Posterior heart field and epicardium in cardiac development : PDGFRα and EMT
Bax, N.A.M.
Citation
Bax, N. A. M. (2011, January 13). Posterior heart field and epicardium in cardiac development : PDGFRα and EMT. Retrieved from https://hdl.handle.net/1887/16330
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/16330
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General Discussion
ChAPter 8
the role of Pdgfrα in the posterior heart field
In this thesis we first described the development of cardiac structures derived from the second heart field. We studied in this respect the role of Pdgfrα, a gene which participates in the formation of the arterial and venous pole of the heart (Chapter 2-4).
We especially focussed on the role of Pdgfrα in the development of cardiac structures at the venous pole of the heart (Chapter 3 and 4). In our animal models we observed expression in the posterior heart field (PHF)-derived sinus venosus myocardium, including the sinoatrial node (SAN), the venous valves (VV), myocardium of the atrial septum (AS), dorsal atrial wall and the myocardial wall of the cardinal (CaV) and pulmonary veins (PV) (Chapter 2-4). In our Pdgfrα deficient mouse model, we describe a hypoplastic SAN, AS and dorsal atrial wall which was be suggested to be caused by diminished contribution of PHF and by increased expression of Nkx2.5 (Chapter 3). At the sinus venosus region, the wall of the PV develops and we observed malformations in the development of the PV related to altered addition of secondary myocardium and smooth muscle cells from the PHF due to lack of Pdgfrα (Chapter 3 and 4).
In our animal models we also described development of the PHF-derived proepicardial organ (PEO) and its derivates such as epicardium. Through a process of epithelial-to- mesenchymal transformation (EMT) these epicardial cells develop into epicardium-derived cells (EPDCs) (Chapter 2 and 3). In the Pdgfrα deficient mice we showed altered EMT with PEO- and EPDC-associated cardiac malformations such as a hypoplastic PEO, abnormal epicardial adhesion and ventricular compact myocardial hypoplasia. The observed malformations are related to impair epicardial-myocardial interaction and increased expression of Wilm’s tumor 1 (WT1) (Chapter 3).
In the second part of this thesis we focused on the role of EPDCs in the maturation of cardiomyocytes. Furthermore we investigated the process of EMT and the electrophysiological properties of human adult epicardial cells as these cells might be suitable for cell-based therapy in cardiac repair.
In the following paragraphs we will discuss the role Pdgfrα in the development of the PHF-derived cardiac structures related to clinical implications. Several congenital heart malformations (CHD) in the human population seem to be related to diminished PHF contribution and this suggest a role of Pdgfrα in the development of CHD. Further more we will discuss the role of PHF-derived PEO, epicardium and EPDCs in regenerative medicine.
Posterior heart field in Atrial septal defects
Atrial septal defects (ASD) are one of the commonest forms of CHD seen in children 1. The formation of atrial septa is initiated by the formation of the primary atrial septum (PAS) which grows from the dorsal atrial wall towards the atrioventricular (AV) cushions. Before
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the PAS fuses with the AV cushions small perforations develop in the upper part of the PAS and subsequently this will form the ostium secundum. A secondary septum develops and grows over the ostium secundum in the right atrium. Blood supply from the right to the left atrium passes a small passageway between the septum secundum and the PAS through the ostium secundum. This is called the foramen ovale and functions as a bypass for the premature pulmonary circulation. This foramen will close after birth 2,3. The most common form of ASD affects the region of the foramen ovale and is referred to as the atrial septum secundum defect (ASD II) 2-4. This defect develops from deficient formation of the PAS as well as insufficient formation of the secondary atrial septum 2-4.
Nkx2.5 was the first gene identified to be related to nonsyndromic ASDII 3. Both increase and decrease in transcriptional activity of this gene are involved in the development of ADSII 5. Two other genes that are involved in the development of ASDII are the Tbx5 and GATA4 gene. Mutations in Tbx5 produce the Holt-Oram syndrome (HAS), an autosomal disorder associated with CHD which is associated with ASDII 3.
A role for Pdgfrα is suggested by the observation that the atrial septum is incomplete 6 and hypoplasia of the atrial septum in Pdgfrα was suggested to be due to diminished addition of myocardium from the PHF (Chapter 3). Furthermore, our data suggested that Pdgfrα in transduction pathways may lead to repression of Nkx2.5 (Chapter 3). The repression of the Nkx2.5 gene regulated by Pdgfrα may lead to ADSII (Chapter 3) 3. Pdgfrα-signalling might also be important in the development of a primary atrial septum defect (ASDI). At the beginning of atrial septation there is an embryonic right to left connection via the ostium primum. This ostium is normally closed by the fusion of the mesenchymal cap on the PAS with the dorsal mesenchymal protrusion (also referred as spina vestibuli) and the AV cushions, as part of atrial septation 2,7-10. In our Pdgfrα deficient mouse model we observed hypoplasia of the mesenchymal cap on the PAS and the DMP (Chapter 3 and 4), which could cause altered fusion to the AV cushion and thereby contribute to the development of ASDI (Chapter 3)10.
