From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells
Winter, E.M.
Citation
Winter, E. M. (2009, October 15). From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells. Retrieved from https://hdl.handle.net/1887/14054
Version: Corrected Publisher’s Version
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From
Cardiogenesis to Cardiac
Regeneration
Liesbeth Winter
Focus on
Epicardium-
Derived Cells
From
Cardiogenesis to Cardiac
Regeneration Focus on
Epicardium- Derived Cells
Liesbeth Winter
Colophon
From Cardiogenesis to Cardiac Regeneration.
Focus on Epicardium-Derived Cells Elizabeth Martha Winter
Thesis Leiden University Medical Center
©2009 Elizabeth M. Winter, Leiden, the Netherlands. All rights reserved. No part of this book may be reproduced or transmitted, in any form or by any means, without written permission of the author.
Cover studio Leclercq, Rotterdam Layout studio Leclercq, Rotterdam Printed by NPN Drukkers
ISBN 978-90-9024546-1
From Cardiogenesis to Cardiac Regeneration Focus on Epicardium-Derived Cells
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus Prof. Mr. P.F. van der Heijden,
volgens besluit van het College voor Promoties te verdedigen op donderdag 15 oktober 2009
klokke 16.15 uur
door
Elizabeth Martha Winter geboren te Haarlem
in 1979
Promotiecommissie
Promotores Prof. Dr. A.C. Gittenberger-de Groot Prof. Dr. R.E. Poelmann
Co-promotores Dr. M.J. Goumans Dr. D.E. Atsma Overige leden Prof. Dr. J.A. Romijn
Dr. R. Passier
Prof. Dr. R.A. Bank (Rijksuniversiteit Groningen)
The work presented in this thesis was carried out at the Department of Anatomy and Embryology of the Leiden University Medical Center and was supported by a grant from the Interuniversity Cardiology Institute of the Netherlands (05.303).
Financial support by the Netherlands Heart Foundation and the Interuniversity Cardiology Institute of the Netherlands for the publication of this thesis is gratefully acknowledged.
The realization of this thesis was also financially supported by the Matty Brand Stichting, J.E.
Jurriaanse Stichting, Boehringer Ingelheim, Astellas Pharma and Astra Zeneca.
Maximus in Minimis
Chapter 1
General Introduction
Chapter 2
Epicardium-derived cells in cardiogenesis and cardiac regeneration.
Cellular and Molecular Life Sciences, CMLS. 2007; 64(6): 692-703
Chapter 3
Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart.
Circulation. 2007; 116(8): 917-927
Chapter 4
The potential of epicardium in the first week after myocardial infarction.
Submitted for publication
Chapter 5
A new direction for cardiac regeneration therapy: application of synergistically acting epicardium-derived cells and cardiomyocyte progenitor cells.
Circulation: Heart Failure. 2009; in press
Chapter 6
Mesenchymal stem cells from ischemic heart disease patients improve left ventricular function after acute myocardial infarction.
American Journal of Physiology. 2007; 293(4): H2438-H2447.
Contents
9-22
25-42
43-70
71-94
95-130
133-152
Part 1
Epicardium-derived cells and their potential in the infarcted adult heart.
Part 2
Mesenchymal stem cells as source for
cardiac regeneration
Chapter 7
Left ventricular function in the post-infarct failing mouse heart by magnetic resonance imaging and conductance catheter: a comparative analysis.
Acta Physiologica. 2008; 194(2): 111-122
Chapter 8
Cell tracking using iron-oxide fails to distinguish dead from living transplanted cells in the infarcted heart.
Magnetic Resonance in Medicine. 2009; in press
Chapter 9 General Discussion
Abbreviations Summary Samenvatting Publications Curriculum Vitae Dankwoord 155-172
173-182
183-199
200 206 210 214 220 222
Part 3
Functional assessment of the infarcted
mouse heart
General Introduction
1
Background
Embryonic heart development Cardiac regeneration therapy 1. Embryonic versus adult cells
2. Adult cells harvested from extracardiac structures 3. Adult cells derived from the heart
Functional assessment of the infarcted mouse heart Chapter Outline
General
Introduction
Background
The epidemic proportions of cardiovascular disease, with coronary artery occlusions and its consequences ranking first in the mortality list of the Western world 1, warrant the development of more appropriate treatment modalities. Promising results of recent innovative stem cell studies direct ischemic heart disease research into an entirely novel field. To accurately design such new experiments, and to fully understand the implication of the outcome, knowledge about embryonic development of the heart might be of benefit. Cardiogenesis and cardiac regeneration therapies will be discussed, emphasizing the outer layer of the heart, the epicardium, which might have promising potential in the adult setting.
