Capillary cell-type specialization in the alveolus

Abstract

In the mammalian lung, an apparently homogenous mesh of capillary vessels surrounds each alveolus, forming the vast respiratory surface across which oxygen transfers to the blood¹. Here we use single-cell analysis to elucidate the cell types, development, renewal and evolution of the alveolar capillary endothelium. We show that alveolar capillaries are mosaics; similar to the epithelium that lines the alveolus, the alveolar endothelium is made up of two intermingled cell types, with complex 'Swiss-cheese'-like morphologies and distinct functions. The first cell type, which we term the 'aerocyte', is specialized for gas exchange and the trafficking of leukocytes, and is unique to the lung. The other cell type, termed gCap ('general' capillary), is specialized to regulate vasomotor tone, and functions as a stem/progenitor cell in capillary homeostasis and repair. The two cell types develop from bipotent progenitors, mature gradually and are affected differently in disease and during ageing. This cell-type specialization is conserved between mouse and human lungs but is not found in alligator or turtle lungs, suggesting it arose during the evolution of the mammalian lung. The discovery of cell type specialization in alveolar capillaries transforms our understanding of the structure, function, regulation and maintenance of the air-blood barrier and gas exchange in health, disease and evolution.

Extended Data Figure 1.. Molecular characterization and mapping of the two alveolar capillary cell types.

a, Feature plots showing log-transformed expression of marker genes used to annotate five molecularly distinct clusters (see tSNE plot in Fig. 1b) derived by unsupervised clustering of lung endothelial cells in the Tabula Muris scRNAseq Smart-seq2 data for adult mouse lung. We identified two endothelial cell clusters as capillaries based on expression of carbonic anhydrases (Car4, Car14) that catalyze the conversion of bicarbonate to carbon dioxide, Gpihbp1, a lipoprotein binding protein that localizes to the luminal membrane of alveolar capillaries, and lipoprotein lipase (Lpl), which is transported to the capillary lumen by Gpihbp1. b, Heatmap showing log-transformed expression of selected cluster markers in individual pulmonary artery, vein, lymphatics, and alveolar capillary (aCap, gCap) endothelial cells identified in the Tabula Muris Smart-Seq2 data. c, Co-expression of alveolar capillary cell type markers Apln (aCap) and Aplnr (gCap) in a single alveolar capillary intermediate (IM) endothelial cell (arrow; marked by Cldn5) in adult mouse lung, as detected by smFISH. d, Co-expression of aCap markers (Apln and Ednrb) in a subset (aCap cells) of alveolar endothelial cells (marked by Cldn5) in adult mouse lung. e, Co-expression of aCap markers Ednrb and Car4 but not Aplnr, a gCap marker, in a subset (aCap cells) of adult mouse lung alveolar endothelial cells. e’, Boxed region from e at higher magnification. Filled arrowhead indicates aCap cell expressing high levels of Car4. Open arrowhead points to gCap cell expressing low levels of Car4. f, g, Co-expression of gCap markers Gpihbp1 (f) or H2-Ab1 (g), a major histocompatibility complex (MHC) class II gene, and Aplnr in a subset (gCap cells) of adult mouse lung alveolar endothelial cells. h, i, Quantification of the relative abundance (in % of capillary endothelial cells) of the two alveolar capillary populations (aCap, gCap) and rare cells (IM) that co-express aCap and gCap markers at the pleura and in intra-acinar regions (h), or in different lobes (i, left vs. right cranial) in lungs from 3-month old mice. Data shown as mean ± s.d.; n=500 cells scored per mouse; n=3 mice; p-values comparing cell type abundance by two-sided Wilcoxon rank sum test: aCap (h, 0.2; i, 0.2), gCap (h, 0.4; i, 0.2), IM (h, 0.7; i, 0.8). n.s., not significant. j, aCap (marked by Ednrb) and gCap (marked by Ptprb) cells in lungs from 3- and 24-month old mice. Endothelial cells are marked by Cldn5. k, l, Co-expression of tdTomato lineage label (asterisks) and either aCap marker Ednrb but not gCap marker Aplnr in an Apln-CreER; Rosa26-tdTomato lung (k) or gCap marker Aplnr but not aCap marker Apln in an Aplnr-CreER; Rosa26-tdTomato lung (l) harvested one month after mature aCap (k) or gCap (l) cells were lineage labeled. Lineage labeled cells (asterisks) continue to express the marker (Ednrb, aCap; Aplnr, gCap) of the labeled population. m, n, Quantification of percent of lineage labeled cells in Apln-CreER; Rosa26-tdTomato (m) or Aplnr-CreER; Rosa26-tdTomato (n) lungs that continue to express the aCap marker, gCap marker, or markers of both cell types (IM) after 48 hours, 1, 6, or 14 months (500–2000 cells scored at each time point from multiple regions in each of two separate lobes from one lung). Blue (c-g, j-l), DAPI. Scale bars, 10 μm.