Although the exact role of Pdgfrα-signalling in the development of ASD has to be further investigated. Based on the expression pattern of PDGFR-α and the abnormalities that occur by deficiency, we suggest that there is most probably role for Pdgfrα in the development of ASD.
Deficiency of Pdgfrα in Atrioventricular septal Defects
When the ostium primum is not closed like in ASDI, this is mostly occurring in combination with insufficient fusion of the superior and inferior atriventricular cushions, which results in an atrioventricular septal defect (AVSD) 2. The key features of AVSD are the absence of an atrioventircular septum and a common atrioventricular orifice 11. AV cushions contribute to both the formation of the AV septum and AV valves, and failure of fusion of
AV cushions with the atrial septum (ASDI) and the muscular portion of the ventricular septum is often the mechanism related to AVSD 11.
In our Pdgfrα knockout mouse embryos, we observed AVSD (Chapter 3)10. Previous there was described that mutations in Pdgfrα resulted in the hypoplasia of the membranous and the absence of the subconal portions of the ventricular septum, resulting in AVSD 6,12.
Another link for the role of Pdgfrα in the development of AVSD, is the relation between transforming growth factor beta (TGFβ) and PDGF-signalling. TGFβ is able to induce the expression of Pdgfrα but also can downregulate this expression 13-15. And from previous studies, the importance of TGFβ-signalling in cardiac development is known, as the TGFβ2 knockout mouse 16 suffers from cardiac malformation like AVSD. Another link between AVSD and Pdgfrα is found in humans with trisomy 21 or Down syndrome patients 7. In human foetuses with Down syndrome it has been shown that a deficiency of the vestibular spine or DMP contributes to the occurrence of AVSD 7. The role of the DMP in the development of AVSD was also suggests in a Sonic hedgehog (Shh) deficient mouse model 17. And as mentioned previously in the development of ASDI, Pdgfrα is important for the development of the DMP (Chapter 3 en 4)10.
The exact role of Pdgfrα-signalling in the development of AVSD has to be further investigated. Although expression expression pattern of PDGFR-α and the abnormalities observed in Pdgfrα knockout mice, suggest that there is a possible role for Pdgfrα in the development of AVSD. The transcriptional regulation of the development of AVSD probably will contain several pathways including TGFβ, Shh, Pdgfrα and several other genes.
Pdgfrα in the development of the cardiac conduction system in relation to putative sites of clinical arrythmias
The cardiac conduction system (CCS) is responsible for the coordinated contraction of the heart. The CCS comprises the SAN, the atriventricular node (AVN), the bundle of His or atrioventricular (AV) bundle, bundle branches (BBs) and the Purkinje fibres 18,19. The cells of the CCS originate form precursor cells which have the potency to form myocardial and conduction cells 20. The contribution of the splanchnic mesoderm to the development of the conduction cells as well as the myocardium of the primary heart tube was confirmed by several lineage tracing studies for several molecular markers for CCs development 18,21. The contribution and role of neural crest cells (NCC) 22,23 and epicardium-derived cells (EPDCs) is more complicated 24.
Although the expression of PDGFR-α is only detected in the SAN and not in the AVN, bundle of His or bundle branches (Chapter 2 and 3), a role of Pdgfrα-signalling in the development of the cardiac conduction system can not be excluded. In Pdgfrα deficient mice, the sinus venosus myocardium is hypoplastic (Chapter 3) and therefore the SAN
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and also the wall of the pulmonary veins (PV) are hypoplastic (Chapter 3 and 4) 10. Furthermore, the myocardialization around the PV is diminished and in older embryonic stages the layer of smooth muscle cells lining the PV and inner atrial wall are thin in the mutant compared to the wildtype embryos (Chapter 4) 10. With regard to CCS abnormalities the hypoplastic SAN and altered development of the PV is related to diminished contribution of the posterior heart field. The increased expression of Nkx2.5 in the SAN may also contribute to CCS abnormalities. During normal cardiac development the SAN is negative for Nkx2.5 to prevent it to differentiate into working myocardium 25,26. The increased expression of Nkx2.5 observed in the Pdgfrα mutant mice (Chapter 3) could indicate abnormal differentiation of the sinoatrial node as well as disturbed pacemaker function of the node 25-27. The abnormal SAN and the increased expression of Nkx2.5 in the SAN in this model might provide more insight into the development of clinical syndromes such as sick sinus syndrome.