Embryonic heart development
Early in gestation, two crescent-shaped cardiogenic plates arise from the anterior splanchnic mesoderm and fuse to form the primitive single-chambered heart tube 2,3 (Figure 1a, b). This straight tube undergoes a complex process of looping and segmentation, orchestrated by a cascade of genes that are important for left-right programming and cardiomyocyte differentiation, before a four- chambered heart is formed. During this process various cells from the so-called second heart field (SHF) add to and grow into the primitive heart, which was initially formed solely by derivatives of the first heart-forming field (FHF)4-10. Within the cardiac crescent the splanchnic mesoderm destined to form either of these two heart fields can already be distinguished, with the precursors of the SHF located at the centre (Figure 1a). The contribution of the SHF is crucial for proper cardiogenesis. The FHF-derived structure does not contain the entire assembly of necessary elements to build a complete heart.
Initially, before cell populations from the SHF are added, the primitive heart tube consists of only endocardium and myocardium with a cardiac jelly in between (Figure 1c). During the looping of the heart this cardiac jelly becomes unevenly distributed, resulting in large protrusions at the sites of the atrioventricular (AV) junction and outflow tract (OFT). The initially acellular structures become invaded by mesenchymal cells derived from the endocardium as a result of epithelial-mesenchymal transformation (EMT) 11. These early cushions will give rise to the cardiac valves in the mature heart, but are also instrumental in cardiac septum-formation. At the moment that the cushions become colonized, SHF-derived cells and cardiac neural crest cells (CNCs) start to populate the outflow and inflow portions of the heart (Figure 1d). The CNCs play likewise an important role in cardiac septation and in cushion development 12-15. Since the majority of the CNCs is destined for apoptosis 12,13, their contribution is suggested to be mainly instructive, rather than constructive.
The SHF contribution is both instructive as well as constructive. The SHF is divided in an anterior heart field (AHF), which adds to the OFT or arterial pole 4,8,10, and a posterior field (PHF), from which the derivatives are located at the inflow tract (IFT) or venous pole (Figure 1d) 9. Besides a considerable contribution to the IFT myocardium 9, the PHF delivers the epicardial progenitors to the heart by forming the pro-epicardial organ (PEO) 16.
The PEO consists of mesothelial protrusions and villi in the splanchnic mesoderm located near the venous pole of the heart (Figure 1d). During the looping stages of cardiac development, cells cross the coelomic cavity as free-floating cell aggregates (mammals and fish)17,18 or guided by tissue bridges (avians)19,20, although a combination is also suggested 21. At first, the epicardial (progenitor) cells attach to the naked heart tube at the dorsal side of the atrioventricular sulcus. From that point, they spread over the heart to cover it with its epicardial layer 22.
It has only recently been discovered that the role of the outermost layer of the heart, the epicardium,
is much more extensive than being the ‘cover’ that enables friction-free movement in the pericardial cavity 17,23-26. The derivatives of the epicardium, the epicardium-derived cells (EPDCs), contribute in both a constructive as well as an instructive way to normal cardiac development. EPDCs are generated soon after the heart has been enveloped entirely by epicardium, as a result of EMT 27. From the subepicardial space, where the EPDCs are located at first, they migrate into the myocardium. They add to the AV cushions, the cardiac interstitium, and the fibrous heart skeleton by delivering fibroblasts
27-30. EPDCs also stabilize the coronary arteries as smooth muscle cells and adventitial fibroblasts
26,28-31. As regulators of cardiogenesis, EPDCs appear to be important for the formation of the compact
myocardium 30,32-34 and for proper ingrowth of coronary arteries into the aorta 35. Cardiac looping 32,36 and development of the Purkinje fiber system 37 are also dependent on EPDC-generated signaling. It has not yet been investigated whether EPDCs remain situated in the cardiac interstitium or in the wall of coronary arteries once they have migrated there. Besides their ultimate destiny, their function in the adult setting remains to be studied since EPDCs hold great potential for application in cardiac regeneration therapy (discussed further below).
