Distinct populations of lung capillary endothelial cells and their functional significance

The role of the lung's microcirculation and capillary endothelial cells in normal physiology and the pathobiology of pulmonary diseases is obviously 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 a more heterogeneous nature 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 major populations of gCaps that express Scn7a(Na⁺) and Clic4(Cl^-) ion transporters form the arterial-to-vein phenotypic transition. 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 disconnected from the zonation represent a high level of Fabp4, other metabolically active genes, and tip-cell markers showing angiogenesis-regulating capacity. The hypoxia-induced models demonstrated that "root" cells exhibit a marked expansion in hypoxia, supporting their role in vascular regeneration and neocapillarization. We also showed a developmental time-course analysis demonstrating an evolution of progenitor (FoxM1+ ) cells, which are progressively replaced by "root" cells during lung maturation, revealing a switch in vascular homeostasis. The discovery of these populations will translate into a better understanding of the involvement of capillary phenotypes and their communications in lung disease pathogenesis.