Extended Data Figure 2.. Alveolar capillary cell morphologies.

a-d, Additional examples of single cytoplasmic YFP- or RFP-expressing plexus cells at E12.5 (a), aerocytes at E18.5 (b), gCap cells at P0 (c), and aerocytes at P0 (d). Plexus and gCap cells were labeled in Aplnr-CreER; Rosa26-Confetti lungs; aerocytes were labeled in Apln-CreER; Rosa26-Confetti lungs. Pores, present in some aerocytes by E18.5, but not plexus or P0 gCap cells, are marked by dots. e, f, Additional examples of single gCap cells (e) or aerocytes (f) expressing cytoplasmic RFP or YFP in adult Aplnr-CreER; Rosa26-Confetti (e) or Apln-CreER; Rosa26-Confetti (f) lungs. Dotted circles outline two alveoli spanned by a single aerocyte (f). Note that gCap cells are less morphologically diverse than aerocytes. g, h, Quantification of individual plexus, aerocyte (aCap), or gCap cell surface area (g) and pore number (h) at indicated times. n>10 cells scored for each cell type at each time point from n=2 lungs; see Methods for exact cell number. Blue, elastin fibers highlighting the alveolar entrance. Grey bar, mean value. Scale bars, 10 μm.

Extended Data Figure 3.. Capillary cells in the lung and other organs.

a, Bronchial (systemic) capillary cell immunostained for tdTomato (red) in adult Aplnr-CreER; Rosa26-tdTomato lung. Airway epithelium is immunostained for E-cadherin (blue). b-f, Single capillary cells labeled with membrane targeted CFP (b) or cytoplasmic YFP (c-f) in brain (b), heart (c), small intestine (d), kidney glomerulus (e), or thyroid (f), where capillaries are arranged in baskets similar to pulmonary alveoli, from an adult Cdh5-CreER; Rosa26-Confetti mouse. Capillaries labeled with tomato lectin (blue, b-f). e’, Cell in boxed region in e at higher magnification, along with other examples of labeled glomerular endothelial cells. g, Single endothelial tip cell expressing cytoplasmic YFP in developing (P7) Apln-CreER; Rosa26-Confetti mouse retina. Filled arrowheads, filopodia. h, i, Scatter plots of alveolar aCap and gCap signature scores assigned to annotated lung, heart, and brain capillary endothelial cells identified in the Tabula Muris scRNAseq Smart-seq2 data (h) or annotated lung, kidney glomerular, and mammary gland capillary endothelial cells identified in the Tabula Muris Senis droplet data for 1, 3, 18, 21, and 30 month old mice (i). Lung capillary cells segregate into two clusters, with annotated gCap cells having high gCap and low aCap signature scores, and annotated aCap cells having low gCap and high aCap signature scores. Heart (h), brain (h), glomerular (i), and mammary gland (i) capillary cells have high gCap and low aCap signature scores and each form a single cluster near gCap but not aCap cells, suggesting that they are more similar to gCap cells and any heterogeneity within these other capillary cell populations is different from that in the lung. j-l, Some aCap and gCap markers (aCap, Ednrb (j, k); gCap, Ptprb, j, or Gpihbp1, k) are broadly co-expressed in glomerular endothelial cells (Pecam1) in adult mouse kidney whereas expression of others (l: Apln, aCap or Aplnr, gCap) is detected only in small numbers of cells or not at all. Dotted lines outline glomeruli. j’-l’, Boxed regions from j-l at higher magnification. Arrows (j’, k’) point to cells co-expressing the markers. Blue (j-l), DAPI. Scale bars, 10 μm.

