A Translational Mouse Model for NASH with Advanced Fibrosis and Atherosclerosis Expressing Key Pathways of Human Pathology

Figures

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  Figure 1. Representative images of perigonadal white adipose tissue (WAT) cross-sections (A) stained with hematoxylin–phloxine–saffron (HPS) and WAT weight of different adipose tissue depots (B) from Ldlr−/−.Leiden mice fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat for 28 weeks. Perigonadal WAT inflammation was analyzed by measuring the number of crown-like-structures (CLS)/mm2 (C). Values represent mean ± SEM for ≥8 mice per group. ** p < 0.01 and *** p < 0.001 vs. chow; # p < 0.05 and ### p < 0.001 FFD vs. HFD.

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  Figure 2. Representative images of liver cross-sections stained with hematoxylin and eosin (H&E) or Sirius Red (A) and quantitative analysis (B–F) from Ldlr−/−.Leiden mice fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat for 28 weeks. Macrovesicular (B) and microvesicular (C) steatosis as a percentage of total liver area, inflammation as number of inflammatory aggregates per mm2 microscopic field (D), and hepatic fibrosis were analyzed as percentage Sirius Red of surface area (E) or biochemically analyzed hepatic collagen/mg protein (F). Values represent mean ± SEM for ≥8 mice per group. ** p < 0.01 and *** p < 0.001 vs. chow; # p < 0.05, ## p < 0.01 and ### p < 0.001 FFD vs. HFD.

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  Figure 3. Quantitative analysis of non-alcoholic steatohepatitis (NASH) from Ldlr−/−.Leiden mice fed a healthy chow diet for 28 weeks or fed a fast food diet (FFD) containing milk fat for 12, 22, or 28 weeks. Data of FFD for 12 weeks were obtained by taking a liver biopsy of the mice that continued on the FFD for 22 weeks and are from a separate group of animals than mice that were fed the different diets for 28 weeks. In addition, mice were fed the FFD and treated with a relatively low dose of obeticholic acid (OCA) (10 mg/kg/d) from week 18 to 28. Macrovesicular (A) and microvesicular (B) steatosis as the percentage of total liver area, inflammation as the number of inflammatory aggregates per mm2 microscopic field (C), and hepatic fibrosis as the percentage of Sirius Red of surface area (D) were analyzed. Values represent mean ± SEM for ≥8 mice per group. ** p < 0.01 and *** p < 0.001 vs. chow or * p < 0.05 FFD + OCA vs. FFD t = 28 (as indicated by bar).

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  Figure 4. Venn diagram (A) illustrating the overlap of differentially expressed pathways (pathway pval < 0.01) of livers between Ldlr−/−.Leiden mice fed a high-fat diet (HFD) vs. healthy chow diet (blue circle) or fed a fast food diet (FFD) vs. healthy chow diet (green circle) for 28 weeks. N ≥ 8 mice per group. The shared top 10 of significantly enriched biological processes (−log (p-value)) for the genes affected by HFD (blue bars) and FFD (green bars) vs. chow (B). Differences between HFD and FFD in downregulation of ATP synthases in complex V of oxidative phosphorylation (C). Quantification of 4-hydroxynonenal (4-HNE) marker of oxidative stress using stained liver sections ** p < 0.01 and *** p < 0.001 vs. chow or * p < 0.05 FFD + OCA vs. FFD t = 28 (as indicated by bar) (D). Effect of HFD and FFD on different inflammatory pathways, expressed as median (2logR) of all genes in the listed inflammatory pathway (E). Heatmap showing expression of genes differentially regulated in human non-alcoholic steatohepatitis (NASH) vs. normal controls [6] in Ldlr−/−.Leiden mice fed the HFD or FFD for 28 weeks relative to chow (F). Blue color indicates downregulation, and red color indicates…

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  Figure 5. Representative images of atherosclerotic plaques in aortic root section (A) stained with hematoxylin–phloxine–saffron (HPS) from Ldlr−/−.Leiden mice fed a healthy chow diet for 28 weeks or fed a high-fat diet (HFD) for 28 weeks or fed a fast food diet (FFD) for 22 or 28 weeks. In addition, mice were fed the FFD and supplemented with a relatively low dose of OCA (10 mg/kg/d) from weeks 18 to 28. Total lesion area per cross-section was quantified (B) and lesion severity (C) was assessed, categorized as mild lesions (type I–III) and severe lesions (IV–V). Values represent mean ± SEM for ≥8 mice per group. ** p < 0.01 and *** p < 0.001 vs. chow.