Clinical anatomy and embryology of heart valves

Clinical anatomy and embryology of heart valves

CHAPTER 1 Clinical anatomy and embryology of heart valves Richard L. Goodwin1, Stefanie V. Biechler2 1 Biomedical Sciences, University of South Caro...

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CHAPTER 1

Clinical anatomy and embryology of heart valves Richard L. Goodwin1, Stefanie V. Biechler2 1

Biomedical Sciences, University of South Carolina School of Medicine, Greenville, SC, United States; 2Director of Marketing Collagen Solutions PLC Minneapolis, MN, United States

Contents 1.1 Atrioventricular valves 1.1.1 Embryology 1.1.2 Morphology 1.1.3 Histology 1.2 Semilunar valves 1.2.1 Embryology 1.2.2 Morphology 1.2.3 Histology 1.3 Epigenetic factors in heart valve formation References

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1.1 Atrioventricular valves 1.1.1 Embryology The heart is first formed as a simple tube from anterior lateral splanchnic mesoderm as the flat, trilaminar embryonic disc rolls into a cylinder. The growing prosencephalon and the closing gut tube endoderm bring the left and right lateral mesoderms together ventrally at the midline of the developing embryo [1]. At this stage or even a bit before, the primitive myocardium begins to spontaneously contract. The formation of the primitive heart tube is critical to further development of the embryo as it relies on effective hemodynamics to support the ontogenesis of other structures. Though the cardiac valves play a central role in the maintenance of unidirectional blood flow for the entire cardiovascular system, other tissues have valves, including some veins and lymphatic vessels. It is important to note that a valve-like structure is formed, transiently, between the left and right atria known as the foramen ovale. This structure allows placentally derived oxygen- and nutrient-rich blood to pass from the right atrium to the left atrium, allowing it to be distributed systemically during fetal development. Following the first breath and perfusion of the pulmonary vascular, the blood pressure of the right side of the circulation drops below that of the systemic left side blood pressures, physiologically closing the foramen. Over time, the septum primum Principles of Heart Valve Engineering ISBN 978-0-12-814661-3, https://doi.org/10.1016/B978-0-12-814661-3.00001-0

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and the septum secundum fuse, leaving a thumbprint-shaped indentation on the atrial septum known as the fossa ovale. Failure of this foramen to close results in atrial septal defects of varying degree and severity. The acelluar cushions are largely composed of the glycosaminoglycans hyaluronan and chondroitin sulfate, which yield a soft, jelly-like consistency, giving it the name, cardiac jelly (Fig. 1.1). Nonetheless these soft, pliable atrioventricular (AV) cushions do contribute to unidirectional blood flow in the early embryonic circulation. The myocardium of the AV junction produces the initial extracellular matrix (ECM) of the cushions. This provides the substrate that cells will use to migrate into the cushions and produce the tissues of the mature valves and supporting structures. The majority of the cells populating the AV cushions are derived from endocardial cells of the inferior and superior AV cushions as well as significant contribution of epicardially derived cells that have undergone an epithelial-to-mesenchymal transformation (EMT) (Fig. 1.1C). During this process, cells detach from the simple epithelium that lines the interior and exterior of the heart and migrate into the matrix-filled cushions. The cells of this newly formed mesenchyme become VICs, which remodel and maintain the ECM into the complex, stratified valve leaflets [2]. The endocardial cells that cover the valves have been reported to retain their ability to undergo EMT throughout adult life [3]. Under pathological conditions, these endothelial cells transform and migrate into the mesenchyme of the valve leaflets and adversely contribute to valve disease. The roles that other cell types, such as macrophages and other immune cells, play in development and disease of valve tissues are beginning to gain increased attention by investigators, as they appear to be key regulators of homeostasis and pathology [4]. The inferior and superior AV cushions fuse, forming the septum intermedium, which physically separates the left (systemic) and right (pulmonary) sides of the circulatory system. As development continues, lateral AV cushions emerge on the left and right sides and fuse with the inferior and superior endocardial tissues, providing the cells that will go on to form the AV septum, AV valve leaflets, and supporting tissues. In lineage tracing studies, neural crest cells were detected in the AV septum and shown to have migrated from the top of the neural tube into the heart via pharyngeal arches 3, 4, and 6. The roles that specific cells play in the differentiation and their contributions to eventual adult cardiac structures are not clear despite intensive and ongoing efforts. It is critical that these studies be brought to their fruition, as defects in the AV valvuloseptal tissues are amongst the most lethal. During normal development of the AV septum, the ostia of the atria anatomically align with the AV valves and the ventricular chambers. Subsequent fibroadipose development of the AV septum provides a foundation for the remodeling of the endocardial cushions into the valve leaflets. The AV septum and its fibrous cardiac skeleton also act as an electrical insulator that allows for the atrial, ventricular delay of the cardiac cycle. Housing the AV node of the cardiac conductance pathway, malformations of this region impact cardiac rhythm and function and are thus critically pathological.