Extended Data Figure 4.. Functional compartmentalization of the alveolus.

a, Transmission electron micrographs of alveolar walls from adult mouse lungs, pseudocolored to highlight cells and connective tissue (extracellular matrix (ECM) or fibers). Apln-CreER; Rosa26-tdTomato (to label aerocytes) or Aplnr-CreER; Rosa26-tdTomato (to label gCap cells) lungs were immunostained for tdTomato (detected with DAB and NiCl₂, heavy black stain). Labeled aerocytes but not gCap cells are associated with thin regions of the air-blood barrier (delimited by dashed lines), where the epithelium is tightly apposed to the endothelium. Some thin regions do not contain labeled aerocytes since aerocyte labeling using Apelin-CreER is inefficient. Thick regions, where the epithelium is separated from the endothelium by fibers or other cells, can be associated with either aerocytes or gCap cells. AT1 cell, alveolar type 1 epithelial cell; AT2 cell, alveolar type 2 epithelial cell. Micrographs in top left, middle, and bottom panels are shown without pseudocoloring in Fig. 2g-i. b, c, gCap cells associate with fibroblasts. Alveoli with labeled aerocytes (aCap) in Apln-CreER; Rosa26-tdTomato (b) or gCap cells in Aplnr-CreER; Rosa26-tdTomato (c) lungs immunostained for integrin α8 to show alveolar fibroblasts. Asterisks mark aerocytes that do not associate with fibroblasts (in b) and gCap cells that do (in c). Example regions of alveoli not covered by fibroblasts are dotted. Note that the dotted region in b is occupied by an aerocyte not overlaid by fibroblasts, whereas fibroblasts overlay gCap cells in c. d, Selected genes with known functions, which are differentially expressed between the two alveolar capillary cell types in adult mouse lung. Pro, procoagulants; anti, anticoagulants^,. e, Summary of signaling interactions between capillary cell types and surrounding cells in the mouse alveolus. Arrows indicate direction of signaling. f, Schematic representation of an alveolus highlighting proposed specialized functions and signaling interactions of alveolar capillary cell types. AT1, alveolar type 1 epithelial cell; AT2, alveolar type 2 epithelial cell. Scale bars, 2 μm (a), 10 μm (b, c).

Extended Data Figure 5.. Proliferation of gCap cells following elastase injury.

a, Elastin fibers (black), labeled with fluorescent hydrazide, in lungs of wild-type mice treated with elastase or mock-treated with saline as control. Loss of elastin fibers in injured areas (dashed outlines) is apparent one day after intratracheal instillation of elastase. At one month after injury, regions containing abnormally thickened elastin fibers and enlarged airspaces are evident. Boxed areas are shown at higher magnification. b, Scheme for detecting proliferation of alveolar capillary cells following elastase injury. aCap and gCap cells were lineage labeled in adult Apln-CreER; Rosa26-tdTomato (for aCap cells) or Aplnr-CreER; Rosa26-tdTomato (for gCap cells) mice 3 weeks prior to elastase administration, then EdU was administered in drinking water and capillary cells were analyzed at the indicated times after injury. c-f, Proliferation analyzed by cumulative EdU incorporation in lineage labeled (red) aCap cells (top panels in c-f) and gCap cells (bottom panels in c-f) at 3 days (c, d), 1 week (e), or 6 weeks (f) after instillation of saline (as control; c) or elastase (d-f). Boxed areas are shown at higher magnification, with cell bodies of individual capillary cells outlined. Following treatment with elastase, gCap cells proliferated in injured areas. White, elastin fibers. Scale bars (c-f), 10 μm.

