Single Cell Multiomics Identifies Cells and Genetic Networks Underlying Alveolar Capillary Dysplasia

Abstract

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Keywords: FOXF1; alveolar capillary dysplasia; pulmonary microvasculature.

Figure 1.

Human lungs with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) with a spectrum of phenotype severity used for single-nucleus RNA and assay for transposase-accessible chromatin sequencing. (A) Lung histology of patient biopsy (ACD1) and explants (ACD2 and ACD3) was diagnostic of ACDMPV in all subjects and markedly differed from control lungs. ACDMPV lungs had deficient alveolar development with thickened alveolar septa containing a paucity of abnormally patterned capillaries (arrows) highlighted by PECAM1 (platelet and EC adhesion molecule 1) immunohistochemistry (IHC). Marked arterial hypertensive changes were present, with medial smooth muscle thickening of the small arteries (a) highlighted by ACTA2 (actin alpha 2, smooth muscle) IHC and pentachrome stains. ACDMPV lungs also had atypical placement of dilated veins (v) adjacent to arteries and bronchioles (b), as opposed to the veins’ being restricted to the pleura and interlobular septa in control lung (6 months of age). Explanted lungs from ACD2 and ACD3, who underwent transplantation at 3.5 years and 9 months of age, respectively, contained patchy ACDMPV features (ACD2, arrow) interspersed with more normally developed alveoli separated by thin septa containing capillaries immediately subjacent to the alveolar epithelium as seen in control lungs and highlighted by PECAM1 IHC (ACD2, arrowheads). Histology of ACD4 and ACD5 ACDMPV lungs at autopsy is shown in Figure E1. (B) FOXF1 (forkhead box F1) IHC showing diffuse alveolar septal (arrow), airway smooth muscle cell (arrowhead), arterial (a) and venous (v, inset) endothelial cell (EC) staining in control lung (6 months of age). FOXF1 expression was absent in arterial and venous ECs in ACDMPV lungs, with patchy alveolar septal (arrows) and airway smooth muscle cell staining (arrowhead). (C) Immunofluorescence staining for FN1 (fibronectin 1), PECAM1, and ACTA2 showing bronchovascular structures in control lung and misalignment of dilated veins (v) adjacent to bronchioles (b) and arteries (a), with hypertensive changes including thickened mural smooth muscle in ACDMPV lungs. Scale bars, 100 μm. (D) Deficient alveolar development and a paucity of alveolar septal capillaries in ACDMPV lungs compared with control lungs are highlighted by PECAM1 staining with diffuse deficiency of the capillary network in subject ACD1, with the most severe phenotype, and patchy areas with a more extensive capillary network in subject ACD2, with the least severe phenotype. Scale bars, 100 μm.

Figure 2.

