Developmental defects of coronary vasculature in rat embryos administered bis-diamine.

BACKGROUND: Conotruncal anomalies are often associated with abnormal coronary arteries. Although bis-diamine is known to induce conotruncal defects, its pathological effects on coronary vascular development have not been demonstrated. This study sought to assess the teratogenic effects of bis-diamine on coronary vascular development and the pathogenesis of this anomalous association. METHODS AND RESULTS: A single 200 mg dose of bis-diamine was administered to pregnant Wistar rats at 10.5 days of gestation. Fifty-two embryos from 10 mother rats underwent morphological analysis of the coronary arteries. Three embryos each were removed from four mothers on embryonic days (ED) 14.5, 15.5, 16.5, and 17.5 and used for immunohistochemical studies using the anti-vascular cell adhesion molecule (VCAM)-1 antibody. Conotruncal anomalies were detected in 48 of 52 embryos, and an aplastic or hypoplastic left coronary artery was found in all of them. In control embryos at ED 16.5, VCAM-1-positive epicardial cells were transformed into mesenchymal cells in vascular plexus, which appeared to differentiate into the endothelial cells of coronary vasculature. In the heart at ED 17.5, coronary vasculature was well developed and connected with coronary ostia near the aorta. However, poor epicardial-mesenchymal transformation and subsequent differentiation was revealed in bis-diamine-treated embryos at EDs 16.5 and 17.5, causing abnormal development of the coronary vasculature and incomplete connections with coronary ostia of the aorta. CONCLUSIONS: Anomalous coronary arteries in the bis-diamine-treated embryos are induced by the disruption of epicardial-mesenchymal transformation and subsequent poor development of coronary vasculature. Incomplete hatching of the coronary ostium is associated with abnormal truncal division.

In vitro model for mouse coronary vasculogenesis.

To analyze the molecular mechanisms of coronary vessel formation, we performed in vitro experiments on explant cultures of proepicardial organs (PEOs) excised from embryos taken from 9.5-day pregnant mice. When plated on coverglasses coated with rat tail collagen I, fibronectin, or laminin, PEO cells spread and formed an epithelial sheet. When PEOs were cultured on collagen gel in the presence of fetal calf serum (FCS), small projections were seen around the explants 3 days after plating. Around day 6, cord-like structures began to grow from the explants, gradually elongating, increasing in number, and forming a branching network. Histological sections demonstrated that the cells migrated into the gel and formed tube-like structures similar to the vascular channels of the embryonic heart. The cells lining the lumen of the tube-like structures were positive for platelet endothelial cell adhesion molecule (PECAM). Reverse transcriptase-polymerase chain reaction analyses demonstrated that the expression of PECAM, basic fibroblast growth factor (bFGF), and smooth muscle 22-alpha (SM22alpha) was upregulated in association with the tube formation, whereas the expression of Flk-1, Flt-1, and hepatocyte growth factor (HGF) was gradually downregulated. Vascular endothelial growth factor (VEGF) was continuously expressed during the culture. These changes were not observed when PEOs were explanted without FCS. Furthermore, addition of any one or combinational addition of the growth factors, including bFGF, VEGF, or HGF, did not induce tube formation. These results suggest that PEOs contain precursor cells of coronary vasculature and that vasculogenesis may be simultaneously regulated by multiple factors.

Association of aortic arch anomalies and subclavian artery supply disruption with neurofibromatosis.

We report an 8-year-old Japanese girl with von Recklinghausen disease, who presented with aortic arch anomalies and left hemilateral oculo-otolaryngeal abnormalities including strabismus, blepharoptosis, a dysplastic external ear and hearing loss. The aortic arch anomalies including subclavian artery obstruction that appeared to be a consequence of the neurofibromatosis, and the hemilateral oculo-otolaryngeal abnormalities could be explained by disruption of the subclavian artery supply during embryogenesis.

Teratogenic effects of bis-diamine on the developing cardiac conduction system.