Clinical anatomy and embryology of heart valves

Development of the valve leaflets and tension apparatus of AV valves is generally thought to be driven by a remodeling process in which cushion cells differentiate into ECM-producing VICs that create the stratified fibroelastic connective tissue of the valve leaflets and the fibrocartilage-like chordae tendineae. This remodeling occurs in humans during infancy and early childhood. The mechanisms that drive the differentiation of cells into VICs versus cells of the chordae tendineae are not clear [5]. Hemodynamically driven differentiation is an attractive, though, not well-tested mechanism. Malformations of these structures include prolapse, stenosis, and atresia. The molecular mechanisms that create and maintain the tissues of the cardiac valves have a long history of investigation. Decades of research studies using a variety of model systems have delineated the molecular signaling pathways that are critical for the induction, differentiation, and maturation of cardiac valves [2,5]. These processes can be divided into four stages: endocardial cushion formation, endocardial transformation, growth and remodeling, and stratification (Fig. 1.2). AV valve formation is initiated when the myocardium of the AV canal produces the cardiac jelly that fills the superior and inferior AV cushions. Along with the ECM proteins, these cells secrete morphogens that activate overlying endocardial cells to disconnect from neighboring endothelial cells and migrate into the ECM of the cushions. Myocardially derived BMP2 signals initiate transformation of the AV canal endocardial cells, while canonical Wnt and TGF-b signaling are critical for sustaining EMT [6]. Endocardially derived Notch and VEGF signaling are also required for EMT, and several other well-characterized signaling pathways that are summarized in Fig. 1.2. In addition to the molecules above, transcription factors Twist1 and Tbx20 are critical for the proliferation and differentiation of newly formed mesenchymal cells. Interestingly, VEGF becomes a negative regulator of VIC proliferation at the post-EMT stage of valve development [6]. During this stage of valve development, the matricellular protein, periostin, becomes highly expressed in the developing cushions and is necessary for the differentiation of VICs into ECM-producing fibroblasts within valve cushions [2]. As its name denotes, periostin is also involved in bone development. In fact, valve development involves a number of molecules that have been implicated in the development of bone and cartilage. Another similarity between bone and cardiac valve development is the BMP-driven expression of Sox9 [6]. However, there is a tendon-like gene expression pattern in the differentiation of the chordae tendineae of AV valves, involving Fibroblast Growth Factor (FGF), scleraxis, and tenascin. As the valve leaflets mature, they become more complex with specific combinations of ECM proteins deposited in different locations within the valve [2]. This results in the formation of three distinct layers within valves, which are discussed in detail below. Here, it is important to note that the bone-like expression pattern remains in the collagen-rich fibrosa layer, which is dependent on NFATc1, whereas a cartilage-like expression pattern has been found in the proteoglycan- and glycosaminoglycan-rich spongiosa layer.

