Inhibition of osteoclast formation by 3-methylcholanthrene, a ligand for arylhydrocarbon receptor: suppression of osteoclast differentiation factor in osteogenic cells.

We investigated the effects of 3-methylcholanthrene (3MC), a ligand for arylhydrocarbon receptor (AhR), on osteoclastogenesis. Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) produced by ST2 cells in response to 1alpha,25(OH)(2) Vitamin D(3). 3MC dose-dependently inhibited the formation of mono- and multinuclear osteoclast-like cells. However, 3MC did not inhibit the formation of osteoclast-like cells from mouse spleen cells which was supported by the exogenous soluble RANKL and M-CSF. 3MC did not affect the formation of an actin ring and pits on slices of dentine by osteoclast-like cells, both of which are typical indices of osteoclast activity. These results suggest that 3MC affects osteoclast-supporting cells such as ST2 cells but not osteoclast precursor cells and mature osteoclastic cells. When we measured the expression levels of RANKL mRNA in ST2 cells, 3MC dose-dependently decreased the level of this mRNA. However, 3MC did not affect levels of mRNAs for osteoprotegerin (OPG), M-CSF, and the receptor of 1alpha,25(OH)(2) Vitamin D(3) in ST2 cells. Furthermore, soluble RANKL was able to counteract the inhibitory effect of 3MC on the formation of osteoclast-like cells. Our findings indicate that 3MC inhibits osteoclastogenesis via the inhibition of RANKL expression in osteoblastic cells.

Remodeling of coronary artery lesions due to Kawasaki disease: comparison of arteriographic and immunohistochemical findings.

Since the original report of Kawasaki disease in 1967 more than 150,000 cases have been reported in Japan. Although there have been no nationwide epidemics in Japan since 1987, more than 6,000 newly diagnosed cases are reported every year, and the number has been increasing year by year despite the decreasing birth rate. The etiology of the disease is still unknown. High dose intravenous gammaglobulin is currently used during the acute phase in 84% of the patients in Japan with a concomitant decrease in coronary arterial sequelae. However, 7-13% of the patients still have persistent coronary artery aneurysms after the acute stage. The aneurysms are seen mostly in the proximal coronary arteries, and are often associated with aneurysms in the distal coronary artery segments (Figure 1A, 2A). Most of the patients show a decrease in the size of aneurysms soon after the acute phase (Figure 1B). However, the aneurysms may progress to obstructive lesions even after initial regression (Figures 1C, D, 2B). Such obstructive lesions may cause sudden death or myocardial infarction. Long term follow-up of coronary artery lesions has revealed several characteristic features, including progressive localized stenosis (Figure 1D), extensive recanalizations (Figure 2D) and development of collateral arteries. Progressive increases in aneurysm size and the appearance of new aneurysms in the late phase have also been reported. The basic mechanisms of the coronary arterial remodeling in Kawasaki disease have not yet been elucidated. Only recently has immunohistochemical staining in formalin-fixed specimens become feasible. This is a major technical breakthrough since it is almost impossible to obtain fresh frozen specimens of coronary artery lesions of Kawasaki discase. In this paper, we compare immunohistochemical findings in coronary artery lesions with the corresponding coronary angiographic findings, and attempt to make inferences as to the mechanism of remodeling both in early and late phases of the disease based on the expression of vascular growth factors.

Active remodeling of the coronary arterial lesions in the late phase of Kawasaki disease: immunohistochemical study.

BACKGROUND: Remodeling of the coronary artery lesions in Kawasaki disease has been observed in longitudinal angiographic studies. However, mechanisms of such remodeling have not yet been elucidated. METHODS AND RESULTS: We examined formalin-fixed specimens of the coronary arteries immunohistochemically by using antibodies against vascular growth factors (GFs) and their receptors in 7 children with Kawasaki disease, 9 children with no coronary disease, and 3 adults with atherosclerosis. In the thickened intima at stenotic sites and at recanalized vessels with Kawasaki disease, extensive expression of vascular GFs, such as transforming GF-beta(1), platelet-derived GF-A, and basic fibroblast GF, was observed both within and surrounding smooth muscle cells. Vascular endothelial GF was observed within smooth muscle cells. Furthermore, all of these GFs were strongly expressed in the newly formed microvessels within the intima. In the thinned media, these GFs were focally and weakly expressed. In contrast, these GFs were expressed only in the media in the control children. In cases of adult atherosclerosis, GFs were expressed diffusely in the media but focally and weakly if at all in the intima. CONCLUSIONS: Active remodeling of the coronary artery lesions in Kawasaki disease continues in the form of luxuriant intimal proliferation and neoangiogenesis for several years after the onset of the disease. This process is distinct from adult-onset atherosclerosis.