Single-nucleus transcriptome atlas of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) and control human lungs. (A) UMAP embedding of snRNA-seq of nuclei (n = 65,809) from ACDMPV (n = 5), preterm neonates (n = 3; 1–4 d of age and 29–31 wk of gestational age), and control lungs (n = 3; 3 yr old). (B) Dot plot of average expression amounts and expression percentage of FOXF1 RNA in each cell type in each lung. Expression with percentage ⩾2% is shown. (C) Dot plot of RNA expression of marker genes in single-nucleus RNA–identified cell types in A. Gene expression with percentage ⩾5% is shown. ABCA3 = ATP binding cassette subfamily A member 3; ACKR1 = atypical chemokine receptor 1; AEC = arterial endothelial cell; AF1 = alveolar fibroblast 1; AF2 = alveolar fibroblast 2; AGER = advanced glycosylation end-product specific receptor; AM = alveolar macrophage; ASCL1 = achaete-scute family BHLH transcription factor 1; ASMC = airway smooth muscle cell; AT1 = alveolar type 1 cell; AT2 = alveolar type 2 cell; CAP1 = capillary 1 cell; CAP2 = capillary 2 cell; CCL21 = C-C motif chemokine ligand 21; CCR7 = C-C motif chemokine receptor 7; CD96 = cluster of differentiation 96; cDC1 = classical dendritic cell subset 1; cDC2 = classical dendritic cell subset 2; CLDN4 = claudin 4; CLEC = C-type lectin domain containing; COL15A1 = collagen type XV α 1 chain; COL2A1 = collagen type II α 1 chain; DC = dendritic cell; DKK2 = Dickkopf WNT signaling pathway inhibitor 2; F13A1 = coagulation factor XIII A chain; FCGR3A = Fc gamma receptor IIIa; FCN = ficolin; FOXF1 = forkhead box F1; FOXJ1 = forkhead box J1; GNLY = granulysin; HBB = hemoglobin subunit β; HPGD = 15-hydroxyprostaglandin dehydrogenase; IM = interstitial macrophage; iMON = inflammatory monocyte; ITGBL1 = integrin subunit β like 1; LAMC3 = laminin subunit gamma 3; LEC = lymphatic endothelial cell; LGR6 = leucine rich repeat–containing G 55protein–coupled receptor 6; maDC = mature dendritic cell subset; MARCO = macrophage receptor with collagenous structure; MFAP5 = microfibril associated protein 5; MS4 = membrane spanning 4-domains; MyoFB = myofibroblast; MZB1 = marginal zone B and B1 cell-specific protein; NK = natural killer; NTRK3 = neurotrophic receptor tyrosine kinase 3; pDC = plasmacytoid dendritic cell; pMON = patrolling monocyte; PNEC = pulmonary neuroendocrine cell; RASC = respiratory airway secretory cell; SCGB3A = secretoglobin family 3A; snRNA-seq = single-nucleus RNA sequencing; SVEC = systemic vascular endothelial cell; TCF21 = transcription factor 21; TP53 = tumor protein P53; UMAP = uniform manifold approximation and projection; VEC = venous endothelial cell; VSMC = vascular smooth muscle cell.

Figure 3.

Cell and gene expression changes in lung endothelial cells (ECs) in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). (A) Uniform manifold approximation and projection embedding of ECs from snRNA-seq of ACDMPV (n = 5), control (n = 3; 3 yr old), and preterm neonate (n = 3; 1–4 d of age and 29–31 wk of GA) lungs. Cells are colored by the predicted EC types. (B) Dot plot of expression of marker genes for EC types. Gene expression with percentage ⩾5% is shown. (C) Changes in EC proportions in snRNA-seq of ACDMPV compared with control infant or preterm neonate lungs. P value represents the significance of the difference in cell proportions using a two-tailed Wilcoxon rank sum test. (D) Expression of FOXF1 and KIT RNA in capillary 1 (CAP1) cells of individual donors. Gene expression with percentage ⩾1% is shown. (E and F) RNAscope analysis validating the decrease in HPGD/cadherin 5 (CDH5) coexpressing capillary 2 (CAP2) cells in ACDMPV (n = 6) (ACD1 shown) versus control (n = 7) (1-day-old shown) lungs (E) and increase in COL15A1/CDH5 SVECs (F) in ACDMPV (n = 5) (ACD3 shown) versus control (n = 4) (13-month-old shown) lungs. Scale bars, 5 μm. (G) Ingenuity Pathway Analysis identified pathways significantly associated with genes downregulated in CAP1 (left) or in CAP2 (right) cells in snRNA-seq of ACDMPV versus control lungs. Shown are the top 10 signaling pathways ranked by P value. Downregulated genes satisfied the following criteria: differentially expressed in ACDMPV versus control snRNA cells (P < 0.05, fold change ⩾ 1.5, and expression percentage ⩾ 20%), selectively expressed in the selected cell type in either ACDMPV or control lung snRNA-seq data (P < 0.05, fold change ⩾ 1.2, and expression percentage ⩾ 10%), and not a mitochondrial gene. The two-tailed Wilcoxon rank sum test was used for differential expression analysis. Boxplots represent 25%, 50%, and 75% quantiles. ABCB1 = ATP binding cassette subfamily B member 1; ACKR1 = atypical chemokine receptor 1; AEC = arterial endothelial cell; CA4 = carbonic anhydrase 4; CCL21 = C-C motif chemokine ligand 21; CLDN5 = claudin 5; COL15A1 = collagen type XV α 1 chain; CPE = carboxypeptidase E; DKK2 = Dickkopf WNT signaling pathway inhibitor 2; EDNRB = endothelin receptor type B; ERK = extracellular signal-related kinase; FAK = focal adhesion kinase; FCN3 = ficolin 3; FOXF1 = forkhead box F1; GA = gestational age; GJA5 = gap junction protein α 5; GTPase = guanosine triphosphatase; HPGD = 15-hydroxyprostaglandin dehydrogenase; ID1 = inhibitor of DNA binding 1; KIT = KIT proto-oncogene, receptor tyrosine kinase; LEC = lymphatic endothelial cell; MAPK = mitogen-activated protein kinase; PECAM1 = platelet and endothelial cell adhesion molecule 1; PROX1 = Prospero homeobox 1; PTEN = phosphatase and tensin homolog; snRNA-seq = single-nucleus RNA sequencing; SLC6A4 = solute carrier family 6 member 4; STAT3 = signal transducer and activator of transcription 3; SVEC = systemic vascular endothelial cell; VEC = venous endothelial cell.

