This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gittenberger-de Groot, A. C. Articles by Poelmann, R. E. Search for Related Content PubMed PubMed Citation Articles by Gittenberger-de Groot, A. C. Articles by Poelmann, R. E. Related Collections Angiogenesis Animal models of human disease Pediatric and congenital heart disease, including cardiovascular surgery Cardiac development

(Circulation Research. 2000;87:969.)
© 2000 American Heart Association, Inc.

Reports

Epicardial Outgrowth Inhibition Leads to Compensatory Mesothelial Outflow Tract Collar and Abnormal Cardiac Septation and Coronary Formation

Adriana C. Gittenberger-de Groot, Mark-Paul F.M. Vrancken Peeters, Maarten Bergwerff, Monica M.T. Mentink, Robert E. Poelmann

From the Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, the Netherlands.

Correspondence to Prof Dr A.C. Gittenberger-de Groot, Department of Anatomy and Embryology, Leiden University Medical Center, PO Box 9602, 2300 RC Leiden, the Netherlands. E-mail acgitten{at}lumc.nl

	Abstract

Top Abstract Introduction Material and Methods Results and Discussion References

 
In the present study, we investigated the modulatory role of the epicardium in myocardial and coronary development. Epicardial cell tracing experiments have shown that epicardium-derived cells are the source of interstitial myocardial fibroblasts, cushion mesenchyme, and smooth muscle cells. Epicardial outgrowth inhibition studies show abnormalities of the compact myocardial layer, myocardialization of cushion tissue, looping, septation, and coronary vascular formation. Lack of epicardial spreading is partly compensated by mesothelial outgrowth over the conotruncal region. Heterospecific epicardial transplant is able to partially rescue the myocardial development, as well as septation and coronary formation.

Key Words: epicardium • coronary vasculature • myocardial differentiation • ablation • chimerization

	Introduction

Top Abstract Introduction Material and Methods Results and Discussion References

 
The important role of the proepicardial organ (PEO) and its epicardium-derived cells (EPDCs) in cardiac development has recently been recognized. Data from both chicken-quail chimera techniques,^{1 2 3} retroviral lineage tracing,⁴ and in vitro data⁵ show that EPDCs are the source of coronary smooth muscle cells, cardiac interstitial fibroblasts, and mesenchymal cells in the atrioventricular cushions. Chimera studies^{6 7} showed that the coronary endothelial cells (ECs) originate from the endothelium of the liver region.

Our present study applying microsurgery to inhibit outgrowth of the PEO was set up to investigate the possibility that coronary ECs were not only derived from the sinus venosus region but could also be recruited under abnormal circumstances from the pharyngeal arch region, which explains aberrant coronary artery origin in neonates.⁸ The second aim was to investigate the role of EPDCs in the myocardium, the endocardial cushions, and the coronary vasculature.

	Material and Methods

Top Abstract Introduction Material and Methods Results and Discussion References

 
We used embryos of the Japanese quail (Coturnix coturnix japonica) and the White Leghorn chicken (Gallus domesticus) for two sets of experiments. In the first set, inhibition of outgrowth of the epicardium in quail embryos was obtained as described by Männer.² In 13 embryos, complete or partial absence of outgrowth of the epicardium from the venous pole was found (Table), which was confirmed in 8 embryos by cytokeratin staining. The second set consisted of 10 chicken host embryos (HH15) in which, after inhibition of outgrowth of the host PEO, a quail PEO (HH15 to HH17) was implanted⁶ resulting in a 1-day delay in outgrowth. In all embryos (4n:HH29, 2n:HH30, and 4n:HH35), quail epicardium covered the greater part of the heart. Embryos were serially sectioned and subjected to standard immunohistochemical procedures^{6 7 9} including the TUNEL technique for detection of apoptosis.¹⁰

View this table: [in this window] [in a new window]   Table 1. Quail Embryos Studied After Epicardial Outgrowth Inhibition