Mesp1 expression is the earliest sign of cardiovascular development.

Understanding the molecular mechanism leading to formation of the heart and vasculature during embryogenesis is critically important because malformation of the cardiovascular system is the most frequently occurring type of birth defect. While the hearts of all vertebrates are derived from bilateral paired fields of primary mesodermal cells that are specified to the cardiac lineage during gastrulation, the mechanism for lineage restriction, and the origin of the myocardium and endocardium have not been defined. Recently, we found that a transcription factor, Mesp1, is expressed in almost all precursors of the cardiovascular system and plays an essential role in cardiac morphogenesis. Mesp1 may play a key role in the early specification for cardiac precursor cells.

Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism.

Pitx2, a bicoid-related homeobox gene, is involved in Rieger's syndrome and the left-right (L-R) asymmetrical pattern formation in body plan. In order to define the genomic structure and roles of Pitx2, we analyzed the genomic structure and generated Pitx2-deficient mice with the lacZ gene in the homeobox-containing exon of Pitx2. We were able to show that among three isoforms of Pitx2, Pitx2c shows asymmetrical expression whereas Pitx2a, Pitx2b and Pitx2c show symmetrical expression. In Pitx2(-)(/)(-) embryos there was an increase in mesodermal cells in the distal end of the left lateral body wall and an amnion continuous with the lateral body wall thickened in its mesodermal layer. These changes resulted in a failure of ventral body wall closure. In lung and heart in which Pitx2 is expressed asymmetrically, right pulmonary isomerism, atrioventricular canals with prominent swelling, and juxtaposition of the atrium were detected. The hearts failed to develop tricuspid and mitral valves and a common atrioventricular valve forms. Further, dysgenesis of the Pitx2(-)(/)(-) extraocular muscle and thickening of the mesothelial layer of cornea were observed in the ocular system where Pitx2 is expressed symmetrically, and these resulted in enophthalmos. The present study shows that Pitx2 expressed in various sites participates in morphogenesis through three types of actions: the involvement of asymmetric Pitx2 expression in the entire morphogenetic process of L-R asymmetric organs; the involvement of asymmetric Pitx2 expression in the regional morphogenesis of asymmetric organs; and finally the involvement of symmetric Pitx2 expression in the regional morphogenesis of symmetric organs.

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.

Cardiac neural crest cells provide new insight into septation of the cardiac outflow tract: aortic sac to ventricular septal closure.

A great deal is unclear about the process of cardiac outflow septation. Much controversy exists regarding the precise details of tissue origins and movements of various components. The contribution of the cardiac neural crest to aorticopulmonary and distal truncal septation has been described; however, the distribution of the neural crest in the proximal outflow and heart is unknown. The present study describes the movement of cardiac neural crest cells from the caudal pharyngeal arches into the outflow tract and base of the heart during the period of outflow septation. Using quail-chick chimeras we found that the cardiac neural crest was distributed to all levels of the outflow tract and into the base of the heart. Septation of the outflow tract lumen occurred by two different processes that involved the cardiac neural crest directly. Cardiac neural crest cells were also distributed to regions of the outflow tract that correlated with sites of remodeling, such as the aortic sac as it was remodeled into the base of the ascending aorta and pulmonary trunk, the distal truncus that was patterned into the two semilunar valves and in the proximal conotruncus where muscularization of the ridges and septum occurred. Additionally, cardiac neural crest cells were found at the site of closure of the ventricular septum, in the wall of the pulmonary infundibulum, and transiently in the wall of the aortic vestibule. Contrary to current thinking, not all of the condensed mesenchyme in the outflow tract during septation was derived from neural crest.

Accelerated maturation of fetal ductus arteriosus by maternally administered vitamin A in rats.