Extended Data Figure 6.. Development, maturation, and aging of specialized alveolar capillary cell types.

a, Expression of Aplnr throughout plexus (labeled by Pecam1) surrounding a developing airway at E12.5, detected by smFISH. b, Expression of mature aerocyte markers Apln and Ednrb in plexus (labeled by Pecam1) surrounding a developing airway at E12.5. Plexus cells expressing Apln but not Ednrb are marked by asterisks. Ednrb is expressed at high levels in Pecam1-negative (stromal) cells, but rarely and only at low levels in Pecam1-positive endothelial cells (arrow) at this stage. c, The subpopulation of Apln-expressing cells within the Aplnr+ plexus gives rise to both capillary cell types, as shown by expression of tdTomato transcripts in both aerocyte (filled arrowheads, marked by Ednrb) and gCap (open arrowheads, marked by Aplnr) cells of P21 Apln-CreER; Rosa26-tdTomato lung lineage labeled at E12.5, demonstrating that even plexus cells that express an aerocyte marker are uncommitted. c’, Lineage labeled aerocyte (filled arrowhead) and gCap (open arrowheads) cells shown at higher magnification. d, Alveolar capillary clone in a P25 Aplnr-CreER; Rosa26-Confetti lung composed of both aCap and gCap cells (higher magnification at right) derived from a single YFP-expressing plexus cell labeled at E14.5. e, Composition of Aplnr-CreER; Rosa26-Confetti clones induced at E14.5 and analyzed at P25, ordered by clone size. All analyzed clones, with the exception of clone number 1, contained both aCap and gCap cells. Some clones also contained cells located in larger vessels (LV). Both coherent clones (in which all cells are touching) and dispersed clones were observed, suggesting that there can be cell movement during capillary development. The number of cells of each type was scored from 3D renderings of confocal z-stacks, as described in the Methods. Clone 4 is shown in Fig. 4e; clone 28 is shown in Extended Data Fig. 6d and Supplementary Video 4. RCr, right cranial lobe; RAc, right accessory lobe, RCd, right caudal lobe; L, left lobe. f, The Aplnr⁺ population remains uncommitted even after birth. The population labeled at P7 in an Aplnr-CreER; Rosa26-tdTomato lung gives rise to both aCap and gCap cells, as shown by co-expression of tdTomato lineage label and either aCap marker Apln or gCap marker Aplnr at P21, detected by smFISH. f’, f’’, Lineage labeled aerocyte and gCap cells shown at higher magnification. g, Expression of aerocyte markers Car4, Ednrb, and Apln in developing mouse lung at indicated stages. Aerocytes (asterisks), identified by marker co-expression, begin to emerge at E17.5. Insets, cells in boxed regions at higher magnification. h, Plexus and capillary cells identified in scRNAseq data for developing mouse lung arranged as tree-shaped branched developmental trajectory inferred using Monocle2. Cells collected at the indicated stages are colored to show plexus located along the stem before the branchpoint, maturing and mature aerocytes along the bottom branch, and maturing and mature gCap cells along the top branch. Note that cells from a single late embryonic or early postnatal time point are found at multiple positions along the trajectory. This analysis is consistent with that presented in ref. . Each cell type undergoes distinct and asynchronous molecular and morphological maturation (see Extended Data Fig. 2), suggesting that the two capillary cell specialization programs are likely under separate genetic control. i, Heatmap showing log-transformed expression levels in individual plexus and capillary cells for selected genes differentially expressed during aerocyte emergence and maturation. Many aerocyte markers are expressed in emerging aerocytes; others are only expressed in mature aerocytes. j, Developmental trajectory plot (top) and feature plots showing log-transformed expression for selected gCap markers expressed only in mature gCap cells (upper branch tip). k, l, Expression of Vwf in the gas exchange region at 3 (k) and 24 months (l), as detected by smFISH. k’, l’, Boxed regions from k and l shown at higher magnification. Endothelial cells are marked by Cldn5 expression. At 24 months, Vwf is induced in gCap cells (marked by Ptprb, open arrowheads), but not aerocytes (filled arrowhead). m, Schematic representation of Vwf induction in gCap cells with aging. At 24 months, Vwf is more broadly expressed in alveolar capillaries than at 3 months but only in gCap cells (marked by Ptprb). Blue, DAPI (a-c, f, g, k’, l’). Scale bars, 10 μm.