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Figure 1.1 Overview of cardiac development. (A) The heart initiates as a tube composed of endothelial cells (ECs), cardiac jelly (CJ), and myocyte cells (MCs). The tube is initially linked to the foregut (FG) via the dorsal mesocardium (DM), but this connection later breaks away as looping occurs. (B) As the tube bends and twists, cushions filled with CJ form. Atrioventricular cushions (AVCs) form in the AV canal, and outflow cushions (OFCs) form at the heart outlet where they receive a cellular contribution from neural crestederived cells (NCCs). The future right and left ventricles, RV and LV, and the future right and left atria, RA and LA, are defined. (C) (i) Signaling from the MCs to the ECs induce EMT.

Clinical anatomy and embryology of heart valves

The third layer, which is on the flow facing side of the valve leaflet, has a smooth muscle-like ECM, which contains elastin and collagen [2]. However, the molecular regulation of this layer has yet to be clearly defined.

1.1.2 Morphology As discussed above, the AV valves differ from the semilunar valves in that the AV valve leaflets have a tension apparatus that consists of the chordae tendineae and the papillary muscles. The chordae tendineae are string-like extensions off of the valve leaflets that connect the AV leaflets to the papillary muscles of the ventricles (Fig. 1.3). The papillary muscles are invested with conductive tissue that are closely connected to the branch bundles and thus are amongst the earliest regions to contract in the ventricles, tensing the leaflet and preparing the structure to withstand systole. Failure to do so results in regurgitation of blood back into the atrium, resulting in loss of cardiac output. Generally, the orifice of the left ostia is bigger than the right, though this can change as a result of malformation or pathology. The mitral valve, or bicuspid valve, separates the left atrial and ventricular chambers and has two valve leaflets, the anterior (aortic) and posterior (mural). The chordae tendineae of these leaflets coalesce into two well-defined papillary muscles located near the apex of the left ventricular chamber. During systole, the two leaflets have one zone of apposition that seals off the AV ostia and prevents regurgitation back into the left atria. Clinically, this crescent-shaped zone is divided into the anterolateral commissure and the posteromedial commissure, which enables anatomical description of areas of regurgitation or prolapse [7]. The tricuspid valve separates the right atrial and ventricular chambers and has three valve leaflets: the anterior; posterior; and the mural. The chordae tendineae from these leaflets coalesce into three clusters of papillary muscles in the right ventricle. The papillary muscles of the right ventricle are less organized and more variable than those of the left

= (ii) Activated ECs lose their cellecell junctions and invade the CJ. (iii) The activated ECs begin to express mesenchymal cell (MesC) markers. (D) The future heart chambers are in their final location when the outflow tract (OFT), atria, and ventricles begin to septate. (E) Muscular protrusions grow from the heart wall to form the atrial septum (AS) and ventricular septum (VS). The AS protrusion has a cap, the dorsal mesenchymal protrusion (DMP), that connects with the AVCs. (F) After EMT, the AVCs elongate into leaflets that are lined with valvular endothelial cells (VECs) and valvular interstitial cells (VICs). (G) Blood flows through the developed heart in the following order: vena cava, RA, tricuspid valve (TV), RV, pulmonary valve (PV), pulmonary artery, lungs, pulmonary veins, LA, mitral valve (MV), LV, aortic valve (AoV), aorta, body (H) Atrioventricular (AV) valves are composed of three layers: the elastin-rich atrialis, the water-rich spongiosa, and the collagenous fibrosa. The leaflet tip is tethered to the heart wall via the chordae tendineae. (I) Semilunar (SL) valves have the same three layers, but the elastin-rich layer is referred to as the ventricularis. The leaflet cusps end in thick, fibrous tips known as the nodules of Arantius in the AoV or nodules of Morgagni in the PV. The trilaminar leaflets and associated support structures function to withstand flow-induced forces.