	Results and Discussion

Top Abstract Introduction Material and Methods Results and Discussion References

 
Epicardial Outgrowth Inhibition
No living embryos beyond HH32 were harvested after epicardial outgrowth inhibition. In most cases (Table), there was a remarkable compensatory outgrowth of the mesothelium from the pharyngeal arch area over the conotruncal region. Normally, the epicardium reaches up to the arterial-myocardial borderline.⁹ In outgrowth-inhibited embryos, there is a collar of cytokeratin-positive mesothelium that not only covered the great arteries but also the myocardial outflow tract up to the level of the inner curvature (Figure, a and b).

View larger version (110K): [in this window] [in a new window]   Figure 1. a, Ventral view of mesothelial collar (cytokeratin-positive: brown) after PEO outgrowth inhibition (Inh) crossing arterial-myocardial borderline (hatched line). b, Sagittal section of a. The adventitial vessel network (CAP) connects (arrow) to the aorta (Ao). The cytokeratin-positive collar (arrowhead) ends over myocardium. c, Sagittal section of immature heart tube after PEO outgrowth inhibition with absent ventricular septation and a wide inner curvature (asterisk). Outflow tract (OT) septation by fusion of endocardial cushion tissue. The deficient atrioventricular cushions (AVCu) have not fused. d, Section of normal compact (CM) and trabecular (TM) myocardium (M) compared with abnormally thin CM in c and e (detail of c). f, Well-developed CM in rescue heart, with peculiar trabeculations. All (c through f) before functional coronary vascularization. g, Quail transplant ECs (brown) invade myocardium and form a peritruncal ring (arrowhead); arrow indicates eggshell membrane. h, EPDCs in ventricular wall (brown stain). i, Absent myocardialization (arrow in box k, detail of i) of OT cushion tissue (Cu), with deficient ventricular septation (VS). j, Normal myocardialization of Cu (see arrows); compare with k. Bar=250 µm. EP indicates subepicardium.

In the experimental embryos, the compact layer of the ventricular myocardium, lacking epicardium, proved to be abnormally thin (compare Figure, c and e with d and f). Focally, there was myocardial necrosis. The thin myocardium did not show altered apoptosis compared with normal.¹¹ The cardiomyocytes were rounded and not well organized in contrast to normal myocardium.¹² The outflow tract myocardium, covered by the collar, did not show marked abnormalities. These findings point to a role for the early intramyocardial EPDC population¹ in inducing normal myocardial development. This is supported by data from ₄ integrin¹³ and vascular cell adhesion molecule-1¹⁴ knockout mice with a similar myocardial phenotype. In addition, because no coronary network is formed, myocardial undernutrition might play a role.¹²

There were variable degrees of abnormal ventricular inlet and outflow tract septation. The heart tube was still primitive, with deficient looping and a wide inner curvature (Figure, c), and presented in all cases with a double-inlet, double-outlet configuration (Figure, c and i). In 3 of 13 cases, there was fusion of the endocardial outflow tract ridges. All others still presented with a common arterial trunk. Myocardialization of these ridges, normally taking place from stage 31 onward,¹⁰ was absent (Figure, j and k). The deficient atrioventricular cushions (Figure, c and e) had not fused to form a tricuspid and a mitral orifice but presented as a common atrioventricular canal. Formation of the interventricular septum was deficient (Figure, i) or absent (Figure, c), which concurs with the aforementioned mice knockout data.¹⁴ Because none of the embryos survived beyond HH32, we could not evaluate the definitive heart malformations. The malformations correlate with early stages of the recently described GATA cofactor FOG2^–/– hearts.¹⁵ In the FOG2^–/– phenotype, there is epicardial formation with lack of coronary formation, which could point toward a defective endothelial outgrowth from the transverse septum in a deficiently differentiated epicardium.

