Abstract
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.
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Data availability
All scRNAseq data associated with this manuscript are deposited at Genome Sequence Archive (GSA) with accession ID- PRJCA047802 and submission ID subPRO070142. All other associated data can be found at Dataverse. Fig 1b- https://doi.org/10.7910/DVN/1LSWWO. Fig. 2b- https://doi.org/10.7910/DVN/JNEZCJ, Fig. 4D- https://doi.org/10.7910/DVN/P9PACR, Supplementary data and numerical source data for graphs can be found at https://doi.org/10.7910/DVN/X0VAXH. The raw spatial sequence data (Fig. 5c, d) reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in the National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA014847), which are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/browse/HRA014847.
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Acknowledgements
This work was supported by NIH grants R01HL151447 and R01HL132918 (R.R.), R01HL133085 and R01HL160666 (O.R.), R01HL158596 and R01HL162794 (Z.D.), the AHA postdoctoral fellowship 834220 and NIH K99HL171869 (J.J.) and AHA fellowship 831538 (M.V.), the AHA Transformational award 969574 (OR), the AHA Career Development Award 23CDA1050843 (M.N.). We thank Paulo Pires for the critical reading of the manuscript. We acknowledge the Microscopy Core at Indiana University and the Indiana Center for Biological Microscopy for their support in confocal imaging. We thank the Center for Medical Genomics at Indiana University School of Medicine and the University of Arizona Genetics Core for performing the barcoding and sequencing. We also thank Yukiko T. Matsunaga from the Department of Bioengineering at the University of Tokyo for providing silicone chips used in vessel-on-chip experiments. Additionally, we extend our gratitude to the Pulmonary Hypertension Breakthrough Initiative (grant to PHBI, NHLBI R24 HL123767) for providing tissue OCT blocks for spatial transcriptomics.
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Conception and design: R.R., O.R.; Experimentation, cells isolations, scRNAseq analysis, and interpretation: A.D., O.S.L., N.M., J.J., M.N., M.V., D.B., M.P.R., D.Y., Z.D., H.G., S.K., T.S.; Drafting the manuscript for important intellectual content: R.S.T., O.G., J.J., O.R., R.R.
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James, J., Dekan, A., Kacar, S. et al. Distinct populations of lung capillary endothelial cells and their functional significance. Commun Biol (2025). https://doi.org/10.1038/s42003-025-09420-x
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DOI: https://doi.org/10.1038/s42003-025-09420-x