Maturation of fetal ductus arteriosus is associated with increased constriction in response to maternally administered indomethacin. Recently retinoic acid has been shown to be important in development of the fetal ductus arteriosus. To determine whether retinoid might be of value in the treatment of patent ductus arteriosus in premature infants, we studied the response of fetal ductus arteriosus to indomethacin with and without pretreatment with vitamin A (1 mg (3000 IU)/kg, intramuscular injection) in near-term and preterm rats. Maturation of the ductus arteriosus was studied by measuring the inner diameters of the ductus arteriosus (D) and main pulmonary artery (P) to get D/P ratio 4 h after orogastric administration of 1 mg/kg indomethacin. D/P was 1.0 in the fetus before administration of indomethacin. In near-term fetuses on the 21st d without vitamin A, D/P decreased to 0.54 with indomethacin, whereas it decreased to 0.27 (p < 0.05) in those with pretreatment with vitamin A on the 19th and 20th d. In preterm fetuses on the 20th d without pretreatment with vitamin A, D/P decreased to 0.82 with indomethacin, whereas it decreased to 0.66 (p < 0.05) in those with pretreatment with vitamin A on the 19th d. It is concluded that maternally administered vitamin A accelerates maturation of the ductus arteriosus in fetal rats.

Pathological study of Japanese quail embryo with acid alpha-glucosidase deficiency during early development.

A pathological study was performed on the tissues of 11 Japanese quail embryos with type II glycogen storage disease (GSD II) between incubation day (ID) 3 and ID 15. Accumulation of glycogen in vacuoles derived from lysosomes was first seen in cardiac muscle at ID 3, in liver at ID 5, in wing muscle at ID 7, and in pectoral muscle at ID 10. The number and size of the glycogen vacuoles gradually increased during development, especially in cardiac muscle. Cytoplasmic glycogen particles, showing the same density as membrane-enclosed glycogen particles, were first seen as masses in cardiac muscle at ID 3, in liver at ID 5, in pectoral muscle at ID 10, and in wing muscle at ID 15. The area of cytoplasm occupied by the glycogen particles increased during development. Myofibrillar degeneration was not seen, although myofibrils appeared in disarray in the early stages, as in normal embryos. This is the first study of the development of embryonic tissues of Japanese quails with GSD II. GSD II in the Japanese quail appears to be clinically analogous to the human late (juvenile)-onset disease, although the disorder starts at very early stages in quail embryos. Therefore, Japanese quails with GSD II can provide a model for elucidating the pathogenetic process of human GSD II.

Morphological observations on the pathogenetic process of transposition of the great arteries induced by retinoic acid in mice.

BACKGROUND: The pathogenesis of complete transposition of the great arteries (TGA) is still controversial because useful animal models have not been established. We previously reported that all-trans retinoic acid induced complete TGA at a high proportion in mice. The aim of the present study was to clarify the morphogenesis of the cardiac outflow tract in the retinoic acid-treated embryos destined to develop TGA. METHODS AND RESULTS: We first examined the morphology of TGA in mouse fetuses treated with retinoic acid to establish an animal model of TGA (experiment 1) and then examined the retinoic acid-treated embryonic hearts by means of ink injection and histology (experiment 2). All mouse fetuses and embryos showed visceroatrial situs solitus and d-ventricular loop. In experiment 1, among 45 embryos treated with retinoic acid 70 mg/kg at day 8.5 of gestation, 35 (78%) had TGA and 3 (6.7%) had a double-outlet right ventricle with a subpulmonary ventricular septal defect. In experiment 2, all hearts already exhibited d-loop at gestation day 8.5. At gestation day 9.5, conus swellings, composed of acellular cardiac jelly, where hypoplastic, and the conotruncal cavity was nonspiral or tubular. At gestation day 11.0, aberrant conus swellings located anteroposteriorly to give a straight orientation to the conotruncal cavity. At gestation day 12.0, side-by-side great arteries were transposed in that the aorta arose from the right ventricle and the pulmonary artery arose above the interventricular foramen. CONCLUSIONS: These results suggest that a reproducible animal model of TGA can be produced in mice by treatment with retinoic acid; that there was no loop anomaly, such as an A-loop or L-loop, in our model; and that hypoplasia of the conus swellings appears to be the primary event leading to TGA.

Retinoic acid ambivalently regulates the expression of MyoD1 in the myogenic cells in the limb buds of the early developmental stages.

