Differential Biological Effects of Dietary Lipids and Irradiation on the Aorta, Aortic Valve, and the Mitral Valve

Figures

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  Figure 1. Effect of lard- and cholesterol-enriched diets on the aorta structure. (A) Representative pictures of aortic wall sections stained with hematoxylin-eosin, sirius red, Movat's pentachrome, and alizarin red, and CT imaging per rabbit group as indicated. Scale bars = 250 μm. Calcifications observed by CT are indicated by asterisks. (B) Percentage of rabbits from each group that presented CT-detected calcification in the different portions of the aorta (Ctrl, n = 13; Vit.D2, n = 7; CHT, n = 17; Lard, n = 13; IR, n = 7; IR/CHT, n = 15). I, indicates intima; M, indicates media.

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  Figure 2. Effect of lard- and cholesterol-enriched diets on the aortic valve leaflet histology. (A) Representative pictures of aortic valve leaflet sections stained with hematoxylin-eosin, sirius red, Movat's pentachrome, and alizarin red per rabbit group as indicated. Magnified areas depict neutrophil infiltrates in valvular ventricularis. Scale bars = 100 μm. (B) Violin plots represent aortic valve leaflet area measured on hematoxylin-eosin-stained aortic valve sections (each violin box represents one rabbit, 4–8 slides per rabbit). Four-group comparisons were performed by using a nested-GLM model and post-hoc SIDAK test on log-transformed data. (C) The graph represents the positive area for Alizarin Red staining on the aortic valve sections. Data are presented as medians with P25-P75 interquartiles. Four-group comparisons were performed by ANOVA and post-hoc Dunnett's test on log-transformed data (Left: Ctrl, n = 5; Vit.D2, n = 7; CHT, n = 7; Lard, n = 6; Right: Ctrl, n = 5; IR, n = 6; CHT, n = 4; IR/CHT, n = 6). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; #P ≤ 0.05; ##P ≤ 0.01; ###P ≤ 0.001. F, indicates fibrosa side; V, indicates ventricular side.

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  Figure 3. Effect of cholesterol-enriched diets on the mitral valve leaflet histology. (A) Representative pictures of mitral valve leaflet sections stained with hematoxylin-eosin, sirius red, and Movat's pentachrome per rabbit group as indicated. Magnified areas depict neutrophil infiltrates in valvular atrialis. Scale bars = 100 μm. (B) Violin plots represent mitral valve leaflet area measured on the histological mitral valve sections (each violin box represents one rabbit, 4–8 slides per rabbit) stained with hematoxylin-eosin. Four-group comparisons were performed by using a nested-GLM model and post-hoc SIDAK test on log-transformed data. P = non-significant. A, indicates atrial side; V, indicates ventricular side.

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  Figure 4. Immuno-detection of RAM-11- and αSMA positive cells in the aorta and heart valves. (A) Representative pictures of RAM-11 and αSMA immunofluorescence costaining performed on the aorta, the aortic valve, and the mitral valve sections. Nuclei are stained with DAPI and appear in blue. αSMA- and RAM-11-positive cells are shown in green and red, respectively. Scale bars = 250 μm for aorta and 100 μm for the aortic and mitral valves. (B) Quantification of αSMA-positive cells in terms of fluorescence area percentage in the aorta (left), aortic valve (middle), and the mitral valve (right). (C) Quantification of RAM-11-positive cells in terms of fluorescence area percentage in the aorta (left), aortic valve (middle), and the mitral valve (right). Data are presented as median with P25–P75 interquartiles. Four-group comparisons were performed by ANOVA and post-hoc Dunnett's test on log-transformed data. Aorta: left: Ctrl, n = 3; Vit.D2, n = 4; CHT, n = 6; Lard, n = 4; right: Ctrl, n = 3; IR, n = 5; CHT, n = 4; IR/CHT, n = 6. Aortic valve: left: Ctrl, n = 3; Vit.D2, n = 6; CHT, n = 6; Lard, n = 3; right: Ctrl, n = 3; IR, n = 5; CHT, n = 4; IR/CHT, n = 6. Mitral valve: left: Ctrl, n = 4;…

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  Figure 5. RNA sequencing (RNAseq) analysis of differentially expressed genes (DEGs) in the aorta, the aortic valve, and the mitral valve induced by cholesterol-enriched diet (CHT), alone or combined with irradiation (IR/CHT), and lard-enriched diet as compared to chow diet (Ctrl) (Ctrl, n = 7; CHT, n = 6; Lard, n = 7; IR/CHT, n = 7). Venn diagrams represent numbers of specific and common DEGs between tissue pairs for each comparison. Lollipop plots of gene ontology (GO) terms depict the top 10 biological processes affected by specific and common DEGs, with corresponding gene frequency and -log10 (P-value) (Wald test in DESeq2 package). Detailed statistical methods are provided in the Methods section. (A) Comparison of DEGs and affected biological processes in the aorta and aortic valve following 16-week treatment with a cholesterol-rich diet. (B) Comparison of DEGs and affected biological processes in the aortic and mitral valves following 16-week treatment with a cholesterol-rich diet. (C) Comparison of DEGs and affected biological processes in the aorta and aortic valve following 16-week treatment with a cholesterol-rich diet combined with irradiation. (D) Comparison of DEGs and affected…

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  Figure 6. Effect of cholesterol- and lard-enriched diets, and irradiation on the circulating biomarkers of calcification, platelet activation, inflammation, oxidative stress, and on circulating nucleosomes. (A) Bubble plot represents concentration fold-change of biomarkers of interest between week 16 (W16) and baseline. Over time evolution of neutrophil count. (B) 15-PGF2 (C) and circulating nucleosomes (D,E). Graphs represent median with P25-P75 interquartiles. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 (Wilcoxon signed-rank test; Ctrl, n = 7–13; Vit.D2, n = 7; CHT, n = 10–15; Lard, n = 7–12; IR, n = 7; IR/CHT, n = 15).