BACKGROUND: Congenital heart defects, including conotruncal anomalies, are often associated with arrhythmias. Bis-diamine induces conotruncal anomalies in embryos when administered to pregnant female rats. To investigate the mechanism of arrhythmia in conotruncal anomalies, we histologically examined the development of the cardiac conduction system in this animal model. METHODS: A single dose of 200 mg of bis-diamine was administered to pregnant Wistar rats on ED 10.5 of pregnancy. The embryos were removed on each day from ED 11.5 to 15.5. Immunoexpression of HNK-1, connexin40, and connexin43 were examined in serial sections. The distribution pattern of TUNEL-positive cells around the conduction system was also examined. RESULTS: HNK-1 immunoreactivity was evident in interventricular septum, in both the control and the bis-diamine-treated embryos from ED 12.5. Although a chain of connexin40-immunoreactive cells from interventricular septum to trabeculae, corresponding to the His bundle and its branches, was demonstrated at ED 13.5 in the control embryos, this chain was first detected at ED 14.5 in the bis-diamine-treated embryos. Immunoexpression of connexin43 in the working myocardium was also less in the bis-diamine-treated embryos than in the control at ED 13.5. The number of TUNEL-positive cells in the interventricular septum was highest at ED 12.5 in the control and at ED 13.5 in the bis-diamine-treated embryos. Furthermore, these TUNEL-positive cells were HNK-1 negative, vimentin-positive, and alpha smooth muscle actin-positive. CONCLUSIONS: Bis-diamine disturbed the normal development of gap junctions and apoptosis of myofibroblasts around the HNK-1-positive conduction tissue through overall poor myocardial proliferation and growth.

Morphological differences in cardiovascular anomalies induced by bis-diamine between Sprague-Dawley and Wistar rats.

It is known that animals show different responses to the same teratogen between different strains. We examined cardiac malformations in Sprague-Dawley (SD) and Wistar rats induced by bis-diamine, which produced conotruncal anomalies and aortic arch malformations in embryos when administered to the dams, to elucidate the morphological differences and pathogenesis in the two strains. Two hundred milligrams of bis-diamine dissolved in 1% gum-tragacanth was administered to pregnant rats on embryonic day (ED) 9.5, 10.5 and 11.5 in each strain. The embryos were removed on ED 20.5. External appearances, cardiovascular morphology and associated anomalies were examined under a dissecting microscope. An immunohistological study with an anti-N-CAM antibody, an excellent marker for neural crest cells, was performed on ED 12.5 embryos. Isolated aortic arch anomalies were common features of malformations induced by bis-diamine in SD rats and intracardiac defects were found in a small number of the embryos. Wistar rats showed more serious cardiovascular anomalies, such as persistent truncus arteriosus and tetralogy of Fallot, especially when dams were treated on ED 10.5 and isolated arch anomalies were significantly less prevalent than in SD rats. Immunohistology demonstrated that there were fewer N-CAM positive cells in the conotruncal region in Wistar rats than in SD rats. Bis-diamine induced more critical cardiovascular malformations in Wistar rats because neural crest cells, which play an important role in conotruncal septation, were more extensively damaged. Different susceptibility to bis-diamine and/or different time of neural crest cell emigration from the hindbrain might explain those morphological differences.

Teratogenic effects of bis-diamine on the developing myocardium.

BACKGROUND: Bis-diamine induces conotruncal anomalies and disproportional ventricular development in rat embryos when administered to the mother. To evaluate the mechanisms of disproportional ventricular development in the anomalous heart, we analyzed the morphology of the embryonic heart and investigated cardiomyocytic DNA synthesis and apoptosis. METHODS: A single dose of 200 mg of bis-diamine was administered to pregnant rats Wistar on day 9.5 of pregnancy. The embryos were removed on each embryonic day from 10.5 to 18.5. Expression of cardiotrophin-1 and hepatocyte growth factor was investigated on the sections, and cardiotrophin-1, hepatocyte growth factor and myocyte enhancer factor 2 mRNA expression was examined by reverse transcriptase-polymerase chain reaction. Myocardial DNA synthesis was investigated using 5-bromo-2'-deoxyuridine and the labeling index was calculated for each heart. Apoptosis was also analyzed using TUNEL reaction and electrophoresis of DNA fragmentation. RESULTS: The embryos treated with bis-diamine had conotruncal anomalies associated with thin left ventricular wall in the later stage. The labeling index on embryonic day 15.5 and 16.5 was significantly lower than those in the controls. Hepatocyte growth factor and cardiotrophin-1 mRNA expression was upregulated on embryonic day 12.5 and 15.5 in bis-diamine-treated hearts. Fewer apoptotic cells were detected in the hearts of bis-diamine-treated embryos than in control hearts from embryonic day 14.5 to 16.5. CONCLUSIONS: The ventricular disproportion in the bis-diamine-treated heart may be caused by the early myocardial differentiation delay and poor proliferation and reduced apoptosis associated with anomalous circulatory condition in the later stage.