Activin A and ALK4 Identified as Novel Regulators of Epithelial to Mesenchymal Transition (EMT) in Human Epicardial Cells

The epicardium, the mesothelial layer covering the heart, is a crucial cell source for cardiac development and repair. It provides cells and biochemical signals to the heart to facilitate vascularization and myocardial growth. An essential element of epicardial behavior is epicardial epithelial to mesenchymal transition (epiMT), which is the initial step for epicardial cells to become motile and invade the myocardium. To identify targets to optimize epicardium-driven repair of the heart, it is vital to understand which pathways are involved in the regulation of epiMT. Therefore, we established a cell culture model for human primary adult and fetal epiMT, which allows for parallel testing of inhibitors and stimulants of specific pathways. Using this approach, we reveal Activin A and ALK4 signaling as novel regulators of epiMT, independent of the commonly accepted EMT inducer TGFβ. Importantly, Activin A was able to induce epicardial invasion in cultured embryonic mouse hearts. Our results identify Activin A/ALK4 signaling as a modulator of epicardial plasticity which may be exploitable in cardiac regenerative medicine.

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SRSF3 is a key regulator of epicardial formation

10.1101/2021.11.25.470003 ◽

2021 ◽

Author(s):

Irina-Elena Lupu ◽

Andia Nicole Redpath ◽

Nicola Smart

Keyword(s):

Heart Development ◽

Molecular Mechanisms ◽

Epithelial To Mesenchymal Transition ◽

Cellular Proliferation ◽

Rna Binding ◽

Cardiac Development ◽

Cardiac Fibroblasts ◽

Mesenchymal Transition ◽

Epicardial Cells ◽

Epicardial Layer

The epicardium is a fundamental regulator of cardiac development, functioning to secrete essential growth factors and to produce epicardium-derived cells (EPDCs) that contribute most coronary vascular smooth muscle cells and cardiac fibroblasts. The molecular mechanisms that control epicardial formation and proliferation have not been fully elucidated. In this study, we found that the RNA-binding protein SRSF3 is highly expressed in the proepicardium and later in the epicardial layer during heart development. Deletion of Srsf3 from the murine proepicardium using the Tg(Gata5-Cre) or embryonic day (E) 8.5 induction of Wt1CreERT2 led to proliferative arrest and impaired epithelial-to-mesenchymal transition (EMT), which prevented proper formation and function of the epicardial layer. Induction of Srsf3 deletion with the Wt1CreERT2 after the proepicardial stage resulted in impaired EPDC formation and epicardial proliferation at E13.5. Single-cell RNA-sequencing showed SRSF3-depleted epicardial cells were removed by E15.5 and the remaining non-recombined cells became hyperproliferative and compensated for the loss via up-regulation of Srsf3. This research identifies SRSF3 as a master regulator of cellular proliferation in epicardial cells.

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Quantitation analysis by flow cytometry shows that Wt1 is required for development of the proepicardium and epicardium

10.1101/2020.10.06.329151 ◽

2020 ◽

Author(s):

Christine Biben ◽

Bette Borobokas ◽

Mary Kamala Menon ◽

Lynne Hartley ◽

Richard Paul Harvey ◽

...

Keyword(s):

