Heleen Lie-Venema

Origin, Fate, and Function of Epicardium-Derived Cells (EPDCs) in Normal and Abnormal Cardiac Development

During heart development, cells of the primary and secondary heart field give rise to the myocardial component of the heart. The neural crest and epicardium provide the heart with a considerable amount of nonmyocardial cells that are indispensable for correct heart development. During the past 2 decades, the importance of epicardium-derived cells (EPDCs) in heart formation became increasingly clear. The epicardium is embryologically formed by the outgrowth of proepicardial cells over the naked heart tube. Following epithelial-mesenchymal transformation, EPDCs form the subepicardial mesenchyme and subsequently migrate into the myocardium, and differentiate into smooth muscle cells and fibroblasts. They contribute to the media of the coronary arteries, to the atrioventricular valves, and the fibrous heart skeleton. Furthermore, they are important for the myocardial architecture of the ventricular walls and for the induction of Purkinje fiber formation. Whereas the exact signaling cascades in EPDC migration and function still need to be elucidated, recent research has revealed several factors that are involved in EPDC migration and specialization, and in the cross-talk between EPDCs and other cells during heart development. Among these factors are the Ets transcription factors Ets-1 and Ets-2. New data obtained with lentiviral antisense constructs targeting Ets-1 and Ets-2 specifically in the epicardium indicate that both factors are independently involved in the migratory behavior of EPDCs. Ets-2 seems to be especially important for the migration of EPDCs into the myocardial wall, and to subendocardial positions in the atrioventricular cushions and the trabeculae. With respect to the clinical importance of correct EPDC development, the relation with coronary arteriogenesis has been noted well before. In this review, we also propose a role for EPDCs in cardiac looping, and emphasize their contribution to the development of the valves and myocardial architecture. Lastly, we focus on the congenital heart anomalies that might be caused primarily by an epicardial developmental defect.

Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart

Background— Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction. Methods and Results— EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4×105 enhanced green fluorescent protein–labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of α-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (α-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected. Conclusions— After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.

Ets-1 and Ets-2 Transcription Factors Are Essential for Normal Coronary and Myocardial Development in Chicken Embryos

&NA; —In the development of a functional myocardium and formation of the coronary vasculature, epicardium‐derived cells play an essential role. The proepicardial organ contributes to the developing coronary system by delivering mural cells to the endothelium‐lined vessels. In search of genes that regulate the behavior of (pro)epicardial cells, the Ets‐1 and Ets‐2 transcription factors stand out as strong candidates. In the present study, the hypothesis that Ets transcription factors have a role in proper coronary and myocardial development was tested via antisense technology, by targeting Ets‐1 and Ets‐2 mRNAs to downregulate protein expression in chicken embryos. The results suggest that hereby the development of the coronary system is hampered, primarily by defects in the process of epithelial‐mesenchymal transformation of the mesothelia of the primary and secondary heart fields. This was indicated by a lack of periarterial and epicardial mesenchyme, of peripheral coronary smooth muscle cells, and changes in myocardial morphology. A defect in myocardial perfusion caused by the absence of one or both coronary ostia seems to be “solved” by the development of numerous small fistulae connecting the ventricular lumen with the subepicardially located coronary vessels. The presence of coronary vascular aberrations in the antisense‐Ets phenotype enabled us for the first time to study abnormal coronary development in a model that is not lethal to the embryo. (Circ Res. 2003;92:749–756.)

Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: Correlation with abnormal epicardial development.

Epicardium and epicardium‐derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three‐dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium‐derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial–mesenchymal transformation caused by up‐regulation of E‐cadherin, normally down‐regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial–myocardial interaction. Abnormal epicardial differentiation and reduced epithelial–mesenchymal transformation result in deficient epicardium‐derived cells leading to myocardial pathology and cardiac anomalies. Developmental Dynamics 237:847–857, 2008.

Coronary Artery and Orifice Development Is Associated With Proper Timing of Epicardial Outgrowth and Correlated Fas Ligand Associated Apoptosis Patterns

The proepicardial organ provides differentiated cell types to the myocardial wall and facilitates coronary development. Ingrowth of the coronary arteries into the aorta has recently been linked to apoptosis. This study was set up to examine the effect of an inhibition of epicardial outgrowth on apoptotic patterning and coronary development. Epicardial outgrowth was blocked at HH15–17 in quail embryos, which survived until HH25–35 (n=33). Embryos with complete inhibition of outgrowth did not survive after HH29. These embryos presented with thin compact myocardium, devoid of vessels. In embryos with delayed epicardial outgrowth the phenotype was less severe, and surviving embryos were studied up to HH35. In these embryos, myocardial vascularization was poor and apoptosis in the peritruncal region at HH30 was diminished. Embryos at HH35 displayed an abnormal coronary network and absent coronary orifices. In a further set of experiments (n=10), outgrowth was inhibited in chicken embryos at HH15, followed by transplantation of a quail proepicardial organ into the pericardial cavity to rescue cardiac phenotype. These chimeras were studied at HH29 and HH35. Myocardial development was restored; however, in 3 of 4 embryos (HH35), the coronary orifices were absent. Examination of double stainings of quail-chicken chimeras revealed that EPDCs produce Fas ligand as an apoptotic inductor at sites of coronary ingrowth. In the absence of proper timing of epicardial outgrowth, myocardial development and vascularization are disturbed. Also apoptosis in the peritruncal region is diminished. During later development, this leads to defective or absent connections of the coronary system to the systemic circulation.