Our epicardial ablation embryos showed obvious deviations from normal coronary endothelial plexus formation. In most cases, the small vessels arising from the sinus venosus used the small patch of epicardium at the venous pole to reach the ventricular myocardium, where in contrast to normal development, connections with the ventricular lumen were established. The remainder of the myocardium lacked coronary vasculature.

At the arterial pole, a compensatory mechanism was seen in which in the submesothelium or adventitia of the pharyngeal arch arteries, an extensive endothelial vessel plexus, reached the arterial-myocardial border (Figure, b and g). In two cases, this network penetrated the aortic wall forming a coronary arterial orifice (Figure, b, and Table). These data support the potential for origin of coronary arteries from thoracic arteries in humans.⁸

Epicardial Rescue Experiments
Before HH29, an outflow tract mesothelial collar was seen comparable to the inhibition series. At HH35, the quail-derived donor epicardium had moved up to the myocardial-arterial borderline. We assume that this change is due to the shortening and remodeling of the outflow tract as a relatively late process in cardiac development, as was also suggested by Männer.² Quail EPDCs participated in all cell types as described in normal development.¹

Our findings are unique in that we show that epicardial grafting rescued the cardiac phenotype. The compact myocardium was well developed already, before functional coronary vascularization was established (Figure, f). The cushions of both outflow tract and atrioventricular levels had fused, and myocardialization had taken place and cardiac septation was normal.

However, in the HH35 embryos, we found two cases with coronary abnormalities. In one, a coronary artery had connected to the pulmonary orifice, as in the Bland White Garland syndrome.⁸ In the other, the connection of both main coronary arteries to the aorta was missing but was replaced by two ventriculo-coronary communications (fistulae). This supports an important clinical notion that fistulae, accompanying pulmonary atresia without ventricular septal defect,¹⁶ may develop as a primary structural coronary abnormality before obliteration of the pulmonary orifice, instead of being caused secondarily by increased right ventricular pressure resulting from pulmonary obstruction.

	Acknowledgments

 

This work was supported by grants D96.017 and 99.022 from the Netherlands Heart Foundation.

Received October 3, 2000; revision received October 23, 2000; accepted October 23, 2000.

	References

Top Abstract Introduction Material and Methods Results and Discussion References

 
1. Gittenberger-de Groot AC, Vrancken Peeters M-PFM, Mentink MMT, Gourdie RG, Poelmann RE. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res. 1998;82:1043–1052.[Abstract/Free Full Text]

2. Männer J. Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium. Anat Rec. 1999;255:212–226.[Medline] [Order article via Infotrieve]

3. Vrancken Peeters M-PFM, Gittenberger-de Groot AC, Mentink MMT, Poelmann RE. Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium. Anat Embryol. 1999;199:367–378.[Medline] [Order article via Infotrieve]

4. Dettman RW, Denetclaw W, Ordahl CP, Bristow J. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol. 1998;193:169–181.[Medline] [Order article via Infotrieve]

5. Landerholm TE, Dong X-R, Lu J, Belaguli NS, Schwartz RJ, Majesky MW. A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells. Development. 1999;126:2053–2062.[Abstract]

6. Poelmann RE, Gittenberger-de Groot AC, Mentink MMT, Bökenkamp R, Hogers B. Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras. Circ Res. 1993;73:559–568.[Abstract/Free Full Text]

7. Vrancken Peeters M-PFM, Gittenberger-de Groot AC, Mentink MMT, Hungerford JE, Little CD, Poelmann RE. The development of the coronary vessels and their differentiation into arteries and veins in the embryonic quail heart. Dev Dyn. 1997;208:338–348.[Medline] [Order article via Infotrieve]

8. Angelini P, Villason S, Chan AV Jr, Diez JG. Normal and anomalous coronary arteries in humans. In: Angelini P, ed. Coronary Artery Anomalies: A Comprehensive Approach. Philadelphia, Pa: Lippincott Williams & Wilkins; 1999:27–150.

