Embryological origin of the endocardium and derived valve progenitor cells: From developmental biology to stem cell-based valve repair

Biochimica et Biophysica Acta 1833 (2013) 917–922

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Embryological origin of the endocardium and derived valve progenitor cells: From developmental biology to stem cell-based valve repair☆ Michel Pucéat Cardiogenesis and stem cells, University Paris Descartes, INSERM UMR 633, Evry, Paris France

a r t i c l e

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Article history: Received 4 July 2012 Received in revised form 26 September 2012 Accepted 29 September 2012 Available online 16 October 2012 Keywords: Valvulogenesis Atrioventricular canal Stem cell EMT

a b s t r a c t The cardiac valves are targets of both congenital and acquired diseases. The formation of valves during embryogenesis (i.e., valvulogenesis) originates from endocardial cells lining the myocardium. These cells undergo an endothelial–mesenchymal transition, proliferate and migrate within an extracellular matrix. This leads to the formation of bilateral cardiac cushions in both the atrioventricular canal and the outflow tract. The embryonic origin of both the endocardium and prospective valve cells is still elusive. Endocardial and myocardial lineages are segregated early during embryogenesis and such a cell fate decision can be recapitulated in vitro by embryonic stem cells (ESC). Besides genetically modified mice and ex vivo heart explants, ESCs provide a cellular model to study the early steps of valve development and might constitute a human therapeutic cell source for decellularized tissue-engineered valves. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The heart functions by pumping out oxygenated blood to the body through the aortic valve and the aorta, and deoxygenated blood to the lungs through the pulmonary valve and artery. During the cardiac cycle, blood originating from the lung is pumped via the pulmonary vein into the left atria and then into the left ventricle through the mitral valve. Similarly, deoxygenated blood is pumped via the vena cava into the right atria and flows through the tricuspid valve to reach the right ventricle. The architecture of both the semilunar valves (aortic and pulmonary valves) and the atrioventricular valves (tricuspid or mitral valves) allows for unidirectional opening and, in turn, blood flow. The valves feature two leaflets or cusps that allow for their opening and closure. The opening is surrounded by a fibrous ring or annulus fibrosus. The leaflets are prevented from prolapsing into the wrong direction by the action of chordae tendineae. The chordae tendinae are tendon chords linking the papillary muscles to the tricuspid valve in the right ventricle and the mitral valve in the left ventricle. They are attached to the posterior spongious region of the valve [1,2] (Fig. 1). The structure of the valve (Fig. 1) is the result of complex developmental events including specification and determination of cell lineages, endothelial to mesenchymal transition (EMT) of endocardial cells and cell migration, as well as morphogenetic and biomechanical (shear stress) phenomena [3]. ☆ This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. E-mail address: [email protected]. 0167-4889/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2012.09.013

Cardiac valves form between E9.5 (cushion formation) and E14.5 (maturation of leaflets) and are then functional in the mouse embryo as recorded by Doppler echocardiography (Fig. 2) which reveals unidirectional blood flow. In humans, valves are formed between the 5th and the 8th weeks of fetal life. Valves are affected in up to one third of cardiac congenital pathologies [4]. The valves open more than 2.5 billion times in the life of an adult individual. This subjects the thin valve leaflets to high mechanical strains which ultimately lead to aging of the tissue, a physiological event often exacerbated by pathologies which primarily affect the myocardium. In adults, the prevalence of valvular heart diseases increases dramatically with age reaching 13% of patients who are 75 years old and older [5–7]. One of the therapeutic solutions to circumvent a valvular dysfunction is the replacement of the valve by a prosthetic device [8]. However, these prosthetic valves neither grow nor remodel. This approach is thus limiting in children undergoing a surgery at birth while their bodies will further grow. Engineered materials that promote native developmental biology cues and signaling are currently under investigation to provide mechanical integrity and plasticity once implanted [9]. Alternatively, cell therapy (i.e., bringing new cells including endothelial cells or valvular fibroblasts) has also been proposed in combination to valve bioengineering to repair myxomatous valves [10]. Such a therapeutic approach ideally requires the generation of the appropriate human cell type, and thus a better understanding of the developmental pathways governing the formation of both embryonic and mature valves. The first steps of valvulogenesis take place in the atrio-ventricular canal (AVC) and the outflow tract (OFT). The AVC and OFT develop when the heart tube elongates and then loops, forming the primary