Cardiac regeneration therapy
Compared to the effective, mainly curative treatments of cardiac arrhythmias (catheter ablations, pacemakers and implantable defibrillators), cardiac valve dysfunction (artificial valves), and coronary obstructions (dotter procedures, stents and surgical bypasses), common therapy of the infarcted heart might be considered somewhat old-fashioned. Currently, general treatment aims at limiting the region of injury by reopening the blocked vessel, and at reducing workload of the remaining tissue with pharmaceutics. But real healing or revival of the dead tissue is not yet possible, and homograft transplantation is no first-line therapy because of the small size of donor organ pool, high risk of complications, and costs. Reasonably, the exciting data from the study by Orlic et al 38 which
Figure 1. Schematic picture illustrating consecutive stages of cardiac development. The cardiac crescent, which is derived from the splanchnic mesoderm (a) first forms the primary heart tube (PHT, pink, b) and atrioventricular endocardial cushions (EC, blue, d). The yellow area within the cardiogenic plates (a) and behind the PHT (c) depicts the second heart field (SHF) that in later stages adds myocardium to the developing heart (d). The subset of the SHF contributing to the arterial pole (AP) of the heart is called the anterior heart field (AHF), and the part that adds to the venous pole (VP) the posterior heart field (PHF, d). The pro-epicardial organ (PEO), which is a subpopulation of the SHF that can be distinguished as a lobulated protrusion at the VP of the heart delivers the epicardial cells to the heart (i.e. the epicardium, d). Cardiac neural crest cells (CNCs, dark blue cells) migrate from the neural tube into the heart, thereby passing through the SHF mesoderm (d). G: gut, C: coelomic cavity, Ep: epicardium.
Modified after Gittenberger-de Groot AC et al, Fetal Cardiology 3rd edition, in press.
demonstrated that bone marrow-derived stem cells could regenerate the injured area and improve cardiac function, energized many cardiovascular scientists and instigated even more new research projects in this field.
1. Embryonic versus adult cells
A large variety of cell populations can be applied for cell-based therapy of the infarcted heart, each having its own profile of advantages, limitations and feasibility. Cell types used can be sorted along their developmental potential. The most potent cells in an organism regarding differentiation are the ‘totipotent’ blastomeres. They form the embryo and also the placenta. Later, during the blastocyst stage, the structure giving rise to the placenta (trophoblast) can be distinguished from the source of the three germ layers of the embryo, the inner cell mass. The cells from the inner cell mass are ‘pluripotent’ and are referred to as embryonic stem cells, with the capacity to self-renew, to differentiate into all lineages 3940,41, and to grow clonogenic 42. Stem cells that reside within the adult somatic tissue are already committed to a specific lineage, and are only able to differentiate into a more selected variety of differentiation pathways, making them ‘multipotent’ stem or progenitor cells.
In current basic fundamental animal research as well as in clinical experimental treatments mainly adult stem or progenitor cells are used, in comparison to the more scarce application of embryonic stem cells. Arguments for this imbalance could be that i) appropriate handling of embryonic stem cells is intricate, ii) usage of embryonic stem cells is ethically challenging, and iii) includes the risk of teratoma formation, iv) adult stem cells can be derived from the patient itself and thus rejection problems are obviated, v) some adult cells, like bone marrow-derived cells, are easily available in large amounts. Drawbacks of adult cells are, however, that possible genetic defects might impair function of the cells to be applied, that they can not be expanded unlimitedly, and that many types are not immediately available at the moment of e.g. acute infarction. Recently described induced pluripotent cells (iPS) might combine best properties of both. These are reprogrammed adult cells in which pluripotency can be induced in vitro by overexpressing a combination of specific transcription factors
43-46. However, their feasibility in vivo has not yet been revealed, leaving them to be dreamed of for the
future.
