doi: 10.1161/hq1201.100260
© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Ezetimibe, a Potent Cholesterol Absorption Inhibitor, Inhibits the Development of Atherosclerosis in ApoE Knockout Mice
From the Department of Central Nervous System and Cardiovascular Research, Schering-Plough Research Institute, Kenilworth, NJ.
Correspondence to Harry R. Davis, Jr, K-15-2/2600, Schering-Plough Research Institute, 2015 Galloping Hill Rd, Kenilworth, NJ 07033. E-mail harry.davis{at}spcorp.com
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Abstract |
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Ezetimibe (SCH58235) is a potent, selective, cholesterol absorption inhibitor. The objective of this study was to determine whether ezetimibe reduces plasma cholesterol and inhibits atherogenesis in apolipoprotein E knockout (apoE-/-) mice. Cholesterol absorption was inhibited by >90% at doses of ezetimibe >3 mg/kg in apoE-/- mice. Atherosclerosis and lipoprotein changes were determined in apoE-/- mice fed a high-fat (0.15% cholesterol) "western" diet, a low-fat (0.15% cholesterol) diet, or a semisynthetic cholesterol-free diet with or without ezetimibe (5 mg/kg per day) for 6 months. Ezetimibe reduced plasma cholesterol levels from 964 to 374 mg/dL, from 726 to 231 mg/dL, and from 516 to 178 mg/dL in the western, low-fat, and cholesterol-free diet groups, respectively. The reductions occurred in the very low density and low density lipoprotein fractions, whereas high density lipoprotein cholesterol levels were increased by ezetimibe treatment. Ezetimibe reduced aortic atherosclerotic lesion surface area from 20.2% to 4.1% in the western diet group and from 24.1% to 7.0% in the low-fat cholesterol diet group. Ezetimibe reduced carotid artery atherosclerotic lesion cross-sectional area by 97% in the western and low-fat cholesterol groups and by 91% in the cholesterol-free group. Ezetimibe inhibits cholesterol absorption, reduces plasma cholesterol, increases high density lipoprotein levels, and inhibits the progression of atherosclerosis under western, low-fat, and cholesterol-free dietary conditions in apoE-/- mice. Although apoE-/- mice are more hypercholesterolemic than are humans and low density lipoprotein reductions with ezetimibe are not as pronounced clinically, ezetimibe may inhibit atherogenesis in individuals consuming restricted-fat or western diets.
Key Words: cholesterol absorption • ezetimibe • atherosclerosis • hypercholesterolemia
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Introduction |
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The abnormal metabolism and elevation of plasma cholesterol and lipoproteins are well-documented risk factors for the development of atherosclerosis. Evidence from clinical trials indicates that reducing plasma cholesterol by dietary and/or pharmacological means leads to reductions in the incidence of death from cardiovascular disease.1–4 Present pharmacological interventions include the inhibition of cholesterol biosynthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (statins) either alone or in combination with other agents. Ezetimibe (SCH58235) is the first of a new class of selective cholesterol absorption inhibitors that may represent a new option for the pharmacological treatment of hypercholesterolemia.5,6 Ezetimibe selectively inhibits the intestinal uptake and absorption of dietary and biliary cholesterol at the brush border of small intestinal enterocytes, confining cholesterol to the intestinal lumen for subsequent excretion.6,7 Results from preclinical studies in various animal models have demonstrated the lipid-lowering properties of ezetimibe as a single agent5–9 and the synergistic cholesterol-lowering effects of ezetimibe when combined with a statin.10,11 This class of cholesterol absorption inhibitors has been shown to selectively reduce the cholesterol content of chylomicrons from monkeys in the postprandial state.9
ApoE is a crucial component of various plasma lipoproteins, including VLDLs, chylomicrons, and chylomicron remnants, and it is an essential ligand for the uptake and clearance of these atherogenic lipoproteins.12 Inactivation of the apoE gene in mice, a species normally resistant to the development of atherosclerosis, is associated with severe hypercholesterolemia, loss of normal resistance to cholesterol feeding, and premature development of atherosclerotic lesions.12–16 These factors have contributed to our understanding of the role of apoE in lipid transport and physiology and to the validity of the apoE knockout (apoE-/-) mouse model in characterizing the impact of dietary factors and other agents on the development and progression of atherosclerotic disease.
The present study evaluated the effects of ezetimibe on cholesterol absorption, plasma lipoprotein profiles, and the development of aortic and carotid atherosclerosis in the apoE-/- mouse, a well-established animal model for atherosclerosis.13–17 Long-term (6-month) assessments examined the effect of chronic cholesterol absorption inhibition by ezetimibe on the development and progression of atherosclerosis in apoE-/- mice fed cholesterol-containing western and low-fat diets and a cholesterol-free diet.