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Tbx20

atrialis fibrous spongious ventricularis

anterior leaflet

Hey BMP2

Chordae tendinis

TGFb2

Notch Twist

Commisural leaflet

papillary muscle

Twist, Tbx20, Msx1,2 Sox9, Sox5,6

posterior leaflet

myocardium

en

EMT

Fig. 1. Two perspectives of the atrioventricular valve structure. Component of a mature valve including the fibrosa (dense collagen), spongiosa (enriched in proteoglycan and glycosaminoglycan) and the atrialis or the ventricularis including smooth muscle cells in atrialis and valve endothelial cells in both (loose fibroelastic structure). The chordae tendinae links the leaflet with the papillary muscle.

ventricle and atrium. Endothelial cells of the endocardium are separated from the myocardium by a space filled by an extracellular matrix (ECM), or cardiac jelly, mainly composed of hyaluronan and chondroitin sulfates [11]. The myocardium also secretes ECM at discrete locations of the AVC and OFT. Endocardial cells located in the neighborhood respond to local signals including BMP2, TGFβ and notch signaling pathways, delaminate and migrate while undergoing EMT. Post-EMT VIC cells express among other genes, Sox9, Sox5, Sox6, Msxs, Tbx20, Twist and Snail and Slug. This process leads to the formation of the cardiac cushions [4,11,12] (Fig. 3). Various cardiac embryonic lineages contribute to the early steps of valvulogenesis which is still a poorly understood developmental process [4,13]. Chicken and mouse embryos have provided some cues to this process. Embryonic stem cells originating from the blastocyst recapitulate many early developmental events including the formation of endocardial cells [14]. Directed differentiation of these cells might also bring useful information in order to better comprehend the first steps of valvulogenesis. This overview updates the origin of cell lineages which contribute to the endocardium, the signaling (both biological and mechanical) pathways which play a role in the determination of these lineages as well as the formation of the cardiac cushions. I will further discuss how human embryonic stem cells contribute to a better understanding of the developmental biology of valves and provide potential therapeutic cells for myxomatous valves. 1.1. Embryological origin of the endocardium Endocardial cells are endothelial cells that form the inner layer of the heart in continuity with the endothelium of the vessels. The endocardial

20 deg

Snail

1mm

5

10

Fig. 2. Continuous wave Doppler recording of the blood flow through the aortic valve in E14.5 embryo in utero.

VIC Growth

do

ca

rdi

um

BMP2

myocardium Fig. 3. Transcriptional pathways underlying the process of EMT and formation of cardiac cushion. Myocardial BMP works together with Notch and TGFb on endocardial cells to induce their EMT. The endocardium-derived cells, VIC express a specific set of genes that includes Twist, Tbx20, Soxs, and Msxs. VIC: valve interstitial cell.

