Smooth Muscle Cell Populations of Differing Embryonic Origins in the Ascending Aorta Exhibit Minor Differences in Angiotensin II-driven Transcriptional Alterations in Mice

Ascending thoracic aortopathy is a life-threatening disease significantly influenced by angiotensin II (AngII). Thoracic aortopathy exhibits regional heterogeneity with the ascending region being susceptible. Smooth muscle cells (SMCs), a major component of the aortic wall, originate from two embryonic origins in the ascending aorta: second heart field (SHF) and cardiac neural crest (CNC). However, functional differences between the origins in AngII-induced thoracic aortopathy formation remain unknown. The present study determined transcriptomic differences between origins in response to AngII by single-cell RNA sequencing using the lineage tracing approach. Mef2c-Cre +/0 mT/mG mice were infused with AngII (1,000 ng/kg/day). To investigate causative mechanisms, ascending aortas were harvested after 3 days of AngII infusion, representing the prepathological phase of thoracic aortopathy. Aortic samples were also harvested from Mef2c-Cre +/0 mT/mG mice without AngII infusion as a control. Following single-cell suspension, cells were sorted based on their origin using mTotamto and mGFP signals. mGFP proteins were present on Mef2c-Cre-driven cells indicating the cells were derived from the SHF, while cells with mTomato signal were not derived from the SHF (nSHF). After sorting cells by origin, single-cell RNA sequencing was performed. Two-way ANOVA analysis identified 1718 differentially expressed genes (DEGs) in the interaction between origin and infusion. Among these DEGs, 1207 genes significantly differed between origins in response to AngII infusion. However, the magnitude of difference in most of these DEGs was modest, ranging between -0.05 and 0.05 Log2FC. Commonly studied molecules, such as TGF-ß, SMC contraction, and extracellular matrix molecules, were undetectable or modestly different. In conclusion, transcriptomic differences in SMCs between origins in response to AngII were modest in the pre-pathological phase of AngII-induced thoracic aortopathy. Overall design: Comparative gene expression analysis of scRNAseq data to determine transcriptomic differences in response to AngII infusion between cells derived from the second heart field and other origins in the ascending aorta of mice.

升胸主动脉病（Ascending thoracic aortopathy）是一类危及生命的疾病，其发生发展显著受血管紧张素II（Angiotensin II, AngII）调控。胸主动脉病存在区域异质性，升主动脉为疾病易感部位。作为主动脉壁主要组成成分的平滑肌细胞（Smooth muscle cells, SMCs），在升主动脉中存在两种胚胎起源：第二心区（Second heart field, SHF）与心脏神经嵴（Cardiac neural crest, CNC）。但目前两种起源的平滑肌细胞在AngII诱导的胸主动脉病形成过程中的功能差异仍未明确。
本研究采用谱系示踪技术结合单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），解析了不同起源的平滑肌细胞在AngII刺激下的转录组差异。实验中，向Mef2c-Cre +/0 mT/mG小鼠输注AngII（1000 ng/kg/天）；为探究致病机制，于AngII输注3天后收集升主动脉样本，此时样本处于胸主动脉病的病理前阶段；同时设置未输注AngII的Mef2c-Cre +/0 mT/mG小鼠主动脉样本作为对照。
制备单细胞悬液后，根据mTomato与mGFP荧光信号按细胞起源进行分选：由Mef2c-Cre驱动表达的mGFP阳性细胞为第二心区起源的平滑肌细胞，而mTomato阳性细胞则为非第二心区起源细胞（nSHF）。完成起源分选后，对细胞进行单细胞RNA测序。
双因素方差分析（Two-way ANOVA）显示，细胞起源与AngII输注的交互作用共筛选出1718个差异表达基因（differentially expressed genes, DEGs）。其中，1207个基因在AngII输注后不同起源的细胞间存在显著差异，但多数此类差异基因的表达变化幅度较小，Log2FC区间仅为-0.05至0.05。常规研究关注的分子如转化生长因子-β（TGF-β）、平滑肌收缩相关分子及细胞外基质分子等，要么未被检测到，要么仅存在轻微表达差异。
综上，在AngII诱导的胸主动脉病病理前阶段，不同起源的平滑肌细胞对AngII刺激的转录组差异较为有限。整体实验设计：通过对单细胞RNA测序数据开展比较基因表达分析，明确小鼠升主动脉中第二心区起源与其他起源的平滑肌细胞在AngII输注后的转录组差异。