2. Adult cells harvested from extracardiac structures
The most extensively studied adult stem cell populations at the moment are those derived from the bone marrow 47. This is a rich source of various immature cells that are applied in basic and clinical experimental cardiac regeneration therapy, being hematopoietic stem cells (HSCs) 38,48-50, a subset of these, the so-called side population cells 51-53, mesenchymal stem cells (MSCs) or stromal cells 54-56, multipotent adult progenitor cells (MAPCs) 57,58, which are a subset of MSCs, and bone marrow-derived multipotent stem cells (BMSCs) 59. MAPCs and BMSCs are able to differentiate into all three germ layers 57,59 a property which has always been considered to be exclusive for embryonic stem cells.
Commonly, especially in clinical trials, a mixture of these subpopulations is used, which are then referred to as bone marrow-derived mononuclear cells (BMCs) 60. Bone marrow-derived cells do not necessarily need to be harvested from the bone marrow, some can also be isolated from the blood, like the circulating progenitor cells (CPCs) 61,62 which include the endothelial progenitor cells (EPCs)
63. Furthermore, potential cardiotherapeutic progenitor cell populations can be derived from adipose tissue 64,65, skeletal muscle 66-68, placental cord blood 69, and probably many other less obvious tissues.
3. Adult cells derived from the heart
It seems logical to expect cell populations derived from the target-organ being the most appropriate cell source for its own tissue regeneration, supposing these cells are present in the organ of interest.
With regard to the heart, the presumed cardiac derived progenitor cells would probably be committed
to the cardiac lineage, adapted to the specific environment and they might have been programmed to excrete factors of use for the heart. Research of the last decade focused to find progenitor cells in the adult heart revealed indeed several progenitor populations, although the heart has long been considered as an organ without regenerative potential. The heart was demonstrated to possess endogenous regenerative potential 70 and to harbor small multipotent cells that could differentiate into cardiomyocytes. These cells were recognized based on their expression of stem cell markers stem cell antigen-1 (sca-1) 71-76 (for human tissue referred to as cardiomyocyte progenitor cell or CMPC), c-kit 77, or the primitive SHF marker islet1 (isl1) 78,79. But they were also phenotyped by their expression profile in combination with adherence properties (cardiospheres) 80, or their ability to efflux the Hoechst dye 81. Of special interest are the CMPCs which can be isolated from human fetal and adult cardiac tissue, since they can differentiate into cardiomyocytes in vitro without the necessity of being co-cultured with neonatal cardiomyocytes 74. The different multipotent residents described by various groups might, however, represent closely related subpopulations of one major cardiac stem cell pool. If these different cell types with cardiomyocyte differentiation potential characterize isolated populations resident in the heart, it is expected that ischemic injury would at least partly be mended by the organ itself. Although adult stem and progenitor cells from non-cardiac tissues are already widely tested for their therapeutic applicability, stem and progenitor cells resident in the heart itself are currently only of modest interest when evaluated for their usage in clinical studies.
Knowing that the adult heart harbors cardiomyocyte-precursors leads to the expectation that other lineage-destined ‘remnants’ of early development are also present in the postnatal heart, including EPDCs. As mentioned before, EPDCs are progenitors of the cardiac interstitial fibroblasts and medial and adventitial cells of the coronary arteries with extensive instructive properties during cardiogenesis. It might be speculated that adult EPDCs could recapitulate their embryonic properties, for example in case of ischemic injury. It is, however, not known whether EPDCs are indeed present and still potent in the adult heart.
In this thesis it is studied whether EPDCs could be derived from the adult epicardium 82, and if these cells could be of benefit for cardiac repair of the adult ischemic heart. It is hypothesized that the adult EPDCs improve cardiac function after myocardial infarction through paracrine signaling like they demonstrate during embryogenesis. This presumed supportive and instructive effect of the adult EPDCs on the surrounding injured host tissue is also elucidated. It is tested whether their profit could be further increased by co-transplanting complementary CMPCs. Being mainly located in the atrium 83, CMPCs are suggested to originate from the SHF like EPDCs, designating them as ontogenetically closely related. A further mesenchymal cell type is studied, the bone marrow-derived MSC. It is examined whether their potential is retained if they are derived from ischemic heart disease patients, as opposed to healthy donors.
