Single cell transcriptomic profiling of microvascular endothelial cell heterogeneity in congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a neonatal anomaly that includes pulmonary hypoplasia and hypertension. We hypothesized that differences in microvascular endothelial cell (EC) heterogeneity may be present during CDH lung development and contribute to nderdevelopment and remodeling of the lungs. Time mated rats were gavaged nitrofen to induce CDH. Left lungs were harvested from healthy control (2HC), nitrofen exposed (NC) and nitrofen-exposed with CDH (CDH) fetuses at E21.5. Transcriptomic analysis was performed using single-cell RNA sequencing, and unbiased clustering revealed 3 distinct microvascular EC clusters. The general microvascular EC cluster (mvEC) had a transcriptomic signature suggestive of increased inflammation in CDH. mvEC differential gene expression (DGE) between NC and CDH revealed downregulation of Ca4, Apln and Ednrb, which define a subpopulation of microvascular ECs important to lung development, gas exchange and repair of alveolar injury (mvCa4+). mvCa4+ ECs were significantly reduced between 2HC (22.6%), NC (13.1%) and CDH (5.3%), demonstrating an impact from lung compression. Gene ontology and Ingenuity Pathway Analysis demonstrated that mvCa4+ ECs are primed for vasculogenesis but have DGE suggestive of impaired cell division. These data suggest that inflammation driven by mvECs and absence of mvCa4+ ECs may contribute to CDH pathogenesis.先天性膈疝（Congenital diaphragmatic hernia, CDH）是一类伴随肺发育不全与肺动脉高压的新生儿先天畸形。我们提出假说：CDH肺发育过程中，微血管内皮细胞（microvascular endothelial cell, EC）的异质性差异可能存在，并可导致肺部发育不全与结构重塑。本研究通过对定时交配的孕鼠灌服硝基酚（nitrofen）以构建CDH模型，于胚胎发育第21.5天（E21.5）分别采集健康对照组（2HC）、硝基酚暴露组（NC）以及伴CDH的硝基酚暴露组（CDH）胎鼠的左肺组织。采用单细胞RNA测序进行转录组学分析，经无偏聚类后得到3个特征迥异的微血管内皮细胞簇。通用型微血管内皮细胞簇（mvEC）的转录组特征显示，CDH模型中该细胞簇的炎症水平升高。对NC组与CDH组的mvEC进行差异基因表达（differential gene expression, DGE）分析，发现Ca4、Apln及Ednrb表达下调；这三个基因是对肺部发育、气体交换及肺泡损伤修复至关重要的微血管内皮细胞亚群（mvCa4+）的标志性基因。mvCa4+内皮细胞在健康对照组（22.6%）、硝基酚暴露组（13.1%）及CDH组（5.3%）中的占比依次显著降低，提示肺部受压对该细胞亚群存在影响。基因本体（Gene Ontology, GO）富集分析与Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）结果显示，mvCa4+内皮细胞本应具备血管生成潜能，但差异基因表达特征提示其细胞分裂功能受损。上述研究结果表明，mvEC介导的炎症反应以及mvCa4+内皮细胞的缺失，可能参与了CDH的发病机制。