layer appears very early during evolution in the Agnathans ( lamprey) [15] and correlates with the formation of a valve between the two cardiac chambers providing a unidirectional blood flow [16]. Surprisingly, despite these evo-devo data and early focused developmental biology studies in the 1960s, the embryonic origin of the endocardium has not yet been fully elucidated [17]. Pioneer studies in the chicken and quail embryos from Mikawa's and Markwald's laboratories have strongly suggested an early segregation of endocardial and myocardial progenitors prior to or at the onset of gastrulation. Using a cell lineage tracing with a retroviral vector encoding a gene reporter to infect cells in the rostral portion of HH stage 3 chick primitive streak (PS), Mikawa's laboratory observed that a specific region of the PS generates a daughter population that migrates into the bilateral heart regions, and differentiates into either endocardial or myocardial cells. No cells remain bipotent at this PS stage [18,19]. These findings argue against a common progenitor between myocardial and endocardial cells at this stage of development. In the quail, using labeling against the endothelial marker QH-1, Sugi and collaborators identified an endocardial endothelial cell population in the precardiac mesoderm from stage 5 embryos [20]. Determination of endocardial cell fate thus occurs before the formation of the primary heart field. In the mouse, endocardial cells are first detected at E7.5 as a subpopulation of mesodermal cardiogenic cells that down-regulate N-cadherin [21]. Genetic lineage tracing studies in the mouse [22] reported that a Flk1 + progenitor emerges when epiblast cells exit the PS and differentiate into, besides other mesodermal derivatives, both endocardial and some myocardial cells. Thus, endocardial and myocardial cells are already determined when they together form the primary heart field in the mouse embryo. Besides the early segregation of endocardial cells among mesodermal cells, Isl1Cre labeled cells [23,24] and Mef2c-AHF-Cre labeled cells [25] were also shown to give rise to both the myocardium and the endocardium. Thus, the cells of the second heart field (i.e., Flk1-cells) contribute at least partially to the formation of the endocardium by re-expressing Flk1 before reaching the endothelial layer. In the cardiac crescent, the Ets-family protein Etv2 has been identified as an Nkx2-5 target and a key gene for endothelial–endocardial specification [26], confirming that endocardial cells arise from a de novo process of vasculogenesis. In a recent paper [13], the authors used a live imaging approach in quail embryos and lineage tracing in mouse. They found that the endocardium derives from a vascular endothelial lineage as also suggested by Rasmussen et al. [27]. Flk1 + mesodermal cells are thus a key cell population in forming the endocardium. However, this layer originates from both an early Flk1 +/Isl1 and a late sub-set of Flk1 -/Isl1 + lineages, pointing to two embryonic origins for these cells [13] (Fig. 4). Nevertheless, the exact timing of segregation of endocardial and myocardial lineages in both bilateral

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heart regions before the formation of the first heart field and within the second heart field is still unknown. The extracellular matrix (ECM) contributes to the cell determination process and setting of the endocardial cells lining the myocardium. The endocardial progenitors move early into an ECM-rich environment located between the splanchnic mesoderm and the endoderm. This ECM includes collagen I and IV, fibronectin, laminin and fibrilllins 1 and 2. Later, the endocardial and myocardial cells of the primary heart field forming the cardiac crescent, will build up the cardiac tube while remaining separated from each other by an acellular ECM or cardiac jelly [12,28]. Several cell types contribute to the formation of the cardiac jelly. These include early endodermal and lateral plate mesodermal cells and myocardial cells [29,30]. Both fibronectin and fibrillin-2 bind morphogens such as TGFβ [31] and VEGF [32], suggesting a potential role of the matrix and jelly in determining cell fate and proliferation as well as, later on, in the modeling of the cardiac cushions. Besides the early determination of the endocardium-derived valvular cells, a late contribution of epicardium-derived cells to the formation of the leaflets of semilunar valves has been reported [33,34] in chicken embryo. This epicardial contribution to the valves was recently confirmed in the mouse embryo [35] (Fig. 4). Cells migrating from the cardiac neural crest also specifically participate in the formation of semilunar valves [36]. Further experiments using an alternative approach are required to fully delineate the cell lineages contributing to the endocardial layer. A retrospective clonal analysis using LaacZ embryos [37,38] should shed light on this issue. 1.2. EMT of endocardial cells and formation of cardiac cushions EMT is an ancient biological process which is characterized by a loss of cell adhesion proteins and adherent junctions of epithelial cells that acquire the morphology of mesenchymal cells [39]. Both cardiac progenitors and more differentiated cells result from several processes of EMT that occur during their embryonic life. Epiblast cells first convert into mesenchymal cells forming part of the lateral plate mesoderm (LPM) at the origin of the first prospective cardiac cells. The LPM forms two new epithelial cell sheets, i.e. the somatopleure and