Functional assessment of the infarcted mouse heart
The extensive interest in the cardiac stem-cell field, with the general aim to cure the adult heart by regenerative cellular therapies, has resulted in the emergence of numerous basic animal studies.
Although small rodents are phylogenetically less related to human than large mammals, mouse and rat are most commonly applied for basic animal studies, since their genetic background is well known and their usage is less complicated and expensive than that of larger animals. To evaluate the effect of the cell type of interest in vivo, i.e. to test whether it can stimulate cardiac performance, functional assessment is needed. This requires highly specialized techniques, in particular for the mouse, considering the extreme small size of the heart (7 mm) and high heart rate (~500 beats per minute).
Nonetheless, magnetic resonance imaging (MRI), echocardiography and invasive pressure-volume (PV) loop measurements have become available for cardiac phenotyping of small animals.
In this thesis several aspects of MRI, which is the method of choice for longitudinal stem cell experiments 84, are described. Additional applications of MRI, being infarct size measurements and identification of transplanted cells in the living heart by usage of iron labeling, are discussed. Also, MRI and PV loop measurements, which each address different aspects of left ventricular function, are compared for their reliability in the infarcted mouse heart.
Chapter Outline
Chapter 1 provides background information regarding embryonic heart development and stem cell therapy for cardiac regeneration. Technical issues in functional basic experimental stem cell research are shortly introduced.
Chapter 2 describes the contribution of EPDCs to the developing heart. It speculates on the role of these cells in the adult heart, in specific their potential in the infarcted heart.
Chapter 3 translates the knowledge from the embryonic situation to the adult diseased heart, investigating whether EPDCs harvested from the adult heart can contribute to functional improvement after myocardial infarction.
Chapter 4 addresses potential underlying mechanisms that might explain the positive effect of adult EPDCs on the infarcted adult heart. It focuses on histological and functional differences between normal scar development and cardiac healing after adult EPDCs have been transplanted.
Chapter 5 reports on the interaction between CMPCs and EPDCs. Their mutual effect is studied parallel in the in vitro and in vivo situation, from which the latter tests the hypothesis that combined transplantation will be of a synergistic benefit for the infarcted heart.
Chapter 6 concerns the applicability of MSCs from ischemic heart disease patients to cure the infarcted heart. Engraftment and differentiation of transplanted MSCs are addressed, as well as their potential to improve LV function.
Chapter 7 compares two methods used for heart function determination of small laboratory animals.
Reliability of MRI and PV–loop measurements for assessment of cardiac function of the failing mouse heart is analyzed.
Chapter 8 describes methodological issues around iron-oxide labeling for tracing of stem cells in the mouse heart.
Chapter 9 provides a general discussion on the concept of using cardiogenesis as basis for cardiac regeneration therapy. Application of adult EPDCs to improve cardiac function after myocardial infarction is addressed, as are potential mechanisms which might explain the detected profit. Other mesoderm-derived cell types are conferred, and technical issues in functional assessment of the infarcted heart are placed in perspective.
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Part 1
Epicardium-derived cells
and their potential in the
infarcted adult heart
Dept. of Anatomy & Embryology, Leiden University Medical Center, Leiden, The Netherlands
Winter EM and Gittenberger-de Groot AC
Epicardium-derived cells in cardiogenesis and cardiac regeneration
Cellular and Molecular Life Sciences, CMLS. 2007; 64(6): 692-703
2
Abstract
During cardiogenesis, the epicardium grows from the proepicardial organ to form the outermost layer of the early heart. Part of the epicardium undergoes epithelial-mesenchymal transformation, and migrates into the myocardium. These epicardium-derived cells differentiate into interstitial fibroblasts, coronary smooth muscle cells, and perivascular fibroblasts. Moreover, epicardium- derived cells are important regulators of formation of the compact myocardium, the coronary vasculature, and the Purkinje fiber network, thus being essential for proper cardiac development. The fibrous structures of the heart such as the fibrous heart skeleton and the semilunar and atrioventricular (AV) valves also depend on a contribution of these cells during development. We hypothesize that the essential properties of epicardium-derived cells can be recapitulated in adult diseased myocardium. These cells can therefore be considered as a novel source of adult stem cells useful in clinical cardiac regeneration therapy.Introduction
The epicardium consists of mesothelial epithelial tissue that forms the outermost layer of the heart.