Figure 4.

Cell and gene expression changes in lung mesenchymal cells in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). (A) Changes in mesenchymal cell type proportions using snRNA-seq of ACDMPV (n = 5), control (n = 3; 3 yr old), and preterm neonate (n = 3; 1–4 d of age and 29–31 wk of gestational age) lungs. P value represents the significance of the difference in cell proportions using a two-tailed Wilcoxon rank sum test. (B) Gene expression changes between ACDMPV and control lungs in pericytes in snRNA-seq. The criteria were as follows: differentially expressed in ACDMPV (P < 0.05, fold change ⩾ 1.5, and expression percentage ⩾ 20%) and selectively expressed in pericytes in either ACDMPV or control lungs (P < 0.05, fold change ⩾ 1.2, and expression percentage ⩾ 10%). A two-tailed Wilcoxon rank sum test was used. (C) Differential expression of FGF7, HGF, and T-box transcription factors in ACDMPV AF1 cells and pericytes compared with control or preterm neonate lungs. The asterisk denotes gene expression changes satisfying the following criteria: Bonferroni-adjusted P < 0.1, expression percentage ⩾ 20%, and fold change of average expression or expression percentage ⩾ 1.5. Boxplots represent 25%, 50%, and 75% quantiles. AF1 = alveolar fibroblast 1; AF2 = alveolar fibroblast 2; ASMC = airway smooth muscle cell; DEG = differentially expressed gene; FGF7 = fibroblast growth factor 7; HGF = hepatocyte growth factor; LAMC3 = laminin subunit gamma 3; MyoFB = myofibroblast; NR3C1 = nuclear receptor subfamily 3 group C member 1; snRNA-seq = single-nucleus RNA sequencing; TBX = T-box transcription factor; TJP1 = tight junction protein ZO-1; VSMC = vascular smooth muscle cell.

Figure 5.

