Rosuvastatin Attenuates Progression of Atherosclerosis and Reduces Serum IL6 and CCL2 Levels in Apolipoprotein-E-deficient Mice

Abstract

Background/aim: Apolipoprotein E-deficient (Apoe^-/-) mice develop atherosclerotic lesions that closely resemble metabolic syndrome in humans. We sought to investigate how rosuvastatin mitigates the atherosclerotic profile of Apoe^-/- mice over time and its effects on certain inflammatory chemokines.

Materials and methods: Eighteen Apoe^-/- mice were allocated into three groups of six mice each receiving: standard chow diet (SCD; control group); high-fat diet (HFD); and HFD and rosuvastatin at 5 mg/kg/d orally via gavage for 20 weeks. Analysis of aortic plaques and lipid deposition was conducted by means of en face Sudan IV staining and Oil Red O staining. Serum cholesterol, low-density lipoprotein, high-density lipoprotein, plasma glucose and triglyceride levels were determined at baseline and after 20 weeks of treatment. Serum interleukin 6 (IL6), C-C motif chemokine ligand 2 (CCL2) and tumor necrosis factor-α (TNFα) levels were measured by enzyme-linked immunosorbent assay at the time of euthanasia.

Results: The lipidemic profile of Apoe^-/- mice on HFD deteriorated over time. Apoe^-/- mice on HFD developed atherosclerotic lesions over time. Sudan IV and Oil Red O-stained sections of the aorta revealed increased plaque formation and plaque lipid deposition in HFD-fed mice compared with SCD-fed mice and reduced plaque development in HFD-fed mice treated with rosuvastatin compared with mice not receiving statin treatment. Serum analysis revealed reduced metabolic parameters in HFD-fed mice on rosuvastatin compared with non-statin, HFD-fed mice. At the time of euthanasia, HFD-fed mice treated with rosuvastatin had significantly lower IL6 as well as CCL2 levels when compared with HFD-fed mice not receiving rosuvastatin. TNFα levels were comparable among all groups of mice, irrespective of treatment. IL6 and CCL2 positively correlated with the extent of atherosclerotic lesions and lipid deposition in atherosclerotic plaques.

Conclusion: Serum IL6 and CCL2 levels might potentially be used as clinical markers of progression of atherosclerosis during statin treatment for hypercholesterolemia.

Keywords: APOE; CCL2; IL6; Rosuvastatin; atherosclerosis; mouse.

Figure 1. Comparison of serum parameters within each group of Apoe^−I− mice at baseline and after 20 weeks of receiving standard chow diet (SCD), high-fat diet (HFD) or HFD plus rosuvastatin. Total cholesterol (A), low-density lipoprotein (LDL) (B), triglycerides (C), plasma glucose (D) and high-density lipoprotein (HDL) (E) levels within each group at baseline versus after 20 weeks of treatment. All groups: n=6. Data are the mean±standard error of the mean. Significantly different from baseline at: *p<0.05, **p<0.01, and ***p<0.001 (paired t-test).

Figure 2. Effects of rosuvastatin on atherosclerotic lesion formation and lipid deposition in plaques developing in Apoe^−I− mice after 20 weeks of receiving standard chow diet (SCD), high-fat diet (HFD) or HFD plus rosuvastatin. A: Representative en face Sudan IV staining of the aortic arch (magnification, ×50) showed that the rosuvastatin-treated group had fewer atherosclerotic lesions compared with the control (SCD) group. B: Results of en face Sudan IV staining and Oil red O staining in atherosclerotic plaques in the aortic root. Rosuvastatin reduced lipid deposition in plaques compared with the SCD group. All groups: n=6. Data are the mean±standard error of the mean. Significantly different at: *p<0.05, **p<0.01, and ***p<0.001 (ANOVA, Tukey’s test).

Figure 3. Levels of chemokines tumor necrosis factor-α (TNFα), interleukin 6 (IL6) and C-C motif chemokine ligand 2 (CCL2) in serum from Apoe^−I− mice after 20 weeks of receiving standard chow diet (SCD), high-fat diet (HFD) or HFD plus rosuvastatin, as detected by enzyme-linked immunosorbent assay. At 20 weeks of treatment, TNFα levels were comparable among all groups irrespective of treatment. In contrast, IL6 and CCL2 in HFD-fed mice treated with rosuvastatin was significantly lower when compared with HFD-fed mice not receiving rosuvastatin. All groups: n=6. Significantly different at *p<0.05, **p<0.01, and ***p<0.001 (ANOVA, Tukey’s test).

Figure 4. Spearman’s correlation of interleukin 6 (IL6) (A) and C-C motif chemokine ligand 2 (CCL2) (B) in serum from Apoe^−I− mice after 20 weeks of receiving standard chow diet (SCD), high-fat diet (HFD) or HFD plus rosuvastatin with Sudan IV- and Oil red O-positive area. Significant positive correlation was noted for IL6 and CCL2 levels at the time of euthanasia and the extent of atherosclerotic plaques shown by Sudan IV staining, as well as lipid deposition in plaques by Oil red O staining.