Elsevier

Life Sciences

Volume 82, Issues 13–14, 26 March 2008, Pages 708-717
Life Sciences

Atorvastatin has hypolipidemic and anti-inflammatory effects in apoE/LDL receptor-double-knockout mice

https://doi.org/10.1016/j.lfs.2008.01.006Get rights and content

Abstract

Statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins in animal models of atherosclerosis are not very consistent. Thus we wanted to evaluate whether atorvastatin possesses hypolipidemic and anti-inflammatory effects in mice lacking apolipoprotein E/low-density lipoprotein receptor (apoE/LDLR-deficient mice). Two-month-old female apoE/LDLR-deficient mice (n = 24) were randomly subdivided into 3 groups. The control group of animals (n = 8) was fed with the western type diet (atherogenic diet) and in other two groups atorvastatin was added to the atherogenic diet at the dosage of either 10 mg/kg or 100 mg/kg per day for a period of 2 months. Biochemical analysis of lipids, ELISA analysis of monocyte chemotactic protein-1 (MCP-1) in blood, quantification of lesion size and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) in the atherosclerotic lesion by means of immunohistochemistry and Western blot analysis were performed. The biochemical analysis showed that administration of atorvastatin (100 mg/kg/day) significantly decreased level of total cholesterol, lipoproteins (VLDL and LDL), triacylglycerol, and moreover significantly increased level of HDL. ELISA analysis showed that atorvastatin significantly decreased levels of MCP-1 in blood and immunohistochemical and Western blot analysis showed significant reduction of VCAM-1 and ICAM-1 expression in the vessel wall after atorvastatin treatment (100 mg/kg/day). In conclusion, we demonstrated here for the first time strong hypolipidemic and anti-inflammatory effects of atorvastatin in apoE/LDLR-deficient mice. Thus, we propose that apoE/LDLR-deficient mice might be a good animal model for the study of statin effects on potential novel markers involved in atherogenesis and for the testing of potential combination treatment of new hypolipidemic substances with statins.

Introduction

Statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. Upon decrease of elevated levels of low-density-lipoprotein cholesterol (LDL-cholesterol) levels, statins significantly reduces the incidence of coronary heart disease events and mortality in hypercholesterolemic patients. Statins are preferred in patients with combined dyslipidemia because they are more likely to reduce LDL-cholesterol levels to target values and also substantially lower triglyceride levels. However, if target levels are not reached, combination therapy should be considered (McKenney, 2001). Yet statins reduce cardiovascular events by only about 20–40%. Nonstatin therapies (either as monotherapy or in addition to statins) to reduce LDL-cholesterol by mechanisms that do not involve inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase are likely to be useful for patients in need of LDL reduction; particularly those who either cannot take statins or respond only partially or not at all to statins alone (Shah, 2003).

Numerous animal species have been used to study the pathogenesis and potential treatment of the lesions of atherosclerosis. In recent years, there has been an explosion in the number of in vivo studies that is largely attributable to the use of mouse models to study atherogenic mechanism. Mice are highly resistant to atherosclerosis. Only when mice were fed a very high cholesterol, high fat diet that also contains cholic acid did their cholesterol levels rise and after many months on this diet they developed several layers of foam cells (Jawien et al., 2004). Genetic research and the application of transgene techniques and gene targeting in mice resulted in generation of a wide range of mouse strains that are much more suitable atherosclerotic models, including apoE-deficient mice, and LDLR−/− mice (Hofker and Breuer, 1998).

The apoE-deficient mouse is a well-established genetic mouse model of atherogenic hypercholesterolemia, in which mice spontaneously develop hypercholesterolemia and atherosclerosis on chow diet. The mice also develop widespread fibrous plaque lesions at vascular sites typically affected in human atherosclerosis and therefore represent an important model for studies of genetic and environmental influences on the atherosclerotic process (Reddick et al., 1994). The effect of statin treatment on atherogenesis in these mice seems to be time-dependent. Short-term administration of statin did not alter plasma lipids in apoE-deficient mice (Nachtigal et al., 2006b, Sparrow et al., 2001). Surprisingly, long-term administration of simvastatin elevated serum total cholesterol and increased aortic plaque area (Wang et al., 2002).

