Epicardium-derived Progenitor Cells Research Articles

Oxytocin promotes epicardial cell activation and heart regeneration after cardiac injury.

Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide, and frequently leads to massive heart injury and the loss of billions of cardiac muscle cells and associated vasculature. Critical work in the last 2 decades demonstrated that these lost cells can be partially regenerated by the epicardium, the outermost mesothelial layer of the heart, in a process that highly recapitulates its role in heart development. Upon cardiac injury, mature epicardial cells activate and undergo an epithelial-mesenchymal transition (EMT) to form epicardium-derived progenitor cells (EpiPCs), multipotent progenitors that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. In mammals, this process alone is insufficient for significant regeneration, but it might be possible to prime it by administering specific reprogramming factors, leading to enhanced EpiPC function. Here, we show that oxytocin (OXT), a hypothalamic neuroendocrine peptide, induces epicardial cell proliferation, EMT, and transcriptional activity in a model of human induced pluripotent stem cell (hiPSC)-derived epicardial cells. In addition, we demonstrate that OXT is produced after cardiac cryoinjury in zebrafish, and that it elicits significant epicardial activation promoting heart regeneration. Oxytocin signaling is also critical for proper epicardium development in zebrafish embryos. The above processes are significantly impaired when OXT signaling is inhibited chemically or genetically through RNA interference. RNA sequencing data suggests that the transforming growth factor beta (TGF-β) pathway is the primary mediator of OXT-induced epicardial activation. Our research reveals for the first time an evolutionary conserved brain-controlled mechanism inducing cellular reprogramming and regeneration of the injured mammalian and zebrafish heart, a finding that could contribute to translational advances for the treatment of cardiac injuries.

Abstract 509: Oxytocin Mediates Neuroendocrine Reprogramming of the Epicardium in Heart Regeneration

Cardiovascular disease (CVD) is the leading cause of mortality both in the United States and worldwide. CVD often results in the massive loss of contractile cardiac cells and tissue. Critical work in the last two decades demonstrates that lost cells can be partially replenished by the epicardium, the outermost mesothelial layer of the heart. Upon cardiac injury, mature epicardial cells activate and undergo epithelial-mesenchymal transition (EMT) to form epicardium-derived progenitor cells (EPDCs), which are a type of multipotent stem cell that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. This process alone is insufficient for significant regeneration, but its efficiency can be improved by priming with specific factors (e.g., thymosin beta-4). Our group has recently discovered evidence that oxytocin (OT), a hypothalamic neuroendocrine peptide, induces a pro-regenerative phenotype in vitro in human induced pluripotent stem cell (iPSC) derived epicardial cells. We hypothesize that upon cardiac injury, oxytocin is released into the bloodstream, causing activation of the epicardium and mobilization of EPDCs to elicit regeneration of damaged tissue and restoration of function. Here, we show that we can differentiate mature, high-quality epicardial cells from iPSCs and that Ki67 levels and cell counts increase after three days of OT exposure. In addition, the peptide alters gene expression levels of several epithelial, mesenchymal, and EMT markers, indicating a transition to a dedifferentiated gene profile characteristic of EPDCs. Finally, when OT is administered intravenously to mice, it accelerates healing from cardiac injury by inducing epicardial activation. Future studies will aim to further reveal the physiological contribution of OT to heart regeneration in vivo and determine its molecular mechanism of action. Our findings have the potential to uncover a novel mechanism of neuroendocrine reprogramming of the injured heart and yield significant translational advances in the treatment of CVD.

Prokineticin Receptor-1 Signaling Inhibits Dose- and Time-Dependent Anthracycline-Induced Cardiovascular Toxicity Via Myocardial and Vascular Protection

