Fig 1: Gene ontology and localization of EC subtypes elucidates known functions and confirms divergence of clusters 2 and 4.(A) Circos plot shows gene expression and gene ontology (GO) overlap between distinct clusters of pulmonary ECs. A purple line connecting two clusters indicates expression of the same gene in both clusters, while a blue line connecting two clusters indicates expression of different genes found within the same GO category in each cluster. (B) GO biological process enrichment performed for each cluster and displayed in heatmap format demonstrates expression of genes associated with different biological processes in each cluster, including some overlap between clusters. Cluster 2 (green box) shares enrichment of some processes related to angiogenesis and blood vessel development with cluster 0 (blue box) but is distinct in its enrichment of genes related to vasculogenesis. (C) In-depth analysis of gene expression in cluster four indicates that this cluster likely represents macrovascular ECs (maECs) with high expression of Vwf and Vcam1. IHC indicates that these proteins localize mainly to the large vessel endothelium. White arrowhead demonstrates Vcam1 located in nearby mesenchymal cells. (D) Clusters 0, 1, and three represent a heterogeneous population of microvascular ECs (miECs) with high expression of Gpihbp1 and Plvap. IHC indicates that these proteins localize to the alveolar capillary plexus endothelium. Yellow arrowhead demonstrates Plvap present in the large vessel endothelium. (E) Cluster two represents an as-yet uncharacterized population of ECs that localize to the alveolar region and express surface marker Cd34 at a higher level from that in other ECs. These cells also express high levels of Car4. CD34 and Car4 proteins localize to the alveolar space, indicating a similar spatial distribution of cells in cluster two to that of miECs. v, vessel; a, alveolar space; scale bars in (C)-(E), 20 microns.