Flow Cytometry ◽

Epithelial To Mesenchymal Transition ◽

Cardiac Development ◽

Therapeutic Potential ◽

Wilms Tumor ◽

Cardiac Fibroblasts ◽

Coronary Smooth Muscle ◽

Mesenchymal Transition ◽

A Cell ◽

Factor Alpha

ABSTRACTThe epicardium is a cell layer found on the external surface of the heart. During development it has an epithelial identity and contains progenitor cells for coronary smooth muscle and cardiac fibroblasts. The epicardium has been suggested to have therapeutic potential in cardiac repair. Study of epicardial development has been difficult because it is dynamic and morphologically complex. We developed a flow cytometry-based method to quantify cardiac development including the epicardial lineage. This provided accurate and sensitive analysis of (1) the emergence of epicardial progenitors within the proepicardium (2) their transfer to the heart to form the epicardium, and (3) their epithelial-to-mesenchymal transition (EMT) to create the subepicardium. Platelet-derived growth factor alpha (Pdgfra) and Wilms tumor protein (Wt1) have both been reported to be pro-mesenchymal during epicardial EMT. Quantitative analysis with flow cytometry confirmed a pro-mesenchymal role for Pdgfra but not for Wt1. Analysis of Wt1 null embryos showed that they had (1) poor formation of proepicardial villi, (2) reduced transfer of proepicardial cells to the heart, (3) a discontinuous epicardium with poor epithelial identity, and (4) a proportionally excessive number of mesenchymal-like cells. This data shows that Wt1 is essential for epicardial formation and maintenance rather than being pro-mesenchymal.

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Epicardial Cells of Human Adults Can Undergo an Epithelial-to-Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro

Stem Cells ◽

10.1634/stemcells.2006-0366 ◽

2007 ◽

Vol 25 (2) ◽

pp. 271-278 ◽

Cited By ~ 122

Author(s):

John van Tuyn ◽

Douwe E. Atsma ◽

Elizabeth M. Winter ◽

Ietje van der Velde-van Dijke ◽

Daniel A. Pijnappels ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Epithelial To Mesenchymal Transition ◽

Muscle Cells ◽

Mesenchymal Transition ◽

Epicardial Cells ◽

Human Adults

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Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population

Developmental Dynamics ◽

10.1002/dvdy.23711 ◽

2011 ◽

Vol 241 (2) ◽

pp. 270-283 ◽

Cited By ~ 70

Author(s):

Margot Williams ◽

Carol Burdsal ◽

Ammasi Periasamy ◽

Mark Lewandoski ◽

Ann Sutherland

Keyword(s):

Cell Population ◽

Epithelial To Mesenchymal Transition ◽

Primitive Streak ◽

Mesenchymal Transition ◽

A Cell

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Vegfaa instructs cardiac muscle hyperplasia in adult zebrafish

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722594115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8805-8810 ◽

Cited By ~ 20

Author(s):

Ravi Karra ◽

Matthew J. Foglia ◽

Wen-Yee Choi ◽

Christine Belliveau ◽

Paige DeBenedittis ◽

...

Keyword(s):

Heart Development ◽

Epithelial To Mesenchymal Transition ◽

Cardiac Injury ◽

Angiogenic Factor ◽

Regulatory Sequences ◽

Mesenchymal Transition ◽

Epicardial Cells ◽

Myocardial Wall ◽

Tightly Coupled ◽

Spatiotemporal Control

During heart development and regeneration, coronary vascularization is tightly coupled with cardiac growth. Although inhibiting vascularization causes defects in the innate regenerative response of zebrafish to heart injury, angiogenic signals are not known to be sufficient for triggering regeneration events. Here, by using a transgenic reporter strain, we found that regulatory sequences of the angiogenic factor vegfaa are active in epicardial cells of uninjured animals, as well as in epicardial and endocardial tissue adjacent to regenerating muscle upon injury. Additionally, we find that induced cardiac overexpression of vegfaa in zebrafish results in overt hyperplastic thickening of the myocardial wall, accompanied by indicators of angiogenesis, epithelial-to-mesenchymal transition, and cardiomyocyte regeneration programs. Unexpectedly, vegfaa overexpression in the context of cardiac injury enabled ectopic cardiomyogenesis but inhibited regeneration at the site of the injury. Our findings identify Vegfa as one of a select few known factors sufficient to activate adult cardiomyogenesis, while also illustrating how instructive factors for heart regeneration require spatiotemporal control for efficacy.

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Estradiol Induces Epithelial to Mesenchymal Transition of Human Glioblastoma Cells

Cells ◽

10.3390/cells9091930 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1930

Author(s):

Ana M. Hernández-Vega ◽

Aylin Del Moral-Morales ◽

Carmen J. Zamora-Sánchez ◽

Ana G. Piña-Medina ◽

Aliesha González-Arenas ◽

...