ObjectivesThis study investigated how different concentrations of doxorubicin (DOX) can affect the function of cardiac cells. This study also examined whether activation of prokineticin receptor (PKR)-1 by a nonpeptide agonist, IS20, prevents DOX-induced cardiovascular toxicity in mouse models. BackgroundHigh prevalence of heart failure during and following cancer treatments remains a subject of intense research and therapeutic interest. MethodsThis study used cultured cardiomyocytes, endothelial cells (ECs), and epicardium-derived progenitor cells (EDPCs) for in vitro assays, tumor-bearing models, and acute and chronic toxicity mouse models for in vivo assays. ResultsBrief exposure to cardiomyocytes with high-dose DOX increased the accumulation of reactive oxygen species (ROS) by inhibiting a detoxification mechanism via stabilization of cytoplasmic nuclear factor, erythroid 2. Prolonged exposure to medium-dose DOX induced apoptosis in cardiomyocytes, ECs, and EDPCs. However, low-dose DOX promoted functional defects without inducing apoptosis in EDPCs and ECs. IS20 alleviated detrimental effects of DOX in cardiac cells by activating the serin threonin protein kinase B (Akt) or mitogen-activated protein kinase pathways. Genetic or pharmacological inactivation of PKR1 subdues these effects of IS20. In a chronic mouse model of DOX cardiotoxicity, IS20 normalized an elevated serum marker of cardiotoxicity and vascular and EDPC deficits, attenuated apoptosis and fibrosis, and improved the survival rate and cardiac function. IS20 did not interfere with the cytotoxicity or antitumor effects of DOX in breast cancer lines or in a mouse model of breast cancer, but it did attenuate the decreases in left ventricular diastolic volume induced by acute DOX treatment. ConclusionsThis study identified the molecular and cellular signature of dose-dependent, DOX-mediated cardiotoxicity and provided evidence that PKR-1 is a promising target to combat cardiotoxicity of cancer treatments.

Application of Bioengineered Materials in the Surgical Management of Heart Failure.

The epicardial surface of the heart is readily accessible during cardiac surgery and presents an opportunity for therapeutic intervention for cardiac repair and regeneration. As an important anatomic niche for endogenous mechanisms of repair, targeting the epicardium using decellularized extracellular matrix (ECM) bioscaffold therapy may provide the necessary environmental cues to promote functional recovery. Following ischemic injury to the heart caused by myocardial infarction (MI), epicardium derived progenitor cells (EPDCs) become activated and migrate to the site of injury. EPDC differentiation has been shown to contribute to endothelial cell, cardiac fibroblast, cardiomyocyte, and vascular smooth muscle cell populations. Post-MI, it is largely the activation of cardiac fibroblasts and the resultant dysregulation of ECM turnover which leads to maladaptive structural cardiac remodeling and loss of cardiac function. Decellularized ECM bioscaffolds not only provide structural support, but have also been shown to act as a bioactive reservoir for growth factors, cytokines, and matricellular proteins capable of attenuating maladaptive cardiac remodeling. Targeting the epicardium post-MI using decellularized ECM bioscaffold therapy may provide the necessary bioinductive cues to promote differentiation toward a pro-regenerative phenotype and attenuate cardiac fibroblast activation. There is an opportunity to leverage the clinical benefits of this innovative technology with an aim to improve the prognosis of patients suffering from progressive heart failure. An enhanced understanding of the utility of decellularized ECM bioscaffolds in epicardial repair will facilitate their growth and transition into clinical practice. This review will provide a summary of decellularized ECM bioscaffolds being developed for epicardial infarct repair in coronary artery bypass graft (CABG) surgery.

A Prokineticin-Driven Epigenetic Switch Regulates Human Epicardial Cell Stemness and Fate.

Epicardial adipose tissues (EATs) and vascular tissues may both belong to the mesoepithelial lineage that develops from epicardium-derived progenitor cells (EPDCs) in developing and injured hearts. Very little is known of the molecular mechanisms of EPDC contribution in EAT development and neovascularization in adult heart, which the topic remains a subject of intense therapeutic interest and scientific debate. Here we studied the epigenetic control of stemness and anti-adipogenic and pro-vasculogenic fate of human EPDCs (hEPDCs), through investigating an angiogenic hormone, prokineticin-2 (PK2) signaling via its receptor PKR1. We found that hEPDCs spontaneously undergoes epithelial-to-mesenchymal transformation (EMT), and are not predestined for the vascular lineages. However, PK2 via a histone demethylase KDM6A inhibits EMT, and induces asymmetric division, leading to self-renewal and formation of vascular and epithelial/endothelial precursors with angiogenic potential capable of differentiating into vascular smooth muscle and endothelial cells. PK2 upregulates and activates KDM6A to inhibit repressive histone H3K27me3 marks on promoters of vascular genes (Flk-1 and SM22α) involved in vascular lineage commitment and maturation. In PK2-mediated anti-adipogenic signaling, KDM6A stabilizes and increases cytoplasmic β-catenin levels to repress peroxisome proliferator-activated receptor-γ expression and activity. Our findings offer additional molecular targets to manipulate hEPDCs-involved tissue repair/regeneration in cardiometabolic and ischemic heart diseases. Stem Cells 2018;36:1589-1602.