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Methods |
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Animals and Diets
Male apo-/- mice on a C57/BL background and age-matched wild-type (C57/BL +/+) control mice were obtained from Jackson Laboratory (Bar Harbor, Me). Mice (aged 8 weeks) used in the radiolabeled cholesterol absorption experiments were fed a Purina rodent chow diet (No. 5001). The effect of ezetimibe on atherosclerosis in apoE-/- mice was determined in 2 studies. In the first study, male apoE-/- mice (aged 8 weeks) were fed a "western" (40 kcal% butterfat, 0.15% [wt/wt] cholesterol) or a low-fat (10 kcal% corn oil, 0.15% [wt/wt] cholesterol) diet with or without 0.005% ezetimibe (wt/wt) for 6 months (n=8 or 9 per group). In the second study, male apoE-/- mice (aged 8 weeks) were fed a semisynthetic cholesterol-free diet (10 kcal% corn oil) either alone or containing 0.005% ezetimibe (wt/wt) for 6 months (n=12 per group). Body weights and food consumption were monitored to determine the ezetimibe dose delivered. Diets were prepared by Research Diets, Inc. All studies were conducted in a facility accredited by the American Association for Accreditation of Laboratory Animal Care in accordance with the principles and guidelines established by the National Institutes of Health for the humane care and use of laboratory animals. The Schering-Plough Research Institute Animal Care and Use Committee approved all study protocols.
Cholesterol Absorption
The ability of ezetimibe to inhibit the absorption of cholesterol was assessed by the dual fecal isotope ratio method, as previously described.18 ApoE-/- mice and control mice (C57/BL +/+) were gavaged first with ezetimibe (0, 0.3, 1, 3, and 10 mg/kg) and then 30 minutes later with a single dose of radiolabeled cholesterol and sitosterol in corn oil with [14C]cholesterol (1 µCi, 0.1 mg unlabeled cholesterol) and [3H]sitosterol (2 µCi, 0.1 mg unlabeled sitosterol; n=4 or 5 per group). Mice were subsequently dosed once daily with ezetimibe, and feces were collected for 3 days. Fecal [14C]cholesterol and [3H]sitosterol levels were determined by combustion in a Tri-Carb Oxidizer (Packard Instrument Co). Cholesterol absorption was calculated by the fecal ratio method.18
Determination of Plasma Cholesterol, Lipoprotein Fractions, and Triglycerides
Nonfasting terminal plasma samples were collected at the end of both 6-month studies, and total and lipoprotein cholesterol levels were determined by a modification of the cholesterol oxidase method of Allain et al19 with the use of kit reagents (Wako Pure Chemical Industries, Ltd). Triglyceride concentrations were determined by a modification of the lipase–glycerol phosphate oxidase method (GPO-Trinder, Sigma Chemical Co).
Lipoprotein fractions were separated by sequential density ultracentrifugation at the following densities (d values): chylomicrons combined with VLDL (d<1.006 g/mL), IDL combined with LDL (1.006 g/mL<d<1.055 g/mL), and HDL (1.055 g/mL<d<1.21 g/mL).20
Determination of Hepatic Cholesterol
The accumulation of hepatic free cholesterol and cholesteryl esters was used as a surrogate marker of cumulative cholesterol absorption and its inhibition in mice.18 At the end of the 6-month studies, representative samples of liver were collected, and hepatic free cholesterol and cholesteryl esters were extracted by using a method described by Folch et al.21 Lipid extracts were dried under nitrogen into high-performance liquid chromatography sample vials, dissolved in hexane/isopropanol, and assayed for cholesteryl ester and free cholesterol concentrations chromatographically as previously described.22
Determination of Atherosclerosis
Atherosclerotic lesion development and progression were evaluated after 6 months of treatment. After exsanguination and liver sampling, the mice were perfusion-fixed (4% paraformaldehyde in PBS) via a cannula placed in the left ventricle of the heart. The heart and aorta, including the carotid arteries, were excised intact to the iliac bifurcation. The aorta was removed from the heart, opened longitudinally between the intercostal ostia to the iliac bifurcation, pinned open, and photographed. The percentage of intimal surface occupied by grossly discernible atherosclerotic lesions in the arch and in the thoracic and abdominal regions was measured morphometrically (Bioquant Imaging System). Cross sections of the right carotid artery and the base of the aorta (serial sections from the aortic valves) were stained (Gamori trichrome) and evaluated for intimal lesion area by image analysis. Atherosclerotic lesions were quantified on the basis of the size of the cross-sectional areas (in square millimeters) in the aortic intima (cholesterol-free diet study) and the carotid artery (all studies) and the surface areas of the aortic arch, thoracic aorta, and abdominal aorta (cholesterol diet study). The atherosclerotic lesion areas were compared between ezetimibe-treated and the appropriate untreated control groups for each of the 3 study diets.