PDGF-B signaling is important for murine cardiac development: Its role in developing atrioventricular valves, coronaries, and cardiac innervation

We hypothesized that PDGF‐B/PDGFR‐β‐signaling is important in the cardiac contribution of epicardium‐derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf‐b−/− and Pdgfr‐β−/− mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for αSMA, periostin, ephrinB2, EphB4, VEGFR‐2, Dll1, and NCAM was performed on wild‐type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest–related innervation deficits. This implies a role for PDGF‐B/PDGFR‐β‐signaling specifically in the contribution of these cell lineages to cardiac development. Developmental Dynamics 237:494–503, 2008.

Epicardium-Derived Cells in Development of Annulus Fibrosis and Persistence of Accessory Pathways

Background— The developmental mechanisms underlying the persistence of myocardial accessory atrioventricular pathways (APs) that bypass the annulus fibrosis are mainly unknown. In the present study, we investigated the role of epicardium-derived cells (EPDCs) in annulus fibrosis formation and the occurrence of APs. Methods and Results— EPDC migration was mechanically inhibited by in ovo microsurgery in quail embryos. In ovo ECGs were recorded in wild-type (n=12) and EPDC-inhibited (n=12) hearts at Hamburger-Hamilton (HH) stages 38 to 42. Subsequently, in these EPDC-inhibited hearts (n=12) and in additional wild-type hearts (n=45; HH 38–42), ex ovo extracellular electrograms were recorded. Electrophysiological data were correlated with differentiation markers for cardiomyocytes (MLC2a) and fibroblasts (periostin). In ovo ECGs showed significantly shorter PR intervals in EPDC-inhibited hearts (45±10 ms) than in wild-type hearts (55±8 ms, 95% CI 50 to 60 ms, P=0.030), whereas the QRS durations were significantly longer in EPDC-inhibited hearts (29±14 versus 19±2 ms, 95% CI 18 to 21 ms, P=0.011). Furthermore, ex ovo extracellular electrograms (HH 38–42) displayed base-first ventricular activation in 44% (20/45) of wild-type hearts, whereas in all EPDC-inhibited hearts (100%, 12/12), the ventricular base was activated first (P<0.001). Small periostin- and MLC2a-positive APs were found mainly in the posteroseptal region of both wild-type and EPDC-inhibited hearts. Interestingly, in all (n=10) EPDC-inhibited hearts, additional large periostin-negative and MLC2a-positive APs were found in the right and left lateral free wall coursing through marked isolation defects in the annulus fibrosis until the last stages of embryonic development. Conclusions— EPDCs play an important role in annulus fibrosis formation. EPDC outgrowth inhibition may result in marked defects in the fibrous annulus with persistence of large APs, which results in ventricular preexcitation on ECG. These APs may provide a substrate for postnatally persistent reentrant arrhythmias.

Platelet-derived growth factors in the developing avian heart and maturating coronary vasculature.

Platelet‐derived growth factors (PDGFs) are important in embryonic development. To elucidate their role in avian heart and coronary development, we investigated protein expression patterns of PDGF‐A, PDGF‐B, and the receptors PDGFR‐α and PDGFR‐β using immunohistochemistry on sections of pro‐epicardial quail–chicken chimeras of Hamburger and Hamilton (HH) 28–HH35. PDGF‐A and PDGFR‐α were expressed in the atrial septum, sinus venosus, and throughout the myocardium, with PDGFR‐α retreating to the trabeculae at later stages. Additionally, PDGF‐A and PDGFR‐α were present in outflow tract cushion mesenchyme and myocardium, respectively. Small cardiac nerves and (sub)epicardial cells expressed PDGF‐B and PDGFR‐β. Furthermore, endothelial cells expressed PDGF‐B, while vascular smooth muscle cells and interstitial epicardium‐derived cells expressed PDGFR‐β, indicating a role in coronary maturation. PDGF‐B is also present in ventricular septal development, in the absence of any PDGFR. Epicardium‐derived cells in the atrioventricular cushions expressed PDGFR‐β. We conclude that all four proteins are involved in myocardial development, whereas PDGF‐B and PDGFR‐β are specifically important in coronary maturation. Developmental Dynamics 233:1579–1588, 2005.

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton

The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton. Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns. We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff-Parkinson White syndrome with persistent AV myocardial connections.

Ventriculo coronary arterial communications (VCAC) and myocardial sinusoids in hearts with pulmonary atresia with intact ventricular septum: two different diseases

Abstract Pulmonary atresia with intact septum is a cardiac malformation with a variable phenotype. Those cases that present with abnormal perfusion of the myocardium of the hypoplastic right ventricle can be distinguished in cases with myocardial sinusoids and cases with ventriculo coronary arterial communications. The first group most probably develops on the basis of atresia of the pulmonary orifice, a high pressure in the right ventricle with subsequent dilatation of the intertrabecular myocardial spaces and development of endocardial fibroelastosis. There is evidence that in cases with ventriculo coronary arterial communications the primary problem is formed by the coronary vasculature that connects abnormally to the ventricular lumen and in some cases also to the aortic orifice. This is exemplified in single and sometimes even completely absent coronary orifices. The altered haemodynamic circumstances with preferential flow through the communications lead to serious coronary vascular wall pathology with severe intimal thickening and partial obliteration of the subepicardial arteries. These anomalies are already present in the second trimester of pregnancy. Prenatal diagnosis supports furthermore, that the actual atresia of the pulmonary orifice may in some cases be secondary to the coronary abnormalities. Overall the anomaly primarily remains a right heart problem with myocardial, valvular and coronary vascular exponents.