Impaired distal epithelial cell differentiation in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) lungs. (A) Increased overall epithelial cell proportions in ACDMPV lungs. (B) Comparison of epithelial cell proportions in ACDMPV (ACD1, ACD4, and ACD5), control (n = 3; 3 yr old), and preterm neonate (n = 3; 1–4 d of age and 29–31 wk of gestational age) lungs. Cell proportions were calculated using the snRNA-seq data. P values represent the significance of the difference using a two-tailed Wilcoxon rank sum test. Figure E9 shows comparisons using snRNA-seq from all five ACDMPV lungs. Boxplots represent 25%, 50%, and 75% quantiles. (C) RNA velocity analysis of differentiation relationships among alveolar epithelial cells from snRNA-seq of ACD1 lung. (D) Left: dot plot of expression of alveolar type 1 (AT1), alveolar type 2 (AT2), and damage-associated transient progenitor cell markers in AT1, AT2, and AT1/AT2 transitional (AT1/AT2) cells from snRNA-seq of ACDMPV lungs (n = 5). Gene expression with percentage ⩾5% is shown. Right: violin plot of KRT8 and CLDN4 in AT1, AT2, and AT1/AT2 cells from snRNA-seq of ACDMPV lungs (n = 5). (E) Immunofluorescence confocal microscopy of control lung (6-mo-old) and ACDMPV lungs demonstrating loss of mature AT1 and increased HOPX/SFTPC coexpressing AT1/AT2 cells in ACDMPV. Lungs from subjects with the most severe phenotype showed diffuse loss of mature AT1 cells and increased coexpressing AT1/AT2 cells (ACD1). Lungs from subjects with less severe phenotypes had decreased mature AT1 cells with markedly increased AT1/AT2 cells in regions with deficient microvasculature (ACD3) alternating with rare AT1/AT2 cells in regions with preserved capillary development in the subject with the least severe phenotype (ACD2), who received a lung transplant at 3.5 years of age. Scale bars, 40 μm (upper panel) and 20 μm (lower panel). ABCA3 = ATP binding cassette subfamily A member 3; AGER = advanced glycosylation end-product specific receptor; CLDN4 = claudin 4; HOPX = HOP homeobox; KRT8 = keratin 8; LAMP3 = lysosomal associated membrane protein 3; PDPN = podoplanin; PECAM1 = platelet and endothelial cell adhesion molecule 1; PNEC = pulmonary neuroendocrine cell; RASC = respiratory airway secretory cell; RTKN2 = rhotekin 2; SFTPC = surfactant protein C; snRNA-seq = single-nucleus RNA sequencing; UMAP = uniform manifold approximation and projection.

Figure 6.