It has many functions during embryonic development and adult life, which were unknown until 40 years ago. Covering the myocardium, the epicardium serves as a smooth layer which enables the heart to slide over the outer pericardial epithelium. During embryogenesis, the epicardium gives rise to all cellular elements of the subepicardial layer, to interstitial and perivascular fibroblasts, and to smooth muscle cells of the coronary arteries. Moreover, recent data demonstrated that epicardium and epicardium-derived cells (EPDCs) have a crucial stimulatory role in the development of the embryonic compact myocardium, the coronary vasculature and the Purkinje fiber system. Their role in valve and fibrous heart skeleton differentiation is still unresolved. In this review we will discuss the origin of the epicardium and the function of EPDCs and their derivatives in embryonic cardiac development. We thereafter postulate that EPDCs might recapitulate their embryonic capacities when in contact with adult diseased myocardium. In this way they can serve as an adult stem cell for cardiac regeneration.
Origin of the epicardium
Initially, the primary heart develops from two cardiogenic fields of splanchnopleuric mesoderm that differentiate into a myocardial tube, lined on the inside by endocardium 1. Between these layers the cardiac jelly is produced. This structure is called the primary heart tube 2 and protrudes into the coelomic cavity referred to as the pericardio-peritoneal canal. The dorsal mesocardium, during development separated to form arterial and venous pole connections, links the primary heart tube to the dorsal body wall. Later on, the primary heart tube is covered by a layer of epicardium, which arises at the venous pole.
Our current view on epicardial origin was already proposed by Kurkiewicz in 1909. He reported that the myocardium consisted solely of cardiomyoblasts, and that the epicardium was derived from an extracardiac source 3. His findings were disregarded and overlooked for a long time. The prevailing dogma in the middle of last century was that the epicardium formed an inert layer, basically
functioning to protect the myocardium, and itself being derived from the myocardium. This layer was also referred to as the epimyocardium 4,5. Manasek showed, using light and transmission electron microscopy, that the early myocardium consisted of cardiomyoblasts only, and thus did not contain epicardial cells. He hypothesized that the epicardium originated from an extracardiac source, but he did not exclude that the myocardium could dedifferentiate into epicardial cells as well 6,7. Viragh gave the solution, by studying mouse embryos with light and transmission electron microscopy.
He observed that epicardial cells migrated to the heart from somatopleural cells of the transverse septum 8. Ho and Shimada supported the research from Viragh, by demonstrating with scanning electron microscopy (SEM) that epicardial cells and cardiomyocytes were cytologically different. They did not find transitional cardiomyocytes, thereby dismissing the possibility that cardiomyocytes would dedifferentiate into epithelial cells 9. By broader use of SEM, many studies clarified the origin of the epicardium. It was shown in amphibians, reptiles, birds and mammals that epicardial cells are derived from villous protrusions in the region of the venous pole of the heart near the developing transverse septum 9-14. Further knowledge about the origin and attachment of epicardial cells to the heart, their spreading pattern, transformation of the cells into mesenchymal cells, and their derivatives, was derived from experiments in the last decade of the 20th century.
The term proepicardial organ (PEO) was coined to describe the previously mentioned villous protrusions because of its heterogeneous cell structure 15, although it is not a real organ. The PEO arises from the coelomic serosa and its immediately underlying mesoderm. This area is also the source of sinus venosus myocardium. We adhere in this review to the use of the term PEO solely
Figure 1.
Spreading and migration of EPDCs. (a) Whole-mount quail embryo (HH16) stained for HNK1, showing a clearly demarcated proepicardial organ (PEO) at the venous pole of the heart. (b) Schematic representation indicating (arrows) the direction of growth of the epicardium over the myocardial tube. (c-e) Schematic drawing of increasing ages with the migration pattern of the EPDCs. (c) HH24: epicardial cells cover the heart tube, and EPDCs (star shaped, grey) enter the myocardium and, through gaps, the subendocardial layer. The endocardial cushion is still devoid of EPDCs. (d) HH28: the compact myocardium is formed, and EPDCs have entered all cardiac components. Note the contribution to the formed atrioventricular sulcus and the endocardial cushions.