Pdgfrα-signalling is important for the development of the pulmonary veins and dysregulation causes abnormal orientation of the PV connection to the left atrium and inflow tract anomalies including total anomalous pulmonary venous return (TAPVR) (Chapter 3 and 4)10. Patients with abnormal PV development often show conduction abnormalities, as PV have arrhythmogenic capacities 28. This arrhythmogenic capacity has been attributed to sleeves of myocardium surrounding the PV 18,29,30. In the Pdgfrα deficient mouse embryos, the myocardialisation of the PV is disturbed and also the formation and differentiation of the smooth muscle cells of the wall of the PV is altered (Chapter 3 and 4). The altered myocardialisation of the PV and altered differentiation of smooth muscle cells of the wall of the PV is previous described in other mouse models with maldevelopment of the posterior heart field-derived cardiac structures 31. The exact mechanism of the contribution of pulmonary veins to arrhythmogenicicity is still unresolved.
Previous studies describe pacemaker activity in the PV 32,33 and anisotropic arrangement of the myocardium surrounding the PV may form the substrate for re-entry 34,35.
The possible contribution of NCC and EPDCs in the development of the CCS suggests a role for Pdgfrα-signalling. It has been established that disruption of Pdgfrα-signalling causes defects in cardiac NCC populations 36,37 and ablation of NCC results in lack of differentiation of the compact lamellar organization which separates the bundle of His from the working myocardium 22,23.
Pdgfrα is involved in the formation of EPDCs by stimulating proper attachment of the epicardium to the myocardium and by regulating the expression of Wilm’s tumor 1 (WT1).
In Pdgfrα deficient mice, the number of EPDCs in the myocardium is decreased (Chapter 3) which can be linked to abnormal and deficient development of the Purkinje fibres. As previous studies showed that there is a role for EPDCs in the formation of the Purkinje fibres has been reported 24.
Although the exact role of Pdgfrα-signalling in the development of the cardiac conduction system has to be unravelled. Based on the expression pattern of PDGFR-α and the abnormalities that occur by deficiency, we suggest that there is a possible role for PDGFRa as part of a transduction pathway in the development of CCS.
A role of the second heart field in cardiac regeneration
To achieve repair after cardiac injury, several routes are being explored. Transplantation is performed with cardiac progenitors or stem cells, from a variety of sources including embryonic stem cells, induced pluripotent stem cells or endogenous sources. As an alternate to cell transplantation, paracrine factors are injected to mobilize resident cardiac progenitor populations and promote cardiomyocyte renewal and vascularisation of the injured area. And last but not least, engineered cardiac tissues are transplant into the injured area 38.
The opportunity to investigate the possibilities to bypass the need for cell transplantation by stimulated repair by endogenous cells, would overcome one of the major challenges in regenerative medicine. To understand the biology of cardiac progenitors and to develop cardiac regeneration strategies it is important to understand the developmental origin of these cells 38.
Although it has long been thought that a resident cardiac progenitor population does not exist in the postnatal heart 39, emerging evidence suggests that several subpopulation of cardiac progenitor cells reside within the neonatal or adult heart. These cardiac progenitor cells have the ability to differentiate into all constituent cells of the adult heart including cardiomyocytes, vascular smooth muscle cells and endothelial cells 38.
Laugwitz and colleagues report the identification of neonatal Isl1-positive cardiac progenitors in mouse, rat and human. Isl1 is a representative of the second heart field (SHF) also at the venous pole of the heart and this multipotent cardiac progenitor contributes to the formation of a functional heart 40,41. The distribution of Isl1-positive cells though the neonatal heart matches the defined contribution of SHF precursors during embryogenesis. Due to the fact that Isl-1 positive cells are not present in the adult heart, they are not applicable for endogenous cell-based therapy 40,41.
In the adult heart, three populations of adult cardiac progenitor cells, which might be remnants of the first and/or second heart field, have been identified. Self-renewing, clonogenic c-Kit-positive cells can give rise to cadiomyocytes, endothelial cells and smooth muscle cells in vitro and form functional myocardium in vivo 42. Furthermore, transplantation studies with human c-Kit-positive cells and it has been claimed that these cells are able to regenerate myocardium in the infarcted heart 43.