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Figure 1.2 Signaling in cardiac development. Each phase of cardiac development is associated with biochemical signals that are still being fully elucidated. During cushion formation, myocyte cells (MCs) signal to the endothelial cells (ECs) and induce alignment and proliferation. At the same time, the MCs secrete hyaluronic acid (HA) and, in the outflow cushions (OFCs), versican to fill the cardiac jelly (CJ) space. The CJ maintains a gradient of growth factors (GFs), allowing different stages of development to be triggered at different times. Epithelial-to-mesenchymal transformation (EMT) begins with EC activation and MC secretion of fibronectin (FN). The activated ECs phenotypically change as they lose their cellecell junctions and migrate into the CJ. Inside the CJ, these cells begin to express mesenchymal cell (MesC) markers. Flow and proteinases stop EMT and the cushions grow and remodel. The VICs in the CJ have regulated proliferation, and EC proliferation is thought to slow as extracellular matrix (ECM) is increasingly deposited in the CJ. MesCs differentiate into ECMsecreting cells characteristic of mature valves. A mature valve exhibits three distinct layers that are regulated with unique signaling mechanisms, and the atrioventricular (AV) valves have a tendonlike support apparatus that undergoes signaling similar to cartilage and tendons.

Clinical anatomy and embryology of heart valves

Figure 1.3 Clinical anatomy of left heart valves. The semilunar and atrioventricular valves have unique structures that provide support and anchor the valves to the wall. The three leaflets of the semilunar valves have commissures at the wall juncture (depicted for the aortic valve) and the atrioventricular valves are attached to the papillary muscle via a tension apparatus, chordae tendineae (depicted for the mitral valve). ((Left) From Frank H. Netter, Atlas of Human Anatomy e 4th Edition, 2006; (Right) CNRI/Science Photo Library.)

papillary muscles [7]. The moderator band, an important cardiac conductance tissue, is incorporated within the septomarginal trabecula, which is a myocardial structure that connects the anterior papillary muscle of the right ventricle to the interventricular septum. Being trifoliate, there are three zones of apposition and three commissures in the tricuspid valve: the anteroseptal; the anteroposterior; and the posterior.

1.1.3 Histology The tissues that make up valve leaflets of both AV and semilunar valves have a similar overall design. A common feature of all cardiac valve leaflets is that they are organized into three layers (Fig. 1.1, panels H and I). The first layer of cells under the endocardial epithelium on the flow side of the leaflet contains densely packed cells that are surrounded by an elastic connective tissue. This layer is named the atrialis in AV valves and the ventricularis in semilunar valves. Elastic fibers are radially oriented from the hinge of the leaflet to the coapting edge [7]. The composition and organization of the matrix allows for extension and recoil of this layer as the valve opens and closes. The middle layer of valve cells is called the spongiosa and contains sparsely distributed cells embedded in ground substance, which is largely composed of proteoglycans. This layer is thought to carry out a cushioning function for the valves. The layer on the back (nonflow) side of the valve leaflets is called the fibrosa. As its name implies, it is a dense connective tissue containing large bundles of insoluble, fibrous Type I collagen, giving it a comparatively stiff quality. These fibers are circumferentially oriented, providing tensile strength to the leaflet. Together, the three layers of the valve leaflets provide a balanced mix of stiffness, pliability, and recoil, giving it the mechanical properties necessary for healthy valves to be

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competent when closing and compliant when opening. Alterations in the composition and organization of these layers are associated with numerous valve pathologies including valve calcification and myxomatous valves. Another difference between the mitral and tricuspid AV valves, in addition to the number of leaflets and the size of the annuli, is the thickness of the valve leaflets. The mitral valve leaflets are thicker than those of the tricuspid. However, this difference is not evident until after birth, indicating that the increasing hemodynamic load experienced on the systemic side of the circulation is driving this morphogenesis. The annuli of the valves are composed of dense connective tissue and provide a firm foundation to anchor the hinges of the leaflets. Type I collagen is the main ECM protein of the valve annuli, forming the major components of the cardiac skeleton. With the exception of the pulmonary valve, cardiac valve annuli are embedded in the AV septum with the aorta being wedged between the tricuspid and mitral valves, making this a highly complex region of the heart.

