ROS-NFAT：唐氏综合征相关AVSD发生过程中遗传和环境因素共同作用的交汇通路？

Atrioventricular Septal Defect (AVSD) is a common congenital heart disease, which is affected by both genetic and environmental factors. Down syndrome (DS), which is also called 21-trisome syndrome, has a high incidence of AVSD.Exploring the detailed environmental and genetic factors involved in the development of AVSD and their cross-talk mechanism would shed light on how to prevent the incidence of the high-risk people. Our undergoing findings using DS mouse model reveals that the additional copy of SOD1 in the 21 chromosome contributes to the reduced ROS and subsequent onset of AVSD. However, the genetic aberration is not sufficient as seen from the only about 15% incidence, suggesting that AVSD can be influenced by the environmental factors. By screening the effects of the common chemicals targeting the well-known signaling molecules on the incidence of AVSD in DS model mice, we found that NFAT inhibitor could increase the incidence of AVSD in the DS embryonic mice. These observation raises the question: what role of NFAT in the AVSD? How can it integrate in the ROS signaling? Previous literatures have shown that ① ROS are one of the most important regulators of NFAT activity and ② NFAT is important in the regulation of cardiac development. Taken together, we assume the following hypothesis: The ROS-NFAT pathway exists and plays an important role during the embryonic heart development. And for the DS model mice or the clinical patients, some of the environmental factors together with the genetic reasons reduce the endogenous ROS level to the threshold, and thus cause the AVSD by decreasing the NFAT activity. The proposed project here will test whether the ROS-NFAT pathway plays a convergent role in the AVSD by both detrimental environmental and genetic factors in the DS context. Our study here will shed light on the mechanism of AVSD and its prevention.

房室隔缺损（AVSD）是一种受遗传和环境共同决定的先天性心脏畸形，多见于唐氏综合征。课题组利用唐氏模型小鼠筛选常见小分子化合物对AVSD发生率的影响时发现：正常条件下唐氏模型小鼠AVSD发生率仅为15%左右，该过程与房室间隔区细胞内ROS减少有关；而NFAT的抑制剂FK506能显著增加唐氏小鼠AVSD发病率。那么，NFAT在唐氏相关AVSD发生过程中究竟扮演什么角色？与ROS又是什么关系？既往研究表明：ROS是NFAT活性的重要调节分子，而NFAT又是房室间隔发育重要的调控分子。综上，我们推测ROS-NFAT通路对正常心脏发育至关重要，唐氏遗传相关因素和某些环境因素共同抑制这条通路，当其活化低于某个阈值，即可促发AVSD。本项目拟利用DS小鼠模型和临床AVSD引产儿心脏样本，结合分子生物学和影像学手段，探讨ROS-NFAT通路是否在环境和遗传因素共同导致AVSD过程中发挥交汇通路的作用。

在该基金的支持下，我们完成了项目既定研究内容，达到了预期目标，主要取得了以下进展：1）通过基因芯片检测和生物信息学分析，发现唐氏模型胎鼠心脏Wnt、Notch、TGFβ、MAPK、NFAT等心脏发育关键信号出现一定程度的表达异常，qPCR检测证实基因芯片检测结果的可信度；2）唐氏模型胎鼠心脏发育畸形不仅和遗传有关，同样受到母体健康状态的影响，糖尿病和肥胖等显著增加唐氏胎鼠心脏发育和其它系统发育异常的风险，表明遗传和环境能够协同作用，导致胚胎发育异常，遗传和环境互作存在“阈值”效应，即当两者共同影响发育信号通路达到一定程度，即可导致胚胎发育出现异常；3）糖尿病和肥胖等显著增加母体和胚胎多个系统的ROS水平，过度的ROS会导致Wnt信号、NFAT和PI3K-AKt信号受限，是细胞增殖和存活能力减弱的重要原因；4）除了经典的信号分子受到遗传和环境影响，糖尿病等系统性发育环境因素还可以导致外泌体改变，糖尿病会导致母体循环血中外泌体数量和内容物的改变，RNA-seq分析发现了一组与ROS、Wnt、NFAT信号等有关的miRNA在糖尿病母体循环外泌体中表达改变；上述外泌体可以进入胎盘，甚至过胎盘进入胚胎组织；尾静脉注射糖尿病母体来源外泌体显著增加胎鼠罹患心脏畸形的概率；5）胎盘是连接胚胎和母体的重要器官，在母胎信息交流中发挥重要作用，胎盘发育的异常同样是妊娠相关母-胎疾病的重要机制。我们发现正常胎盘同样是外泌体产生的重要脏器，分离获得的胎盘外泌体能够显著降低脂肪和系统的炎症因子表达，高度提示胎盘外泌体是胎盘生理功能的重要参与者。糖尿病条件下，胎盘外泌体同样出现异常，特别是miRNA表达差异显著，提示其生理功能异常。