Stem Cell Repair for Cardiac Muscle Regeneration: A Review of the Literature

The notion that the human adult heart is a quiescent organ incapable of self-regeneration has been successfully challenged. It is now evident that the heart possesses a significant ability for repair and regeneration. Stem cells of endogenous cardiac origin are currently considered to possess the greatest ability to differentiate into cardiomyocytes. The major types of cardiac stem cells that show a promising potential to replace damaged cardiomyocytes include C-KIT positive (C-KIT+) cardiac progenitor cells, cardiosphere-derived progenitor cells, islet-1 (Isl1+) cardiac progenitor cells, side-population cardiac progenitor cells, epicardium-derived progenitor cells and stem cell antigen-1 (SCA1+) cardiac progenitor cells. Moreover, stem cells of extra-cardiac origin are also thought to restore contractility and vascularization of the myocardium. These include skeletal myoblasts, bone marrow mononuclear cells, mesenchymal stem cells, endothelial progenitor cells as well as embryonic stem cells. The need for further investigation on cardiac stem cell therapeutic strategies still remains.

Myocardin-related transcription factors control the motility of epicardium-derived cells and the maturation of coronary vessels.

An important pool of cardiovascular progenitor cells arises from the epicardium, a single layer of mesothelium lining the heart. Epicardium-derived progenitor cell (EPDC) formation requires epithelial-to-mesenchymal transition (EMT) and the subsequent migration of these cells into the sub-epicardial space. Although some of the physiological signals that promote EMT are understood, the functional mediators of EPDC motility and differentiation are not known. Here, we identify a novel regulatory mechanism of EPDC mobilization. Myocardin-related transcription factor (MRTF)-A and MRTF-B (MKL1 and MKL2, respectively) are enriched in the perinuclear space of epicardial cells during development. Transforming growth factor (TGF)-β signaling and disassembly of cell contacts leads to nuclear accumulation of MRTFs and the activation of the motile gene expression program. Conditional ablation of Mrtfa and Mrtfb specifically in the epicardium disrupts cell migration and leads to sub-epicardial hemorrhage, partially stemming from the depletion of coronary pericytes. Using lineage-tracing analyses, we demonstrate that sub-epicardial pericytes arise from EPDCs in a process that requires the MRTF-dependent motile gene expression program. These findings provide novel mechanisms linking EPDC motility and differentiation, shed light on the transcriptional control of coronary microvascular maturation and suggest novel therapeutic strategies to manipulate epicardium-derived progenitor cells for cardiac repair.

Epicardial Origin of Resident Mesenchymal Stem Cells in the Adult Mammalian Heart

The discovery of stem and progenitor cells in the adult mammalian heart has added a vital dimension to the field of cardiac regeneration. Cardiac-resident stem cells are likely sequestered as reserve cells within myocardial niches during the course of embryonic cardiogenesis, although they may also be recruited from external sources, such as bone marrow. As we begin to understand the nature of cardiac-resident stem and progenitor cells using a variety of approaches, it is evident that they possess an identity embedded within their gene regulatory networks that favours cardiovascular lineage potential. In addition to contributing lineage descendants, cardiac stem cells may also be stress sensors, offering trophic cues to other cell types, including cardiomyocytes and vasculature cells, and likely other stem cells and immune cells, during adaptation and repair. This presents numerous possibilities for endogenous cardiac stem and progenitor cells to be used in cell therapies or as targets in heart rejuvenation. In this review, we focus on the epicardium as an endogenous source of multi-potential mesenchymal progenitor cells in development and as a latent source of such progenitors in the adult. We track the origin and plasticity of the epicardium in embryos and adults in both homeostasis and disease. In this context, we ask whether directed activation of epicardium-derived progenitor cells might have therapeutic application.

Thymosin β4 and Cardiac Regeneration: Are We Missing a Beat?

Epicardial resident stem cells are known to differentiate into cardiomyocytes during cardiac development, amongst other cell types. Whether epicardium-derived progenitor cells (EPDCs) retain this plasticity in the adult heart has been the topic of heated scientific debate. Priming with thymosin beta 4, a peptide which has been suggested to be critical for cardiac development and to have cardio-protective properties, was recently shown to induce differentiation of EPDCs into cardiomyocytes in a small animal model of myocardial infarction. This finding is in stark contrast to another recent study in which thymosin beta 4 treatment following myocardial infarction did not induce cardiomyocyte differentiation of EPDCs. While EPDCs seem to exhibit overall cardio-protective effects on the heart following myocardial infarction, they have not been shown to differentiate into cardiomyocytes in a clinically relevant setting. It will be important to understand why the ability of one therapeutic agent to induce cardiomyocyte differentiation of EPDCs seemingly depends on a single variable, i.e. the time of administration. Furthermore, in light of a recent report, it appears that thymosin beta 4 may be dispensable for cardiac development.

