Molecular basis of cardiac endothelial-to-mesenchymal transition (EndMT): Differential expression of microRNAs during EndMT
Highlights
► A small molecule inhibitor of TβRI-kinase (prevents Smad phosphorylation and activation), but not MEK inhibitor (prevents ERK phosphorylation), blocks cardiac endothelial-to-mesenchymal transition (EndMT) ► The epigenetic regulator ATp300 is significantly elevated during cardiac EndMT ► Several miRNAs, the epigenetic regulators, are differentially regulated during cardiac EndMT. The level of cellular p53, a target of miR-125b, is downregulated during EndMT ► Epigenetic regulators, specific miRNA and ATp300, may be an ideal target to controlling cardiac EndMT and EndMT-derived fibroblast-like cells-contributed cardiac fibrosis
Introduction
Cardiac fibrosis is a common end-stage pathologic manifestation of several cardiovascular diseases. While fibroblasts are the major source of extracellular matrix (ECM) proteins during tissue repair under normal physiologic conditions as well as during development of cardiac fibrosis under pathologic conditions, the origin of fibroblasts participating in cardiac fibrosis is not well understood. Originally, it was thought that in response to myocardial infarction, intracardiac resident fibroblasts derived from embryonic mesenchymal cells were the primary origin of myofibroblasts contributing to repair processes. However, numerous recent studies suggest that, in addition to resident cardiac fibroblasts, adult fibroblast-like cells also originate from endothelial cells (embryonic origin from splanchnopleuric mesoderm) by endothelial-to-mesenchymal transition (EndMT) [1]. EndMT is a common biologic process during embryonic development of the heart and other organs such as lung [2], [3]. However, in adults, abnormal activation of EndMT and differentiation of EndMT-derived fibroblast-like cells to collagen producing myofibroblasts play a significant role in the development and progression of fibrosis in organs such as heart and lung [3], [4], [5], [6], [7], [8]. EndMT is characterized by endothelial cell disaggregation, morphologic change related to myofibroblast differentiation, and gradual loss of endothelial markers such as CD31, VE cadherin, and vWF, with the gradual appearance of fibroblastic markers such as FSP1, alpha-smooth muscle actin (α-SMA) and collagen. Additionally, different transcription factors such as Snail and β-catenin are also known to participate in the process of EndMT, via suppression of endothelial markers [2], [4], [6], [9]. It is now well documented that elevated transforming growth factor-β (TGF-β) signaling controls endothelial plasticity and plays a significant role in the EndMT process [8]. However, the molecular basis of TGF-β-induced EndMT is poorly understood. Understanding the molecular basis of EndMT and the inhibition of new fibroblast formation from endothelial cells will be an ideal approach to control fibrosis because EndMT-derived fibroblast like cells in the adult myocardium are only associated with pathologic conditions [4], [7].
MicroRNAs (miRNAs) are short, highly conserved, RNA sequences comprised of approximately 22-nucleotides, and are involved in epigenetic regulation of eukaryotic gene expression. Aberrant expression levels of several miRNAs are associated with the pathologic conditions of different cardiovascular diseases such as hypertrophy, cardiac fibrosis, arrhythmia, myocardial infarction, heart failure, and cardiomyopathy [10], [11], [12]. However, the expression levels of miRNAs and their implication in fibrogenesis via activation of EndMT are still unknown. To better understand the molecular basis of EndMT, we examined the effect of a small molecule inhibitor of transforming growth factor-β (TGF-β) receptor type I kinase (TβRI) on EndMT and presented data showing the efficacy of a small molecule inhibitor of TβRI in blocking cardiac EndMT. Along with EndMT-markers such as α-SMA and involved transcription factors like Snail and β-catenin, the epigenetic regulator of profibrotic signaling ATp300 was also elevated during EndMT. Furthermore, we conducted, for the first time, study of the expression levels of miRNAs by miRNA array in EndMT-derived fibroblast like cells and demonstrated differential expression of several miRNAs during cardiac EndMT. We discuss here the significance of these observations on miRNA and EndMT in the light of cardiac endothelial plasticity and cardiac fibrosis.
Section snippets
Mouse cardiac endothelial cells isolation
Mouse cardiac endothelial cells (MCECs) were isolated as described by Lim and Luscinskas [13], with modification. In brief, mouse hearts were collected and minced with scissors. Minced cardiac tissues were treated with collagenase and centrifuged. The cell pellets were resuspended in cold buffer and incubated with anti-mouse CD31 antibody-Dynabeads (Invitrogen, Carlsbad, CA). Dynabead-bound cells were isolated on a magnetic separator, resuspended in media containing 20% fetal bovine serum, and
TGF-β-receptor kinase inhibitor but not MEK inhibitor blunts TGF-β2-induced EndMT in mouse cardiac endothelial cells (MCECs)
TGF-β2 induces endothelial-to-mesenchymal transition [8]. Here, we investigated the molecular mechanism by which TGF-β2 induces EndMT in primary cultures of MCECs. Exposure of isolated low passage primary cultures of MCECs to TGF-β2 for 7 days altered their morphology from an endothelial polygonal cobblestone-like shape to a more spindle shaped fibroblast-like morphology (Fig. 1A, top). Treatment of MCECs with SB431542, a potent inhibitor of TβRI kinase, prevented TGF-β2-induced morphologic
Conclusions
In conclusion, our present study reveals for the first time that i) microRNAs which are dysregulated in cardiovascular diseases, are differentially regulated during cardiac EndMT compared to cardiac endothelial cells; ii) The level of cellular p53, a target of miR-125b and a negative modulator of TGF-β-induced profibrotic signaling, is significantly downregulated during EndMT; iii) The epigenetic regulator ATp300, an essential coactivator of profibrotic signaling, is significantly elevated
Acknowledgements
This work was supported by grants from NIH-NHLBI (HL051387 and 1P01HL108795-01).
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