(e) HH35: the coronary vasculature has grown into the aorta, and EPDCs through epithelial mesenchymal transformation (EMT, cuboid cells) now also contribute to the coronary arterial vascular wall. OT: outflow tract, SV: sinus venosus, PEO: proepicardial organ, AVC: atrioventricular cushion, EPDC: epicardium-derived cell, Ep: epicardium, V: ventricle, A: atrium, AVS: atrioventricular sulcus, EC: endothelial cell, SMC: smooth muscle cell, Fb: fibroblast, CA: coronary artery, Ao: aorta.
for the transient cauliflower like structure which has already differentiated into a purely epicardial direction. In mammals, the PEO consists of bilaterally and symmetrically distributed clusters of mesothelial protrusions and villi, covering the transverse septum 10-12. In avian embryos, the PEO first consists of bilateral protrusions of the right and left sinus horns. Preceded by right sided asymmetric gene expression 16, the left part ceases to develop, leaving only the right protrusion to form into a cauliflower-like structure consisting of mesothelial villi covered by squamous cells 17.
The way in which the epicardial cells translocate from the proepicardial serosa to the heart differs between species. In avian embryos the main pathway through which epicardial cells reach the heart is a tissue bridge between the ventral side of sinus venosus and the dorsal surface of the developing ventricles 14,18,19. This tissue is positioned around, and probably guided by, a bridge of extracellular matrix 20. In mammalian and fish embryos such a sino-ventricular ligament is absent. Free-floating epicardial cell aggregates fuse together to give rise to the epicardial sheet that covers the heart 10-12,21. The epicardial cells cover the developing heart in a spatiotemporal pattern comparable for various species 9,11,13,14,19,22. Embryo stages described for quail can be extrapolated to corresponding stages in other species. At Hamburger Hamilton stage 14 (HH14) 23, the PEO of the quail embryo starts to develop at the ventral surface of the proepicardial serosa 15 (Figure 1a, b). Villi protrude from the surface at HH 15 and 16, giving it its cauliflower-like appearance 10,15,19. At HH17, the tips of the epicardial protrusions reach the dorsal surface of the early heart tube at the atrioventricular sulcus, and form a circular patch of epicardial cells as they migrate radially over the myocardium at stage 18 19. At HH18- HH20 the epicardial cells spread ventrally along the left and right side of the atrioventricular canal to the inner curvature of the heart, and caudoventrally over the ventricular inlet segment. Spreading proceeds at HH20-HH24 from the inner curvature, over the outflow tract, towards the ventriculo- arterial junction. The right atrium is completely covered between HH23 and HH24. At HH25 the left atrium and a part of the outflow tract are the only parts of the heart that are still uncovered. Whole- mount cytokeratin staining patterns show that these parts are covered at HH26, by which stage the epicardial covering of the heart is complete 19, and after which proepicardial structures are no longer seen 15.
There is some conflicting evidence on timing and source of epicardium at the ventriculo-arterial junction, which may be tracing technique dependent. Whole-mount cytokeratin studies show complete covering of the myocardial outflow tract by PEO-derived epicardium at HH26 19. After complete PEO ablation, arterial pole-derived mesothelial cells, also referred to as cephalic
pericardium, cover a myocardial collar of the outflow tract at HH28 24,25. This might be explained by the concurrent addition of secondary heart field myocardium to the outflow tract (for review see
26). From quail-chick chimera techniques, it has been described that the distal part of the outflow tract is covered by a mixed population of arterial pole-derived mesothelium and venous pole-derived epicardium at least until HH35 24,25,27,28.
Origin of epicardium-derived cells (EPDCs)
After the primitive heart has been covered by a layer of epicardial cells, part of the epicardial cells undergoes epithelial-mesenchymal transformation (EMT), thereby acquiring the ability to migrate.
