Deficiency of the vestibular spine in atrioventricular septal defects in human fetuses with down syndrome

Data on the morphogenesis of atrioventricular septal defect (AVSD) in Down syndrome are lacking to support molecular studies on Down syndrome heart critical region. Therefore, we studied the development of complete AVSD in human embryos and fetuses with trisomy 21 using 3-dimensional graphic reconstructions and immunohistochemical markers. Eight trisomic hearts with AVSD and 10 normal hearts, ranging from 5 to 16 weeks' gestation, were examined. In AVSD, the muscular septum primum and venous valves develop normally, and the size and histology of the nonfused endocardial cushions also appear normal. However, the mass of extracardiac mesenchyme (vestibular spine), located at the dorsal mesocardium, is reduced and does not protrude ventrally along the right wall of the common pulmonary vein. As a result of this, the muscular septum primum and the right pulmonary ridge are seen as 2 separate septa that attach to the inferior endocardial cushion. Both the muscular septum primum and the superiorly fused venous valves (septum spurium) converge and are capped by a small rim of mesenchyme, which forms the roof of the persisting ostium primum and connects to cushions and the reduced vestibular spine. At 7 weeks, ventricular septation in AVSD is comparable to 5 to 6 weeks of normal cardiac development. At later stages, the septum spurium forms the anterosuperior limbus of the septum secundum and the mesenchymal cap becomes the bridging tendon that connects the bridging leaflets. Therefore, reduced expansion of the vestibular spine derived from the dorsal mesocardium appears to play an important role in the development of AVSD in Down syndrome.     

Origin of mesenchymal tissue in the septum primum: a structural and ultrastructural study

A structural, ultrastructural and histochemical study in chick embryos indicates that the septum primum mesenchymal tissue originate between 3 and 5 days of development and that their origin may be related to an activation of endocardial cells that cover the septum primum. By day 3, endocardial cells display migratory appendages, cell hypertrophy and an increase in secretory and mitotic activity. In later stages (day 4) hypertrophic endocardial cells undergoing division seem to delaminate and translocate toward the subendocardial space to give rise to free mesenchymal-type cells. These results suggest that the endocardium makes up the bulk of the septum primum mesenchymal tissue as has been demonstrated during mesenchymal tissue formation in the atrioventricular canal and outflow tract. Before and during mesenchymal tissue formation an accumulation of extracellular matrix components like proteoglycans can be visualized using tannic acid. These extracellular components might be related to the promotion of cellular events described during endocardial activation. The fusion of the septum primum with the atrioventricular (AV) endocardial cushions which would obliterate the foramen primum, occurs between mesenchymal tissues. Therefore, any alteration in the normal development of these mesenchymal tissues could be related to pathological cases of persistent atrial communications. Light microscopy preliminary observations of embryonic mouse heart indicate that septum primum mesenchymal tissue formation occurs similarly between mouse and chick embryos.     

Development of the myocardium of the atrioventricular canal and the vestibular spine in the human heart

To establish the morphogenetic mechanisms underlying formation and separation of the atrioventricular connections, we studied the remodeling of the myocardium of the atrioventricular canal and the extracardiac mesenchymal tissue of the vestibular spine in human embryonic hearts from 4.5 to 10 weeks of development. Septation of the atrioventricular junction is brought about by downgrowth of the primary atrial septum, fusion of the endocardial cushions, and forward expansion of the vestibular spine between atrial septum and cushions. The vestibular spine subsequently myocardializes to form the ventral rim of the oval fossa. The connection of the atrioventricular canal with the atria expands evenly. In contrast, the expression patterns of creatine kinase M and GlN2, markers for the atrioventricular and interventricular junctions, respectively, show that the junction of the canal with the right ventricle forms by local growth in the inner curvature of the heart. Growth of the caudal portion of the muscular ventricular septum to make contact with the inferior endocardial cushion occurs only after the canal has expanded rightward. The atrioventricular node develops from that part of the canal myocardium that retains its continuity with the ventricular myocardium.     

