endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) levels may induce in healthy endothelial cells (EC) an endothelial-to-mesenchymal transition (EndMT). The same cytokines are associated with fibrosis development in systemic sclerosis (SSc). Although EndMT has not been definitively shown in SSc, this process, potentially induced by a stimulatory loop involving these 2 cytokines, overexpressed in this disease might contribute to fibroblast accumulation in affected tissues. Macitentan (MAC), an ET-1 receptor antagonist interfering with this loop, might prevent EndMT and fibroblast accumulation. METHODS: EC, isolated from healthy controls (HC) and patients with SSc, were treated with ET-1 and TGF-β and successively analyzed for gene and protein expressions of endothelial and mesenchymal markers, and for Sma- and Mad-related (SMAD) phosphorylation. Further, in the supernatants, we evaluated ET-1 and TGF-β production by ELISA assay. In each assay we evaluated the ability of MAC to inhibit both the TGF-β and ET-1 effects. RESULTS: We showed that both TGF-β and ET-1 treatments induced an activation of the EndMT process in SSc-EC as reported in HC cells. The ELISA assays showed a mutual TGF-β and ET-1 induction in both SSc-EC and HC-EC. A statistically significant increase of SMAD phosphorylation after treatment was observed in SSc-EC. In each assay, MAC inhibited both TGF-β and ET-1 effects. CONCLUSION: Our work is the first demonstration in literature that SSc-EC, under the synergistic effect of TGF-β and ET-1, may transdifferentiate toward myofibroblasts, thus contributing to fibroblast accumulation. MAC, interfering with this process in vitro, may offer a new potential therapeutic strategy against fibrosis.
Authors: María Valero-Muñoz; Albin Oh; Elizabeth Faudoa; Rosa Bretón-Romero; Fatima El Adili; Andreea Bujor; Flora Sam Journal: Circ Heart Fail Date: 2021-08-19 Impact factor: 10.447
Authors: P Cipriani; R Giacomelli; P Di Benedetto; P Ruscitti; O Berardicurti; M Vomero; L Navarini; V Dolo Journal: Clin Exp Immunol Date: 2021-04-18 Impact factor: 5.732