✍🏼 Joel James, Aleksandr Dekan, Maki Niihori, Nolan McClain, Mathews Varghese, Dinesh Bharti, Odunayo Susan Lawal, Marco Padilla-Rodrigez, Dan Yi, Zhiyu Dai, Oleg Gusev, Olga Rafikova, Ruslan Rafikov

🏠 University of Arizona, Tucson, Arizona, USA

📑 Research Square (2023)

Abstract
The role of the lung’s microcirculation and capillary endothelial cells in normal physiology and the pathobiology of pulmonary diseases is unequivocally vital. The recent discovery of molecularly distinct aerocytes and general capillary (gCaps) endothelial cells by single-cell transcriptomics (scRNAseq) advanced the field in understanding microcirculatory milieu and cellular communications. However, increasing evidence from different groups indicated the possibility of more heterogenic structures of lung capillaries. Therefore, we investigated enriched lung endothelial cells by scRNAseq and identified five novel populations of gCaps with distinct molecular signatures and roles. Our analysis suggests that two populations of gCaps that express Scn7a(Na+) and Clic4(Cl−) ion transporters form the arterial-to-vein zonation and establish the capillary barrier. We also discovered and named mitotically-active “root” cells (Flot1+) on the interface between arterial, Scn7a+, and Clic4 + endothelium, responsible for the regeneration and repair of the adjacent endothelial populations. Furthermore, the transition of gCaps to a vein requires a venous-capillary endothelium expressing Lingo2. Finally, gCaps detached from the zonation represent a high level of Fabp4, other metabolically active genes, and tip-cell markers showing angiogenesis-regulating capacity. The discovery of these populations will translate into a better understanding of the involvement of capillary phenotypes and their communications in lung disease pathogenesis.

How the WOLF was used in this study
The authors used the WOLF Cell Sorter to enrich endothelial cells prior to single‑cell transcriptomic analysis. Specifically, after isolating lung cells, they sorted GFP‑expressing endothelial cells by FACS to obtain a purified population of vascular endothelium for downstream single‑cell RNA sequencing, which enabled high‑resolution identification of distinct capillary endothelial subpopulations and novel gCap phenotypes. This sorting step was crucial for reducing sample heterogeneity and ensuring that the resulting scRNA‑seq data accurately reflected the true transcriptional diversity of lung capillary endothelial cells.