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

DIAMANTIS I. TSILIMIGRAS, KALLIOPI THANOPOULOU, MARIA SALAGIANNI, GERASIMOS SIASOS, EVANGELOS OIKONOMOU, DESPINA D. PERREA, NIKOLAOS NIRAKIS, KONSTANTINOS FILIS, KONSTANTINOS TSIOUFIS, DIMITRIOS TOUSOULIS and FRAGISKA SIGALA
In Vivo May 2023, 37 (3) 994-1002; DOI: https://doi.org/10.21873/invivo.13173

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.

Key Words:

Atherosclerosis is a chronic, progressive disease characterized by the hardening and thickening of arterial wall, and the formation of plaques, which comprise immune and mesenchymal cells, lipids and extracellular matrix (1). Despite advances in therapeutic strategies and pharmaceutical agents, cardiovascular disease remains a leading cause of mortality worldwide (1, 2). Besides an imbalanced lipid metabolism, it is now well established that inflammation of the arterial wall and immune responses are key to the development of atherosclerosis (3). The infiltration of the vessel wall with monocytes but also other immune cells, including neutrophils and T cells, is considered a hallmark of atherosclerosis (3, 4). The recruitment of these cells is mediated by adhesion molecules along with chemokines and their receptors, including vascular cell adhesion molecule 1, intercellular adhesion molecule 1, C-C motif chemokine ligand 2 (CCL2; formerly known as monocyte chemoattractant protein-1), and interleukin-6 (IL6) (3, 4). Tumor necrosis factor-α (TNFα) has also been postulated to promote the inflammatory cascade within the arterial wall during development of atherosclerosis by activating endothelial cells to express adhesion molecules and release inflammatory cytokines (5).

Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that inhibit the mevalonate pathway. Depending on their properties, different statins (such as atorvastatin, lovastatin, pravastatin, simvastatin, or rosuvastatin) have differential metabolic effects (6). They are all unequivocally able to effectively lower the low-density lipoprotein cholesterol levels, thus reducing the risk of atherosclerotic cardiovascular diseases (7). To date, a number of clinical studies have demonstrated the efficacy of statins in delaying atherosclerosis in both clinical (i.e., humans) and preclinical (i.e., mouse models of laboratory-induced atherosclerosis) settings (8-10). Statins improve endothelial function, diminish oxidative stress and platelet adhesion, and enhance atherosclerotic plaque stability (11, 12). Besides directly regulating lipid metabolism, statins also exert pleiotropic effects, including attenuating inflammatory responses that contribute to the progression of atherosclerosis (11, 12). Rosuvastatin is a newer generation of statins that is well known for its hypolipidemic properties (13). Nevertheless, less is known regarding its anti-inflammatory and immunomodulatory effects. To this end, the objective of this study was to further investigate the effect of rosuvastatin in a high-fat diet fed mouse model that lacks apolipoprotein E (Apoe−/−) that is prone to developing atherosclerotic lesions. In addition, we sought to examine the effect of rosuvastatin on certain chemokines including IL6, CCL2 and TNFα along with the correlation of these chemokines with the development and extent of atherosclerotic plaques in Apoe−/− mice.

Materials and Methods

Animal model. A total of 18 male Apoe−/− mice were obtained from the Jackson Laboratory, Bar Harbor, ME, USA. Mice were kept separately in the Laboratory for Experimental Surgery and Surgical Research (Athens, Greece) in a controlled environment at 20°C±2°C in cages that complied with European standards (Tecniplast, Buguggiate, Italy) with 55% relative humidity, central ventilation (15 air changes/hour), and an artificial 12-hour light–dark cycle. Mice from all groups had unrestricted access to food and water. All animals received appropriate care in compliance with the “Guide for the Care and Use of Laboratory Animals” (14). The experimental protocol was approved by the Institutional Animal Care and Use Committee of Athens University Medical School and the Veterinary Directorate of the Athens Prefecture in agreement with EU legislation (EL 25 BIO 05).

Grouping and interventions. The Apoe−/− mice were kept on a standard chow diet (SCD) (Conigli Svezzamento, S.I.V.A.M. Società Italiana Veterinaria Agricola Milano S.P.A., Casalpusterlengo, Italy) until they became 8 weeks old. At that time, they were allocated into three distinct groups of six mice each receiving: SCD-only; a commercial high-fat diet (HFD) only (45% fat; Dieta Speciali, Mucedola SRL, Milan, Italy) and HFD plus rosuvastatin (AstraZeneca, Athens, Greece) given orally via gavage at a daily dosage of 5 mg/kg for 20 weeks, as previously described (15, 16).

