Analysis of coronary vascular development in an animal model of congenital heart disease - in vivo and in vitro studies to detect a role of proepicardial organ derived cells

我们检查了鹌鹑来源的心外膜前器官（PEO）细胞在按照曼纳描述的程序产生的鸡-鹌鹑嵌合胚胎心脏中的分布模式。鹌鹑PEO细胞在培养后第5天形成心外膜，观察PEO细胞上皮间充质转化（EMT）。孵育后第6天和第9天，分别在心肌和冠状动脉未来口周围检测到鹌鹑PEO细胞。在与鸡发育阶段一致的小鼠胚胎中，tenascin-C在PEO中表达，但当PEO细胞移动到心脏表面并迁移到心肌时，tenascin-C被抑制。Tenascin-C在EMT中表达上调。小鼠PEO细胞在有胎牛血清存在的胶原凝胶上培养形成管状结构。从培养的PEO中提取的rna进行RT-PCR分析，发现表达多种血管生长因子，包括bFGF、HGF、VEGF、Flt-1、Flk- 1、Ang-1、Ang-2和Tie-2，但在培养基中加入双二胺后，这些生长因子的表达下调。大鼠胚胎在胚胎日14.5时心外膜和心肌之间形成血管丛，胚胎日15.5时心肌血管结构已被识别。双二胺在妊娠期10.5时给予母鼠胚胎心脏血管丛分散，血管结构未形成。免疫组织学研究显示，双二胺处理的大鼠胚胎中血管生长因子的表达延迟。这些结果表明，双二胺通过直接影响PEO细胞的生长，干扰EMT和血管生长因子的表达而引起冠状动脉血管的异常发育。

We examined distributional patterns of quail-derived proepicardial organ (PEO) cells in the hearts of chick-quail chimeric embryos produced following the procedure described by Manner. Quail PEO cells formed epicardium at day 5 after incubation, beneath which epithelial-mesenchymal transformation (EMT) of PEO cells were observed. Quail PEO cells also were detected in the myocardium and around the future ostia of coronary arteries at day 6 and at day 9 after incubation, respectively. In the mouse embryos at the coincident developmental stage with chick, tenascin-C was expressed in PEO but was suppressed when PEO cells moved to the cardiac surface, migrating into the myocardium. Tenascin-C expression was upregulated in EMT. Mouse PEO cells cultured on the collagen gels in the presence of fetal calf serum formed tube structures. RT-PCR analyses of RNAs extracted from cultured PEO demonstrated expression of various vascular growth factors including bFGF, HGF, VEGF, Flt-1, Flk- 1, Ang-1, Ang-2 and Tie-2, however, the expression of these growth factors were downregulated when bis-diamine was added into the culture medium. In rat embryos, vascular plexus was formed between the epicardium and myocardium at 14.5 embryonic day (ED) and vascular structure was recognized in the myocardium at ED 15.5. The vascular plexus was dispersed and vascular structures were not formed in the embryonic hearts when bis-diamine was given to mother rats at ED 10.5. Immunohistological studies showed delayed expression of the vascular growth factors in those rat embryos treated with bis-diamine.These results suggested that bis-diamine caused the abnormal development of coronary vasculature by making a direct effect on growth of PEO cells and by disturbing EMT and expression of vascular growth factors.