Background: Despite its importance in health and disease, the cellular diversity of the lung endothelium has not been systematically characterized in humans. Here we provide a reference atlas of human lung endothelial cells (ECs), to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium, both in health and disease. Methods: We reprocessed control single cell RNA sequencing (scRNAseq) data from five datasets of whole lungs that were used for the analysis of pan-endothelial markers, we later included a sixth dataset of sorted control EC for the vascular subpopulation analysis. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by immunohistochemistry and in situ hybridization. Signaling network between different lung cell types was studied using connectomic analysis. For cross species analysis we applied the same methods to scRNAseq data obtained from mouse lungs. Results: The six lung scRNAseq datasets were reanalyzed and annotated to identify over 15,000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including pan-endothelial, pan-vascular and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial and venous ECs we found previously indistinguishable subpopulations; among venous EC we identified two previously indistinguishable populations, pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary EC we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1 and TBX2 and general capillary EC. We confirmed that all six endothelial cell types, including the systemic-venous EC and aerocytes are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-Receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. Our manuscript is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusion: Our integrated analysis provides the comprehensive and well-crafted reference atlas of lung endothelial cells in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

中文翻译：

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人肺内皮细胞综合单细胞图谱

背景：尽管肺内皮细胞在健康和疾病中具有重要意义，但尚未在人类中对其进行系统表征。在这里，我们提供人肺内皮细胞（EC）的参考图集，以促进对健康和疾病中包含肺内皮的细胞的表型多样性和组成的更好了解。方法：我们从五个全肺数据集中的全肺内皮细胞标记物数据中重新处理了对照单细胞RNA测序（scRNAseq）数据，之后又包括了用于血管亚群分析的第六个分类对照EC数据集。EC人口的特点是通过迭代聚类和随后的差异表达分析。通过免疫组织化学和原位杂交验证标记基因。使用连接组分析研究了不同肺细胞类型之间的信号网络。对于跨物种分析，我们将相同的方法应用于从小鼠肺部获得的scRNAseq数据。结果：重新分析并注释了六个肺scRNAseq数据集，以鉴定来自73个个体的15,000多个血管EC细胞。EC的差异表达分析揭示了与内皮细胞谱系相对应的特征，包括泛内皮，泛血管和亚群特异性标记基因集。除了广泛的细胞类型的淋巴，毛细血管，动脉和静脉内皮细胞外，我们还发现了以前无法区分的亚群。在静脉EC中，我们确定了两个以前无法区分的人群，肺静脉ECs（COL15A1neg）定位于肺实质，而全身静脉ECs（COL15A1pos）定位于气道和内脏胸膜；在毛细血管内皮细胞中，我们证实了它们的分类为最近发现的以EDNRB，SOSTDC1和TBX2为特征的气细胞以及一般的毛细血管内皮细胞。我们确认小鼠中存在所有六种内皮细胞类型，包括全身静脉EC和细胞，并确定了人类和小鼠中保守的内皮标记基因。配体-受体连接体分析揭示了EC与其他肺部驻留细胞类型的重要稳态平衡串扰。我们的手稿随附在线数据挖掘工具（www.LungEndothelialCellAtlas.com）。结论：