Based on expression of Sca-1, a population of cardiac cells was found to have the potential for self-renewal and differentiation into cardiomyocytes in vitro 44. Transplantation with
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Sca-1-positive cardiac progenitor cells can migrate to and engraft the site of injured myocardium by fusing with resident cardiomyocytes 44. A recent described group of Sca- 1-positive cardiac progenitor present in the adult heart are the cardiomyocyte progenitor cells (CMPCs) 45,46. CMPCs are able to differentiate efficiently into spontaneously beating cardiomyocytes in vitro after stimulation with 5-aza-cytidine and TGFβ without the coculture with cardiac fibroblasts or myocytes 45,46. Transplantation of CMPCs into the injured myocardium prevented progression of dilatation and maintained ejection fraction of the heart. Transplanted foetal CMPCs were able to differentiate in vivo in to cardiomyocytes, endothelial cells and smooth muscle cells 45.
Another source of cardiac progenitors which is present in the adult heart which can contribute to diverse lineages are the stem-cell like side population or SP cells. These cells are identified by their expression of ABCG2 (ATP-binding cassette sub-family G member 2) and have the ability to efflux Hoechst dye 47. Cardiac SP cells can proliferate and differentiation into both cardiac and hematopoietic lineages 48.
Despite the wealth of published studies on adult cardiac stem/progenitor cells, there is relatively little known about the developmental origin of these cells and their contribution to myocardial homeostasis and/or repair after injury.
Another cell source which is derived from the second heart field or more specific from the posterior heart field and is an attractive population to be used in cardiac regeneration are the epicardial cells and epicardium-derived cells (EPDCs) 49-53. These cell populations are indispensible for cardiac development as they stimulate the formation of the compact myocardium (Chapter 5) 54,55. Furthermore, they stimulate correct mechanical and electrical coupling and alignment of cardiomyocytes (Chapter 5) which has a beneficial effect on contraction of the cardiomyocytes (Chapter 5). Previous studies already showed that EPDCs are important for the proper development of the coronary vasculature 56. These findings implicate that EPDCs are ideal candidates for cell-based cardiac therapy.
Addition of EPDCs to the infarcted myocardium showed beneficial effect for cardiac function mediated by increased vasculature and increased wall thickness 51,53. Recent studies show that the endogenous epicardium is reactivated after myocardial infarction and the re-activated epicardial cells undergo epithelial-to-mesenchymal transformation thereby supporting their migration into the injured myocardium where they contribute to regeneration 49,52,57,58. After transplantation of EPDCs or reactivation of endogenous epicardial cells, EPDCs are able to differentiate into interstitial and adventitial fibroblasts and into smooth muscle cells 55,59-62. Regarding the debate about the endocardial and myocardial fate of embryonic EPDCs 61,63-67, it must be notified that the adult transplanted exogenous and reactivated endogenous EPDCs were never detected within the vessel lining and never expressed cardiac markers 51,53. Therefore, it is we suggest that contribution of epicardial cells to cardiac regeneration lies in the ability to promote
endogenous cardiac progenitor cell expansion and neovascularisation by paracrine signalling to the surrounding cells.
Future research on the regenerative role of EPDCs should include assessment of factor that contribute to reactivation of endogenous epicardium e.g. thymosin beta 4 49 and Wilm’s tumor 1 (WT1) 52,53. Furthermore, factors that are involved in the process of epithelial-to-mesenchymal transformation, which stimulate migration into the injured myocardium and is the onset of differentiation (Chapter 6), should be further investigated.
SHF-derived cardiac progenitors and epicardial cells might be important for cardiac regeneration. We questioned if there was also a role for Pdgfrα in cardiac regeneration as this gene is know the be a marker for SHF-derived cardiac progenitors (Chapter 3)68 and it is involved in epicardial development and EMT probably via regulation of WT1 (Chapter 3). From previous studies it is know that PDGF-signalling critically regulates postinfarction repair. Expression of PDGFR-α is increased to stimulate pathways that promote collagen deposition in the infarct 69. PDGFR-α stimulates fibroblast migration and these fibroblasts are probably epicardium-derived as Pdgfrα is involved in epicardial EMT and the onset of differentiation (Chapter 6) 70. Our data show that there is a relation between Pdgfrα-signalling and WT1 (Chapter 3). WT1 is known to inhibit apoptosis, stimulate capillary development and increase proliferation 71-73.
Details on the role of Pdgfrα-signalling in cardiac regeneration via addition of SHF-derived cardiac progenitors or via activation of the epicardium still have to be investigated. Based on the fact that myocardial injury increases the expression of PDGFR-α and the included data on the of Pdgfrα role in epicardial development and EMT, this suggests that there is a possible role for Pdgfrα-signalling in cardiac repair. Further investigation into the role of Pdgfrα in the activation, migration and differentiation of adult epicardial cells would be beneficial for the development of cell-based cardiac regeneration therapy.
Future research in cardiac repair will include data on cell-based therapy for cardiac repair by injecting suitable cells or growth factors that can activate endogenous progenitor cells to stimulate cardiac proliferation and vascularisation.
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221 General Discussion
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