Stefano Soldano1, Sabrina Paolino1, Carmen Pizzorni1, Amelia Chiara Trombetta1, Paola Montagna1, Renata Brizzolara1, Claudio Corallo2, Nicola Giordano2, Alberto Sulli1, Maurizio Cutolo3. 1. Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, IRCCS AOU San Martino Hospital, Genoa, Italy. 2. Department of Medicine, Surgery and Neurosciences, Scleroderma Unit, University of Siena, Italy. 3. Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, IRCCS AOU San Martino Hospital, Genoa, Italy. mcutolo@unige.it.
Abstract
OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor β1 (TGFβ1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10μM) or macitentan (1μM, 10μM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFβ1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFβ1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.
OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor β1 (TGFβ1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10μM) or macitentan (1μM, 10μM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFβ1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFβ1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.
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