Pulmonary Arterial Hypertension Induces a Metabolic and Inflammatory Hepatopathy

Figures

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  Figure 1. snRNA-seq identified alterations in the cellular landscape in PAH, NASH, and FALD livers. (A) UMAP visualization of 8 cell types identified in PAH livers with unsupervised clustering. Validated marker genes used to identify cell types in PAH. UMAP and relative abundances of each cell type in control and PAH livers, with arrows indicating relative abundance change in PAH relative to control. (B) UMAP visualization of 6 cell types identified in NASH livers with unsupervised clustering. Validated marker genes used to identify cell types in NASH. UMAP and relative abundances of each cell type in control and NASH livers, with arrows indicating relative abundance change in NASH relative to control. (C) UMAP visualization of 6 cell types identified in FALD livers with unsupervised clustering. Validated marker genes used to identify cell types in FALD. UMAP and relative abundances of each cell type in control and FALD livers, with arrows indicating relative abundance change in FALD relative to control.

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  Figure 2. PAH hepatocyte metabolic alterations, pro-proliferative phenotype, and suppressed cell adhesion signaling were distinct from NASH and FALD. (A) UMAP visualization of PAH, NASH, and FALD hepatocyte clusters and respective controls. (B) Relative pathway activity displayed by violin plots of module scores calculated from relevant enriched/suppressed pathways in PAH, NASH, and FALD hepatocytes. Median values with interquartile range are displayed, and P values were calculated by either Student’s t test or Wilcoxon’s rank-sum test, as appropriate. (C) Top 5 most dysregulated cellular communication pathways based on total signaling strength across PAH, NASH, and FALD hepatocytes. Cell signaling pathways are ranked based on magnitude of the log2FC in PAH, with red bars indicating increased pathway signaling in PAH relative to control hepatocytes and blue bars indicating decreased pathways. No statistical test was applied; pathways are ranked by absolute log2FC. (D) Normalized protein abundances in control and PAH human livers across the WikiPathways aerobic glycolysis pathway and the KEGG fatty acid metabolism, oxidation by cytochrome P450, and HIF-1 signaling pathways. Median values…

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  Figure 3. PAH ECs exhibited Warburg-like metabolism and alterations in cell adhesion and barrier function pathways. (A) UMAP visualization of PAH, NASH, and FALD EC clusters and respective controls. (B) Relative pathway activity displayed by violin plots of module scores calculated from relevant enriched/suppressed pathways in PAH, NASH, and FALD ECs. Median values with interquartile range are displayed, and P values were calculated by either Student’s t test or Wilcoxon’s rank-sum test, as appropriate. (C) Top 5 most dysregulated cellular communication pathways based on total signaling strength across PAH, NASH, and FALD ECs. Cell signaling pathways are ranked based on magnitude of the log2FC in PAH, with red bars indicating increased pathway signaling in PAH relative to control ECs and blue bars indicating decreased pathways. No statistical test was applied; pathways are ranked by absolute log2FC.

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  Figure 4. Upregulated HIF-1 and PI3K/Akt signaling characterized HSC signaling in PAH. (A) UMAP visualization of PAH, NASH, and FALD HSC clusters and controls. (B) Relative pathway activity displayed by violin plots of module scores calculated from relevant enriched/suppressed pathways in PAH, NASH, and FALD HSCs. Median values with interquartile range are displayed, and P values were calculated by either Student’s t test or Wilcoxon’s rank-sum test, as appropriate. (C) Top 5 most dysregulated cellular communication pathways based on total signaling strength across PAH, NASH, and FALD HSCs. Cell signaling pathways are ranked based on magnitude of the log2FC in PAH, with red bars indicating increased pathway signaling in PAH relative to control HSCs and blue bars indicating decreased pathways. Pathways are ranked by absolute log2FC. (D) Representative images and quantification of median perivascular fibrosis (%) surrounding the central vein (CV) in control (n = 4) and PAH (n = 5) livers using trichrome staining (blue indicates fibrosis). 4–5 central veins were quantified per patient. Data are presented as median values with range, and P values were calculated using Mann-Whitney tests.…

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  Figure 5. Proinflammatory complement signaling with suppressed JAK/STAT signaling and cell–cell communication defined PAH macrophages. (A) UMAP visualization of PAH, NASH, and FALD macrophage clusters and respective controls. (B) Relative pathway activity displayed by violin plots of module scores calculated from relevant enriched/suppressed pathways in PAH, NASH, and FALD macrophages. Median values with interquartile range are displayed, and P values were calculated by either Student’s t test or Wilcoxon’s rank-sum test, as appropriate. (C) Top 5 most dysregulated cellular communication pathways based on total signaling strength across PAH, NASH, and FALD macrophages. Cell signaling pathways are ranked based on magnitude of the log2FC in PAH, with red bars indicating increased pathway signaling in PAH relative to control macrophages and blue bars indicating decreased pathways. No statistical test was applied; pathways are ranked by absolute log2FC. (D) Representative images and quantification of median CD206+ cells per area in control (n = 4) and PAH (n = 5) livers. 4 discrete images were quantified per section. Orange indicates CD206 staining, gray indicates wheat germ agglutinin, and…

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  Figure 6. PAH altered the expression of vasoactive peptides in multiple cell types, heightened IL-6 production in HSCs, and suppressed ketone metabolism in hepatocytes. (A) Relative log2FC in expression between control and PAH for relevant pro-proliferative and vasoactive genes across hepatocytes, ECs, and HSCs. P values were calculated using a Wilcoxon’s rank-sum test. Graphical representation of predicted consequences stemming from dysregulated expression of selected vasoactive genes in the liver on pulmonary vascular remodeling in PAH. (B) Relative log2FC in expression between control and PAH for IL-6 production across all liver cell types. P values were calculated using a Wilcoxon’s rank-sum test. (C) Relative ketone metabolism activity displayed by a violin plot of the hepatocyte module scores for the ketone metabolism pathway. Median values with interquartile range are displayed, and significance was assessed using a Wilcoxon’s rank-sum test.