Background/Purpose: Endothelin-1 (ET-1) has been shown to activate myofibroblasts and to enhance the synthesis of extracellular matrix proteins (ECM),  such as collagen type I (COL-1) and fibronectin (FN), leading to tissue fibrosis in systemic sclerosis (SSc) progression (1, 2). Macitentan is a new molecule able to antagonize ET receptors. in plasma, besides macitentan, a circulating pharmacologically active metabolite (ACT-132577) has been identifyed (3, 4). We investigated the effects of macitentan and ACT-132577 on myofibroblast activation, by evaluating α-smooth musce actin (α-SMA) expression, and COL-1 and FN synthesis together with their  gene expression, induced by ET-1 in cultured SSc and normal skin fibroblasts (Fbs). A comparison between the in vitro effects of macitentan, or ACT-132577 or bosentan was carried out. 

Methods: Cultured human skin Fbs were obtained from biopsies of clinically involved skin of 6 SSc patients (4 females and 2 males, mean age 64.8±6.9 years) and 4 age-matched healthy subjects (3 females, 1 male) during diagnostic procedures (Dermatological Clinic, University of Genoa) and after signing informed consent. Following serum starvation (18 hrs), cultured human skin Fbs at 3^rd culture passage were treated for 48 hrs with ET-1 (100nM), macitentan (concentration range from 1nM to 10μM), or ACT-132577 (concentration range from 10nM to 100µM), or bosentan (10μM) (Actelion Pharmaceuticals) in RPMI 1640 medium at 5% of fetal bovine serum. Cells were also pre-treated (1 hr) with macitentan, or ACT-132577 or bosentan, respectively, before treatment with ET-1 for 48 hrs. Untreated SSc and normal skin Fbs were used as controls. α-SMA expression was investigated by immunofluorescence (IF). COL-1 and FN synthesis and gene expressions were investigated by immunocytochemistry (ICC) and quantitative real time-polymerase chain reaction (qRT-PCR). Statistical analysis was performed by Mann-Whitney non-parametric t test.

Results: Macitentan and ACT-132577, at the concentration of 10µM, contrasted the increase in α-SMA expression induced by ET-1 in cultured human SSc skin Fbs, displaying same effects already showed by bosentan. Macitentan and ACT-132577 significantly antagonized the increase in COL-1 and FN synthesis induced by ET-1 (p<0.01; p<0.01 for macitentan; p<0.05; p<0.05 for ACT-132577 vs. only ET-1-treated cells). Bosentan significantly antagonized the ET-1 mediated increase in COL-1 and FN synthesis in SSc skin Fbs vs. only ET-1-treated cells (p<0.01; p<0.05). Data were confirmed by qRT-PCR. Same results were obtained in normal skin Fbs. Not significant differences were observed between macitentan, ACT-132577 and bosentan effects.

Conclusion: Macitentan and its active metabolite might down regulate the myofibroblast activation and the increased ECM protein synthesis induced by ET-1 in cultured human SSc skin Fbs at the same concentrations of bosentan.

References. 1.Bhattacharyya S. et al. Nat Rev Rheumatol. 2012;8:42-51. 2.Abraham D. et al. Arthrit Res Ther. 2007;9:S2. 3.Iglarz M. et al. J Pharmacol Exp Ther 2008;327:736-45. 4.Sidharta P. et al. Eur J Clin Pharmacol 2011;67:977-84.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/effects-of-macitentan-and-its-active-metabolite-in-modulating-extracellular-matrix-synthesis-in-cultured-human-systemic-sclerosis-and-normal-skin-fibroblasts/