Normal development of the pulmonary veins in human embryos and formulation of a morphogenetic concept for sinus venosus defects

A sinus venosus defect is a form of interatrial communication associated with abnormal drainage of the right pulmonary veins. Its morphogenesis still remains unclear. We therefore studied the normal development of pulmonary veins in human embryos in relation to the sinus venosus and the dorsal mesocardium using graphic reconstructions and HNK-1 immunohistochemistry. Twenty embryos, ranging from 4 to 7 weeks' gestation, were examined. At 4 weeks, the orifice of the nonlumenized common pulmonary vein is visible as an endothelial invagination within the sinus venosus segment. Development of the muscular septum primum and the ventral proliferation of extracardiac mesenchyme from the dorsal mesocardium positions the common pulmonary vein (CPV) eventually into the left atrium. The right wall of the CPV contributes to the posterior part of the atrial septum and is continuous with the dorsal sinuatrial fold (the future left venous valve). With the use of HNK-1 antigen expression as a marker for sinus venosus myocardium, this common wall between the right-sided sinus venosus and the CPV is demonstrated, and at 7 weeks the proximal part of the right upper pulmonary vein also becomes part of this common wall. This study demonstrates that the CPV develops within the sinus venosus segment and that later a common myocardial wall is present between the sinus venosus in the right atrium and the CPV. A deficiency of this wall explains the development of sinus venosus defects.     

Mechanisms of deficient cardiac septation in the mouse with trisomy 16

It used to be thought that the atrioventricular septum was predominantly the product of the atrioventricular endocardial cushions. In a previous study, we have shown that multiple developmental primordia are of importance in its formation. With this in mind, we have evaluated cardiac morphogenesis in the mouse with trisomy 16, an animal model with a high incidence of atrioventricular septal defects. Normal and trisomic fetuses from an Rb(11.16)2H/Rb(16.17)7Bnr x C57BL/6J cross were collected on days 10 to 15 of gestation and examined by scanning electron microscopy and histological serial sectioning. No evidence was found to suggest that atrioventricular septal defect could be explained simply on the basis of "failure of fusion" between the atrioventricular endocardial cushions. Rather, our findings supported two other developmental elements as being important in the genesis of atrioventricular septal defect. The first is an alteration in the configuration of the heart tube, with inadequate remodeling of the inner heart curvature. This resulted in the failure of the atrioventricular junction to expand to the right, with subsequent malalignment of the atrioventricular endocardial cushions with the proximal outflow cushions. The second is a variability in the connection of the primary atrial cardiac segment to the body of the embryo, the so-called dorsal mesocardium, which influences its relationship to the extracardiac mediastinal mesoderm. There appeared little difference in the connection between normal and trisomic embryos at the stage of 20 to 25 somites, but the area subsequently showed marked changes. In most trisomic embryos, the connection with the mediastinal mesoderm of the body was over a larger area than seen in normal embryos. As this area of attachment encloses the pulmonary pit, the entry point of the pulmonary vein, this gives potential for variation in the connection of the pulmonary vein. In addition, in the majority of trisomic embryos, the right pulmonary ridge (the spina vestibuli) did not accumulate extracardiac mesoderm, nor did it undergo the pronounced forward growth seen in normal embryos of equivalent stages. Consequently, the trisomic embryos show incomplete formation of both the atrial and the atrioventricular septal structures.     

Lineage and morphogenetic analysis of the cardiac valves

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.     

Influence of the mesenchymal microenvironment on myocardial and endocardial cell behaviour in experimental interaction with chick limb mesenchyme

In an attempt to clarify the possible influence of the mesenchymal microenvironment in the differentiation of myocardial and endocardial cells, an "in vivo" transplantation experiment was performed in which the ventricular region of the heart (chick or quail) was placed in close association to the mesenchyme of the anterior chick limb to create an experimental interaction between myocardium and foreign mesenchyme. The results showed that after 48 h of tissue interactions the ventricular myocardium is incorporated into the mesenchyme of the anterior chick limb, changing its organization and cytological appearance. The myocytes tend to dissociate, exhibiting a less organized myofibrillar pattern. In addition, abundant extracellular matrix components made up of granular and fibrillar material were observed associated with the myocardial and the mesenchymal cell membranes as well as distributed in their surrounding microenvironment. The endocardium became discontinuous, due to detachment of the cells and emitted multiple pseudopodia and filopodia. These observations indicate that the mesenchyme from the anterior chick limb modifies the cellular behaviour and organization of the neighbouring myocardium and endocardium with which it interacts. We suggest that this might occur through participation of extracellular matrix components such as glycosaminoglycans, fibronectin and collagen which are known to act as macromolecular mediators in cell to cell interactions, cell migration and differentiation.     