Extended Data Figure 7.. Emergence and maturation of the alveolar capillary cell types in developing mouse lung.

a, tSNE plots with plexus and capillary endothelial cells from embryonic, postnatal, and adult stages (n=3094 cells) identified in scRNAseq MARS-Seq data for developing mouse lung. Cells from each indicated stage are colored black, except emerging/maturing aerocytes (aCap), first evident at E18.5 in this dataset, light blue; mature aerocytes, dark blue; mature gCap cells, dark green. See also feature plots in Supplementary Data 3. b, Branched heatmap showing gene expression changes during the transformation of plexus into mature alveolar capillary cell types. Normalized expression values (z-scores) are plotted for genes with branch-dependent expression, identified by branched expression analysis modeling (BEAM) (n=1119 genes at q-value < 0.05), and genes that vary as a function of pseudotime, identified by differential expression analysis as implemented in Monocle2 (n=3734 genes at q-value < 0.05), in plexus and capillary cells. Cells are ordered by ascending pseudotime values with plexus in the middle, mature aerocytes (aCap) on the left, and mature gCap cells on the right. Genes (n=4129) are grouped by expression pattern and selected genes are indicated on the heatmap. Branchpoint genes change their expression where the stem splits into aCap and gCap branches. Capillary genes are expressed in both aCap and gCap cells, but not plexus. An uncropped version of the heatmap is shown in Supplementary Data 4. c, Developmental trajectory plot (top) and feature plots (below) showing log-transformed expression for selected genes in plexus (cells located along the stem before the branchpoint), aerocytes (bottom branch), and gCap cells (top branch).

Extended Data Figure 8.. Specialized alveolar capillary cell types in the human lung.

a, Alveolar capillary network in adult human lung immunostained for PECAM1 and VE-Cadherin. Boxed region shown at higher magnification. b, tSNE plot of annotated artery, vein, lymphatics, bronchial endothelial, aerocyte (aCap) and gCap cell clusters identified in scRNAseq droplet data for adult human lung (75 year old male). c, Heatmap showing log-transformed expression of selected cluster markers in individual endothelial cells. L., lymphatics. d-g, Expression of aerocyte (EDNRB, d, or APLN, f) and gCap (EDN1, e, g or VWF, g) markers in alveolar endothelial cells (marked by CLDN5, d-f), detected by smFISH in adult human lung. d’-g’, d’’-g’’, Aerocytes and gCap cells shown at higher magnification. h, As in the mouse lung (see Extended Data Fig. 1c), occasional human alveolar capillary cells co-express aerocyte (APLN) and gCap (EDN1) markers. h’, Boxed region shown at higher magnification with clustered capillary intermediate cells (arrows) near a large vessel (dotted line in h) and a nearby single cell. i, Quantification of the relative abundance (in % of capillary endothelial cells) of the two alveolar capillary populations (aCap, gCap) and intermediate (IM) cells that co-express aCap and gCap markers in adult human lung (n=2 individuals; 69 and 75 year old males; 500–600 capillary cells scored per lung; data as mean). j, j’ Co-expression of aerocyte markers EDNRB, APLN, and CA4 in emerging aerocytes (dotted outlines) but not plexus cells (asterisks) in fetal human lung (23 weeks gestational age, corresponding to E16.5–17.5 in mouse). At 23 weeks, 6% of CA4-expressing cells also express APLN and EDNRB at high levels (emerging aerocytes; 5 or more puncta per cell), compared to 0% at 17 weeks (500 CA4+ cells scored at each time point from one lung). Inset, adjacent haematoxylin and eosin (H&E)-stained section. j’, Boxed region from j at higher magnification. Blue (a, d-h, j), DAPI. Scale bars, 10 μm.

Extended Data Figure 9.. Altered capillary cell patterns in mouse and human lung tumors.

