Identification and role of microRNAs in endothelial-to-mesenchymal transition

Abstract

Studies in fibrotic diseases demonstrate that myofibroblasts may be derived from cell types other than fibroblasts. Sources include endothelial-to-mesenchymal transition (EndMT), generating 27% to 35% of myofibroblasts in cardiac fibrosis and around 10% in kidney fibrosis. Over the last decade, miRNAs have increasingly been described as key regulators in biologic processes but their profile remains mainly undescribed in EndMT. Therefore, we have investigated the possible role of miRNA signatures in EndMT models relevant to cardiac and kidney fibrosis. EndMT was modelled in human umbilical vein endothelial cells (HUVEC) by treatment with TGFβ2 (10ng/mL) and IL1β (10ng/mL). Significantly decreased expression of endothelial markers such as vWF and increased levels of mesenchymal markers such as fibronectin were observed by qPCR in HUVEC after 48 hours of treatment (p<0.05). Similarly, immunofluorescence showed increased expression of fibronectin and decreased expression of VE-cadherin in HUVEC 6 days post-treatment. In parallel, miRNAs were profiled with an nCounter assay in HUVEC. Profiles in untreated cells were compared to cells treated with TGFβ2 and IL1β. In these profiles, miR-126-3p was found down-regulated 24 hours’ post-treatment. Over-expression of miR-126-3p in HUVEC by transfection restored the expression of CD31 and repressed the expression of fibronectin induced by TGFβ2 and IL1β treatment, protecting the cells from EndMT induction. EndMT in vivo was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice. Mice expressing YFP specifically in endothelial cells underwent myocardial infarction or unilateral ureteral obstruction. After sacrifice, lineage tracing showed expression of mesenchymal markers in endothelial derived cells, indicating the presence of EndMT in cardiac and kidney fibrosis. In addition, insitu hybridisation revealed the presence of miR-126-3p mainly in endothelial cells in mouse heart and kidney. We conclude that miR-126-3p may have a role in EndMT and this may have therapeutic potential in cardiac and kidney fibrosis.