Each mouse was assigned a unique number and was kept in a separate cage labeled with that number. The mice also carried articular badges with imprinted numbers. The mice were inspected daily by a specialized veterinarian. Blood draws were performed at baseline and after they completed the 20-week experimental period at the Laboratory for Experimental Surgery and Surgical Research, Athens, Greece. Blood draws were performed at a specific time in the morning (9:00 a.m.) following a 12-hour fasting period. Capillary tubes were introduced into the medial retro-orbital venous plexus under light ether anesthesia (17) and blood was collected in Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ, USA). Serum was separated by centrifugation at 1,000-2,000×g for 10 min and was stored at −20°C until further analysis. Following 20 weeks of the interventions, all mice were euthanized under ketamine-xylazine anesthesia followed by cervical dislocation, as previously reported (17).

Serum measurements and enzyme-linked immunosorbent assay. Serum total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and glucose concentrations were determined enzymatically with commercially available kits (Biosis Biotechnological Applications, Athens, Greece). The calculation of low-density lipoprotein (LDL) cholesterol was performed using the Friedewald formula (18). Serum TNFα, IL6 and CCL2 levels were measured by enzyme-linked immunosorbent assay using commercially available kits (BioLegend, Amsterdam, the Netherlands).

Quantification of atherosclerotic lesions and lipid deposition. Following euthanasia, both the heart and the entire aorta were removed. The thoracic aorta was opened longitudinally and fixed with 10% neutral buffered formalin. To quantify atherosclerotic lesions in the aortic arch, we performed en face Sudan IV staining, as previously described (19). Aortic images were captured with a Nikon digital camera and analyzed using ImageJ software v1.48 (National Institutes of Health, Bethesda, MD, USA).

Each heart was cut along a horizontal plane between the lower tips of the right and left atria. The aortic root was then sectioned serially (5-mm intervals) from the point where the aortic valves appeared in the ascending aorta, until the valve cusps were no longer visible. Accumulated lipids on aortic sinus were determined by Oil red O staining. The percentage lesion area was calculated as the total lesion area stained divided by the total surface area.

Statistical analysis. Continuous data are presented as the mean±standard error of the mean. Individual statistics of dependent samples were performed by paired t-test, and, when not normally distributed, by the Wilcoxon signed-rank test. For multiple between-group comparisons, one-way analysis of variance followed by Tukey post hoc test was performed. Correlation coefficient between enzyme-linked immunosorbent assay results and Sudan IV and Oil Red O staining results was assessed using the Spearman correlation coefficient. The level of statistical significance for all tests was set at α=0.05. All statistical analyses were performed using Graphpad Prism v4 (Graphpad Prism Inc., La Jolla, CA, USA) or JMP v14 (SAS Institute Inc., Cary, NC, USA) statistical packages.

Results

Rosuvastatin improved the metabolic profile of HFD-fed ApoE−/− mice. To examine the effect of rosuvastatin on the biochemical profile of Apoe−/− mice, mice were fed with SCD, or HFD with or without rosuvastatin for 20 weeks. Baseline blood draw did not reveal differences in metabolic parameters (i.e., blood glucose, total cholesterol, triglycerides, HDL, and LDL) among the three groups at the start of the study period (all p>0.05). Repeated biochemical analysis revealed increasing total cholesterol and LDL in all three groups over time (0 vs. 20 weeks), although differences were more pronounced in HFD-only mice (all p<0.01, Figure 1A and B). Triglyceride levels increased in SCD (p=0.02) and HFD-only mice (p<0.001), however, mice fed HFD and given statin did not develop significant increases in triglyceride levels over time (p=0.11) (Figure 1C). No significant differences were noted over time when examining blood glucose levels in mice from the SCD (p=0.51) and HFD plus statin (p=0.50) groups; however, HFD-only mice had progressively increasing blood glucose levels without statin treatment (p=0.045, Figure 1D). No differences were noted in serum HDL levels over time irrespective of treatment (p=0.79, p=0.07 and p=0.64, respectively, Figure 1E).

Figure 1.
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).

After 20 weeks of treatment, rosuvastatin was shown to improve metabolic parameters among mice fed with HFD, including reducing the levels of total cholesterol, triglycerides, and LDL (Table I). No differences were noted among the three groups in serum HDL levels after 20 weeks of treatment. In addition, no differences in mouse weight were noted among the three groups at the end of the study period (Table I).

View this table:
Table I.

Metabolic parameters of Apoe−/− mice after 20 weeks of receiving standard chow diet (SCD), high-fat diet (HFD) or HFD plus rosuvastatin.

Rosuvastatin reduced the development of atherosclerotic plaques in ApoE−/− mice. To examine the effect of rosuvastatin on the progression of atherosclerotic plaques, mice were euthanized after 20 weeks of treatment and their aortas were examined with en face IV Sudan and Oil red O staining. Rosuvastatin attenuated atherosclerotic lesion progression in the aortic arch of HFD-fed mice as determined by en face Sudan IV staining (p<0.05, Figure 2A). The SCD-fed group had fewer lesions compared with HFD-fed groups with and without rosuvastatin (both p<0.05, Figure 2B). Oil red O staining demonstrated reduced lipid deposition in atherosclerotic plaques of HFD-fed mice treated with rosuvastatin compared with HFD-fed mice not receiving rosuvastatin (Figure 2B).

