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. 4 samples, 2 healthy controls, 1 treated non-diseased, 1 treated diseased