Korean Circ J.  2012 Aug;42(8):543-550. 10.4070/kcj.2012.42.8.543.

Simvastatin and Losartan Differentially and Synergistically Inhibit Atherosclerosis in Apolipoprotein E-/- Mice

• Lee BS
• Choi JY
• Kim JY
• Han SH
• Park JE

• KMID: 2225004
• DOI: http://doi.org/10.4070/kcj.2012.42.8.543

Abstract

BACKGROUND AND OBJECTIVES
Since statins and angiotensin receptor blockers are a frequently prescribed combination in patients with atherosclerotic cardiovascular diseases, we tested the interactive effects of simvastatin and losartan on atherosclerosis in apolipoprotein E (apoE)-/- mice.
MATERIALS AND METHODS
Apolipoprotein E-/- mice were fed a high-fat, high-cholesterol (HFHC) diet for 12 weeks, with and without simvastatin (40 mg/kg) and/or losartan (20 mg/kg). The mice were divided into 5 groups and were fed as follows: regular chow (control diet, n=5), HFHC diet (n=6), HFHC diet with losartan (n=6), HFHC diet with simvastatin (n=6), and HFHC diet with both losartan and simvastatin (n=6).
RESULTS
Losartan treatment in apoE-/- mice significantly decreased atherosclerotic lesion areas in whole aortic strips stained with Oil Red O. The plaque area measured at the aortic sinus level was reduced significantly by 17% (HFHC; 346830.9+/-52915.8 microm2 vs. HFHC plus losartan; 255965.3+/-74057.7 microm2, p<0.05) in the losartan-treated group. Simvastatin and simvastatin plus losartan treatments reduced macrophage infiltration into lesions by 33% (HFHC; 183575.6+/-43211.2 microm2 vs. HFHC plus simvastatin; 120556.0+/-39282.8 microm2, p<0.05) and 44% (HFHC; 183575.6+/-43211.2 microm2 vs. HFHC plus simvastatin and losartan; 103229.0+/-8473.3 microm2, p<0.001, respectively). In mice fed the HFHC diet alone, the smooth muscle cell layer in the aortic media was almost undetectable. In mice co-treated with losartan and simvastatin, the smooth muscle layer was more than 60% preserved (p<0.05). Given alone, losartan showed a slightly stronger effect than simvastatin; however, treatment with losartan plus simvastatin induced a greater inhibitory effect on atherosclerosis than either drug given alone. Serum lipid profiles did not differ significantly among the groups.
CONCLUSION
Losartan displayed anti-atherosclerotic effects in apoE-/- mice that were equivalent to or greater than the effects of simvastatin. Combined treatment with these drugs had greater effect than either drug alone.

Keyword

Atherosclerosis; Losartan; Simvastatin; Mice, knockout; Models, animal

MeSH Terms

Angiotensin Receptor Antagonists
Animals
Apolipoproteins
Atherosclerosis
Azo Compounds
Cardiovascular Diseases
Diet
Humans
Losartan
Macrophages
Mice
Mice, Knockout
Models, Animal
Muscle, Smooth
Myocytes, Smooth Muscle
Simvastatin
Sinus of Valsalva
Angiotensin Receptor Antagonists
Apolipoproteins
Azo Compounds
Losartan
Simvastatin

Figure

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  Fig. 1 Effects of simvastatin and/or losartan on lipid accumulation in aorta and aortic sinus in apoE-/- mice fed a high-fat, high-cholesterol (HFHC) diet. Mice were fed an HFHC diet for 12 weeks of HFHC with or without 40 mg/kg simvastatin and/or 20 mg/kg losartan twice weekly. A control group was fed regular chow for 12 weeks. A: representative en face Oil Red O-stained aorta from each group of apoE-/- mice is shown. B: representative Oil Red O-stained area in aortic sinus from each group of apoE-/- mice is shown. C: Oil Red O-positive areas of the aortic sinus were quantitatively analyzed by computer-assisted morphometery. All results are shown as the mean±SD. *p<0.05 compared with the HFHC group. S: simvastatin, L: losartan, S+L: simvastatin plus losartan, apoE-/-: apolipopretein E.

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  Fig. 2 Effects of simvastatin and/or losartan on macrophage infiltration into atherosclerotic lesions in apoE-/- mice fed an HFHC diet. Mice were fed the HFHC diet for 12 weeks of HFHC with or without 40 mg/kg simvastatin and/or 20 mg/kg of losartan twice weekly. A control group was fed regular chow for 12 weeks. Tissue sections from the aortic sinuses of apoE-/- mice from each group were stained with rat monoclonal antibody to mouse macrophages (MOMA-2). A: representative MOMA-2-stained aortic sinuses from each group are shown. B: the MOMA-2-positive areas of the aortic sinus were quantitatively analyzed by computer-assisted morphometery. All results are shown as the mean±SD. *p<0.05, †p<0.01 compared with the HFHC group, respectively. S: simvastatin, L: losartan, S+L: simvastatin plus losartan, HFHC: high-fat, high-cholesterol, apoE-/-: apolipoprotein E.

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  Fig. 3 Effects of simvastatin and/or losartan on smooth muscle cell destruction in HFHC-fed apoE-/- mice. Three sections of the aortic sinus from each animal were stained with alpha-SMC actin antibody. Mean staining values were expressed as a percentage of the total lesion area. A: representative examples of aortic sinus staining are shown for each group. B: the preserved medial layer was quantified by expressing the area positively stained with alpha-SMC actin antibody as a fraction (%) of the entire vessel wall area. All results are shown as the mean±SD. *p<0.01 compared with the HFHC group. S: simvastatin, L: losartan, S+L: simvastatin plus losartan, HFHC: high-fat, high-cholesterol, apoE-/-: apolipopretein E, SMC: smooth muscle cell.

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  Fig. 4 Effects of simvastatin and/or losartan on MMP9 and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in the aortic sinus of HFHC-fed apoE-/- mice. Each section of the aortic sinus was stained with rabbit anti-mouse MMP9 or co-stained with goat anti-mouse SMC antibody and rabbit anti-mouse TIMP1 antibody followed by Alexa 488 donkey anti-goat IgG and Alexa 568 donkey anti-rabbit IgG. DAPI was used for nuclear staining. Immunofluorescence images were obtained by confocal microscopy (LSM700; ZEISS, Jena, Germany). A: MMP9. B: SMC & TIMP. S: simvastatin, L: losartn, S+L: simvastatin plus losartan. MMP9: metalloproteinase 9, HFHC: high-fat, high-cholesterol, apoE-/-: apolipopretein E, SMC: smooth muscle cell, DAPI: 4'-6-diamidino-2-phenylindole.

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  Fig. 5 Effect of simvastatin and/or losartan on heat shock protein 27 (HSP27) expression in the aortic sinuses of HFHC-fed apoE-/- mice. Tissue sections were stained with rabbit anti-mouse HSP27 followed by Alexa-488 goat anti-rabbit IgG. Nuclei were stained with DAPI. Immunofluorescence images were obtained by confocal microscopy (LSM700; ZEISS, Jena, Germany). S: simvastatin, L: losartan, S+L: simvastatin plus losartan, HFHC: high-fat, high-cholesterol, apoE-/-: apolipopretein E, DAPI: 4'-6-diamidino-2-phenylindole.

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