Maternal Diet Supplemented with Methyl-Donors Protects against Atherosclerosis in F1 ApoE^−/− Mice

<div><p>Atherosclerosis is an inflammatory condition of the arterial wall mediated by cells of both innate and adaptive immunity. T lymphocytes play an important role in orchestrating the pathogenic immune response involved in the acceleration of atherosclerosis. Previously, we have shown that a prenatal methyl-donor supplementation diet (MS), when fed to dams during pregnancy and lactation, decreased the T cell-mediated pro-inflammatory cytokine and chemokine response in F1 mice. In the current study, we report feeding Apolipoprotein E (ApoE^−/−) deficient dams with the MS diet during pregnancy reduces atherosclerotic plaques in F1 mice that were fed high fat diet (HFD) after weaning. F1 mice from dams on the MS diet exhibited increased global T cell DNA methylation. T-cell chemokines and their receptors (in particular CCR2, CCR5, and CXCR3) play important roles in the inflammatory cell recruitment to vascular lesions. MS diet significantly reduced Ccr2 mRNA and protein expression in CD3+ T cells but not in CD11b+ monocytes in MS F1 mice relative to controls. F1 litter size, HFD consumption, body weight, and body fat were similar between control and MS diet groups. Moreover, serum thiol metabolite levels were similar between the two groups. However, MS diet is associated with significantly higher serum HDL and lower LDL+VLDL levels in comparison to F1 mice from dams on the control diet. Inflammatory cytokines (IL-17, TNF-α, IL-6) were also lower in MS F1 mice serum and conditioned media from T-cell culture. Altogether, these data suggest that the MS diet ameliorates development of atherosclerosis by inhibiting the T-cell Ccr2 expression, reducing inflammatory cytokines production and increasing serum HDL:LDL ratio.</p> </div

MS F1 mice have less atherosclerotic plaque in the aorta than control mice.

<p>(<b>A</b>) Representative images of control and MS F1 aortas at 17 wk, 28 wk and 34 wk of age. Oil Red O color indicates presence of atheroma. Panels <b>B</b> shows quantification of plaque surface area as a percentage of total aorta surface area in 17 wk, 28 wk and 34 wk F1 mice. Data presented is mean percent of control ± SEM. 17 wk: N = 5 control and 5 MS mice; 28 wk: N = 9 control and 7 MS mice; 34 wk: 7 control and 7 MS mice *<i>p</i><0.05, ns = not significant.</p

MS diet decreases CCR2 expression in F1 T cells.

<p>Splenic CD3+ T cells and CD11b+ cells from F1 ApoE^−/− mice were harvested at the age of 17 wk, 28 wk and 34 wk. mRNA was isolated and Ccr2 levels were measured by qRT-PCR in T cells (<b>A</b>) or monocytes (<b>B</b>). <b>C.</b> Protein from ApoE^−/− mice at the age of 17 wk, 28 wk, and 34 wk were used to check the level of CCR2 in CD3+ T cells. <b>D.</b> Quantitative analysis of panel C. Results are mean ± SEM. A, B, D) 17 wk: N = 26 control, 23 MS mice; 28 wk: N = 28 control, 23 MS mice. 34 wk: N = 9 control, 9 MS mice *<i>p</i><0.05, ns = not significant.</p

T cells and Liver from MS F1 mice are hypermethylated relative to control mice.

<p>T cell (<b>A</b>) and Liver (<b>B</b>) DNA were isolated from 4 wk, 17 wk, 28 wk or 34 wk old F1 ApoE^−/− mice and the level of methylation measured by ELISA. For each age group, the percent of methylated DNA (5-mC) in total DNA are shown. Results are mean ± SEM. (A) 4 wk: N = 5 control, 6 MS mice; 17 wk: 26 control, 23 MS mice; 28 wk: 28 control, 23 MS mice; 34 wk: 9 control, 9 MS mice. (B) 4 wk: N = 5 control, 6 MS mice; 17 wk and 28 wk: 10 control, 10 MS mice.</p

T cells and monocytes from MS F1 spleens are hypermethylated relative to control mice.

<p>CD3+ T cells (<b>A</b>) or CD11b+ cells (<b>B</b>) isolated from 17 wk or 28 wk old F1 ApoE^−/− mice were stained with anti-methylcytidine antibody and subjected to FACS analysis. Results shown are representative of 3 independent experiments. Data presented is mean ± SEM. 17 wk: N = 7 control and 5 MS mice; 28 wk: N = 9 control and 8 MS mice *<i>p</i><0.05.</p

MS diet decreases production of pro-inflammatory cytokines in circulation.

<p>Serum was collected and the level of TNF-α and IL-6 was measured as described in Methods. Results are mean ± SEM. N = 4 control and 4 MS mice *<i>p</i><0.05, **p<0.01.</p

MS diet modulates the expression of hepatic genes involved in <i>de novo</i> synthesis, regulation, and transport of cholesterol.

<p>Liver mRNA from control and MS-F1 ApoE^−/− mice was harvested at the age of 17 wk, and 28 wk and levels of HMGCoR, PPAR-γ, LDLr, and SR-B1 were measured by qRT-PCR. Results are mean ± SEM. 17 wk: N = 7 control, 5 MS mice; 28 wk: N = 6 control, 5 MS mice. *<i>p</i><0.05, **<i>p</i><0.01, ns = not significant.</p

Analysis of small thiol metabolites involved in methionine-homocysteine pathway in serums from 28-wk-old control or MS F1 mice.

<p>N = 10 control and 10 MS mice.</p

Weight, chow consumption, pup litter size and body composition of dams and F1 mice. A.

<p>Weight changes of dams over the weeks following mating. <b>B.</b> Pup litter size from dams on the control and MS diets. <b>C.</b> Body weight of control and MS F1 mice over time. Pups from control and MS fed dams were weaned at 4 wk of age onto a high fat (42%) diet (HFD). Weight of mice was measured at 4 wk, 17 wk, 28 wk, and 34 wk of age. <b>D.</b> Amount of HFD consumed by F1 mice per week over a 24 week period. <b>E.</b> Body fat percentage of control and MS F1 mice over time as measured by NMR. Data presented is mean ± SEM. A) N = 5 control and 5 supplement dams. B) N = 23 control litters and 22 MS litters. C) 4 wk: N = 14 control and 13 MS mice; 17 wk: N = 23 control and 21 MS mice; 28 wk: N = 27 control and 18 MS mice; 34 wk: N = 9 control and 9 MS mice. D) Total chow consumed per week per cage was divided by the number of mice per cage to determine average chow consumed per mouse. N = 11 control and 15 MS cages containing 1–5 mice per cage. E) 17 wk: N = 10 control and 9 MS mice; 28 wk: N = 21 control and 18 MS mice; 34 wk: N = 9 control and 9 MS mice. *<i>p</i><0.05, ns = not significant.</p

MS diet increase serum HDL and lowers LDL/VLDL in F1 mice.

<p>Serum was collected and cholesterol levels were measured fluorometrically as described in Methods. Results are mean ± SEM. 17 wk: N = 9 control and 9 MS mice; 28 wk: N = 11 control and 11 MS mice; 34 wk: N = 9 control and 9 MS mice. ***<i>p</i><0.001, ns = not significant.</p