1.2 Semilunar valves 1.2.1 Embryology The cushions that go on to contribute to the semilunar valves appear just after the cushions of the AV canal. These conotruncal cushions form as oppositely opposed ridges that spiral down the truncus arteriosus, which is the single outflow vessel, or arterial pole, of the tubular heart. This single outflow tract is divided into the pulmonary and aortic arteries as the conotruncal cushions become populated with cells, grow, and fuse at midline, creating the septum intermedium, which physically separates left and right sides of the arterial pole of the heart and sets the stage for complete aorticopulmonary septation [2]. Septation of the truncus arteriosus occurs from the inside out, with the tunica intima of the two newly formed vessels forming first, followed by the generation of their tunica medias, and, finally, their adventitias. In this way, the single outflow vessel is divided into two completely formed great arteries. The completeness of this separation is particularly evident in the transverse pericardial sinus, which separates the infundibulum of the pulmonary trunk from the aorta. Failure of proper formation and progression of the aorticopulmonary septum can result in life-threatening lesions including persistent truncus arteriosus or subclinical lesions such a small ventricular septal defect. This is because the aorticopulmonary septum fuses with the muscular ventricular septum that arises between the left and right ventricular chambers, becoming the membranous component of the ventricular septum. Neural crest cells have been found to contribute to both the AV valves and the semilunar valves; however, their role in morphogenesis of the outflow tract is particularly critical. Failure of neural cells to populate and migrate with the aorticopulmonary septum results in persistent truncus arteriosus.

Clinical anatomy and embryology of heart valves

In combination with the remodeling of the conotruncal cushions of the embryonic outflow tract, the leaflets of the semilunar valves develop from another set of cushions in the outflow tract, the intercalated cushions, which form adjacent to the conotruncal cushions. Once again in a manner similar to the AV valves, cardiac jelly filled swellings appear between the myocardium and endocardium, become cellularized by endocardial EMT, and undergo ECM remodeling over time into the highly organized valve leaflets. Much remains unknown about mechanisms that regulate the number and positioning of semilunar valve leaflet anlagen that differentiate into the trifoliate adult semilunar valves. Defects in these structures result in bicuspid and stenotic semilunar valves.

1.2.2 Morphology The two semilunar valves have similar structures with three pocket-like leaflets arranged such that they are competent without the tension apparatus that is found in the AV valves. The three leaflets of the semilunar valves have three commissures, which act as anchoring points to support the juncture to the wall at the base of the leaflets (Fig. 1.3). The semilunar leaflet geometry creates spaces behind the leaflets known as the sinuses of Valsalva. The U-shaped base of the semilunar valve leaflets creates triangular-shaped areas in the walls of the great arteries that are not occupied by either valve tissue or valve sinuses. Thickened nodes are present at the tip of each leaflet, known as the nodules of Arantius in the aortic valve and the nodules of Morgagni in the pulmonary valve. These nodules exhibit an enlarged spongiosa layer making them characteristically elastic structures that can act to support the extreme hemodynamic forces present at the point of valve closure or the valvular orifice (Fig. 1.11I). The pulmonary valve differs from the aorta in that it has a column of myocardium, the infundibulum, to support its root. However, the aortic root is embedded in the connective tissue of AV septum. The aortic valve leaflets are thicker than those of the pulmonary, but again, this difference appears to develop postpartum as a response to the increased load that is required for systemic circulation. Another difference between this class of cardiac valves is the presence of coronary artery ostia in two of the aortic valve sinuses. The aortic valve is situated in the middle of the AV septum, wedged between the mitral and tricuspid annuli and wrapped by the pulmonary infundibulum. The left and right sinuses of the aorta have the openings of the left and right coronary arteries. Therefore, the sinus of the posterior leaflet is known as the noncoronary sinus. Interestingly, this leaflet is the only aortic leaflet that does not appear to have any contribution of neural crest cells; however, this may be due to the fact this leaflet is deeply embedded in between the mitral and tricuspid annuli and may be inaccessible to neural crest cells migrating down the aorticopulmonary septum during development. As mentioned above, the aortic valve, unlike the pulmonary valve, is surrounded by fibrous tissues. A portion of the aortic valve is continuous with the fibrous aspects of

