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Outflow tract development of the heart is complex. The presence, differential growth and interactions of the various tissues through space and time contribute to the final development of the tract. This paper presents a novel interpretation of observations of outflow tract development, in particular of the aortic and pulmonary semilunar valves in embryos from the Shaner Collection at the University of Alberta. Three-dimensional reconstructions assist in the visualization of the spatial relationships of the developing valve tissues. In some embryos the aortic intercalated valve swelling is displaced proximally, giving rise to a bicuspid aortic semilunar valve more distally. In addition, the developing valve tissue first appears external to the myocardial cuff. The pulmonary semilunar valve regions appear to be more normal. This paper thus proposes a novel mechanism for generating a bicuspid aortic valve and also supports the idea that there is some independence of the aortic and pulmonary regions from each other during development., (Copyright © 2017 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Objectives: The purpose of this study was to determine whether the morphologic characteristics and area of the semilunar valves in healthy fetuses and fetuses with cardiac defects can be visualized by using spatiotemporal image correlation (STIC)., Methods: Spatiotemporal image correlation volumes from 74 healthy fetuses were recorded in 5 examinations between the 15th and 36th weeks of pregnancy. Second, we recorded STIC volumes from 64 fetuses with various cardiac defects. The quality of the volumes was rated. The areas of the aortic and pulmonary valves were measured in systole by rendering the valves on 4-dimensional sonography. The number of leaflets was examined. Longitudinal data analysis using linear mixed models was performed., Results: Two hundred ninety-three volumes from normal hearts were examined. In 82.5%, the quality of the normal volumes was sufficient. Visualization of the valve opening was feasible in 96.1% of the normal hearts and 97.4% of the abnormal hearts. The success rate of visualization of the pulmonary and aortic valve leaflets was dependent on the gestational age, with the highest percentage (72.1% in normal hearts) at 19 to 24 weeks. Longitudinal regression analysis showed a positive relationship of the aortic and pulmonary valve areas with gestational age (P < .0001) and fetal biometric measurements (P < .0001). Fifty-eight abnormal volumes were examined. Cardiac defects with abnormal valve areas due to aortic and pulmonary stenosis could be clearly visualized by using STIC., Conclusions: Examination of the morphologic characteristics of the semilunar valves using STIC is feasible, which is difficult when using 2-dimensional sonography. With increasing implementation of 4-dimensional sonography, the understanding of rendered images might be useful for anyone practicing fetal echocardiography., (© 2016 by the American Institute of Ultrasound in Medicine.)

Objective: To develop reference ranges of fetal aortic and pulmonary valve diameter derived from volume datasets of spatio-temporal image correlation (STIC)., Methods: A cross-sectional study was undertaken on low-risk pregnancies with well-established data from 14 to 40 weeks. Volume datasets of STIC were acquired for subsequent off-line analysis. Aortic and pulmonary valve diameters were measured in STIC multiplanar view using 4D-View version 9. Normal Z scores and centile reference ranges were constructed from these measurements against gestational age (GA) and biparietal diameter (BPD) as independent variables, using regression models for both mean and SD., Results: A total of 606 volume datasets were successfully measured. Normal reference ranges for predicting mean values and SD of aortic and pulmonary valve diameter were constructed based on best-fit equations (linear function) as follows: mean aortic diameter (mm) was modeled as a function of GA (weeks) and BPD (mm) as - 2.4838 + 0.2702 × GA, (SD = 0.1482 + 0.0156 × GA) and - 1.5952 + 0.0989 × BPD (SD = 0.1672 + 0.00572 × BPD). Mean pulmonary diameter was modeled as - 2.5924 + 0.2935 × GA (SD = 0.2317 + 0.01524 × GA) and - 1.6830 + 0.1083 × BPD (SD = 0.1971 + 0.0059 × BPD)., Conclusion: We have provided nomograms and Z scores of fetal aortic and pulmonary valve diameters. These reference ranges may be a useful tool in the assessment of fetal cardiac abnormalities., (Copyright © 2011 John Wiley & Sons, Ltd.)

