OP0296 Comparison of Bosentan and Macitentan Effects on Endothelial-To-Mesenchymal Transition Process Induced by ET-1 in Cultured Human Microvascular Endothelial Cells
Authors: S. Soldano, P. Montagna, A.C. Trombetta, R. Brizzolara, A. Sulli, C. Pizzorni, S. Paolino, M. Cutolo
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> In systemic sclerosis (SSc), the damage of microvascular endothelial cells (ECs) and their phenotype alteration are considered early events responsible for myofibroblast activation and the development of fibrosis (1). Endothelin (ET)-1 and transforming growth factor (TGF)β1 induce ECs to acquire features of extracellular matrix-producing myofibroblasts through the induction of the endothelial-to-mesenchymal transition process (EndoMT) (2). <h3>Objectives</h3> To compare the ability of ET-1 receptor antagonists (ET<sub>A/B</sub>RAs), bosentan and macitentan, to support the <i>in vitro</i> endothelial damage repair and to contrast the EndoMT induced by ET-1 in cultured human microvascular ECs (HMVECs). <h3>Methods</h3> HMVECs, at 3rd culture passage, were grown in endothelial cell medium (EGM2-MV). A part of these cultured HMVECs was untreated or treated with either ET-1 (100nM) or TGFβ1 (10ng/mL) for 6 days. Another part of cultured cells was treated for 1hr with bosentan (10μM) or macitentan (1μM) and then stimulated with ET-1 for 6 days (2,3). At the end of treatments, cultured HMVECs were scraped with a 200μL tip to create a linear injury, whose area was determined at baseline and after 24hrs of culture in EGM2-MV by ImageJ analysis software, to evaluate the <i>in vitro</i> endothelial damage repair. The gene expression of specific markers of the EndoMT, such as α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), fibronectin (FN), platelet endothelial cell adhesion molecule (CD31), and vascular endothelial (VE)-cadherin was evaluated by quantitative real time polymerase chain reaction (qRT-PCR). Statistical analysis was performed by Man-Whitney non-parametric <i>U</i>-test. <h3>Results</h3> ET-1 treated HMVECs showed a significantly lower ability to promote the <i>in vitro</i> endothelial damage repair compared to untreated cells (p&lt;0.05). A similar effect was observed in TGFβ1-treated cells (p&lt;0.01). On the contrary, cultured HMVECs treated with ET<sub>A/B</sub>RAs significantly improved the <i>in vitro</i> endothelial damage repair compared to ET-1-treated cells (p&lt;0.05 for bosentan and macitentan). ET-1 significantly up-regulated the gene expression of α-SMA, S100A4, and FN (p&lt;0.0001 for all proteins) and down-regulated that of CD31 and VE-cadherin compared to untreated cells (p&lt;0.05, p&lt;0.01) as observed by qRT-PCR. Bosentan contrasted the up-regulation of α-SMA as well as significantly contrasted the up-regulation of S100A4 and FN induced by ET-1 (p&lt;0.01, p&lt;0.05 vs. ET-1 treated cells). Macitentan significantly reduced the ET-1-induced increase in α-SMA, S100A4, and FN (p&lt;0.01; p&lt;0.01; p&lt;0.05), as well as it antagonized the down-regulation of CD31 and VE-cadherin compared to ET-1 treated cells (p&lt;0.05 for both proteins), as observed by qRT-PCR. <h3>Conclusions</h3> Both macitentan (at very low concentration) and bosentan seem to allow <i>in vitro</i> endothelial damage repair and to efficiently contrast the EndoMT induced by ET-1 in cultured HMVECs. Therefore, ET<sub>A/B</sub> receptor antagonism might contrast the damage of ECs and their phenotype transition that seems to contribute the early phases of the fibrotic process in SSc. <h3>References</h3> Denton CP. Clin Exp Rheumatol.2015;33 (Suppl 92):S3–7. Cipriani P et al. J Rheumatol.2015;42:1808–16. Cutolo M et al. J Rheumatol.2015;42:456–63. <h3>Disclosure of Interest</h3> None declared