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the mitral valve, including its anterior leaflet. The left fibrous trigone of the aortic root is also continuous with the mitral valve. The right fibrous trigone is continuous with the membranous portion of the ventricular septum, which is derived from the aorticopulmonary septum. The aortic root is bulbous in appearance and contains the annulus and sinuses of the valve leaflets. At the distal attachments of the valve leaflets, the aorta becomes cylindrical and is called the sinotubular junction. This marks the end of the aortic valve and the beginning of the ascending portion of the aorta. The pulmonary valve is positioned at a distinctly different angle than the other three cardiac valves. The elongated, funnel-shaped infundibulum of the right ventricular outlet wraps around the aortic root. This sleeve of myocardium acts to support the pulmonary valve root. The three leaflets of the pulmonary valve are the left, right, and anterior. Common malformations of this valve include atresia and stenosis, which can be associated with other cardiac lesions, as in the case of tetralogy of Fallot, and result in cyanosis.

1.2.3 Histology As described above, the histological organization of the semilunar valve leaflets is similar to that of the AV valves. They share a common tissue architecture, having the three tissue layers of the fibrosa, spongiosa, and elastic layer, known as the ventricularis in the semilunar valves. The semilunar leaflets are significantly thinner than the AV leaflets. Another difference between the AV and semilunar leaflets is that the distal edges of the semilunar leaflets are thick and contain a bulbous structure in the middle of the free edge known as the nodule of Arantius. These modifications and their overall geometry allow for the semilunar valves to seal as they close during diastole, preventing regurgitation. Recently a new anatomical structure has been described in the root of the aorta. This “prelymphatic” organ appears to be distinct from the adventitia and is thought to provide a “shock absorber” function. This structure contains a series of interconnected “vessels” that are produced by bundles of fibrous collagen and sporadically lined with CD34 þ cells that have not been well-characterized [8]. This initial report did not appear to investigate whether this structure is present in the pulmonary trunk.

1.3 Epigenetic factors in heart valve formation As previously indicated, hemodynamics is thought to play an important and fundamental role in the morphogenesis of the cardiac valves. In particular, the shear stresses and pressures of the developing cardiovascular system appear to play a formative role in the remodeling of the EMC of the cardiac cushions as they morph into the fibrous tissues of the valves [9]. While substantial clinical and experimental literature support the “no flow/no grow” hypothesis, the specific molecular mechanisms that are used to transduce these mechanical signals into distinct cellular activities are not well-characterized.

Clinical anatomy and embryology of heart valves

These studies are frequently confounded by the chicken/egg paradox. For instance, individuals with bicuspid aortic valves have a high risk of developing calcified leaflets that become incompetent. Is this pathological calcification caused by the abnormal geometry that results in an aberrant aortic flow field or by the same process that drove the altered anatomy? New 3D in vitro model systems in which mechanical forces can be carefully controlled are coming on line that will allow for the direct testing of mechanotransduction pathways in specific mechanical environments [10,11]. These studies will be important in designing new therapies for these deadly valve diseases. Other investigations using surgically created hemodynamic abnormalities have been carried out on avian embryos and have revealed that at its earliest stages, valve development is regulated by blood flow by affecting EMT [12]. Not surprisingly, similar approaches have found that altered hemodynamics drives alterations in the expression and deposition of critical ECM proteins [13]. The mechanisms by which developing valve tissues sense and transduce mechanical signals have been aided by the discovery of primary cilia on VICs within the developing valve cushions [14]. Primary cilia have been implicated in valve development previously, but only in early endocardial cells [15]. Primary cilia have long been known as cellular structures that sense and mediate responses to mechanical forces. Thus, their discovery in cushion mesenchyme indicates that forces other than shear stress, such as deformation-inducing pressures, could be important regulators of valve development. Gestational diabetes has recently been reported to be an epigenetic regulator of valve development. Fetal hyperglycemia results in the increase of reactive oxygen species, which has been reported to result in cardiac malformations. Specifically, malformations of the outflow tract were associated with hyperglycemia and decreased nitrous oxide signaling [16]. Much progress has been made in delineating the genetic pathways that are critical in the progression of valve development and disease. However, the roles that epigenetic factors play in these processes are in their infancy and require more intense study.