Gittenberger-de Groot and co-workers called these cells that undergo EMT epicardium-derived cells or, for short, EPDCs 29. The EPDCs migrate into the, originally acellular, subepicardial space and subsequently into the myocardium, where they differentiate into various cell types. EMT involves cytoskeletal reorganization, observed in epicardial cells both in vivo 30-32 and in vitro 33. Proepicardial and epicardial cells contain the keratin tonofilament bundles ‘cytokeratin’ 15,19. These bundles are replaced by filaments of vimentin during the process of transformation. This substitution process is not
instantaneous. Therefore, a coexpression of vimentin and cytokeratin is observed in the proepicardial cells that will undergo EMT, and in the EPDCs recently derived from the epicardial layer 30,31.
Insight into timing of EPDC invasion into the developing heart can be gained by quail-chick chimera studies 34,35, viral tracing experiments 33,36, and EPDC reporter gene studies in mice 37. The results of the tracing studies, however, focus mostly on subsequent EPDC differentiation which will be dealt with later on in this review. Normal quail-chick chimera experiments in which quail PEO is added to chick PEO 34,35 are superior to blocking of the PEO by an eggshell membrane 18, because delay in PEO outgrowth is initiated in the latter. Quail-chick experiments provide evidence that already at HH19, immediately after the onset of spreading over the myocardial surface, EMT is seen and EPDCs migrate into inner curvature myocardium. This area seems to be specifically permissive at this time point as other myocardial areas are not yet invaded 35. Thereafter, invasion of the still thin atrial and ventricular myocardium is seen, with a specific migration to the subendocardial layer through myocardial gaps from HH20-24 29 (Figure 1c). With formation of compact myocardium these gaps disappear and EPDCs are found throughout both the compact and trabecular myocardium (Figure 1d).
At HH28, invasion of the atrioventricular endocardial cushions is seen as well as abundant filling of atrioventricular and periarterial mesenchyme 27,29. These data are recently supported by results from studies with epicardium-restricted LacZ expression in transgenic mice 37. At the time of ingrowth of the coronary vasculature into the aorta (HH32) (Figure 1e), abundant EMT is seen adjacent to the developing coronary orifices 32. It is unknown whether this process of EMT continues throughout development, initial hatching or birth, or even into postnatal stages.
Molecular processes involved in epicardium and EPDC formation
Although important regulators of EMT and differentiation of EPDCs have been recently described, only little is known about these processes. Most of the factors discovered to be important for epicardial outgrowth and EPDC formation were used as manipulative targets to study the role of the EPDC in cardiac development. In this paragraph we will discuss the principal molecular processes known to date.
Factors involved in adhesion of epicardial cells
Interaction between vascular cell adhesion molecule (VCAM-1) and α4 integrin is essential for adhesion and spreading of the epicardium 38-40. These surface molecules are expressed in a reciprocal fashion in the myocardium and epicardium, respectively, and mediate cell-cell adhesion. VCAM-1 and α4 integrin null mice show a remarkably comparable phenotype, being absence of epicardium, absence of subepicardial vessels with subsequent cardiac hemorrhage 38,39, and hampered compaction of the ventricular myocardium 38. Yang et al showed that α4 integrin is not essential for initial adhesion of epicardium to the myocardium, but that it is crucial for the maintenance of epicardial integrity.
In contrast, a more recent study showed that α4 integrin is not only essential for maintaining the epicardium, but that it is also involved in the earlier process of outgrowth of the epicardium from the PEO and the subsequent spreading of the epicardium over the heart 40. It was also described that normal levels of α4 integrin promote adhesion of epicardial cells and restrain EMT and migration, while inhibition of α4 integrin leads to stimulation of EMT 41. Spreading of epicardial cells and maintenance of epicardial integrity therefore depend on a balanced interaction between VCAM-1 and α4 integrin.
Factors involved in outgrowth and differentiation of EPDCs
Essential for the initial steps in EMT are the homologous transcription factors Snail and Slug, expressed in mammalian and avian embryos, respectively 42-44. Slug, expressed by the proepicardium, epicardium and undifferentiated EPDCs 45, can trigger EMT in epithelial cells by repression of cell adhesion molecules, including E-cadherin 43,44,46. It would be interesting to study the relation between Slug and α4 integrin, because it has been demonstrated as mentioned above that inhibition of α4