Distribution of the neural cell adhesion molecule (NCAM) during heart development

The neural cell adhesion molecule, NCAM, was localized in the embryonic chick heart from Hamburger-Hamilton stage 14 up to hatching and in the adult heart. A monoclonal antibody directed to NCAM was used with the indirect antibody technique to stain frozen sections with immunoperoxidase. The myocardium showed immunoreactivity at stages 15 and 21, with little to no staining of epicardium, endocardium or atrioventricular endocardial cushion tissue. At stage 22, additional immunoreactivity was found in the endocardium of both the atrial septum and the atrial and ventricular surfaces of the atrioventricular cushions. Endocardial-derived mesenchymal cells within the cushions were also immunostained for NCAM. A gradient of NCAM staining was evident in the ventricular wall by stage 16. The staining intensity in the myocardium subjacent to the epicardium was less than found near the ventricular lumen. Biochemical analyses revealed that the embryonic heart expresses polysialylated NCAM. Upon desialylation with the endoneuraminidase Endo-N, the predominant heart NCAM has an apparent molecular weight of 155 to 160 kDa, which is distinct in size from the predominant forms found in embryonic chick nervous system (180, 140 and 120 kDa). NCAM expression is regionally regulated in the heart. The pattern of its expression is consistent with our hypothesis that it is involved in (1) differentiation of the atrial and ventricular walls, (2) fusion of the atrial septum with the endocardial cushions, (3) fusion of the endocardial cushions, and (4) formation and remodeling of ventricular trabeculae.     

Pleomorphic surface features of mammalian endocardium: fine structure of canine bicuspid valves

SEM and TEM have demonstrated normal canine endocardium to consist of a somewhat irregularly undulating, continuous sheet of cells characterized by plasmalemmal micro-appendages, rounded and raised nuclear bulges and variably discernible, overlapping cell margins. The endocardium covering the atrioventricular valves presented the most pleomorphic surface features in that our observations of both the atrial and ventricular aspects of the valvular leaflets revealed obvious differences in cell density, micro-appendage population, nuclear prominence and glycocalyx. Cells from the ventricular surface of these valves possessed few, if any, micro-appendages and more flattened surface contours; whereas those cells over the atrial surface demonstrated numerous bleb-like to microvillous-like processes extending from distinctly rounded and raised surface contours. Valvular specimens exposed to such catonic dyes as ruthenium red, alcian blue and cetylpyridinium chloride, with the latter two accompanied by lanthanum nitrate en bloc staining, revealed an obvious surface reactivity differential between the two sides that implies a compositional difference in the glycocalyx.     

Postnatal endocardial fibroelastosis of the valve of the foramen ovale

The valve of the foramen ovale of the heart changes at birth from a prenatal shunting device for the transseptal flow of systemic venous return to a postnatal active barrier to backflow of left atrial blood at higher pressure. To investigate the morphologic response to this functional change in the valve of the foramen ovale, we examined 200 hearts from embryos, fetuses and autopsy patients spanning over a millionfold range of heart weights (0.001 to 1,230 g). The relation of valve thickness to log heart weight was studied by a stepwise linear regression procedure that fitted the data to two linear regressions, one including observations made before birth and the other including observations made after birth. Analysis of variance tables demonstrated that the data are significantly better explained (p < 0.001) by two linear regressions that abruptly separate from one another at the time of birth than by a single linear regression. Histologic study showed that the prenatal valve consists of a thin myocardial layer with delicate endocardium. The postnatal thickening of the valve tissue occurs mainly by fibroelastotic thickening of the endocardium. The endocardial fibroelastosis of the valve develops after it closes and becomes a component of the left atrial wall. The observations support the concept that fibroelastosis is a nonspecific response of the lining of the cardiovascular system to increased mural tension.     