During the fetal-to-neonatal transition, transvalvular pressures (TVPs) on the aortic and pulmonary valves change dramatically-but differently for each valve. We have examined changes in the molecular stability and crosslinking of collagen during this transition. Aortic and pulmonary valves were harvested from fetal and neonatal cattle. Using differential scanning calorimetry (DSC), denaturation of valvular collagen was examined and, using HPLC, the types and quantities of enzymatic crosslinks were examined. No difference in hydrothermal stability was found between the collagens in the fetal aortic and pulmonary valves; this was expected since the TVP is approximately the same across both valves before birth. Only in the neonatal samples was the collagen from aortic valves (higher TVP) less stable than that from pulmonary valves (lower TVP). Surprisingly, the enthalpy of denaturation did not differ either between valve type or with age, suggesting an entropic mechanism of altered molecular stability. A significant difference in immature-to-mature crosslink ratio was found between neonatal aortic and pulmonary valves: a difference absent in fetal valves. This ratio-indicative of remodeling rate-parallels (and may be a function of) the changing in vivo load. This study highlights the relationship between in vivo load and both (i) molecular stability and (ii) collagen remodeling in heart valves.

The great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 mum x 2 mum x 2 mum and 3 mum x 3 mum x 3 mum) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.

The development of cartilage in the aortic and pulmonary valves of chick and quail was studied using histological, histochemical and immunohistochemical techniques. In both species, the earliest evidence of chondrogenesis is the formation of smooth muscle alpha-actin-negative prechondrogenic (type II collagen-negative) cellular condensations in the tunica media of the proximal aorta and pulmonary trunk, in front of or slightly distal to the valvular commissures. Such condensations are present as early as stage 37 of Hamburger-Hamilton in the aortic and pulmonary valves of the chick. In quail embryos, they form somewhat later, namely, at stage 38 in the aortic valves and stage 39 in the pulmonary valves. In the chick, synthesis of type II collagen starts in the central core of the aortic cellular condensations at stage 38. In the pulmonary valves of chick and aortic and pulmonary valves of quail, production of type II collagen does not begin until stage 40. This production then gradually increases toward the periphery of the condensations, which remain devoid of perichondrium prior to hatching. After birth, the condensations become transformed into hyaline cartilaginous foci. In the aortic valves of some chickens and quails, more or less extensive deposits of hyaline cartilage or fibrocartilage form along the attachments of the leaflets to their supporting sinuses. They develop later than the commissural cartilages. The present findings, together with previous data from the literature, suggest that the aortic and pulmonary valve cartilages differentiate from neural crest-derived nonmuscular cells.

Atrioventricular and semilunar valve abnormalities are common birth defects, but how cardiac valvulogenesis is directed remains largely unknown. During studies of genetic interaction between Egfr, encoding the epidermal growth factor receptor, and Ptpn11, encoding the protein-tyrosine-phosphatase Shp2, we discovered that Egfr is required for semilunar, but not atrioventricular, valve development. Although unnoticed in earlier studies, mice homozygous for the hypomorphic Egfr allele waved-2 (Egfrwa2/wa2) exhibit semilunar valve enlargement resulting from over-abundant mesenchymal cells. Egfr-/- mice (CD1 background) have similar defects. The penetrance and severity of the defects in Egfrwa2/wa2 mice are enhanced by heterozygosity for a targeted mutation of exon 2 of Ptpn11 (ref. 3). Compound (Egfrwa2/wa2:Ptpn11+/-) mutant mice also show premature lethality. Electrocardiography, echocardiography and haemodynamic analyses showed that affected mice develop aortic stenosis and regurgitation. Our results identify the Egfr and Shp2 as components of a growth-factor signalling pathway required specifically for semilunar valvulogenesis, support the hypothesis that Shp2 is required for Egfr signalling in vivo, and provide an animal model for aortic valve disease.