Figure 2.
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).

Rosuvastatin reduced the serum levels of IL6 and CCL2 but not of TNFα. Baseline serum levels of IL6, CCL2 and TNFα were comparable among all groups (all p>0.05). At the time of euthanasia (20 weeks of treatment), TNFα levels were examined using serum from mice and were found to be comparable among all groups irrespective of treatment (p>0.05, Figure 3A). In contrast, at the same time point, HFD-fed mice treated with rosuvastatin had significantly lower levels of IL6 as well as CCL2 when compared with HFD-fed mice not receiving rosuvastatin (p<0.001, Figure 3B and C).

Figure 3.
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).

A positive correlation was noted for the extent of atherosclerotic plaques (lesion area as determined by en face Sudan IV staining) (Spearman correlation r=0.62, p=0.01) as well as lipid deposition in plaques (Oil Red O staining) (r=0.57, p=0.04) with IL6 level at the time of euthanasia (Figure 4A). Similarly, a positive correlation was noted for the extent of plaques (r=0.68, p=0.004) as well as lipid deposition (r=0.82, p=0.001) with CCL2 levels after 20 weeks of treatment (Figure 4B). No significant correlation was noted between area of atherosclerotic lesions (r=−0.38, p=0.14) and lipid deposition within plaques (r=−0.11, p=0.71) with TNFα levels at the time of euthanasia.

Figure 4.
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.

Discussion

The present study demonstrated that the metabolic profile of Apoe−/− mice worsened over time, especially while on HFD only. Rosuvastatin reduced levels of total cholesterol, LDL, and triglycerides in Apoe−/− mice while on HFD, which resulted in a reduction of atherosclerotic plaques in the aorta, as well as lower lipid deposition in atherosclerotic plaques. In addition, rosuvastatin was shown to reduce serum IL6 and CCL2 levels in Apoe−/− HFD-fed mice, while IL6 and CCL2 levels positively correlated with the extent of atherosclerotic lesions and the percentage of lipid deposition in the atherosclerotic plaques.

The Apoe−/− mouse line is a well-established, genetically altered animal model of atherosclerosis which spontaneously develops hypercholesterolemia and atherosclerotic lesions closely resembling the metabolic syndrome in humans (20). As such, Apoe−/− mice represent a useful experimental mouse model that has long been used in the field of cardiovascular research. The current study utilized this mouse model to facilitate the development of atherosclerosis in mice. We noted increased metabolic parameters in all three groups of mice (SCD, HFD only, HFD plus rosuvastatin) over time. This is in line with previous reports suggesting that Apoe−/− mice develop fibrous plaques after the age of 20 weeks even under a normal diet (15). Of note, the lowest increases in biochemical parameters were noticed in Apoe−/− mice fed with SCD, while the highest differences were shown in mice fed only with HFD for 20 weeks. Treatment with rosuvastatin reduced metabolic parameters of HFD-fed mice, especially total cholesterol and LDL, yet not to the level of SCD-fed mice. By the age of 28 weeks (20 weeks of interventions), atherosclerotic plaques had developed with distinct differences in the extent of atherosclerotic lesions based on the type of diet and the receipt of statin treatment. The current study revealed that administration of HFD accelerated the development of atherosclerotic plaques, while rosuvastatin appeared to be protective against the development of atherosclerotic lesions in Apoe−/− HFD-fed mice.

Atherosclerosis is a complex inflammatory process that is characterized by the presence of monocytes, macrophages and T-cells in the atheroma (12). It is currently well established that inflammation is key to the development of atherosclerosis. Inflammatory cytokines secreted by macrophages and T-lymphocytes can modify endothelial function, smooth-muscle cell proliferation, collagen degradation, and thrombosis (4). An early step in atherogenesis involves monocyte adhesion to the endothelium and penetration into the subendothelial space (4). Inflammatory cytokines and adhesion molecules have been implicated in this process. CCL2, also known as monocyte chemoattractant protein 1, plays a significant role in the pathogenesis of atherosclerosis by promoting the migration of monocytes into the injured endothelium, which is key to the development of the atherosclerotic lesions (21). Increased CCL2 expression has been noted in atherosclerotic lesions, highlighting the role of this chemokine in the recruitment of monocyte-macrophages during atherosclerosis (22). In addition, TNFα activates endothelial cells to express adhesion molecules and proinflammatory cytokines, thereby recruiting activated leukocytes to an inflammatory lesion (5, 23). IL6 also has been shown to be correlated with endothelial dysfunction, arterial stiffness and, in turn, the extent of sub-clinical atherosclerosis (24).