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splanchnopleure under the control of the ectoderm [40]. The splanchnopleure undergoes a second EMT to form the splanchnic mesoderm at the origin of both myocardial (first heart field) and endocardial cells. Endocardial endothelial valve prospective cells (VEC) undergo a third EMT under the action of BMP2 in the AVC or of BMP4 in the OFT, both secreted by the myocardium, in order to form the cardiac cushions. A BMP signaling pathway in the endocardium is activated through ALK3 and ALK6 and regulates, through a Notch-dependent pathway, downstream TGFβ2 secretion which also participates in the initiation of EMT (Table 1; Fig. 3; for review: [41,42]). Indeed, TGFβ2 knock-out mice, but not TGFβ1 or TGFβ3 knock-out mice, feature a deficient EMT in the cardiac cushions [43] (Table 1). Tbx2, specifically expressed under the tight regulation of BMP2 in both the myocardium and the endocardium of the AVC, prevents expression of cardiac chamber genes [44] as well as EMT within the ventricular region [45]. This gene has been expressed early in the evolution. The lamprey, the first organism to develop an endocardial layer as well as cardiac valves, expresses LjTbx2/3A in the non-chambered myocardium, supporting the idea that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multi-chambered hearts and in turn the necessary valves [46]. The VIC (fibroblasts) generated by the EMT process invade the cardiac jelly, proliferate under the action of VEGF and form the cushions. A few genes including Twist, Tbx20, and Sox9 and their downstream targets Sox5 and Sox6 are instrumental to regulate this proliferative stage (Fig. 3). Periostin, a fascilin domain including protein, is expressed in the cardiac mesenchyme of the cushions. The periostin knock-out mouse features shortened and thicker valve leaflets, revealing a role of periostin in the differentiation of mesenchymal cushions (for review [47]). A series of transgenic mouse lines generated in the last decade display deficient cardiac cushions or, for a minority of them, cushion enlargement due to dysregulation of EMT (Table 1). These mice led to the identification of transcriptional and signaling pathways involved in EMT of specific endocardial cells. While we now better understand the EMT process thanks to the 3D collagen hydrogel ex-vivo approach [48] and this series of transgenic mice (Table 1), the remaining question is why only a subset of NFATc, Snail, Slug, Sox9, Msx1,periostin,filamin

VEC

GATA5,Cx37,NFATc,Tbx20, Etv2,N1lCD Smad6, Isl1, VE-cadherin, CD31, Tie

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ES cells Pr A

Embryonic

Endocardium

EMT

Flk1high Flk1low HF

LPM

SHL FHL

CCM PM

Flk1

high

AVC

Nkx2.5 Isl1 Tbx1 Tbx20 FGF8/10 Nkx2.5 Mef2c Tbx5

Valves

OFT

Endocardial progenitor

CC Endoderm Me sP 1

Blastocyst

P D Embryonic

ICM

Extra

cardiac cushions

aortic pulmonary

VIC VIC mitral tricuspid

interstitial fibroblast

Wt1+, Tbx18+

EPDC Ectoderm

Cardiac Neural Crest

Fig. 4. Cell lineages contributing to the formation of the endocardium and valves. Endocardial cells derive both from a mesodermal Flk1+ cell and a Flk1low cell originating from the second heart field. Both progenitors participate in the formation of the leaflets. Epicardium derived cell as well as cardiac neural crest cells also contribute to the valve maturation at a later stage. CC: cardiac crescent; CCM: cranial cardiac mesoderm; EPDC: epicardium derived cell. HF: head fold; ICM: inner cell mass; LPM: Lateral plate mesoderm; PM: paraxial mesoderm; VEC: valve endothelial cell progenitor; VIC: valve interstitial cell.

