肺动脉特异DNA甲基化位点决定其生理特性的机制研究

Neo-aortic construction is one of the most common surgical treatments for arterial diseases in newborns. Neo-aortic root dilation frequently happens when original pulmonary artery is overloaded with systematic blood pressure. However, it is unclear whether cellular origin of arterial smooth muscle cells contributes to vascular physiology and susceptibility of the disease. Previously, our lab discovered smooth muscle cells (SMCs) in pulmonary artery (PA) trunk were derived from mesodermal second heart field progenitors, while ascending aorta (AO) SMCs were originated from ectodermal neural crest cells. Using whole genome bisulfite sequencing, we identified a group of differentially methylated regions between PA and AO SMCs. Furthermore, mesodermal specific transcription factor, GATA4, was highly expressed in PA compared to AO, and has potential to regulate genes involved in PA physiological functions including MMP2. Here we propose to identify active enhancer regions that regulate GATA4 expression using differential DNA methylation regions as molecular marker. We will investigate GATA4 downstream target genes that establish PA physiological features. Moreover, we will manipulate these regulatory enhancers to validate their biological function in PA. In conclusion, this study will shed lights on lineage-specific regulatory mechanism controlled by DNA methylation, providing a novel therapeutic strategy for treating neo-aortic root dilation.

新主动脉构建术后，来源于肺动脉的组织长期承受体循环高压，容易发生新主动脉根部扩张，其原因尚不清楚。我们前期研究发现主、肺动脉平滑肌分别起源于外、中胚层祖细胞，并各自具有与祖细胞来源对应的特征性基因表达和DNA甲基化修饰。中胚层发育关键基因GATA4在肺动脉平滑肌内高表达，其非启动子区域具有DNA低甲基化位点。且GATA4能驱动MMP2等肺动脉特征性基因表达。据此我们推测肺动脉平滑肌细胞内GATA4非启动子区域特征性低甲基化维持其高表达，而GATA4能驱动肺动脉特征性基因表达，是决定主、肺动脉平滑肌的生理特性差异的关键原因之一。本项目我们拟研究肺动脉平滑肌中GATA4基因特异位点DNA低甲基化状态对其表达的影响和相关的分子机制，确认其和肺动脉特征性基因表达的关系。本项目将阐明主、肺动脉平滑肌因发育路径不同导致生理功能差异的机制，解释新主动脉根部扩张产生的原因，为未来分子治疗提供新思路。

新主动脉构建术后，来源于肺动脉的组织长期承受体循环高压，容易发生新主动脉根部扩张，其原因尚不清楚。我们前期研究发现主、肺动脉平滑肌分别起源于外、中胚层祖细胞，并各自具有与祖细胞来源对应的特征性基因表达和DNA甲基化修饰。中胚层发育关键基因GATA4在肺动脉平滑肌内高表达，其非启动子区域具有DNA低甲基化位点。且GATA4能驱动MMP2等肺动脉特征性基因表达。本研究课题以小鼠为研究模型，对野生型小鼠中主动脉、肺动脉和突变体的动脉干进行DNA甲基化测序，发现流出道异常动物模型中具有差异的DNA甲基化区域，解析永生动脉的发生机制。我们还在人流出道平滑肌细胞中验证小鼠结果, 利用人鼠之间保守的DMR对人类和小鼠胚胎时期的样本进行聚类，发现人类主动脉和肺动脉与小鼠有着相同的分化来源，人类动脉干与小鼠动脉干具有相似发病机制。我们还利用空间转录组结合单细胞转录组对小鼠胚胎发育阶段心脏流出道细胞异质性进行系统分析, 并使用小鼠谱系示踪模型, 对小鼠胚胎阶段流出道分隔的生物学过程进行研究。我们的研究结果揭示了肺动脉平滑肌中GATA4基因特异位点DNA低甲基化状态对其表达的影响和相关的分子机制，确认其和肺动脉特征性基因表达的关系。此外, 通过对第二心域祖细胞谱系示踪, 我们还意外发现第二心域来源细胞是构成心脏流出道和心外膜下淋巴管系统的主要细胞类型，在急性缺血性心脏损伤及心脏后负荷增加模型中发挥重要的调控功能。本项目阐明了主、肺动脉平滑肌因发育路径不同导致生理功能差异的机制，解释新主动脉根部扩张产生的原因，为未来分子治疗提供新思路。