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[4] Hulin A, Anstine LJ, Kim AJ, Potter SJ, DeFalco T, Lincoln J, Yutzey KE. Macrophage transitionsinheart valve development and myxomatous valve disease. Arterioscler Thromb Vasc Biol March 2018;38(3):636e44. PMID: 29348122. [5] Koenig SN, Lincoln J, Garg V. Genetic basis of aortic valvular disease. Curr Opin Cardiol February 2, 2017. https://doi.org/10.1097/HCO.0000000000000384. PMID: 28157139. [6] Hinton RB, Yutzey KE. Heart valve structure and function in development and disease. Annu Rev Physiol 2011;73:29e46. https://doi.org/10.1146/annurev-physiol-012110-142145. Review. PMID: 20809794. [7] Spicer DE, Bridgeman JM, Brown NA, Mohun TJ, Anderson RH. The anatomy and development of the cardiac valves. Cardiol Young December 2014;24(6):1008e22. https://doi.org/10.1017/ S1047951114001942 [Review]. [8] Benias PC, Wells RG, Sackey-Aboagye B, Klavan H, Reidy J, Buonocore D, Miranda M, Kornacki S, Wayne M, Carr-Locke DL, Theise ND. Structure and distribution of an unrecognized interstitium in human tissues. Sci Rep March 27, 2018;8(1):4947. https://doi.org/10.1038/s41598-018-23062-6. [9] Wu B, Wang Y, Xiao F, Butcher JT, Yutzey KE, Zhou B. Developmental mechanisms of aortic valve malformation and disease. Annu Rev Physiol February 10, 2017;79:21e41. Review. PMID: 27959615. [10] Tan H, Biechler S, Junor L, Yost MJ, Dean D, Li J, Potts JD, Goodwin RL. Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves. Dev Biol February 15, 2013;374(2): 345e56. PMID: 23261934. [11] Biechler SV, Junor L, Evans AN, Eberth JF, Price RL, Potts JD, Yost MJ, Goodwin RL. The impact of flow-induced forces on the morphogenesis of the outflow tract. Front Physiol June 17, 2014;5:225. 2014. [12] Menon V, Eberth JF, Goodwin RL, Potts JD. Altered hemodynamics in the embryonic heart affects outflow valve development. J Cardiovasc Dev Dis 2015;2(2):108e24. Epub 2015 May 15. [13] Rennie MY, Stovall S, Carson JP, Danilchik M, Thornburg KL, Rugonyi S. Hemodynamics modify collagen deposition in the early embryonic chicken heart outflow tract. J Cardiovasc Dev Dis December 20, 2017;4(4). pii: E24. PMID: 29367553. [14] Toomer KA, Fulmer D, Guo L, Drohan A, Peterson N, Swanson P, Brooks B, Mukherjee R, Body S, Lipschutz JH, Wessels A, Norris RA. A role for primary cilia in aortic valve development and disease. Dev Dynam August 2017;246(8):625e34. https://doi.org/10.1002/dvdy.24524. Epub 2017 Jun 28. PMID: 28556366. [15] Egorova AD, Khedoe PP, Goumans MJ, Yoder BK, Nauli SM, ten Dijke P, Poelmann RE, Hierck BP. Lack of primary cilia primes shear-induced endothelial-to-mesenchymal transition. Circ Res April 29, 2011;108(9):1093e101. https://doi.org/10.1161/CIRCRESAHA.110.231860. PMID: 21393577. [16] Basu M, Zhu JY, LaHaye S, Majumdar U, Jiao K, Han Z, Garg V. Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease. JCI Insight October 19, 2017;2(20). https://doi.org/10.1172/jci.insight.95085. pii: 95085.