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). Created with BioRender.com/d23b733. 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 (n=5). 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). F) Gene Set Enrichment Analysis (GSEA) of all significantly regulated transcriptomics and proteomics targets between donor PASMC and PAAF using the gene ontology (GO) database. Parent- to-root node visualization (intermediate terms omitted) with node size reflecting significance. Highlighted nodes depict significantly altered GO overview terms being higher expressed in 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.

Preserved lineages and distinct PASMC/PAAF 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) T score plots separating predictive variability (x-axis), attributed to biological grouping, and non-predictive variability (technical/inter-individual, y-axis). Monofactorial OPLS-DA model for separation according to cell type or disease. C) Bifactorial OPLS-DA model considering cell type and disease simultaneously. Ellipse depicting the 95% confidence region, Q2 denoting model’s predictive power (significance: Q2>50%) and R2Y representing proportion of variance in the response variable explained by the model (higher values indicating better fit). D) Immunoflourescent and in situ hybridization localization of PASMC (ACTA2, RGS5, IGFBP5) and PAAF markers (PDGFRA, ADH1C, CFD, SCARA5) in IPAH human lungs (n=5). White bar depicting 50 μm (5 μm for zoomed in panels). E) Dot plot showing relative expression of omics-identified PASMC/PAAF markers in published single cell transcriptomics dataset of fresh human pulmonary arteries of donors and IPAH patients (GSE210248).

Phenotypic correlates of PASMC/PAAF cell state in idiopathic pulmonary arterial hypertension (IPAH).

A) Top 5 biological process terms from the gene ontology enrichment analysis that are up- or downregulated in IPAH. B) 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. C) Representative Alcian blue (glycosaminoglycans – blue) with Verhoeff’s staining (elastic fibers – black/gray) of donor and IPAH lungs. Scale bar 100 µm. Annotated regions depicting adventitia (white), media (yellow), neointima (red). D) Quantitative image analysis of Alcian blue staining intensities in adventitial, medial and neointimal regions of pulmonary arteries from donors (n=10) and IPAH patients (n=10). 2-way ANOVA followed by Sidak’s multiple comparisons test (*p<0.05). E) Proliferative response of passage 3 cells measured by [3H]- thymidine incorporation assay upon serum stimulation. Dots represent mean values (n=5-6 donors/IPAH) with bars showing standard errors of mean. Interaction effects of stimulation, cell type, and disease state on cellular proliferation were analyzed by 3-way ANOVA. Significant interaction effects are indicated as follows: * for stimulation × cell type interactions and # for cell type × disease state interactions (both *, # p<0.05). F) Absolute cell counts measured after 24h growth. Mann-Whitney test, p<0.05. G) 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. H) Comparison of gene expression changes in IPAH cells compared to healthy state in three conditions (fresh - GSE210248; passage 1 - GSE255669; and passage 3 - GSE144274 or passage 6 - GSE144932). Dotplot of gene set enrichment analysis for selected biological process terms with dot size depicting absolute value of the normalized enrichment score (absNES), color intensity showing significance level (log10p) and color-coding for up- or downregulation of the biological process.

Mitochondrial dysfunction as an intersecting phenotypic characteristic of PASMC and PAAF in IPAH.

A) Euler 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) Scatter 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) Visualization of STRING-based interaction and regulatory network with gray nodes representing identified putative upstream regulators. D) Pathway analysis performed in Enrichr using the BioPlanet database with a matrix annotating the main genes involved with color coding reflecting the IPAH dependent regulation. 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. Cell numbers from each condition and cell type denoted by N on the graph (n=3 independent donors/IPAH) (E-G). H) Representative immunofluorescent localization of apoptotic cells (TUNEL marker). PASMC (ACTA2 marker) and PAAF (PDGFRA marker) in health (donor) and diseased (IPAH) lungs (n=6). DAPI nuclear counterstain, 25 μm scale bar (5 μm for zoomed in panel).

Cell type specific IPAH dependent transcriptomic and proteomic changes

A) Dot plot 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. Color depicting significantly regulated elements in omics dataset. D) Top 20 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) STRING physical interaction network for PASMC expressed receptors (red, ITGA2 and ITGB1) and PAAF expressed ligands (blue, COL3A1 and LAMA1). Edge thickness indicating strength of data support. C) Representative image of crystal violet-stained attached cells on collagen-I or laminin coated plates. 200 µm scale bar. D) Attachment assay for donor and IPAH PASMC and PAAF (n=5-6) 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) for each cell type. E) Gap closure assay on collagen-1 (donor/IPAH PASMC n=3/5, donor/IPAH PAAF n=5/6) or laminin-coated (donor/IPAH PASMC n=2/3, donor/IPAH PAAF n=6/6) plates. Mean values (presented as % of gap area) over time with standard error mean. F) Mean gene expression values in p1 cells of integrin subunits functioning as collagen and laminin receptors (GSE255669, Data S1). G) Relative expression of integrin subunits in published single cell transcriptomics dataset of fresh human pulmonary arteries of donors and IPAH patients (GSE210248). H) Versican gene expression in PASMC stimulated 24h with active complement components C3a or C5a (100 ng/mL). I) Decorin gene expression in PAAF stimulated 24h with active complement components C3a or C5a (100 ng/mL). Mann-Whitney test (*p<0.05).

Skewed expression profile of PASMC cell-state markers mediated by IPAH-dependent changes in PAAF ligands.

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. Created with BioRender.com/f24i687. D) Gene expression of health (calponin: CNN1, Ras Homolog Family Member A: RHOA, peroxisome proliferator-activated receptor gamma: PPARG, osteoprotegerin: TNRFSF11B) and disease (versican: VCAN, biglycan: BGN, cyclin D1: CCDN1) state phenotypic markers in donor PASMC (upper row) and donor PAAF (lower row) following their co-culture with IPAH cells. E) Top 10 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 cell state markers in donor and IPAH PASMC (n=6) treated 24h with pentraxin-3 (5 μg/mL) or hepatocyte growth factor (HGF, 25 ng/mL). Smooth muscle myosin heavy chain: MHY11. Friedman test followed by Dunn’s multiple comparisons test (*p<0.05).