The expression of MyoD1 in myogenic cells located in the muscle prospective region of the limb bud at stage 20-22 was highly sensitive to retinoic acid. Unlike RAR-beta, the expression of MyoD1 mRNA in the muscle precursor cells was significantly increased by retinoic acid at lower concentrations (0.1-10 nM), but inhibited by it at higher concentrations (0.1-1 microM). The ambivalent modulation of MyoD1 expression suggested that MyoD1 expression is regulated by not only the retinoic acid receptor and its response element, but also by other factors. Retinoic acid may be involved in the differentiation of the myogenic cells during early development.

Cellular retinoic acid binding protein type II was preferentially localized in medium and posterior parts of the progress zone of the chick limb bud.

The expression and distribution of cellular retinoic acid binding protein II (CRABP II) was examined in chick limb buds. CRABP II was detected in the limb buds at Hamburger and Hamilton (1) stage 21 and the amount of CRABP II was gradually increased during stages 21-27 and thereafter decreased. CRABP II was mainly located in the progress zone, and the dorsal and ventral premuscular mass in the proximal region of the limb buds at stage 23. CRABP II was preferentially localized in the medium and posterior parts rather than the anterior part of the progress zone; The content of CRABP II in the medium and posterior parts was 8-9 times more than that in the anterior part.

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.

Fetal cardiovascular morphology of truncus arteriosus with or without truncal valve insufficiency in the rat.

BACKGROUND: Recent advances in fetal echocardiography have necessitated further study on fetal in situ cardiovascular morphology of truncus arteriosus and the effects of truncal valve insufficiency. METHODS AND RESULTS: We studied 55 fetal rats with truncus arteriosus among 300 fetuses from 40 virgin females treated with 200 mg fertilysin on the 10th day of pregnancy. After rapid whole-body freezing on the 21st day, the fetuses were studied by means of serial cross-sectional photographs of the frozen thorax. Thirty-five fetuses with a normal heart treated with fertilysin served as controls. Truncus arteriosus was characterized by a large ventricular septal defect, a solitary artery (truncus arteriosus) overriding the ventricular septum, the right and left pulmonary arteries originating from the truncus arteriosus with or without a common trunk (main pulmonary artery), and absent ductus arteriosus. Fetuses with truncal valve insufficiency had thick truncal valves, a large truncus arteriosus, and large ventricles. The subgroup of 12 fetuses with a large truncus (truncal diameter greater than 160% of the ascending aorta diameter in the controls) showed significantly greater values for right ventricular volume (200% of control) and mass (120% of control), left ventricular volume (170% of control) and mass (110% of control), right (120% of control) and left (110% of control) atrial volume, and pericardial fluid (140% of control) than the controls. These changes were less prominent and ventricular volumes were not increased in the remaining subgroup with a truncal diameter of 160% or less of aorta diameter in the controls. CONCLUSIONS: In fetal truncus arteriosus, truncal valve insufficiency was associated with increased ventricular volume load and incomplete cardiac compensation in rats.

Development of cranial nerves in the chick embryo with special reference to the alterations of cardiac branches after ablation of the cardiac neural crest.

Development of cranial nerve branches in the cardiac region was observed in whole-mount specimens which were stained with a monoclonal antibody, E/C8, after the ablation of the cardiac neural crest. In early embryos, nerve trunks of IX and X were lacking or only poorly developed, while the early development of pharyngeal branch primordia was normal. In day 5 embryos, the nerve trunks of IX-X were present in all the embryos, however; extensive communication was observed between X and XII. On day 6 and later, the spiral pattern of superior cardiac branches was disturbed, as were the blood vessels. Furthermore, the distal branches of XII passed within the superficial layer of cardiac outflow mesenchyme. Vagal branches passed within the deeper layer. There was no apparent change in the development of the sinal branch. Using quail--chick chimeras, it was found that the cardiac neural crest cells formed the Schwann cells of XII, and that they were also associated with the hypobranchial muscle primordium, suggesting that the absence of the cardiac neural crest not only disturbs the development of the cardiac outflow septation, but also affects the normal morphogenesis of the hypobranchial musculature and its innervation. Embryologically, the tongue is located close to the cardiac outflow tract, which is the migration pathway of the cardiac neural crest-derived cells.