Multiomic prediction of cell type–specific FOXF1 regulatory targets. (A) Predicted FOXF1 regulatory targets in capillary 1 (CAP1) cells, capillary 2 (CAP2) cells, and pericytes in normal human lung. The prediction was performed using PECA2, integrating cell type–specific snRNA-seq and snATAC-seq data from control lungs (n = 3; 3 yr old). Shown are targets that satisfied the following criteria: false discovery rate < 0.001 of P value of regulatory potential in at least two snRNA–snATAC sample pairs and selectively expressed (P < 0.05, fold change (FC) ⩾ 1.2, and percentage ⩾ 10%) in the corresponding cell type in snRNA-seq of control lungs. Visualization was generated using the “Interaction Network” function of ToppCluster (https://toppcluster.cchmc.org) using the predicted targets as input gene sets. (B) The predicted regulatory targets overlapped among the three cell types. (C) Expression of BTNL9 (butyrophilin like 9) in snRNA-seq of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) versus control lung (top) and chromatin accessibility near BTNL9 in CAP1 and CAP2 cells in snATAC-seq of control lungs (bottom). (D) Chromatin immunoprecipitation sequencing (ChIP-seq) analysis demonstrated reproducible FOXF1 binding sites (IDR < 0.05) near Btnl9 in E18.5 wild-type mouse lung. (E) Predicted FOXF1 regulatory targets that have nearby mouse lung FOXF1 ChIP-seq binding sites that overlapped with evolutionarily conserved regions (placental mammal base-wise conservation by phyloP). Positive scores (blue) denote sites predicted to be conserved, and negative scores (red) denote sites predicted to be fast evolving. Shown are TBX2 and PTN. An extended list is shown in Figure E11. (F) Metascape analysis of biological processes and pathways significantly associated with the predicted FOXF1 regulatory targets. Shown are the top 20 functional annotation clusters identified. (G) Differential expression of FOXF1 regulatory targets in CAP1 and CAP2 cells and pericytes in ACDMPV versus control lungs. The criteria were as follows: P value of two-tailed Wilcoxon rank sum test < 0.05, FC ⩾ 1.5, and expression percentage ⩾ 20%. A2M = alpha-2-macroglobulin; ADCY4 = adenylate cyclase 4; AEC = arterial endothelial cell; AF1 = alveolar fibroblast 1; AF2 = alveolar fibroblast 1; AM = alveolar macrophage; ARHGAP29 = Rho GTPase activating protein 29; ARRDC2 = arrestin domain containing 2; ASMC = airway smooth muscle cell; AT1 = alveolar type 1 cell; AT1/AT2 = AT1/AT2 transitional cell; AT2 = alveolar type 2 cell; CACNA1H = calcium voltage-gated channel subunit alpha1 H; cCRE = candidate cis-regulatory element; CD93 = cluster of differentiation 93; cDC1 = classical dendritic cell subset 1; cDC2 = classical dendritic cell subset 2; chr = chromosome; COL4A = collagen type IV α; CYP3A5 = cytochrome P450 family 3 subfamily A member 5; DPYSL3 = dihydropyrimidinase like 3; E18.5 = Embryonic Day 18.5; EEPD1 = endonuclease/exonuclease/phosphatase family domain containing 1; ENCODE = Encyclopedia of DNA Elements; EPB41L2 = erythrocyte membrane protein band 4.1 like 2; ERG = ETS transcription factor ERG; ETS1 = ETS proto-oncogene 1, transcription factor; FANCC = FA complementation group C; FIGN = fidgetin, microtubule severing factor; GALNT16 = polypeptide N-acetylgalactosaminyltransferase; GFOD1 = glucose-fructose oxidoreductase domain containing 1; GO = Gene Ontology; GTPase = guanosine triphosphatase; HMBOX1 = homeobox containing 1; IDR = irreproducible discovery rate; IM = interstitial macrophage; ITGA = integrin subunit α; JAM3 = junctional adhesion molecule 3; KDR = kinase insert domain receptor; LEC = lymphatic endothelial cell; MAPRE2 = microtubule associated protein RP/EB family member 2; MKLN1 = muskelin 1; MOCS1 = molybdenum cofactor synthesis 1; MyoFB = myofibroblast; NEDD4L = NEDD4 like E3 ubiquitin protein ligase; NK = natural killer; NRG3 = neuregulin 3; NRP1 = neuropilin 1; OBSCN = obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF; PALD1 = phosphatase domain containing paladin 1; PALMD = palmdelphin; pct = percentage; pDC = plasmacytoid dendritic cell; PDLIM1 = PDZ and LIM domain 1; PHLDB1 = pleckstrin homology like domain family B member 1; PITPNM2 = phosphatidylinositol transfer protein membrane associated 2; PNEC = pulmonary neuroendocrine cell; PRICKLE2 = prickle planar cell polarity protein 2; PTN = pleiotrophin; SH3RF3 = SH3 domain containing ring finger 3; SLCO2A1 = solute carrier organic anion transporter family member 2A1; SMURF2 = SMAD specific E3 ubiquitin protein ligase 2; snATAC-seq = single-nucleus assay for transposase-accessible chromatin sequencing; snRNA-seq = single-nucleus RNA sequencing; STXBP6 = syntaxin binding protein 6; SVEC = systemic vascular endothelial cell; TBX2 = T-box transcription factor 2; TEK = TEK receptor tyrosine kinase; TGFB2 = transforming growth factor β 2; TMOD3 = tropomodulin 3; TOX2 = TOX high mobility group box family member 2; VEC = vascular endothelial cell; VSMC = vascular smooth muscle cell; WWTR1 = WW domain containing transcription regulator 1; ZMIZ1 = zinc finger MIZ-type containing 1; ZNF704 = zinc finger protein 704.

Figure 7.