Formation of the primitive myo- and endocardial tubes in the chicken embryo

The morphogenesis of the mesenchymal cardiogenic plate, the formation of the bilateral heart primordia leading to the primitive heart tube, and also the genesis of the endocardial tube, have been studied in 1 to 13 somite chick embryos. The morphological data were compared with those obtained in electrophysiological studies of the development of the cardiac action potentials (Fujii et al., 1981a). The primordia of the consecutive heart chambers are definable before the appearance of myosin-type filaments and primitive Z bands, which occurs simultaneously with the first spontaneous action potentials in the 7 somite embryo. At the 8 to 9 somite stage, fusion of the lateral heart primordia proceeds to include the outflow tract and atrial primordia; the pacemaker site migrates into the atrial wall and subsequently into the sinus venosus, this process occurring simultaneously with the progressive transformation of mesenchymal cells into cardiomyocytes. Proendocardial cells are first detected detaching individually or in small groups from the cardiogenic plate to become attached by fine filamentous material to the basal surface of the foregut endoderm, on which they "stream", establishing an anastomosing V-shaped array with respect to the ventral mesoderm. This array coalesces first at the atrioventricular groove to form double endocardial tubes, which in turn fuse into a single tube with the establishment of the single myocardial tube. Evidence suggests a key role for the ventral mesocardium and its transient attachment zone with the foregut, in providing a line focus for migration of proendocardial cells and primitive capillaries from the lateral splanchnic mesoderm, from which they derive.     

Combined loss of Hey1 and HeyL causes congenital heart defects because of impaired epithelial to mesenchymal transition

Congenital heart defects affect almost 1% of human newborns. Recently, mutations in Notch ligands and receptors have been found to cause a variety of heart defects in rodents and humans. However, the molecular effects downstream of Notch are still poorly understood. Here we report that combined inactivation of Hey1 and HeyL, two primary target genes of Notch, causes severe heart malformations, including membranous ventricular septal defects and dysplastic atrioventricular and pulmonary valves. These defects lead to congestive cardiac failure with high lethality. We found both genes to be coexpressed with Notch1, Notch2 and the Notch ligand Jagged1 in the endocardium of the atrioventricular canal, representing the primary source of mesenchymal cells forming membraneous septum and valves. Atrioventricular explants from Hey1/HeyL deficient mice exhibited impaired epithelial to mesenchymal transition. Although epithelial to mesenchymal transition was initiated regularly, full transformation into mesenchymal cells failed. This was accompanied by reduced levels of matrix metalloproteinase-2 expression and reduced cell density in endocardial cushions in vivo. We further show that loss of Hey2 leads to very similar deficiencies, whereas a Notch1 null mutation completely abolishes epithelial to mesenchymal transition. Thus, the Hey gene family shows overlap in controlling Notch induced endocardial epithelial to mesenchymal transition, a process critical for valve and septum formation.     

Mitral and tricuspid lesions associated with polypoid atrial tumors, including myxoma

The related atrioventricular valves in 11 instances of polypoid atrial tumors were examined grossly and histologically. Nine of the tumors involved the left atrium and, of these, seven were myxomas. The two right atrial tumors were myxomas.

Histologically evident lesions of the mitral valve were present in seven of the nine left atrial tumors. The most common change consisted of fibrous thickening of the endocardium in the angle between the left atrium and posterior mitral leaflet and fibrous thickening of the atrial surfaces of the leaflet. Less common changes were a fibrous thickening of the chordae and of the left ventricular mural endocardium in relation to chordae inserting into the posterior leaflet. The changes described were also evident at gross examination in two of the nine cases with left atrial tumors.

In the two cases of right atrial myxoma, both showed fibrous thickening of the atrial surfaces of the tricuspid leaflets; one also exhibited fibrous thickening of the right ventricular endocardium beneath the septal leaflet of the tricuspid valve.

The lesions described are considered responses to the effects of friction by the tumor upon the surfaces involved. It is significant that grossly evident lesions may evolve from this rubbing action.     

Differentiative potential of human metanephric mesenchymal cells

OBJECTIVE: To evaluate the ability of mesenchymal cells derived from nonhematopoietic organs to form blood and other tissues in vitro and in vivo. MATERIALS AND METHODS: Because of its mesodermic derivation, human fetal kidney was used as a source of mesenchymal cells. Two populations of kidney cells were studied at a nonclonal level: a crude preparation, and an adherent fraction that was derived from the first by propagation in vitro (MNMC). Both populations were transplanted into sheep fetuses and analyzed at intervals for the presence of human cells in different organs by flow cytometry, PCR, immunohistochemistry, and in situ hybridization. Secondary transplantation studies were performed using human hematopoietic cells obtained from the bone marrow (BM) of primary recipients. RESULTS: MNMC were Thy-1(+), CD51(+), CD44(+), CD45(-), and vimentin(+), a phenotype consistent with that of metanephric mesenchyme. The crude population displayed the same phenotype but was contaminated with 0.4% CD34(+)CD45(+) cells. Cells with hepatocyte-like morphology and phenotype were obtained from the MNMC after culture in specific inducing media. After transplantation, both populations of cells produced multilineage hematopoietic engraftment and gave rise to CD34(+) cells. Successful hematopoietic engraftment in secondary recipients demonstrated the generation of long-term engrafting hematopoietic stem cells from MNMC. PCR analysis confirmed human hematopoietic engraftment and revealed that human cells were also present within other organs. Liver sections of transplanted animals contained human albumin-producing hepatocyte-like cells. CONCLUSION: A human metanephric mesenchymal cell population simultaneously gave rise to human blood and liver-like cells, suggesting that mesenchymal cells may represent a broad population of putative stem cells in multiple adult organs.     