Keyword(s):

Cell Migration ◽

Epithelial To Mesenchymal Transition ◽

Epithelial Mesenchymal Transition ◽

Estrogen Receptor Α ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Selective Agonist ◽

A Cell ◽

Human Gbm ◽

Emt Markers

The mesenchymal phenotype of glioblastoma multiforme (GBM), the most frequent and malignant brain tumor, is associated with the worst prognosis. The epithelial–mesenchymal transition (EMT) is a cell plasticity mechanism involved in GBM malignancy. In this study, we determined 17β-estradiol (E2)-induced EMT by changes in cell morphology, expression of EMT markers, and cell migration and invasion assays in human GBM-derived cell lines. E2 (10 nM) modified the shape and size of GBM cells due to a reorganization of actin filaments. We evaluated EMT markers expression by RT-qPCR, Western blot, and immunofluorescence.We found that E2 upregulated the expression of the mesenchymal markers, vimentin, and N-cadherin. Scratch and transwell assays showed that E2 increased migration and invasion of GBM cells. The estrogen receptor-α (ER-α)-selective agonist 4,4’,4’’-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, 10 nM) affected similarly to E2 in terms of the expression of EMT markers and cell migration, and the treatment with the ER-α antagonist methyl-piperidino-pyrazole (MPP, 1 μM) blocked E2 and PPT effects. ER-β-selective agonist diarylpropionitrile (DNP, 10 nM) and antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazole[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 1 μM) showed no effects on EMT marker expression. These data suggest that E2 induces EMT activation through ER-α in human GBM-derived cells.

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Scleraxis regulates Twist1 and Snai1 expression in the epithelial-to-mesenchymal transition

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00092.2018 ◽

2018 ◽

Vol 315 (3) ◽

pp. H658-H668 ◽

Cited By ~ 12

Author(s):

Danah S. Al-Hattab ◽

Hamza A. Safi ◽

Raghu S. Nagalingam ◽

Rushita A. Bagchi ◽

Matthew T. Stecy ◽

...

Keyword(s):

Transcription Factor ◽

Growth Factor ◽

Epithelial Cells ◽

Epithelial To Mesenchymal Transition ◽

Transforming Growth Factor ◽

Cardiac Development ◽

Cardiac Fibroblasts ◽

Transforming Growth Factor Β ◽

Mesenchymal Transition ◽

E Cadherin

Numerous physiological and pathological events, from organ development to cancer and fibrosis, are characterized by an epithelial-to-mesenchymal transition (EMT), whereby adherent epithelial cells convert to migratory mesenchymal cells. During cardiac development, proepicardial organ epithelial cells undergo EMT to generate fibroblasts. Subsequent stress or damage induces further phenotype conversion of fibroblasts to myofibroblasts, causing fibrosis via synthesis of an excessive extracellular matrix. We have previously shown that the transcription factor scleraxis is both sufficient and necessary for the conversion of cardiac fibroblasts to myofibroblasts and found that scleraxis knockout reduced cardiac fibroblast numbers by 50%, possibly via EMT attenuation. Scleraxis induced expression of the EMT transcriptional regulators Twist1 and Snai1 via an unknown mechanism. Here, we report that scleraxis binds to E-box consensus sequences within the Twist1 and Snai1 promoters to transactivate these genes directly. Scleraxis upregulates expression of both genes in A549 epithelial cells and in cardiac myofibroblasts. Transforming growth factor-β induces EMT, fibrosis, and scleraxis expression, and we found that transforming growth factor-β-mediated upregulation of Twist1 and Snai1 completely depends on the presence of scleraxis. Snai1 knockdown upregulated the epithelial marker E-cadherin; however, this effect was lost after scleraxis overexpression, suggesting that scleraxis may repress E-cadherin expression. Together, these results indicate that scleraxis can regulate EMT via direct transactivation of the Twist1 and Snai1 genes. Given the role of scleraxis in also driving the myofibroblast phenotype, scleraxis appears to be a critical controller of fibroblast genesis and fate in the myocardium and thus may play key roles in wound healing and fibrosis. NEW & NOTEWORTHY The molecular mechanism by which the transcription factor scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epithelial-to-mesenchymal transition and demonstrate that transforming growth factor-β-mediated epithelial-to-mesenchymal transition is dependent on scleraxis, providing a potential target for controlling this process.