9. Vrancken Peeters M-PFM, Mentink MM, Poelmann RE, Gittenberger-de Groot AC. Cytokeratins as a marker for epicardial formation in the quail embryo. Anat Embryol (Berl). 1995;191:503–508.[Medline] [Order article via Infotrieve]

10. Poelmann RE, Mikawa T, Gittenberger-de Groot AC. Neural crest cells in outflow tract septation of the embryonic chicken heart: differentiation and apoptosis. Dev Dyn. 1998;212:373–384.[Medline] [Order article via Infotrieve]

11. Poelmann RE, Molin D, Wisse LJ, Gittenberger-de Groot AC. Apoptosis in cardiac development. Cell Tissue Res. 2000;301:43–52.[Medline] [Order article via Infotrieve]

12. Sedmera D, Pexieder T, Vuillemin M, Thompson RP, Anderson RH. Developmental patterning of the myocardium. Anat Rec. 2000;258:319–337.[Medline] [Order article via Infotrieve]

13. Yang JT, Rayburn H, Hynes RO. Cell adhesion events mediated by ₄ integrins are essential in placental and cardiac development. Development. 1995;121:549–560.[Abstract]

14. Kwee L, Baldwin HS, Min Shen H, Stewart CL, Buck C, Buck CA, Labow MA. Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice. Development. 1995;121:489–503.[Abstract]

15. Tevosian SG, Deconinck AE, Tanaka M, Schinke M, Litovsky SH, Izumo S, Fujiwara Y, Orkin SH. FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium. Cell. 2000;101:729–739.[Medline] [Order article via Infotrieve]

16. Gittenberger-de Groot AC, Sauer U, Bindl L, Babic R, Essed CE, Buhlmeyer K. Competition of coronary arteries and ventriculo-coronary arterial communications in pulmonary atresia with intact ventricular septum. Int J Cardiol. 1988;18:243–258.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				P. Snider, K. N. Standley, J. Wang, M. Azhar, T. Doetschman, and S. J. Conway Origin of Cardiac Fibroblasts and the Role of Periostin Circ. Res., November 6, 2009; 105(10): 934 - 947. [Abstract] [Full Text] [PDF]

				E. M. Winter, A. A.M. van Oorschot, B. Hogers, L. M. van der Graaf, P. A. Doevendans, R. E. Poelmann, D. E. Atsma, A. C. Gittenberger-de Groot, and M. J. Goumans A New Direction for Cardiac Regeneration Therapy: Application of Synergistically Acting Epicardium-Derived Cells and Cardiomyocyte Progenitor Cells Circ Heart Fail, November 1, 2009; 2(6): 643 - 653. [Abstract] [Full Text] [PDF]

				C. Stamm, Y.-H. Choi, B. Nasseri, and R. Hetzer A heart full of stem cells: the spectrum of myocardial progenitor cells in the postnatal heart Therapeutic Advances in Cardiovascular Disease, June 1, 2009; 3(3): 215 - 229. [Abstract] [PDF]

				K. J. Lavine and D. M. Ornitz Shared Circuitry: Developmental Signaling Cascades Regulate Both Embryonic and Adult Coronary Vasculature Circ. Res., January 30, 2009; 104(2): 159 - 169. [Abstract] [Full Text] [PDF]

				R. J. Tomanek, H. K. Hansen, and L. P. Christensen Temporally Expressed PDGF and FGF-2 Regulate Embryonic Coronary Artery Formation and Growth Arterioscler Thromb Vasc Biol, July 1, 2008; 28(7): 1237 - 1243. [Abstract] [Full Text] [PDF]

				D. P. Kolditz, M. C.E.F. Wijffels, N. A. Blom, A. van der Laarse, N. D. Hahurij, H. Lie-Venema, R. R. Markwald, R. E. Poelmann, M. J. Schalij, and A. C. Gittenberger-de Groot Epicardium-Derived Cells in Development of Annulus Fibrosis and Persistence of Accessory Pathways Circulation, March 25, 2008; 117(12): 1508 - 1517. [Abstract] [Full Text] [PDF]

