Background Endothelial/microvascular damage and myofibroblast activation are considered early events in the development of connective tissue diseases, such as systemic sclerosis (SSc) (1). Recent studies showed that vascular endothelial cells may aquire matrix-producing myofibroblast features through the endothelial-to-mesenchymal transition (EndoMT) process (2,3).
Objectives To investigate the possible direct effect of ET-1 in inducing the EndoMT in cultured human dermal microvascular endothelial cells (HMVECs).
Methods HMVECs (Lonza Clonetic, Switzerland) were cultured in endothelial cell growth medium (EGM2-MV, Lonza Clonetic) and treated with or without ET-1 (100nM, EnzoLife Science, UK) for 6 days, according to recent studies (4, 5). For the experiments, the cells were used between the second and fourth passages. Gene expression levels of α-smooth muscle actin (α-SMA) and fibroblast specific protein-1 (S100 calcium binding protein A4, S100A4), two markers of the myofibroblast phenotype, as well as plateled endothelial cell adhesion molecule (PECAM-1 or CD31), a marker of the endothelial phenotype, were evaluated by quantitative real time-polimerase chain reaction (qRT-PCR). Moreover, α-SMA and CD31 protein expressions were evaluated by immunofluorescence (IF) using primary antibodies to human α-SMA (dilution 1:50, Dako Cytomation, Denmark) and to human CD31 (dilution 1:200, CellSignaling Technology, Denver, USA).
Results ET-1 induced the gene expression of α-SMA and S100A4 without modulating the expression of CD31 in cultured HMVECs after 6 days of treatment, as detected by qRT-PCR. The IF analysis supported these results by showing that ET-1 induced the expression of α-SMA but no changes in the expression of CD31 after 6 days of treatment. Results were obtained from three different experiments.
Conclusions These preliminary observations show that ET-1 seems to induce in short term the expression of myofibroblast markers (α-SMA and S100A4) in cultured dermal microvascular endothelial cells, supporting a possible direct involvement in the development of the EndoMT process (6). Longer term observations seem necessary to detect further changes in phenotype markers in treated endothelial cells (i.e. CD31, von Willebrand factor).
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Disclosure of Interest None Declared
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