a, Human lung adenocarcinoma with tumor vessels immunostained for CD34 (brown), an endothelial marker expressed by both aCap and gCap cells. Blue, haematoxylin counterstain. a’, Co-expression of alveolar capillary cell type markers EDNRB (aCap) and PTPRB (gCap) in vessels surrounding tumor acinus (boxed in a), as detected in adjacent section by smFISH. a’’, Boxed region from a’ showing two endothelial cells (marked by CLDN5) co-expressing aerocyte and gCap markers (dotted outlines) at higher magnification. Merged image also shown in Fig. 4l. b-c’, Co-expression (asterisks) of alveolar capillary cell type markers (Ednrb, b, b’, or Apln, c, c’ (aCap), and Ptprb, b, b’, or Aplnr, c, c’ (gCap)) in a subset of endothelial cells (marked by Cldn5, b’) of mouse lung adenomas induced by conditional expression of an activating Kras mutation in AT2 cells. Open arrowheads point to gCap cells. b’, c’, Boxed regions in b and c shown at higher magnification. d, Quantification of the relative abundance (in % of capillary endothelial cells) of the two alveolar capillary populations (aCap, gCap) and intermediate cells (IM) that co-express aCap and gCap markers in adenomas (n=1,332 cells scored in 21 tumor sections from n=2 mice) and alveoli (n=11,219 cells scored in n=5 mice at 3 months). e, Scatter plot of gCap:IM ratios in individual adenomas (n=21). Grey bar, mean value. The fraction of intermediate cells varies from tumor to tumor even within the same lung, perhaps reflecting different stages of tumor development. f, f’, Expression of MHC class II gene H2-Ab1 (normally expressed in gCap cells; see Extended Data Fig. 1g) is lost in gCap cells (open arrowhead) in mouse adenomas, suggesting that they may lose their antigen presentation function. H2-Ab1 is also not expressed by the abundant tumor capillary cells that co-express Aplnr and Ednrb (asterisk). f’, Boxed region in f shown at higher magnification. Scale bars, a, 200 μm; a’-c’, f, f’, 10 μm.

Extended Data Figure 10.. Conserved and species-specific alveolar capillary cell specialization.

a, Dot plots showing log-transformed average expression levels and percent expression in aerocytes (aCap) and gCap cells for selected genes with known physiologic and immune functions, ligands and receptors/co-receptors, and transcription factors, which are differentially expressed by the two capillary cell types in adult mouse lung (Tabula Muris scRNAseq Smart-Seq2 data) or human lung (Human Lung Cell Atlas; droplet (10X) or Smart-Seq2 data; 75 year old male). b, Selected differentially expressed genes in the human lung with known functions. c-e, Diagrams of selected proposed specialized alveolar capillary functions. Selected genes involved in leukocyte trafficking and vasomotor control (see legend to Fig. 3d) show the same specialized expression pattern in mouse and human (c; Type 0), whereas some genes involved in hemostasis show specialized expression only in the human lung (d; Type 1), and some antigen presenting genes show specialized expression that switches cell type between mouse and human (e; Type 2). Blue, genes expressed in aerocytes; green, genes expressed in gCap cells; yellow, genes expressed in both aerocytes and gCap cells; purple, genes expressed in pericytes.

Extended Data Figure 11.. Faveolar capillary cell types in alligator and turtle.

a, Cladogram illustrating phylogenetic relationships between amniote taxa with different lung structures. The relationship between testudines and other diapsids is unresolved (dotted line). Ma, million years.b, h, Internal anatomy of alligator (Alligator mississippiensis; b) and turtle (Emys orbicularis; h) lungs, reproduced from ref. . Alligator and turtle lungs have multiple separate airspaces (‘chambers’), smooth muscle, a branched vasculature, and terminal airspaces known as faveoli where gas exchange occurs across a thick air-blood barrier^,. c, i, Gas exchange regions of American alligator (Alligator mississippiensis; c) and Western painted turtle (Chrysemys picta bellii, i) lungs, stained with haematoxylin and eosin. d, Faveolar capillary network in American alligator lung immunostained for Claudin 5 (red; endothelium). Green, elastin fibers. e, Co-expression (asterisks) of alveolar capillary cell type markers CA4 (aCap) and PTPRB (gCap) in faveolar endothelial cells (marked by CLDN5) in juvenile American alligator lung as detected by smFISH. e’, Faveolar capillary cell in boxed region from e shown at higher magnification. Note that faveolar capillary cells – reminiscent of the intermediate cells found in adult mouse and human lungs – appear to differ in gene expression from the developmental precursors identified in the mouse vascular plexus in which aCap and gCap markers are only rarely co-expressed (Supplementary Data 2). f, Co-expression (asterisks) of alveolar capillary cell type markers EDNRB (aCap) and PTPRB (gCap) in faveolar endothelial cells (marked by CLDN5) in adult American alligator lung. f’, Faveolar capillary cell in boxed region from f shown at higher magnification. g, Alligator faveolar septum immunostained for Claudin 5 (red; endothelium) and E-cadherin (white; epithelium). Dotted line, capillary lumen. j, k, Faveolar capillary network in Western painted turtle lung immunostained for Claudin 5 (red) to label endothelial cells. Neighboring faveoli are separated by septa with a capillary layer on each side of the septal wall (j). A capillary lumen is outlined (j, dotted line). l, Co-expression (asterisks) of alveolar capillary cell type markers (EDNRB, aCap and APLNR, gCap) in faveolar endothelial cells (marked by CLDN5) in adult turtle lung. l’, Two faveolar capillary cells in boxed region from l shown at higher magnification. m, Faveolar septum in turtle lung immunostained for Claudin 5 (red) to label endothelial cells in the capillary layer on each side of septal wall and E-cadherin (white) to label respiratory epithelium. A capillary lumen is outlined (dotted line). Blue, DAPI. Scale bars, 200 μm (c, i), 10 μm (d-g, j-m).