				P. F. van Loo, E. A. F. Mahtab, L. J. Wisse, J. Hou, F. Grosveld, G. Suske, S. Philipsen, and A. C. Gittenberger-de Groot Transcription Factor Sp3 Knockout Mice Display Serious Cardiac Malformations Mol. Cell. Biol., December 15, 2007; 27(24): 8571 - 8582. [Abstract] [Full Text] [PDF]

				Y. Ishii, J. D. Langberg, R. Hurtado, S. Lee, and T. Mikawa Induction of proepicardial marker gene expression by the liver bud Development, October 15, 2007; 134(20): 3627 - 3637. [Abstract] [Full Text] [PDF]

				J. T Butcher and R. R Markwald Valvulogenesis: the moving target Phil Trans R Soc B, August 29, 2007; 362(1484): 1489 - 1503. [Abstract] [Full Text] [PDF]

				E.M. Winter, R.W. Grauss, B. Hogers, J. van Tuyn, R. van der Geest, H. Lie-Venema, R. V. Steijn, S. Maas, M.C. DeRuiter, A.A.F. deVries, et al. Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart Circulation, August 21, 2007; 116(8): 917 - 927. [Abstract] [Full Text] [PDF]

				K. J. Lavine, A. C. White, C. Park, C. S. Smith, K. Choi, F. Long, C.-c. Hui, and D. M. Ornitz Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development. Genes & Dev., June 15, 2006; 20(12): 1651 - 1666. [Abstract] [Full Text] [PDF]

				R. P. Visconti, Y. Ebihara, A. C. LaRue, P. A. Fleming, T. C. McQuinn, M. Masuya, H. Minamiguchi, R. R. Markwald, M. Ogawa, and C. J. Drake An In Vivo Analysis of Hematopoietic Stem Cell Potential: Hematopoietic Origin of Cardiac Valve Interstitial Cells Circ. Res., March 17, 2006; 98(5): 690 - 696. [Abstract] [Full Text] [PDF]

				I. Eralp, H. Lie-Venema, M. C. DeRuiter, N. M.S van den Akker, A. J.J.C. Bogers, M. M.T. Mentink, R. E. Poelmann, and A. C. Gittenberger-de Groot Coronary Artery and Orifice Development Is Associated With Proper Timing of Epicardial Outgrowth and Correlated Fas Ligand Associated Apoptosis Patterns Circ. Res., March 18, 2005; 96(5): 526 - 534. [Abstract] [Full Text] [PDF]

				A. J. Watt, M. A. Battle, J. Li, and S. A. Duncan GATA4 is essential for formation of the proepicardium and regulates cardiogenesis PNAS, August 24, 2004; 101(34): 12573 - 12578. [Abstract] [Full Text] [PDF]

				R. W. Stottmann, M. Choi, Y. Mishina, E. N. Meyers, and J. Klingensmith BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium Development, May 1, 2004; 131(9): 2205 - 2218. [Abstract] [Full Text] [PDF]

				H. Lie-Venema, A. C. Gittenberger-de Groot, L. J.P. van Empel, M. J. Boot, H. Kerkdijk, E. de Kant, and M. C. DeRuiter Ets-1 and Ets-2 Transcription Factors Are Essential for Normal Coronary and Myocardial Development in Chicken Embryos Circ. Res., April 18, 2003; 92(7): 749 - 756. [Abstract] [Full Text] [PDF]

				D. E. Reese, T. Mikawa, and D. M. Bader Development of the Coronary Vessel System Circ. Res., November 1, 2002; 91(9): 761 - 768. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gittenberger-de Groot, A. C. Articles by Poelmann, R. E. Search for Related Content PubMed PubMed Citation Articles by Gittenberger-de Groot, A. C. Articles by Poelmann, R. E. Related Collections Angiogenesis Animal models of human disease Pediatric and congenital heart disease, including cardiovascular surgery Cardiac development