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Table 1 Genes and signaling pathways involved in the formation of cardiac cushions. Genes/signaling pathways

EMT cardiac cushions

References

Smad4 Gata4 Smad7 Cpsg2 RPP-J (Notch) ALK2, ALK3, BMP-2, (redundant roles of BMP-4, -5, -6, -7)

deficient deficient deficient deficient deficient deficient

TGFbRII Cx40 Cx45 CHF1/Hey1 Trisomy 16 Adm19 SHP2 Noonan Tbx20 Endoglin twist Sox9 PDK1 Fog2 Ezh2 Mmp15 snail Msx1/2 Smad6 Slug Irx3,5 RA Tbx2(misexpression in myocardium) MAPK pathway MEKK4 Versican

deficient deficient deficient deficient deficient deficient Enlarged cushion deficient deficient deficient deficient deficient Enlarged cushion deficient deficient deficient deficient deficient deficient deficient Excess EMT deficient deficient

[70,71] [72] [70] [73] [74] [75–79] For recent review: [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [88,90] [91] [92] [93] [54] [94] [95] [96] [97] [98] [99–102] [45] [103] [73]

endocardial cells undergoes EMT in order to form the cardiac cushions. Several lines of research are emerging to address this question. Pioneering work in zebrafish reported the role of micro RNAs in the formation of ECM. Mir23 targets hyaluronic acid synthase 2(Has2), Icat, and Tmem2, thus restricting cushion formation [49]. The involvement of miRs to locally restrict gene expression and in turn the EMT process is appealing. Investigation in the mouse embryo will further tell us whether such a miR-dependent process is conserved in mammals. Then the chicken or egg question emerges: how a localized Mir expression can be explained? What are the upstream morphogens which regulate miRs in this region? A possible gradient of expression of BMP2 or smad in this region could also explain a local high activity of this transduction pathway and a possible expression of a reprogrammation factor at the origin of EMT. Biomechanical events have also been reported to contribute to the localized EMT process. Indeed an elegant paper recently reported that endocardial cells harbor a cilia; such a structure translates local blood flow signal into functional responses, including nitric oxide production likely produced by the ciliated cells [50] as an important cue for valve formation and initiation of gene expression. However cells, submitted to constant high flow, loose their cilia, down-regulate Klf4, stop Klf2 up-regulation, and undergo EMT in a TGFβ-dependent manner [51]. The role of mechanobiology in the process of EMT of the cardiac cushions is just emerging. This question is expected to be more thoroughly investigated in the near future [52,53]. Epigenetic events which occur throughout the genome at the regions of specific gene enhancers are likely to regulate gene expression in cardiac cushions [54] in a space-restricted manner and in specific cells prone to undergo EMT. EMT is indeed associated with cell reprogrammation regulated by epigenetic events [55,56]. Immunoprecipitation of chromatin of AVC cells combined to deep sequencing assays using antibodies directed against markers of active epigenetic events at enhancers (anti-modified histones H3K4m1, anti-p300 …) should identify enhancers and possibly genes that are instrumental in AVC cells before or at the onset of EMT ( E9.5 in mouse embryo).

