Omics-assisted phenotypic characterization of cell states in healthy human pulmonary artery smooth muscle (PASMC) and adventitial fibroblast (PAAF) lineages. A) Schematic representation of the experimental setup using early passage cells (n=4). B) 3D score plots of principal component analyses (PCA), larger nodes represent gravity centers. C) Volcano plots of log2 fold change between donor PASMC and PAAF plotted against significance (-log10(P)). Genes names depicted for the top20 transcriptomics hits and proteins above the threshold (-log10(P)>1.3 │LFC│>1). D) Immunoflourescent localization of PASMC (ACTA2, RGS5) and PAAF markers (PDGFRA, ADH1C) in normal human lungs. E) In situ hybridization localization of PASMC (IGFBP5) and PAAF markers (CFD, SCARA5). DAPI as nuclear counterstaining. White bar depicting 50 μm (5 μm for zoomed in panels in D). F) Gene Set Enrichment Analysis (GSEA) of all significantly regulated transcriptomics and proteomics targets between donor PASMC and PAAF using the gene ontology (GO) dataset. Parent-to-root node visualization (intermediate terms omitted) with node size reflecting significance. Highlighted nodes depict significantly altered GO overview terms associated with either PASMC or PAAF. G) Top GO terms resulting from an Overrepresentation Analysis (ORA) of the omics dataset using more stringent cutoff values (-log10(P) >3 │LFC│>2 transcriptomics;-log10(P) >1.5 │LFC│>0.5 proteomics). H) Calculated change in basal oxygen consumption rate upon addition of UK5099 (glucose/pyruvate mitochondrial uptake inhibitor) and I) etomoxir (long chain fatty acid mitochondrial uptake inhibitor). Mann-Whitney test, p<0.05.

Defining PASMC and PAAF lineage cell states in idiopathic pulmonary arterial hypertension (IPAH). A) 3D score plots of principal component analyses (PCA), larger nodes represent gravity centers. B) Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) scores plots with 95% confidence intervals (elipse) for separation according to cell type (significance Q2>50%) or disease (non-significant). C) Bifactorial OPLS-DA model considering cell type and disease simultaneously. Ellipse denoting the 95% confidence region. D) Volcano plot showing the log2 fold changes between donor and IPAH in relation to the respective significances in PASMC and PAAF achieved via transcriptomic and proteomic analysis (threshold bar set at -log10(P)=1.3). E) Top5 biological process terms from the gene ontology enrichment analysis that are up- or downregulated in IPAH. F) Representative Alcian blue (glycosaminoglycans – blue) with Verhoeff’s staining (elastic fibers – black/gray) of donor and IPAH lungs. Scale bar 100 µm. G) Dimethylmethylene blue (DMMB) assay for quantification of glycosaminoglycan content in isolated pulmonary arteries from donor (n=7) or IPAH (n=8) patients. Mann Whitney test. H) Proliferative response of passage 3 cells measured by [3H]-thymidine incorporation assay upon serum stimulation. Dots represent a mean value (n=5-6 donors/IPAH) with corresponding standard error mean bars. Interaction significance (* for stimulation x cell-type; # for cell-type x disease state, *, # p<0.05.) calculated by 3-way ANOVA (stimulation, cell type and disease state). I) Absolute cell counts measured after 24h growth. Mann-Whitney test, p<0.05. J) Representative immunofluorescent localization of proliferating (PCNA marker) PASMC (ACTA2, yellow arrow) and PAAF (PDGFRA, white arrowhead) in health (donor) and diseased (IPAH) lungs. Immune cells were identified through CD45 expression. DAPI nuclear counterstain, 50 μm scale bar.