Alterations in alveolar niche cell–cell communications in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) lungs. (A) CellChat analysis predicted alterations in ligand receptor–based cell–cell communications among alveolar cell types in ACDMPV (middle) compared with control (left; 3 yr old) and preterm neonate (right; 1–4 d of age and 29–31 wk of gestational age) lungs. Edge thickness is proportional to the number of CellChat-inferred ligand–receptor interactions for each cell–cell pair. Shown are edges with the top 50% number of interactions. Edge color represents the cell type expressing the ligands. Node size is proportional to the number of cells in a cell type. (B) Relative contributions of outgoing signaling by each cell type in ACDMPV, control, and preterm neonate lungs. The relative contribution of a cell type was calculated as the total number of outgoing signals from the cell type divided by the total number of outgoing signals from the cell types of the same lineage. (C) Top: CellChat analysis predicted the loss of VEGFA (vascular endothelial growth factor A)–VEGFR2 (vascular endothelial growth factor receptor 2) signaling from alveolar type 1 (AT1) to capillary 2 (CAP2) cells and an increase in VEGFA–VEGFR2 signaling from AT1 and AT1/AT2 to SVECs in ACDMPV lungs. Node color represents normalized communication probability calculated using CellChat. Label colors represent cell types in ACDMPV (red) or in control (blue) lung. Bottom: differential expression of VEGFA in epithelial cells and KDR in endothelial cells in snRNA-seq of ACDMPV versus control lungs. The asterisk represents gene expression changes satisfying the following criteria: P value of two-tailed Wilcoxon rank sum test < 0.05, expression percentage ⩾ 20%, and fold change of average expression or expression percentage ⩾ 1.5. (D) CellChat analysis predicted an increase in FGF (fibroblast growth factor) signaling from AF1 cells to alveolar epithelial cells in ACDMPV lungs. Top: FGF signaling among alveolar cell types in the control, ACDMPV, and preterm lungs. Edge thickness is proportional to the normalized aggregated communication probabilities of significant FGF ligand–receptor interactions for a cell–cell pair. Edges are from source to target cells. Bottom: significant (P < 0.01) FGF ligand–receptor interactions inferred by CellChat using snRNA-seq of control, ACDMPV, and preterm lungs. Node color represents normalized communication probability. AF1 = alveolar fibroblast 1; AF2 = alveolar fibroblast 2; AT1/AT2 = AT1/AT2 transitional cell; AT2 = alveolar type 2 cell; CAP1 = capillary 1 cell; FGFR=fibroblast growth factor receptor; KDR = kinase insert domain receptor; snRNA-seq = single-nucleus RNA sequencing; SVEC = systemic vascular endothelial cell.

Figure 8.

Schematic showing alveolar structure and cell–cell communications in normal human lung and alterations in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). FOXF1 (forkhead box F1) is expressed in pulmonary mesenchymal cells, including fibroblasts, pericytes, and endothelial cell (EC) progenitors. FOXF1 was required for the differentiation or survival of endothelial and fibroblast progenitors, which in turn influence the growth and differentiation of the pulmonary epithelial progenitors during the formation of the peripheral lung. FOXF1 deficiency in ACDMPV disrupted gene expression in EC progenitors, preventing differentiation or survival of CAP2 ECs, critical for the formation of the alveolar gas exchange, resulting in hypoxemia at birth. Increased expression of VEGFA by atypical alveolar epithelial progenitors (AT1/AT2) is likely to enhance the proliferation and migration of the systemic vasculature (SVECs), a characteristic of ACDMPV. aCAP = aerocyte capillary cell; ACD = alveolar capillary dysplasia; AF1 = alveolar fibroblast 1; AF2 = alveolar fibroblast 2; AT1 = alveolar type 1 cell; AT1/AT2 = AT1/AT2 transitional cell; AT2 = alveolar type 2 cell; CAP1 = capillary 1; CAP2 = capillary 2; FGFR = fibroblast growth factor receptor; gCAP = general capillary; KDR = kinase insert domain receptor; SVEC = systemic vascular endothelial cell; TBX = T-box transcription factor; VEGFA = vascular endothelial growth factor A.