LDL receptor-deficient mice have been created to induce high plasma levels of LDL and IDL lipoproteins. These mice develop no, or only very small lesions on chow diet; however, robust lesions develop on the western type diet (Jawien et al., 2004). Still, the development of these lesions is slow and long-term experiments are needed. Effect of statin in this model of atherosclerosis is inconsistent. The administration of statins in these mice lowered total cholesterol and LDL-cholesterol levels and reduced aortic plaque area (Bisgaier et al., 1997, Wang et al., 2002). In contrast, Chen et al. (2002) showed that simvastatin did not alter plasma cholesterol or triglyceride levels in LDLR receptor-deficient mice. Thus, conflicting results are available for these mouse models of atherosclerosis regarding the effects of statins on atherogenesis.

More recently, apoE/LDLR-deficient mice have been created, representing a new mouse model that develops severe hyperlipidemia and atherosclerosis (Ishibashi et al., 1994). From papers previously published it is known that apoE/LDLR-deficient mice develop spontaneous hypercholesterolemia and atherosclerosis even on chow diet and addition of western type diet just increases cholesterol levels and potentiates atherogenesis in the same way as in other genetic mouse models of atherosclerosis (Bonthu et al., 1997, Ishibashi et al., 1994). Moreover it has been reported that, even on chow diet, the progression of atherosclerosis is more marked in these mice than in mice deficient for apoE or LDL receptor alone (Witting et al., 1999).

To the best of our knowledge the effect of statins on atherogenesis in apoE/LDLR-deficient mice has not been studied. Thus, the aim of this study was to evaluate whether atorvastatin possesses hypolipidemic and anti-inflammatory effects in these mice. We analyzed lipid profile and inflammatory markers in blood and together with expression of inflammatory markers and lesion size in the blood vessel wall.

Section snippets

Animals

The Ethical Committee of the Faculty of Pharmacy, Charles University, approved the protocols of the animal experiments. The protocol of experiments was pursued in accordance with the directive of the Ministry of Education of the Czech Republic (No. 311/1997).

Experimental design

Two-month-old female apoE/LDLR-deficient mice on a C57BL/6J background (n = 24) (Taconic Europe, Lille Skensved, Denmark) were randomly subdivided into 3 groups. Female mice were used in the experiment, because many papers published

Biochemical analysis

Biochemical analysis of blood samples of apoE/LDLR-deficient mice showed that administration of atorvastatin at a dose of 100 mg/kg/day resulted in a significant decrease of total cholesterol (51.90 ± 1.72 vs. 28.23 ± 3.30 mmol/l, P < 0.001) in comparison with control (non-treated) mice. Moreover the administration of this dose of atorvastatin resulted in a significant decrease of VLDL-cholesterol (32.41 ± 0.97 vs. 16.10 ± 2.41 mmol/l, P < 0.001), LDL-cholesterol (18.59 ± 0.81 vs. 11.00 ± 1.04 mmol/l, P < 0.001)

Discussion

The advent of HMG-CoA reductase inhibitors, or statins, has revolutionized the treatment of hypercholesterolemia. Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. In addition a growing body of evidence suggests that statins exert beneficial vascular effects that are independent of their cholesterol-lowering potencies (Calabro and Yeh, 2005). In spite of the fact that statins are strong in decreasing of LDL-cholesterol

Acknowledgments

The authors wish to thank Mrs. P. Jaburkova and Ing. Z. Mullerova for skilful technical assistance, Dr. Jiri Havranek, Zentiva, Czech Republic, kindly provided atorvastatin. This work was supported in part by grants from the Grant Agency of Charles University in Prague (101/2005C), Ministry of Education of the Czech Republic (MZO 00179906), Grant Agency of MSMT (FRVS 51/75105) and Alltracel Pharma Ltd.

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