Figure 1.. Two stable, intermingled alveolar capillary cell types.

a, Alveolar capillaries in adult mouse lung immunostained for Pecam1. b, tSNE plot of endothelial cell populations annotated in scRNAseq data for adult mouse lung. c, Heatmap of expression of capillary subset markers and general endothelial marker Cldn5 in individual capillary cells. d-f, smFISH for capillary subset markers Apln, or Ednrb and Car4 (aCap) and Aplnr (gCap) in adult mouse lung. d’, f, Individual aCap and gCap cells. g, Relative abundance of aCap, gCap, and cells that co-express aCap and gCap markers (IM) in lungs from 3- and 24-month old mice (data as mean; n=500 cells scored per mouse; 2 mice per group). h, i, Co-expression of tdTomato lineage label (asterisks) and aCap marker Ednrb but not gCap marker Aplnr (h) or gCap marker Aplnr but not aCap marker Apln (i) in lungs harvested six months after mature aCap (h) or gCap (i) cells were lineage labeled. Blue, DAPI. Scale bars, 10 μm.

Figure 2.. Specialized alveolar capillary cell types in gas exchange and capillary renewal.

a, b, Single aerocyte (a) or gCap (b) cells in adult Apln-CreER; Rosa26-Confetti (a) or Aplnr-CreER; Rosa26-Confetti (b) lungs. c, Quantification of individual cell volumes. Bar indicates mean (19 aCap and 17 gCap cells scored from n=2 mice). d, aCap and gCap cells in adult Cdh5-CreER; Rosa26-Confetti lung form multicellular tubes (asterisks) within capillaries surrounding a single alveolus (dotted outline). Blue, elastin fibers (a, b, d). e, Schematic of alveolar capillary network. Asterisks indicate multicellular tubes. RBC, red blood cell. f-i, Transmission electron micrographs of adult mouse alveolar walls. f, f’ Thick and thin regions of the air-blood barrier. g-i, Apln-CreER; Rosa26-tdTomato (g, h) or Aplnr-CreER; Rosa26-tdTomato (i) lungs immunostained for tdTomato (heavy black stain). Labeled aerocytes (g, h) but not gCap cells (i) are associated with thin regions (dashed lines). j, Quantification of percent of each labeled cell type associated with thick or thin regions (n=2 mice of each genotype, 21 labeled aCap cells and 24 labeled gCap cells scored). k, Schematic representation of air-blood barrier. AT1, alveolar type 1 epithelial cell. l, m, Analysis of proliferation of lineage labeled aCap (l) or gCap cells (m) during adult homeostasis, detected by cumulative EdU incorporation for 6 weeks. 0.1% (mean) of labeled aCap cells incorporated EdU (l) compared to 7.7% of labeled gCap cells (m) (n=400–4,000 cells scored per lung in n=2 mice of each genotype). n, Quantification of fraction of EdU⁺ lineage labeled aCap or gCap cells during indicated intervals after elastase (El) or mock injury with saline as control (Ctrl; data as mean; n=200–1,600 cells scored per lung in 2–4 mice of each genotype per time point and treatment group; see Methods for exact sample sizes). o, o’ smFISH for lineage label (tdTomato), aCap (Ednrb) and gCap (Aplnr) markers in gCap lineage labeled lung six weeks after elastase injury. Blue, DAPI (l, m, o). p, Quantification of fraction of gCap (left) or aCap (right) lineage labeled cells expressing aCap (Ednrb), gCap (Aplnr or Ptprb), or both markers (intermediate (IM) cells) six weeks after elastase injury in injured and uninjured (control) regions (n=500–1000 cells scored per region; 3 injured and control regions scored in n=2 mice of each genotype). q, Relative abundance of capillary cell types in injured and uninjured (control) regions, six weeks after elastase administration (n=800–5600 cells scored per region; 3 injured and control regions; n=3 mice). Scale bars, 10 μm (a, b, d, l, m, o, o’), 2 μm (f-i).

