Background and Objectives The early events involved in the pathogenesis of many connective tissue diseases, such as systemic sclerosis (SSc) include endothelial/microvascular damage and activation of myofibroblasts responsible for excessive deposition of extracellular matrix components (i.e. collagens) (1-4). Recent studies showed that myofibroblasts may originate from the endothelial-to-mesenchymal transition (EndoMT) process (5).
To investigate the possible effects of endothelin-1 (ET-1) and its receptor antagonist (ETA/BRA) on the EndoMT in cultured human dermal microvascular endothelial cells (HMVECs).
Materials and Methods HMVECs (Lonza Clonetic, Switzerland) were treated with or without ET-1 (100 nM, EnzoLife Science, UK) for 6 days, in accordance with recent studies (6,7). Cells were also pre-treated (1 hr) with ETA/BRA (bosentan, 10µM) before being treated with ET-1. The expression levels of fibroblast specific protein-1 (S100A4, S100 calcium binding protein A4) and α-smooth muscle actin (α-SMA), both markers of the myofibroblast phenotype, and vascular endothelial growth factor receptor (VEGFR) as well as platelet endothelial cell adhesion molecule (PECAM-1 or CD31), markers of the endothelial phenotype, were investigated by quantitative real time-polimerase chain reaction (qRT-PCR). S100A4 expression was also evaluated by immunocytochemistry using primary antibody to human S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas) whereas α-SMA and CD31 expressions were investigated by immunofluorescence using primary antibodies to human α-SMA (dilution 1:50, Dako Cytomation, Denmark) and CD31 (dilution 1:200, CellSignaling Technology, Denver, USA). Data were obtained from 5 different experiments. Statistical analysis was performed using the Mann-Whitney non-parametric t-test and the p-values lower that 0.05 were considered statistically significant.
Results ET-1 induced a statistically significant increase in S100A4 and α-SMA expressions (p = 0.01 vs. untreated cells). Interestingly, ET-1 was able to reduce the VEGFR expression in a statistically significant manner (p = 0.03 vs. untreated cells) without modulating CD31 expression. ETA/BRA contrasted the ET-1-mediated increase in S100A4 expression and, less efficiently, the increase in α-SMA expression. Moreover, ETA/BRA was able to partially contrast the decrease in VEGFR expression induced by ET-1. All these data were obtained by qRT-PCR and confirmed by immunocytochemistry for S100A4 and by immunofluorescence for α-SMA and CD31.
Conclusions Results indicate that ET-1 seem to activate the EndoMT, by inducing the expression of markers of mesenchymal/myofibroblast phenotype (S100A4 and α-SMA), and at the same time, by reducing the expression of endothelial phenotype marker (VEGFR) in cultured human dermal microvascular endothelial cells (8). ETA/BRA might contrast the ET-1-mediated induction of EndoMT.
Wynn TA et al. Nat Med 2012;18:1028-40.
Chaudury V et al. J Cutan Pathol 2007;34:147-53.
Jimenez SA. ISRN Rheumatol 2013 Sep 23;2013:835948.
Cutolo M et al. Nature Rev Rheumat 2010;6:578–87.
Zeisberg EM et al. Nat Med 2007;13:952-61.
Kitao A et al. Am J Pathol 2009;175:616-26.
Wydyantoro B et al. Circulation 2010;121:2407-18.
Soldano S et al. Arthrit & Rheum 2012;64(Supplement).
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.