Following EMT of cardiac cushions, developing valves are composed of multiple cell types, including a subset of them that express the transcription factor scleraxis (scx) and other tendon-associated structural proteins. Scx expression is further correlated with sox9 downregulation. These genes are under the regulation of FGF4/Erk (scx) or BMP/Smad (sox9, agregan …) signaling pathways [57].Thus, EMT does not engage all endocardial cells to the same phenotype. A tight balance between BMP and FGF signaling pathways is set during EMT to drive the cells to chondrogenic or tendinous phenotype ensuring a proper distribution of various cell types that are required to optimal valve function. Such a subtle balance of signaling pathways remains to be better defined in a space- and temporal-manner using in-vivo, ex-vivo and in-vitro experimental settings. 1.3. Embryonic stem cells recapitulate early valvulogenesis and provide potential therapeutic cells While transgenic mice have provided us with invaluable information as to the role of specific genes, or signaling pathways regulating valve formation, the knowledge of early human processes of valvulogenesis is still limited. Furthermore, the first steps of the later process (i.e., cell lineage specification, triggering signals of EMT in cushion formation) are still very elusive both in the mouse and human embryos. Thus, embryonic stem cells bring a cellular model to investigate such events. We have derived a population of MesP1 + cells from human embryonic stem cells (HUESC) [58]. This cell population is obtained by a combination of BMP2 and secreted Wnt3a [59,60]. Guided by the developmental biology approach, this population is further challenged by FGF8 to direct their fate toward the second heart lineage and VEGF to favor the endothelial phenotype [59]. Using some stromal factors of the ECM of embryonic fibroblasts, MesP1 + cells acquire the phenotype of valve endocardial cells (VEC) expressing the proteins CD31, Isl1, Tbx20, VEcadherin, Smad6, Msx1 and NFATc. The HUESCderived cells further undergo EMT under different experimental conditions including high BMP2 treatment, co-culture on AVC mouse explants seeded on collagen gels or injection into the AVC of E10.5 mouse embryos (Neri et al. in revision; [61]). Thus HUESCs, as mouse ESCs [14], recapitulate the formation of the endocardium and further early valvulogenesis. This cell population is very promising from several points of views: The cells do recapitulate step by step the different lineages that contribute to the endocardium which makes them a faithful model to study the cell fate decisions and the EMT process in a human context including the processes of mechanobiology. Several options emerge in cell therapy of valve disease. The cells might be used to restore a population of quiescent fibroblasts (VIC) in congenital valve disease with improper valve formation or in a degenerating leaflet of an aging valve. Such an approach could decrease the stiffness of the leaflet induced by an increased number in myofibroblasts [62]. Such a restoration of the leaflet could be accompanied by a drug and/or growth factor delivery to ensure a better maturation of the cells, for example, toward a tendinous (using FGF and BMP inhibitors) or more chondrogenic (using BMP2 and FGF inhibitors) cell fate. Addition of exogenous cells could also mobilize endogenous cells such as endothelial progenitors from the circulation acting through secreted and chemoattractant factors. Alternatively, HUESC-derived VICs could be used to repopulate a decellularized valve in order to build up a suitable biological valve scaffold that could be more adapted in mitral stenosis or aortic valve disease to replace calcified valves [10]. Indeed the decellularization approach has been applied to valves to test tissue-engineering of valve replacements [63]. The idea to repopulate the decellularized valve has thus been tested in a few laboratories and has proven to be feasible in a prospective clinical arena [64,65] using human valves [66]. This process overcomes the limitations of prosthetic valves, (i.e., lack of growth, repair and of remodeling) and could match with

