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Figure 1. Single-cell transcriptomic profiling showing four pulmonary vascular EC subpopulations. (A) Proportion and numbers of lung cell populations identified in this study. (B) Uniform manifold approximation and projection (UMAP) visualization of pulmonary vascular EC subpopulations, including arterial, venous, general capillary (gCap) and aerocyte capillary (aCap) ECs, as well as lymphatic ECs. (C) Dot plot displaying expression pattern of canonical EC subtype marker genes (Car4, Ednrb, Tbx3, Plvap, Tek, Cav1, Gja5, Bmp5, Fbln1, Ephb4 and Nr2f2) across the identified vascular EC subpopulations. Dot size and color scale reflect the frequency of cells in the indicated clusters expressing the marker genes and average log-transformed expression intensity, respectively. (D) Heatmap showing expression profile of classical EC markers in the EC subpopulations in (C) Shown are arterial EC markers (Gja5, Ednra), venous EC markers (Nr2f2, Ephb4), capillary-associated genes (Plvap, Car4), and additional markers (Tek, Cav1, Ednrb, Tbx2, Tbx3). Distinct expression boundaries indicate accurate annotation of each EC lineage. (E) Dot plot showing Smad4 expression across pulmonary vascular EC subpopulations in Smad4^fl/fl and Smad4^iko mice. Dot size indicates the percentage of cells expressing Smad4, and dot color represents the average expression levels of Smad4. Note that Smad4 is broadly expressed in the four vascular EC subpopulations, and that Smad4 expression is markedly reduced in all subpopulations in Smad4^iko mice compared with Smad4^fl/fl controls, attesting to successful gene deletion. (F) Heatmap showing expression pattern of the indicated signaling components of BMP/ALK1 and TGF-β/ALK5 pathways across pulmonary vascular EC subpopulations. Color scale represents average log-transformed expression intensity. EC: Endothelial cell; Fib: fibroblast; VSMC: vascular smooth muscle cell; ASMC: airway smooth muscle cell; Alveolar_macro: alveolar macrophage; Inter_macro: interstitial macrophage; DC: dendritic cell; NK: natural killer cell; AT: alveolar epithelial cell.