The fibrous skeleton of the heart has featured prominently in anatomical and surgical descriptions, although all its purported components are difficult to demonstrate. In descriptions of the skeleton, there have been repeated references to the presence of a tendon (or ligament) between the aortic and pulmonary roots. Such a tendon is rarely, if ever, discussed in the context of surgical procedures being carried out on the ventricular outflow tracts. Our study was undertaken, therefore, to investigate the existence and nature of such a tendon or ligament. Serial transverse sections were made through roots of aorta and pulmonary trunk in an intact fetal heart. In addition, ten normal adult hearts were dissected to display the components of the fibrous skeleton of the heart. No discrete fibrous or elastic structure could be detected in the tissue plane between the aortic sinuses and the subpulmonary muscular infundibulum, although a fascial strand was observed in one heart. Apart from this specimen, the space between the free-standing muscular subpulmonary infundibulum and the sinuses of aorta hearing the coronary arteries was occupied only by loose fibroareolar tissue. The initial presence of the ligament was described following studies of animal and macerated human hearts. Subsequently, it would seem its existence has been passed down through generations of morphologists and surgeons without its presence being reconfirmed. We have been unable to demonstrate any structure approximating to the initial illustrations.

Two fetuses of approximately 11 weeks development with aplasia of the pulmonary as well as the aortic valve leaflets are reported. Both cases showed additional cardiac malformations. Case 1, with all leaflets missing, also had double-outlet right ventricle, hypoplastic left ventricle, large ventricular septal defect, straddling tricuspid valve, and atretic mitral valve. Case 2, with only one hypoplastic aortic valve leaflet, showed hypoplasia of the mitral valve and the left ventricle, and a subaortic ventricular septal defect. The observations made and data in the literature suggest that aplasia of semilunar valve leaflets reflects an underdevelopment of the endocardial cushion swellings at the ventriculoarterial junction, rather than resulting primarily from a malseptation of the cardiac outflow tract.

The present study analyzes the composition and organization of the extracellular matrix (ECM) and its changes in the course of development of the chick embryo semilunar heart valves. In the present work we have employed chick embryos from stage 29 until hatching, using silver and picrosirius red staining, lectin probes and light and transmission electron microscopy. Our results show that during semilunar valve development a series of elements arise and are organized in the ECM which seem to be more closely related to the maintenance of the structural and biomechanical properties of the valvular leaflets than with morphogenetic processes per se.

The quail-chick chimera method was used to examine whether neural crest cells were associated with the formation of semilunar valves. From the metencephalon to somite 5, or from the otocyst to somite 3, left, right, or bilateral neural folds, including the neural crest, were transplanted. Among embryos used for the experiment, three into which left neural crest cells were transplanted, two into which right neural crest cells were transplanted, and two into which bilateral neural crest cells were transplanted had a morphologically normal heart. In these embryos, neural crest cells were found in all cusps of the aortic and pulmonary semilunar valves. Although neural crest cells have been thought to have no association with the formation of the semilunar valves, our experiment indicates that such association indeed occurs.

Recent ultrastructural studies have revealed that differences exist in endothelial cell shape and cytoskeletal architecture between the arterial and ventricular faces of developing semilunar valves. In the present work we analyzed the morphologic response of the valvular endothelial cells of chick embryos to colchicine by light microscopy, scanning electron microscopy and transmission electron microscopy. The results show that colchicine administration during the stages of valve morphogenesis causes a very conspicuous disruption of the endothelial layer of the arterial face of the valves. The cells appear rounded and show massive surface blebbing. These alterations were not present in the endothelial cells on the ventricular face of the valves at the same stages. On the basis of these results we suggest that a difference in the degree of cell differentiation exists between the endothelial cells of the arterial and ventricular faces of the cusps and that this difference may have morphogenetic significance.

The developmental anatomy of human cardiac outflow was studied in a series of 16 normal embryos (gestational days 29-39, crown-rump length 6-20 mm, stages 14-19). Structural features and kinetics during truncal septation (TS) were described from external photographs, serial histological sections, and computer graphic reconstructions of selected tissues. Early in the period studied, the tubular myocardium ensheathed the single cardiac lumen and spiralling conotruncal ridges, which were filled with mesenchymal cells during days 31-33. As TS began (late stage 16), the aorticopulmonary (AP) septum appeared across the dorsal wall of the aortic sac between arches IV and VI. Mesenchymal condensations formed within the AP septum, crossing the lumen bifurcation to extend along the truncal ridges to the myocardium. During days 35-37, the cephalic margin of the myocardium grew or folded in toward these mesenchymal condensations between the developing valves and within the nearby conal ridges, which appeared to fuse to separate the subvalvular outflow channels by day 39. These observations are consistent with studies in chicks and rats which suggest that mesenchymal condensations or cell death foci interact with the distal myocardial rim during TS to form a structural septation complex dividing the two arterial streams.

