Molecular basis of cardiac endothelial-to-mesenchymal transition (EndMT): differential expression of microRNAs during EndMT

Abstract

Fibroblasts are responsible for producing the majority of collagen and other extracellular matrix (ECM) proteins in tissues. In the injured tissue, transforming growth factor-β (TGF-β)-activated fibroblasts or differentiated myofibroblasts synthesize excessive ECM proteins and play a pivotal role in the pathogenesis of fibrosis in heart, kidney and other organs. Recent studies suggest that fibroblast-like cells, derived from endothelial cells by endothelial-to-mesenchymal transition (EndMT), contribute to the pathogenesis of cardiac fibrosis. The molecular basis of EndMT, however, is poorly understood. Here, we investigated the molecular basis of EndMT in mouse cardiac endothelial cells (MCECs) in response to TGF-β2. MCECs exposed to TGF-β2 underwent EndMT as evidenced by morphologic changes, lack of acetylated-low density lipoprotein (Ac-LDL) uptake, and the presence of alpha-smooth muscle actin (α-SMA) staining. Treatment with SB431542, a small molecule inhibitor of TGF-β-receptor I (TβRI) kinase, but not PD98059, a MEK inhibitor, completely blocked TGF-β2-induced EndMT. The transcript and protein levels of α-SMA, Snail and β-catenin as well as acetyltransferase p300 (ATp300) were elevated in EndMT derived fibroblast-like cells. Importantly, microRNA (miRNA) array data revealed that the expression levels of specific miRNAs, known to be dysregulated in different cardiovascular diseases, were altered during EndMT. The protein level of cellular p53, a bonafide target of miR-125b, was downregulated in EndMT-derived fibroblast-like cells. Here, we report for the first time, the differential expression of miRNAs during cardiac EndMT. These results collectively suggest that TβRI serine-threonine kinase-induced TGF-β signaling and microRNAs, the epigenetic regulator of gene expression at the posttranscriptional level, are involved in EndMT and promote profibrotic signaling in EndMT-derived fibroblast-like cells. Pharmacologic agents that restrict the progression of cardiac EndMT, a phenomenon that is found in adults only in the pathological conditions, in targeting specific miRNA may be helpful in preventing and treating cardiac fibrosis.

Figure 1. Molecular basis of EndMT

A–D. TβR1 kinase inhibitor SB431542 blocks TGF-β2-induced EndMT: Mouse cardiac endothelial cells (MCECs) were treated with DMSO or SB431542 (SB) or PD98059 (PD) in the presence and absence of TGF-β2 for 7 days. Cell morphology was studied by light microscopy (A); receptor-mediated high endocytosis of Dil-Ac-LDL by primary cultures of MCECs was studied by fluorescence microscopy (B), and immunostaining of cells was performed using FITC tagged α-SMA (myofibroblast marker) antibody (C); MCECs were treated with DMSO or SB431542 (SB) or PD98059 (PD) in the presence and absence of TGF-β2 for 7 days and cell lysates were prepared on three plates and pooled. Equal amounts of proteins were subjected to western blot analysis with α-SMA, p-ERK, T-ERK, pSmad2 and actin (loading control) (D).

Figure 2. Differential expression of EndMT marker and regulators during EndMT

MCECs were treated with TGF-β2 for 7 days and total RNA was isolated. mRNA levels of Snail (left), β-catenin (middle) and α-SMA (right) were determined by qPCR using gene specific primers. Data were analyzed using q-gene software. Note: *denotes P<0.05 vs. MCECs ^#denotes P=0.06 vs. MCECs. Yellow bar represents MCECs; Black bar represents EndMT-derived fibroblast-like cells.

Figure 3. Epigenetic regulator ATp300 is upregulated during EndMT

MCECs were treated with TGF-β2 for 7 days and cell lysates were subjected to western blot using ATp300, Snail, β-catenin, PAI-1, α-SMA (mesenchymal markers), CD31 (endothelial marker), pSmad2 (TGF-β-signal transducer), and actin antibodies (left panel). The levels of ATp300 were quantified and presented as fold induction of ATp300 relative to actin in EndMT-derived fibroblast-like cells compared to MCECs (right panel). Yellow bar represents MCECs; Black bar represents EndMT-derived fibroblast-like cells.

Figure 4. Differential expression of microRNAs during EndMT

MCECs were treated with TGF-β2 for 7 days and total RNA was extracted from three plates and pooled. Equal amounts of RNA were subjected to miRNA array (A,B) and qPCR analysis (C). The levels of individual miRNA expression relative to U6 expression were quantified and presented as mean ± SEM. Note: * denotes P<0.05 vs. MCECs. The levels of miR-125b, Let7C, Let-7g, miR-21, miR-30b and miR-195 in miRNA array were upregulated during EndMT (A); the levels of miR-122a, miR-127, miR-196, and miR-375 were downregulated during EndMT (B). The levels of miR-125b in MCECs and TGF-2-induced EndMT-derived cells were further determined by microRNA qPCR and data were presented as fold induction of miR-125b relative to U6 expression in EndMT-derived fibroblast-like cells compared to MCECs (C). Total protein extracted from MCECs (control) and EndMT-derived fibroblast-like cells were subjected to western blot using antibodies against p53, α-SMA and actin. Note: while EndMT marker α-SMA was upregulated, the miR-125b target gene p53 protein level was significantly decreased (D).