Figure 3.. Molecular functions of alveolar capillary cell types.

a, Dot plots showing expression in aerocytes and gCap cells for selected differentially expressed genes. b-f, Diagrams of proposed specialized alveolar capillary cell functions. Aerocyte genes, blue; gCap genes, green; aerocyte/gCap genes, yellow; pericyte genes, purple. d, gCap cells express Edn1, eNOS (encoded by Nos3) and prostaglandin I2 synthase (encoded by Ptgis), making them a unique source of vasomodulators. Edn1 can signal to Ednra expressed on pericytes or to Ednrb expressed on aerocytes, which may feed back to gCap cells (dashed arrow) to regulate vasodilator production. f, Lipoprotein lipase (encoded by Lpl), anchored to the lumen of aerocytes by Gpihbp1, converts circulating lipoprotein triglycerides to monoglycerides, which are broken down to free fatty acids by monoacylglycerol lipase (encoded by Mgll) expressed by gCap cells.

Figure 4.. Development and evolution of specialized alveolar capillary cell types.

a, Plexus surrounding airways in immunostained E13.5 mouse lung. b, Sparsely labeled plexus cells in E12.5 Aplnr-CreER; Rosa26-Confetti lung immunostained for RFP. c, Near complete labeling of alveolar capillaries in Aplnr-CreER; Rosa26-tdTomato lung administered tamoxifen at E12.5. Endomucin, green; tdTomato, red. d, smFISH to detect tdTomato (white) in aCap (Apln, red) and gCap (Aplnr, green) cells in P60 Aplnr-CreER; Rosa26-tdTomato lung lineage labeled at E12.5. e, Clone in Aplnr-CreER; Rosa26-Confetti lung composed of aCap and gCap cells derived from a single YFP-expressing plexus cell labeled at E14.5. f, Schematics depicting the origin of both alveolar capillary cell types from single bipotent cells in the embryonic plexus. g, Individual aerocytes in Apln-CreER; Rosa26-Confetti lungs at indicated stages. Dots, pores. h, Quantification of individual cell volumes at indicated times (n>10 cells scored for each cell type at each time point from n=2 mice; see Methods for exact cell number; adult aCap and gCap cells from Fig. 2c). Bar, mean value. i, Quantification of Vwf-expressing capillary cells in lungs from 3- and 24-month old mice (mean± s.d.; n>500 cells scored per mouse; 3 mice per age group). j, smFISH for capillary cell type markers in alveolar capillary cells in adult human lung (75 year old male). k, Heatmap of expression of cell type markers in individual capillary cells. l, Human adenocarcinoma vessel containing cells co-expressing EDNRB (aCap marker) and PTPRB (gCap marker). See also Fig. ED9a. m-o, Dot plots showing expression in aCap and gCap cells for selected conserved genes (Type 0, leukocyte trafficking; m), human- and mouse-specialized genes (Type 1, hemostasis; n), or genes (Type 2, antigen presentation; o) which switch cell type between species (mouse, human). See Fig. ED10c-e. p, Schematic illustrating alligator lung faveolus. q, Faveolar capillary network in alligator lung immunostained for CLDN5. r, r’ Co-expression (asterisks) of mammalian alveolar capillary cell type markers EDNRB (aCap) and APLNR (gCap) in faveolar capillary cells (CLDN5, white) in alligator lung. Blue, DAPI (d, j, l, r) or elastin fibers (e, g). Scale bars, 10 μm.