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the ten commandments proposed by Dr Harken in order to design the best prosthetic valve [67]. The main issue in this therapeutic approach is to find the right cell population to repopulate the decellularized valve. Many cells, including endothelial or hematopoietic cells, have been tested [68,69] with limited success. A genuine population of human VICs is expected to better fulfill the role of original valvular cells and might have an additional repair benefit. Human ES and iPS cells and their valvular derivatives might provide new strategies in this field of developmental biology research and medical applications. Valvulogenesis is a complex developmental process including genetic, epigenetic, morphogenetic and mechanical events that remain to be better understood. Using the combination of mouse transgenic embryos, ex vivo AVC and OFT explants and pluripotent stem cells, we are in a position to better document this developmental biology field for the benefit of a better understanding and cure (treatment) of congenital and acquired valvulopathies. Acknowledgments The author thanks his team and specifically Dr Tui Neri and Emylie Hiriart for their contribution to the valve project in the laboratory, Dr Hany Nematalla (HEGP, Paris) for echocardiographic recording in embryos, Dr Tom Moore Morris (UCSD) for critical reading and editing the review. This work has been funded by the Leducq Foundation (network MITRAL) and the ANR (National Agency for Research, programme Specistem). References [1] M.E. Silverman, J.W. Hurst, The mitral complex. Interaction of the anatomy, physiology, and pathology of the mitral annulus, mitral valve leaflets, chordae tendineae, and papillary muscles, Am. Heart J. 76 (1968) 399–418. [2] J.J. Silbiger, R. Bazaz, Contemporary insights into the functional anatomy of the mitral valve, Am. Heart J. 158 (2009) 887–895. [3] J.T. Butcher, R.R. Markwald, Valvulogenesis: the moving target, Philos. Trans. R. Soc. Lond. B Biol. Sci. 362 (2007) 1489–1503. [4] M.D. Combs, K.E. Yutzey, Heart valve development: regulatory networks in development and disease, Circ. Res. 105 (2009) 408–421. [5] V.T. Nkomo, J.M. Gardin, T.N. Skelton, J.S. Gottdiener, C.G. Scott, M. EnriquezSarano, Burden of valvular heart diseases: a population-based study, Lancet 368 (2006) 1005–1011. [6] V.L. Roger, A.S. Go, D.M. Lloyd-Jones, E.J. Benjamin, J.D. Berry, W.B. Borden, D.M. Bravata, S. Dai, E.S. Ford, C.S. Fox, H.J. Fullerton, C. Gillespie, S.M. Hailpern, J.A. Heit, V.J. Howard, B.M. Kissela, S.J. Kittner, D.T. Lackland, J.H. Lichtman, L.D. Lisabeth, D.M. Makuc, G.M. Marcus, A. Marelli, D.B. Matchar, C.S. Moy, D. Mozaffarian, M.E. Mussolino, G. Nichol, N.P. Paynter, E.Z. Soliman, P.D. Sorlie, N. Sotoodehnia, T.N. Turan, S.S. Virani, N.D. Wong, D. Woo, M.B. Turner, Heart disease and stroke statistics–2012 update: a report from the American Heart Association, Circulation 125 (2012) e2–e220. [7] R.R. Markwald, R.A. Norris, R. Moreno-Rodriguez, R.A. Levine, Developmental basis of adult cardiovascular diseases: valvular heart diseases, Ann. N. Y. Acad. Sci. 1188 (2010) 177–183. [8] M. Padala, S.N. Powell, L.R. Croft, V.H. Thourani, A.P. Yoganathan, D.H. Adams, Mitral valve hemodynamics after repair of acute posterior leaflet prolapse: quadrangular resection versus triangular resection versus neochordoplasty, J. Thorac. Cardiovasc. Surg. 138 (2009) 309–315. [9] M.K. Sewell-Loftin, Y.W. Chun, A. Khademhosseini, W.D. Merryman, EMT-inducing biomaterials for heart valve engineering: taking cues from developmental biology, J. Cardiovasc. Transl. Res. 4 (2011) 658–671. [10] J.T. Butcher, G.J. Mahler, L.A. Hockaday, Aortic valve disease and treatment: the need for naturally engineered solutions, Adv. Drug Deliv. Rev. 63 (2011) 242–268. [11] A.D. Person, S.E. Klewer, R.B. Runyan, Cell biology of cardiac cushion development, Int. Rev. Cytol. 243 (2005) 287–335. [12] L.M. Eisenberg, R.R. Markwald, Molecular regulation of atrioventricular valvuloseptal morphogenesis, Circ. Res. 77 (1995) 1–6. [13] M. Milgrom-Hoffman, Z. Harrelson, N. Ferrara, E. Zelzer, S.M. Evans, E. Tzahor, The heart endocardium is derived from vascular endothelial progenitors, Development 138 (2011) 4777–4787. [14] H. Narumiya, K. Hidaka, M. Shirai, H. Terami, H. Aburatani, T. Morisaki, Endocardiogenesis in embryoid bodies: novel markers identified by gene expression profiling, Biochem. Biophys. Res. Commun. 357 (2007) 896–902. [15] S. Hatta, Contribution to the morphology of cyclostomata. I. On the formation of the heart in petromyzon, J. Coll. Sci. Imp. Univ. Tokyo 10 (1897) 225–237. [16] Y. Tahara, Normal stages of development in the lamprey, Lampetra reissneri (Dybowski), Zool. Sci. 5 (1988) 109–118. [17] I.S. Harris, B.L. Black, Development of the endocardium, Pediatr. Cardiol. 31 (2010) 391–399.

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