Mitochondrial dysfunction as an intersecting phenotypic characteristic of PASMC and PAAF in IPAH. A) Venn Diagram of the differentially expressed genes summarizing the overlaps and disjoints in regulation (criteria employed: -log10(P)>1.3; and LFC ±│1│for transcriptomics data and ±│0.5│for proteomics data). B) Dot plot graph of log2 fold changes between donor and IPAH in PASMC plotted against changes in PAAF, shaded areas highlighting commonly regulated genes/proteins. C) STRING-based interaction analysis vizualizing the network of shared interactors. D) Pathway analysis performed in Enrichr using the BioPlanet database with a matrix annotating the main genes involved. E) Mitochondrial content measurement using TMRM dye in complete or starvation (no serum) medium. F) TMRM dye (quench mode) based mitochondrial membrane potential measurement following starvation. G) Basal and PDGF-BB-stimulated reactive oxygen species production (ROS) (40minutes, 50 ng/mL) using CellRox DeepRed dye. Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Measurements done on 18-61 cells from each condition and cell type (n=3 independent donors/IPAH) (E-G).

Cell type specific IPAH dependent transcriptomic and proteomic changes A) Dotplot of log2 fold changes between donor and IPAH in PASMC plotted against changes in PAAF, shaded areas highlighting the inversely regulated genes/proteins (criteria employed: -log10(P)>1.3; and LFC ±│1│for transcriptomics data and ±│0.5│for proteomics data). B) A synoptic view of the network analysis performed with all uniquely regulated elements (as highlighted in A)), depicting their initial enrichment in donor cell type. C) Significant terms resulting from a gene ontology enrichment analysis of uniquely regulated elements represented as nodes connecting to related nodes, reflecting which cell type mostly contributed to the changes observed. D) Top20 most significantly regulated genes in IPAH PASMC and E) IPAH PAAF. Color coding reflects the IPAH dependent regulation, and initial enrichment in donor cell type. F) Circular heatmap of IPAH dependent regulation of metabolic, extracellular matrix, immune system and cell cycle elements in PASMC and PAAF. The two outer rings give information of direction and intensity of change in IPAH-PASMC (ring panel III) and IPAH PAAF (ring panel II), while the inner most ring (ring panel I) depicts initial cell-type enrichment at normal condition. Highlights (bold font) are given to significantly differing regulations between PASMC and PAAF under IPAH conditions. The functional association is performed based on data extracted from GeneCards, following a manual curation.

IPAH dependent changes mediated through cell type distinct extracellular matrix response. A) Ligand-receptor interaction analysis for PAAF and PASMC based on transcriptomic and proteomic data set. B) Representative image of crystal violet-stained attached cells on collagen-I or laminin coated plates. 200 µm scale bar. C) Attachment assay for donor and IPAH PASMC and PAAF on collagen-I and laminin coated plates (n=6 for each condition). 2-way ANOVA followed by Dunnett’s multiple comparisons test (*p<0.05). D) Gap closure assay on collagen-1 or laminin-coated plates. Mean values (presented as % of gap area) over time with standard error mean (n=6 for each condition). E) Versican gene expression in PASMC stimulated 24h with active complement components C3a or C5a (100 ng/mL). F) Decorin gene expression in PAAF stimulated 24h with active complement components C3a or C5a (100 ng/mL). Mann-Whitney test (*p<0.05).

IPAH dependent changes mediated through cell type distinct extracellular matrix response. A) Ligand-receptor interaction analysis for PAAF and PASMC based on single cell RNA sequencing dataset. B) Circos plot showing consensus soluble ligand and cognate receptor pairs identified by CellChat and NicheNet analysis of PAAF or PASMC as sender (ligand-expressing) cell type. C) Schematic representation of PASMC-PAAF co-culture experiment to determine the influence of IPAH cells on phenotypic marker expression in donor cells. D) Gene expression of normal (donor) and disease (IPAH) state phenotypic markers in donor PASMC and PAAF following their co-culture with either donor or IPAH cells. E) Top10 gene ontology biological processes enriched in the set of significantly upregulated genes in donor-PASMC co-cultured with IPAH-PAAF compared to reference (source-matched donor-PAAF). F) Representative protein array scans determining the content of soluble ligands secreted by donor or IPAH PAAF over 24 hours in cell culture medium. G) Heatmap of fold change expression of soluble ligands secreted by IPAH PAAF compared to donor PAAF (n=4 for each condition). H) Gene expression changes of contractile markers smooth muscle myosin heavy chain (MHY11), calponin (CNN1), and Ras Homolog Family Member A (RHOA) in PASMC treated 24h with pentraxin-3 (5 μg/mL) or hepatocyte growth factor (HGF, 25 ng/mL).