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. 2013 Dec 12;5(5):1436-42.
doi: 10.1016/j.celrep.2013.11.009. Epub 2013 Dec 5.

Testing the Iron Hypothesis in a Mouse Model of Atherosclerosis

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Free PMC article

Testing the Iron Hypothesis in a Mouse Model of Atherosclerosis

Léon Kautz et al. Cell Rep. .
Free PMC article

Abstract

Hepcidin, the iron-regulatory hormone and acute phase reactant, is proposed to contribute to the pathogenesis of atherosclerosis by promoting iron accumulation in plaque macrophages, leading to increased oxidative stress and inflammation in the plaque (the "iron hypothesis"). Hepcidin and iron may thus represent modifiable risk factors in atherosclerosis. We measured hepcidin expression in Apoe(-/-) mice with varying diets and ages. To assess the role of macrophage iron in atherosclerosis, we generated Apoe(-/-) mice with macrophage-specific iron accumulation by introducing the ferroportin ffe mutation. Macrophage iron loading was also enhanced by intravenous iron injection. Contrary to the iron hypothesis, we found that hepatic hepcidin expression was not increased at any stage of the atherosclerosis progression in Apoe(-/-) or Apoe/ffe mice and that the atherosclerotic plaque size was not increased in mice with elevated macrophage iron. Our results strongly argue against any significant role of macrophage iron in atherosclerosis progression in mice.

Figures

Figure 1
Figure 1. Saa3 mRNA, hepcidin mRNA and liver iron analysis in atherosclerotic mice
Wild-type, Apoe−/−ffe and Apoe/ffe mice were fed for 4 months with either a standard chow (300 ppm Fe, grey bars) or a high-fat diet (300 ppm Fe, black bars). Serum amyloid A-3 (Saa3) (A) and hepcidin (Hamp) (B) mRNA levels were measured by qRT-PCR. Values shown are means of −ΔCt (i.e., Ct Rpl4 – Ct Hamp or Saa3) ± standard deviation. (C) Liver non-heme iron content (mean ± standard deviation). For each gender and diet, statistical analysis was done to evaluate the effect of atherosclerotic genotype: Apoe−/− mice were compared to WT, and Apoe/ffe were compared to ffe mice. ***p<0.001, **p<0.01, *p<0.05 by Student’s t-test (n=5 to 11). None of the comparisons in panel B are statistically significant.
Figure 2
Figure 2. Saa3 mRNA, hepcidin mRNA and liver iron analysis in Apoe−/− mice
Apoe−/− mice were fed for 2 months either a low-fat diet containing 50 ppm Fe (LFD, grey bars) or a high-fat diet containing 50 ppm Fe (HFD, black bars) (n=6 to 10). F=females, M= males. (A) Saa3 mRNA. (B) Hepcidin mRNA. (C) Liver non-heme iron concentrations. Vertical bars show the means, and error bars show standard deviations. Statistical analysis was performed to evaluate the effect of worsening atherosclerosis: mice on low-fat diet were compared to those on high-fat diet by Student’s t-test (***p≤0.001, **p=0.01).
Figure 3
Figure 3. Despite iron-loaded macrophages, Apoe/ffe do not develop more severe atherosclerosis than Apoe−/− mice
Apoe−/− (n=15) and Apoe/ffe (n=19) mice were fed high-fat diet with 300 ppm Fe for 4 months. (A) Non-heme spleen iron content was measured to confirm macrophage iron loading associated with ffe mutation. Means and standard deviations are shown. (B) Atherosclerotic lesions size (μm2, horizontal lines represent the means). (C) Calcification count (mean % slides showing calcification ± standard deviation). For each gender, Apoe−/− mice were compared to Apoe/ffe mice using Student’s t-test (***p<0.001, **p<0.01, *p<0.05). M=males, F=females.
Figure 4
Figure 4. Parenteral iron administration does not worsen atherosclerosis in Apoe−/− and Apoe/ffe mice
Apoe−/− and Apoe/ffe mice were fed for 2 months high-fat diet (50 ppm Fe). One group received weekly iron sucrose injection for 8 weeks (16 mg iron total, HFD+Fe) (n=13 for Apoe−/− and 9 for Apoe/ffe), and the other group did not (HFD) (n=13 for Apoe−/− and 7 for Apoe/ffe). (A) Non-heme liver iron and (B) spleen iron content were quantitated to confirm macrophage iron loading induced by iron sucrose injections. (C) Hepatic Hamp mRNA expression. For A, B and C, means and standard deviations are shown. (D) Histological examination of iron loading in the aortic lesions. Tissue iron was detected by enhanced Perls’ stain (green). Scale bars = 100 μm. Illustrative tissue sections from Apoe−/− mice are shown; Apoe/ffe animals had an equivalent iron loading pattern. (E) Atherosclerotic lesion size (μm2, horizontal lines represent the means). For each genotype (either Apoe−/− or Apoe/ffe), statistical analysis was performed to evaluate the effect of iron sucrose injections. Furthermore, for each condition (either HFD or HFD+Fe), Apoe−/− mice were compared to Apoe/ffe mice. ***p≤0.001, **p=0.01 by Student’s t-test. No statistically significant difference between the groups was observed in (E).

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