Study of atrial conduction tissue in the 17 mm V-C human embryo. Morphological contribution to the pathogenesis of sinoauricular node dysfunction

Is there a specialised conduction pathway connecting the sinoatrial and atrioventricular nodes within the crista terminalis? As lesions of this zone can cause sinus node dysfunction and the sick-sinus syndrome, we decided to study this structure at the beginning of its embryonic development. Serial 10 micronthichness transverse sections of a 17 mm vertex-coccyx (Stage 19) human embryo were studied by light microscopy, anatomical reconstruction (Born) and morphometry. The region of the sinus venosus and venous valves, of the superior vena cava and the atrioventricular region were drawn and reconstructed with 150 X magnification by Born's method using wax plates of the following dimensions: 175 X 95X 1.5 mm. The volume of the region of the sino-atrial node was determined by multiplying the thickness of the sections by the surface of the region (measured by planimetry using the point counting method); its value was 15.4 X 10(6) microns3 with an antero-posterior diameter of 490,0 micron. These results show that this region is already proportionally larger in Stage 19 embryos. Reconstruction showed the sinoatrial node region to be a round bulge with a groove on either side: that of the sulcus terminalis and the superior vena cava on the left and the same vein with the mesocardium on the right. The right venous valve connects this zone with the interatrial septum and comprises two distinct cellular components, on derived from the atrial wall and the other from the muscle of the sinus venous. The artery fo the sinoatrial node was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperplastic conotruncal endocardial cushions and transposition of great arteries in perlecan-null mice

Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific alpha-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.     

Tissue interaction in androgen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone.

In the androgen response of the embryonic mammary rudiment of the mouse, both gland epithelium and surrounding mesenchyme are visibly involved. The question whether this is due to a direct action of testosterone on both tissues was investigated in experimental combination of mammary epithelium and mammary mesenchyme, derived either from normal or from androgen-insensitive (XTfm/Y) embryos. A typical androgen response occurred in combinations of androgen-insensitive epithelium with normal mesenchyme, whereas all combinations of normal epithelium with androgen-insensitive mesenchyme failed to respond. It is therefore concluded that only the mesenchyme of the mammary rudiment is the target tissue for testosterone, and that all changes in the gland epithelium, including its necrosis, are secondarily caused by testosterone-activated mesenchymal cells.     

Cardiac Papillary Fibroelastoma: case report and review of the literature

Primary cardiac tumors are very rare. Cardiac Papillary Fibroelastoma (CPF) is the third most common primary tumor of the heart and most commonly affects cardiac valves. Those tumors appear like a "sea anemone" with multiple papillary fronds attached to the endocardium by a short stalk. It generally occurs as a single structure and is typically found on the atrial aspect of atrioventricular valves or either side of the semilunar valves. We report a case of aortic valve papillary fibroelastoma which was found on autopsy in a patient with normal coronary artery with evidence of myocardial and cerebral ischemic damage and sudden death.     

Contracted form of primary endocardial fibroelastosis in a young adult without congestive heart failure

A case of a 24-year-old man with the contracted form of primary endocardial fibroelastosis diagnosed by left ventricular endomyocardial biopsy showing a markedly thickened endocardium with fibroelastic proliferation is reported. He had no evident symptoms of congestive heart failure except for shortness of breath on moderate exertion. Echocardiogram showed thickened and dense echoes from the left side of the septum and from the posterior left ventricular endocardium. Hemodynamic and angiographic studies revealed marked elevation of right and left ventricular end-diastolic pressures with dip and plateau pressure contours, moderate pulmonary hypertension, left atrial enlargement and mild mitral regurgitation. Further elevation of right and left ventricular diastolic pressures and pulmonary artery pressure was observed at the second evaluation after 5 years. Our patient suggests that primary endocardial fibroelastosis should be included in the differential diagnosis of adult patients with obscure types of cardiac disease.