Muscle for a damaged heart

When cardiac muscle cells die during a heart attack, this can lead to heart failure and even death. It now emerges that stem cells of the 'sheet' enveloping the heart can be coaxed to form new muscle after such an event. See Letter p.640 The prospect of cell-based therapy in cardiovascular regenerative medicine comes a step closer with the demonstration that a peptide can stimulate a progenitor cell population in the adult heart to act as a source of new cardiomyocytes. The stem or progenitor cells, thought to be derived from the epicardium, are activated by the small actin monomer binding protein thymosin β4, which has previously been shown to restore vascular potential to adult epicardium-derived progenitor cells after injury. The discovery of a resident source of myocardial progenitors will stimulate a search for small molecules and other factors that promote optimal progenitor activation and replacement of destroyed myocardium.

De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

Thymosin β4 induces epicardium-derived neovascularization in the adult heart

The inability of the human heart to effectively repair itself after acute ischaemic injury has driven the search for efficacious means of promoting cardiac regenerative growth. Central to this has been the emergence of cell-based strategies to stimulate and augment both myocardial regeneration and neovascularization. Autologous cell transplantation of a variety of adult progenitor cells has been taken forward in clinical trials and, in parallel, investigators have begun to focus on the activation of resident cardiac cell populations as a means to stimulate endogenous repair. The latter approach depends on characterizing native progenitors with self-renewal, clonality, multipotency and arguably an analogous embryological counterpart. Recently, we have focused on adult EPDCs (epicardium-derived progenitor cells), which, when induced by the actin monomer-binding protein Tbeta4 (thymosin beta4), are able to revert to their embryonic phenotype and give rise to endothelial cells and vascular smooth muscle cells ex vivo. Studies are ongoing to determine whether activated adult EPDCs can contribute to bona fide neovascularization in the injured adult mammalian heart proper, as a therapeutic means to support surviving cardiac muscle cells and sustain regenerating myocardium.

Derivation of Epicardium‐Derived Progenitor Cells (EPDCs) from Adult Epicardium

The epicardium has, like the other cell lineages of the terminally differentiated adult heart, long been regarded as quiescent, incapable of migration or differentiation. In contrast, the embryonic epicardium possesses an innate ability to proliferate, migrate, and differentiate into a number of mature cardiovascular cell types, including vascular smooth muscle cells, fibroblasts, cardiomyocytes, and, arguably, some endothelial cells. In recapitulating its essential developmental role, we recognized the ability of the actin-binding peptide thymosin beta4 (Tbeta4) to induce epicardium-derived progenitor cell (EPDC) migration from adult heart and noted the derivation of cell types originating from embryonic epicardium. This protocol provides a means of enabling adult EPDC outgrowth and culture. We establish a model system in which to study the ability of factors to influence the migration of vascular precursors and their differentiation and to move towards screening of small molecules ex vivo prior to clinical trials of therapeutic cardiac repair.

Epicardium-derived progenitor cells require beta-catenin for coronary artery formation.

We have previously identified several members of the Wnt/beta-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of beta-catenin expression affects mouse development at gastrulation with failure of both mesoderm development and axis formation. To circumvent this early embryonic lethality and study the specific role of beta-catenin in coronary arteriogenesis, we have generated conditional beta-catenin-deletion mutant animals in the proepicardium by interbreeding with a Cre-expressing mouse that targets coronary progenitor cells in the proepicardium and its derivatives. Ablation of beta-catenin in the proepicardium results in lethality between embryonic day 15 and birth. Mutant mice display impaired coronary artery formation, whereas the venous system and microvasculature are normal. Analysis of proepicardial beta-catenin mutant cells in the context of an epicardial tracer mouse reveals that the formation of the proepicardium, the migration of proepicardial cells to the heart, and the formation of the primitive epicardium are unaffected. However, subsequent processes of epicardial development are dramatically impaired in epicardial-beta-catenin mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived mesenchymal cells into coronary smooth muscle cells. Our data demonstrate a functional role of the epicardial beta-catenin pathway in coronary arteriogenesis.