Takashi Hanato

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.

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

ABSTRACT  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 effect of bis‐diamine on embryonic rat heart

ABSTRACT  Bis‐diamine induces conotruncal anomalies including persistent truncus arteriosus, tetralogy of Fallot, interruption of the aortic arch, and ventricular septal defect in rat embryos when administered to the mother. Bis‐diamine also induces extracardiac malformations including thymic hypoplasia, facial dysmorphism, forelimb anomalies and diaphragmatic hernia. However, the teratogenic mechanisms of this chemical in early developing rat hearts have not been fully established. Chimeric studies in chick and quail embryos demonstrated that the cranial neural crest cells reached the cardiac outflow tract, contributing to aorticopulmonary and truncal septation. Since an ablation of the cranial neural crest also produced the conotruncal anomalies, bis‐diamine is proposed to disturb the normal migration of cardiac neural crest cells to the heart. Based on our data concerning cardiac anomalies induced by bis‐diamine, we reviewed how the cardiac malformations were morphologically established in early developing rat hearts. Our data showed that 1) cardiovascular anomalies induced by bis‐diamine are time‐ and species or strain‐ dependent. 2) bis‐diamine reduces the number of neural crest cells migrating to participate in the conotruncal septation, 3) bis‐diamine induces anomalous coronary arteries, thin ventricular walls and epicardial defects, and 4) some embryos cultured in the medium containing bis‐diamine had extra‐cardiac abnormalities including abnormal location of the otic placodes and delay in mid brain closure. Conclusively, bis‐diamine does not appear to merely affect the cardiac development, but rather disturbs normal development of all the organs contributed to by neural crest cells.