The developmental changes of collagen and mucopolysaccharides in the semilunar values of the mouse were studied during the embryonic, fetal and postnatal period. The valvular collagen was investigated using Van Gieson and Sirius red-polarization microscopy methods. These procedures showed that the establishment of the fibrosa layer of the cusps does not occur until the second week of the postnatal period. The nature and distribution of the valvular mucopolysaccharides were investigated by staining with Alcian blue at specific pH values and at various critical electrolyte concentrations, with the appropriate enzymatic controls using Streptomyces and testicular hyaluronidase. The results show that hyaluronate and chondroitin 4- and 6-sulphate are the major components during the embryonic and fetal period. In the older fetal stages and during the postnatal period the relative amount of hyaluronate decreases, while chondroitin sulphate increases. It is concluded from this study that the maturation of the valves occurs over a long period of the postnatal life.

The development of the semilunar valves of the great arteries of the mouse is studied by light and scanning electron microscopy. The earliest anlage of the valves is observed at day 11.5 of gestation, as three pairs of tubercles protruding towards the lumen of the truncus arteriosus. These early cusps consist of a core of mesenchymal tissue covered by the endocardium. From day 12.5 to 14 a process of excavation takes place in the arterial face of the cusps resulting in the achievement of their final shape. Our observations suggest that the excavation process is produced by a selective growth of the free edges of the cusps. Numerous pieces of evidence are reported suggesting that the selective growth of the cusps is due to an inductive interaction between the endocardium of the arterial face of the cusps and the underlying mesenchyme. The histogenesis of the cusps takes place very late in development and possibly continues into the postnatal period. The results are compared with studies made in other vertebrates.

In view of recent evidence showing that shape and orientation of endothelial cells is determined by blood flow, the endothelium of the semilunar valves was studied in the developing chick heart using transmission and scanning electron microscopy. The results reveal significant developmental modifications of endothelial morphology and structure. These modifications can be linked to modifications of local blood flow and can also explain several aspects of valvular morphogenesis. The results substantially support the hypothesis of an involvement of hemodynamics in the development of the semilunar valves.

The development of the semilunar valves of the great arteries was studied by light and scanning electron microscopy in the chick embryo. The results show that three distinct developmental periods can be distinguished. The formation of the anlage of the valves takes place in the first period (stages 26--29). These early anlage consist of three pyramidal shaped cusps formed by a core of loosely packed mesenchymal cells covered by a flattened endothelium. In the second period (stages 30--35) the cusps undergo excavation on their distal face. Morphololgical evidence is reported suggesting that this excavation process is produced by an initial solid ingrowth of the endothelium of the arterial face of the cusps which is immediately luminated by detachment of cells towards the bloodstream and by cell death. The histogenesis of the valves takes place in the third period (from stage 36 until hatching). It was observed that during this period some myocardial cells of the outflow tracts of the ventricles invade the valvular tissue and that in the upper part of the cusps a prominent fibrous layer is formed.

The development of human semilunar valves was studied in a range of specimens including embryos before the appearance of cellular semilunar valves at stage 17 and hearts after the attainment of mature fibroelastic valvular structure, which occurs around the time of birth. The valves develop by modification of endocardial cushion material at the downstream end of the cardiac tube and appear to grow at the margins, probably by cellular proliferation into a stagnant zone caused by boundary layer separation of blood flow. The flat endothelium on the ventricular aspect and the cuboidal endothelium on the arterial aspect of the valves correlate, respectively, with expected high and low shear forces produced by surface blood flow. Development of an aorta and pulmonary trunk with tricuspid semilunar valves appears to be contingent on the appearance of separate entwined ventricular ejection streams. The later fibroelastic phases of semilunar valve development show progressive increase in elastic and collagenous fibers, at sites which appear to be subjected, respectively, to fluctuating and static tensions.