The current study demonstrated that Apoe−/− mice treated with rosuvastatin had lower serum levels of CCL2 and IL6, but not of TNFα, compared with HFD-fed Apoe−/− mice not receiving rosuvastatin. This suggests that apart from its lipid-lowering effect, rosuvastatin also attenuates the inflammatory process associated with atherosclerosis. Our findings are in line with those of Saadat et al. who reported that rosuvastatin improved the lipidemic profile and reduced inflammation (as evidenced by reduced IL6 and IL10 levels) in an asthmatic-hyperlipidemic rabbit model (25). Our results are also in line with a previous study that demonstrated that rosuvastatin significantly reduced atherosclerosis to a great extent in the aortic area of Apoe*3Leiden transgenic mice (9).

Perhaps more interestingly, IL6 and CCL2 were strongly correlated with the extent of atherosclerotic lesions and lipid deposition in atherosclerotic plaques in Apoe−/− models. Indeed, CCL2 has been associated with plaque formation and has been postulated to be a direct mediator of plaque instability (24). In addition, IL6 has also been shown to be correlated with endothelial dysfunction, arterial stiffness and, in turn, extent of sub-clinical atherosclerosis (24). A recent meta-analysis performed by the Emerging Risk Factors Collaboration demonstrated that for each standard deviation increase in log IL6 level, there was a 25% increased risk of future vascular event (relative risk=1.25, 95% confidence interval=1.19-1.32) (26). Our study finding suggesting a positive correlation of serum IL6 and CCL2 levels with the extent of atherosclerotic lesions is in line with this. In our study, HFD-fed mice treated with rosuvastatin had lower IL6 levels than HFD-fed mice not receiving rosuvastatin, which also translated to reduced atherosclerotic plaques in the former group. This is also in line with the ASTEROID trial that provided direct ultrasonographic evidence of atheroma regression among patients receiving high-intensity rosuvastatin therapy (27).

The present study has certain limitations. Firstly, a small number of mice were used in each of the groups, which limited the statistical power of our analysis, although significant associations were found. Secondly, we only examined a single dose of rosuvastatin in an attempt to investigate its effect on the progression of atherosclerosis, yet the decision was made based on previous reports that used rosuvastatin in mouse models in the literature (16). In addition, the current study examined biochemical parameters and serum chemokine levels and was not designed to measure local expression of molecules including IL6, CCL2 and TNFα. As such, the exact physiological mechanism behind attenuation of atherosclerotic plaque formation with rosuvastatin treatment was not investigated in the context of this study. Rather, a strong positive correlation was noted of IL6 and CCL2 with the extent of atherosclerotic lesions, which potentially means they might serve as potential markers of the efficacy of statin treatment. Nevertheless, further studies are needed to validate the utility of these markers in the clinical setting.

In conclusion, rosuvastatin reduced progression of atherosclerotic lesions in HFD-fed Apoe−/− mice and reduced serum IL6 and CCL2 levels after 20 weeks of treatment. Serum IL6 and CCL2 levels correlated with the extent of atherosclerotic lesions. Further studies are warranted to determine whether serum IL6 and CCL2 levels might potentially be used as clinical markers of progression of atherosclerosis during statin treatment for hypercholesterolemia.

Acknowledgements

The Authors would like to thank Dr. Evangelos Andreakos for their guidance and support with this study. This research work was supported by the Onassis Foundation — Scholarship ID: G ZO 030-1/2018-2019.

Footnotes

  • Authors’ Contributions

    DIT, KT, MS, GS, EO, DDP, NN, KF, KT, DT, FS all i) substantially contributed to the conception and the design of the study, or in the acquisition, analysis and interpretation of the data; ii) contributed to article drafting or critical revisions on the intellectual content; iii) approved of the final article version to be published; and iv) agreed to be accountable for all aspects of the work, so that any questions relating to research integrity or scientific accuracy in any part of the study are appropriately investigated and resolved. DIT and FS confirm the authenticity of all the raw data.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received March 6, 2023.
  • Revision received March 28, 2023.
  • Accepted March 29, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Rosuvastatin Attenuates Progression of Atherosclerosis and Reduces Serum IL6 and CCL2 Levels in Apolipoprotein-E-deficient Mice
DIAMANTIS I. TSILIMIGRAS, KALLIOPI THANOPOULOU, MARIA SALAGIANNI, GERASIMOS SIASOS, EVANGELOS OIKONOMOU, DESPINA D. PERREA, NIKOLAOS NIRAKIS, KONSTANTINOS FILIS, KONSTANTINOS TSIOUFIS, DIMITRIOS TOUSOULIS, FRAGISKA SIGALA
In Vivo May 2023, 37 (3) 994-1002; DOI: 10.21873/invivo.13173
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