Supplementary Material for: The Effects of Endurance Exercise and Diet on Atherosclerosis in Young and Aged ApoE^–/– and Wild-Type Mice

Background: Atherosclerosis is the leading cause of death worldwide. The disease development is by and large driven by old age and lifestyle factors, such as diet, physical activity, and smoking. In the present study, we have investigated the effect of exercise and diet on the development of atherosclerosis in young and aged mice. Objective: This study aimed at comparing multiple age-dependent factors that may influence atherosclerosis in a transgenic mouse model. Methods: Young (14 weeks) and aged (49–52 weeks) C57BL/6 wild-type (WT) and atherosclerosis-prone ApoE^–/– mice were subjected to physical endurance exercise on a treadmill, with or without a high-fat diet. Five weeks later, the frequencies of regulatory T cells (T_REGs) in lymph nodes were assessed by flow cytometry, plasmatic cytokines (interleukin [IL]-1β, IL-6, IL-10, IL-17, interferon-γ, tumor necrosis factor-α, and transforming growth factor [TGF]-β₁) levels were determined by Luminex assay. Lipids (cholesterol and triglycerides) and anti-heat shock protein 60 (HSP60) autoantibodies were measured by ELISA. Aortic lesion sizes were assessed by en face imaging. Microarray analysis and qPCR of skeletal muscle gene expression were also performed. Results: Exercise leads to a reduction of aortic lesions in young ApoE^–/– and aged WT mice independent of diet. In most groups, this reduction was followed by an increased proportion of T_REGs and TGF-β₁ levels. Moreover, gene expression analysis showed that exercise seems to affect the AMPK signaling pathway. In particular, PGC-1α₁ mRNA was induced in aged WT mice, whereas it was reduced in young ApoE^–/– mice. In addition, GSEA analysis showed a marked reduction in the insulin signaling pathway in aged ApoE^–/– mice. Conclusion: Practicing endurance exercise seems to be enough for reducing early aortic lesion formation, independent of diet. However, this was only true in mice with smaller aortic lesions, since mice with large, advanced, complicated atherosclerotic plaques did not show any reduction in lesion size with exercise training.