Liver-specific deletion of the Plpp3 gene alters plasma lipid composition and worsens atherosclerosis in apoE−/− mice

Figures

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  Figure 1. (A) Genotyping of the floxed and processed Plpp3 locus. A representative PCR screening is shown. Tail tips (A-B) and liver tissue (C-D) of Plpp3f/f apoE−/− Alb-Cre− mice show only the 235 bp band corresponding to the floxed, unprocessed Plpp3 locus. Plpp3f/f apoE−/− Alb-Cre+ mice also display the unprocessed Plpp3 locus band in the tail tips (E-F), but in the liver, both the unprocessed and processed allele (162 bp) bands are present (G-H). Liver PCR amplicons from a wild type Plpp3wt/wt mouse (I), Plpp3f/wt heterozygous mouse (L) and Plpp3f/f parent mouse (M) are shown as controls. Molecular weight marker, 100 bp New England Biolabs (N); (B) Quantitative expression of the Plpp3 mRNA 3′ end. Expression levels of Plpp3 mRNA are shown, quantified by qPCR analysis detecting a region downstream of the Cre recombinase-mediated floxed exons excision site. The values were normalized to the expression of the transcript in each corresponding tissue of the Plpp3f/f apoE−/− Alb-Cre− mice (n = 6 for liver, n = 3 for all the other tissues; *p = 0.0022 in Plpp3f/f apoE−/− Alb-Cre+ vs Plpp3f/f apoE−/− Alb-Cre− mouse liver by Wilcoxon rank-sum test). Wat = white adipose tissue; (C) Western…

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  Figure 2. Representative H&E photomicrographs and quantification of the maximum plaque area at the aortic sinuses in Plpp3f/f apoE−/− Alb-Cre+ and Plpp3f/f apoE−/− Alb-Cre− mice. The atherosclerosis development in the chow fed mice (n = 7–8) was comparable between the two genotypes (A,C,E). Western diet administration significantly worsened atherosclerosis development in Plpp3f/f apoE−/− Alb-Cre+ compared with Plpp3f/f apoE−/− Alb-Cre− mice (n = 16–17) (B,D,F). The data are shown as the mean ± SEM; *p = 0.031 vs Plpp3f/f apoE−/− Alb-Cre− by Wilcoxon rank-sum test. Bar length = 500 μm.

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  Figure 3. Histological and immunohistochemical characterization of plaques at the aortic sinuses in Plpp3f/f apoE−/− Alb-Cre− and Plpp3f/f apoE−/− Alb-Cre+ mice fed a Western diet. Representative photomicrographs of the necrotic core (A,B), macrophages (Mac-2 + cells; C,D), neutral lipids (Oil Red O; E,F), extracellular matrix (ECM; G,H), smooth muscle cells (αSMA + celIs; I,J) and CD3 + cells (K,L). Bar length = 200 μm. Asterisks mark the localization of the necrotic core (A,B).

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  Figure 4. Representative photomicrographs and quantification of plaque area detected by en-face analysis of the aortas of mice fed a Western diet. After 12 weeks of dietary treatment, whole aortas were collected and en-face analysis was performed to quantify the percentage of aortic surface covered by atherosclerotic plaques. Atherosclerotic plaque development in the arches (A) and in the thoracic segments (B) was significantly increased in the aortas from Plpp3f/f apoE−/− Alb-Cre+ mice (white dots) compared with Plpp3f/f apoE−/− Alb-Cre− mice (black dots). Atherosclerosis development at the abdominal segment (C) was not different between the two genotypes. Data are shown as the mean ± SEM; n = 7–8 mice per group, p = 0.0016 in the arch and p = 0.0016 in the thoracic segment vs Plpp3f/f apoE−/− Alb-Cre− mice by Wilcoxon rank-sum test.