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Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition

Blood ◽

10.1182/blood-2002-05-1341 ◽

2003 ◽

Vol 101 (8) ◽

pp. 2973-2982 ◽

Cited By ~ 105

Author(s):

Jalila Chagraoui ◽

Adeline Lepage-Noll ◽

Aurora Anjo ◽

Georges Uzan ◽

Pierre Charbord

Keyword(s):

Stem Cells ◽

Epithelial To Mesenchymal Transition ◽

Fetal Liver ◽

Smooth Muscle Actin ◽

Primary Cultures ◽

Oncostatin M ◽

Fetal Life ◽

Hematopoietic Stem ◽

Mesenchymal Transition ◽

A Cell

Abstract Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work, we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin, osteopontin, collagen I, α smooth muscle actin, thrombospondin-1, EDa fibronectin, calponin, Stro-1 antigens, myocyte-enhancer factor 2C) and epithelial (α-fetoprotein, cytokeratins 8 and 18, albumin, E-cadherin, hepatocyte nuclear factor 3 α) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT), often observed during development, including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation, induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment.

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Bioengineering strategies to control epithelial-to-mesenchymal transition for studies of cardiac development and disease

APL Bioengineering ◽

10.1063/5.0033710 ◽

2021 ◽

Vol 5 (2) ◽

pp. 021504

Author(s):

Dawn Bannerman ◽

Simon Pascual-Gil ◽

Marie Floryan ◽

Milica Radisic

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Cardiac Development ◽

Mesenchymal Transition

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Cardiac Repair: The Intricate Crosstalk between the Epicardium and the Myocardium

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200219105448 ◽

2020 ◽

Vol 15 (8) ◽

pp. 661-673

Author(s):

Laura Pellegrini ◽

Eleonora Foglio ◽

Elena Pontemezzo ◽

Antonia Germani ◽

Matteo Antonio Russo ◽

...

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Heart Diseases ◽

Pericardial Fluid ◽

Cardiac Repair ◽

Bioactive Molecules ◽

Ischemic Heart ◽

Paracrine Factors ◽

Mesenchymal Transition ◽

Ischemic Heart Diseases ◽

Epicardial Cells

Background: Substantial evidences support the hypothesis that the epicardium has a role in cardiac repair and regeneration in part providing, by epithelial to mesenchymal transition (EMT), progenitor cells that differentiate into cardiac cell types and in part releasing paracrine factors that contribute to cardiac repair. Besides cell contribution, a significant paracrine communication occurs between the epicardium and the myocardium that improves the whole regenerative response. Signaling pathways underlying this communication are multiple as well as soluble factors involved in cardiac repair and secreted both by myocardial and epicardial cells. Most recently, extracellular vesicles, i.e. exosomes, that accumulate in the pericardial fluid (PF) and are able to transport bioactive molecules (cytosolic proteins, mRNAs, miRNAs and other non-coding RNAs), have been also identified as potential mediators of epicardial-mediated repair following myocardial injury. Conclusions: This mini-review provides an overview of the epicardial-myocardial signaling in regulating cardiac repair in ischemic heart diseases. Indeed, a detailed understanding of the crosstalk between myocardial and epicardial cells and how paracrine mechanisms are involved in the context of ischemic heart diseases would be of tremendous help in developing novel therapeutic approaches to promote cardiomyocytes survival and heart regeneration following myocardial infarction (MI).

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