Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension

Figures

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  Figure 1. Pulmonary vein banding causes PH. (A) Angiogram of the inferior venous confluence of a representative Cntrl (top) and PVB animal (bottom). (B,C) Picrosirius red staining of the lung upper and lower lobe in Cntrl and PVB animals. (D–F) Mean pulmonary artery pressure (mPAP), total pulmonary vascular resistance index (tPVRi) and pulmonary artery compliance in awake, resting swine. PA—pulmonary artery, PV—pulmonary vein, Br—bronchiole. Values are means ± SEM. * p ≤ 0.05, *** ≤0.001, **** ≤0.0001. Scale bar: 100 μm. PVB vs. Cntrl by Student’s t-test, Cntrl n = 7; PVB n = 6.

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  Figure 2. Proteomic analysis of lung tissue of PVB vs. Cntrl animals. (A,B) Principal component analysis (PCA) of proteome profiles from upper (A) and lower (B) lobe of Cntrl (n = 7) and PVB animals (n = 6). Colored circles indicate individual animals. Red-filled circles are Cntrl and blue-filled circles are PVB animals.

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  Figure 3. Quantitative and functional analysis of upper and lower lobe of PVB vs. Cntrl animals. (A,B) The quantitative proteome alterations are visualized via volcano plots in upper and lower lobes, respectively, from PVB vs. Cntrl pigs. Color-filled circles (blue—upregulated, red—downregulated) indicate differentially abundant proteins (Benjamini–Hochberg corrected p-value ≤ 0.05 and fold change ≥ 1.5). (C) Functional characterization of differences between upper and lower lobes in PVB and Cntrl animals. Heatmap shows GO-term enrichment in PVB compared to Cntrl. Cntrl (n = 7); PVB (n = 6).

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  Figure 4. Metabolomic analysis in the upper and lower lobe of PVB vs. Cntrl animals. (A,B) Three-dimensional PCA analysis based on metabolic profiles of PVB and Cntrl animals in the upper and lower lobe in negative mode (HILIC). (C,D) OPLS-DA analysis based on metabolic profiles of PVB and Cntrl animals in upper and lower lobe in negative mode (HILIC). n = 6 in each group.

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  Figure 5. Metabolomic analysis in the upper lobe of PVB vs. Cntrl animals. (A) Metabolites ranked by Variable importance in the projection score (VIP) > 1 in PVB vs. Cntrl pigs in upper lobes based on OPLS-DA analysis. (B) Pathway analysis based on KEGG database of the enriched metabolites (VIP > 1 and p-value < 0.05 in the upper lobe of PVB vs. Cntrl swine. n = 6 in each group. The size of the dots represents the enrichment ratio and the shade of the color represent the −log 10 (p value).

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  Figure 6. Interaction network analysis of the metabolites and proteins from metabolomics and proteomics. (A) Metabolite–metabolite interaction network for the most enriched metabolites in the upper lobe of PVB vs. Cntrl. Labeled and red-filled circles represent the upregulated metabolites, and green-filled circles represent the downregulated metabolites detected in the PVB group. Metabolites clustered in green background represent metabolites participating in fatty acid metabolism, red background—ROS pathway; blue—ECM remodeling and yellow—glucose metabolism. (B) Gene–metabolite network analysis for the significantly altered proteins and enriched metabolites for upper lobe of PVB vs. Cntrl. The red-filled squares represent the downregulated proteins, and green-filled circles represent the upregulated metabolites detected in the PVB group. Metabolites of VIP > 1 with p-value < 0.05 and proteins with fold change > 1.3 and Benjamini–Hochberg-adjusted p-value < 0.05 were used for the analysis. The proteins and metabolites belonging to one GO term are encircled together.

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  Figure 7. Fatty acid uptake pathway in the upper lobe at the protein and mRNA level. (A) Protein abundance of apolipoproteins that were significantly changed (Benjamini–Hochberg corrected p-value ≤ 0.05) between PVB compared to Cntrls in the upper and lower lobes are depicted as dots. n = 6 per group. (B–E) mRNA level of APOA, APOE, CD36 and LDLR, respectively, are expressed relative to the mean expression of the sham animals (values are mean +/− SEM. * p < 0.05, ** p < 0.01 Students’s t-test n = 5 animals per group).

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  Figure 8. Schematic illustration of the metabolic pathways altered in PVB. The blue-labeled metabolites and proteins are differentially expressed in the upper lobe, which is under high shear stress, and the green-labeled ones are differentially expressed in the lower lobe, which is under low shear stress. Pathways or metabolites/proteins labeled in red are found to be altered in both the lobes. 3PG—3-phosphoglyceric acid, SAM—S-adenosyl methionine, SAH—S-adenosylhomocysteine, NO—nitric oxide, PC—phosphatidyl choline, OAA—oxaloacetic acid.