Statistical Analysis
Results are presented as mean±SEM. Statistical significance among responses in ezetimibe-treated and control groups was assessed by using 1-way ANOVA, the Dunnett multiple comparison test, and the unpaired Student t test. A value of P<0.05 was considered significant.
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Results |
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Cholesterol Absorption
Cholesterol absorption in apoE-/- and age-matched control (C57/BL +/+) mice was determined by the dual fecal isotope method. ApoE-/- mice were found to absorb 55.5% of the [14C]cholesterol, whereas the wild-type mice absorbed 51.2% (Figure 1). Ezetimibe was found to significantly inhibit cholesterol absorption in wild-type and apoE-/- mice at 0.3 mg/kg per day. Cholesterol absorption was inhibited by 90% at 3 mg/kg per day in apoE-/- mice and by >90% at 10 mg/kg per day in C57/BL +/+ and apoE-/- mice (Figure 1). The lack of apoE in the knockout mice did not alter cholesterol absorption or the activity of ezetimibe compared with that in wild-type mice.
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Body Weights and Food Consumption
Male apoE-/- mice were fed cholesterol-containing western or low-fat diets either alone or containing 0.005% ezetimibe for 6 months. Changes in body weight and food consumption were similar between the control and ezetimibe-treated groups fed the cholesterol-containing diets (Table). The calculated dose of ezetimibe consumed over the 6-month study was 5.31 and 5.93 mg/kg per day in the western diet–fed and the low-fat diet–fed mice, respectively. In the second 6-month study, apoE-/- mice were fed a semisynthetic cholesterol-free diet either alone or containing 0.005% ezetimibe. Body weights and food consumption were similar in the control and ezetimibe-treated mice fed the cholesterol-free diet (Table). The calculated ezetimibe dose delivered over 6 months to the cholesterol-free diet–fed apoE-/- mice was 4.65 mg/kg per day. The results of the cholesterol absorption study indicate that cholesterol absorption is inhibited by >90% at ezetimibe doses >3 mg/kg per day in mice fed Purina rodent chow. With the assumption that the activity of ezetimibe was not altered by the 3 different dietary conditions, cholesterol absorption would be predicted to be inhibited by >90% throughout the 6-month studies at ezetimibe doses of 4.65 to 5.93 mg/kg per day.
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Plasma Lipids and Lipoproteins
Plasma lipid and lipoprotein profiles were determined after the apoE-/- mice were fed the test diets for 6 months (Figure 2). The average plasma cholesterol level in the control western diet group was 964 mg/dL, and ezetimibe treatment caused a 61% reduction to 374 mg/dL (Figure 2A). The control low-fat cholesterol diet–fed mice had an average plasma cholesterol level of 726 mg/dL, and ezetimibe treatment caused a 68% reduction to 231 mg/dL. ApoE-/- mice fed a cholesterol-free diet had an average plasma cholesterol level of 516 mg/dL, and the inhibition of biliary cholesterol absorption by ezetimibe caused a 66% reduction in plasma cholesterol levels to 178 mg/dL (Figure 2A). Plasma triglyceride levels were not different between the control and ezetimibe-treated mice fed the low-fat cholesterol diet (94±13 and 102±10 mg/dL, respectively) or the cholesterol-free diet (93±8 and 103±11 mg/dL, respectively) for 6 months. The plasma triglyceride levels in the apoE-/- mice fed the western diet were reduced from 224±40 to 134±11 mg/dL by the 6-month ezetimibe treatment (P<0.05).
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Lipoprotein profile analysis after 6 months of treatment with ezetimibe demonstrated that the plasma cholesterol reductions occurred primarily in the VLDL/chylomicron fractions in all 3 of the diet groups (Figure 2B). Ezetimibe reduced VLDL/chylomicron (d<1.006 g/mL) cholesterol levels by 89%, 80%, and 87%, in the western diet–fed, low-fat cholesterol diet–fed, and cholesterol-free diet–fed mice, respectively. LDL cholesterol levels were also reduced from 53% to 67% by ezetimibe treatment in the 3 diet groups (Figure 2C). HDL cholesterol levels were reduced in the apoE-/- mice compared with the wild-type mice. Ezetimibe treatment for 6 months significantly increased HDL cholesterol levels in mice fed the cholesterol-free diet (P<0.001) and increased HDL levels by >2-fold in mice fed either a low-fat cholesterol or western diet (Figure 2D).
Hepatic Cholesterol
Hepatic cholesterol levels increase in cholesterol-fed animals, and the inhibition of the accumulation of hepatic cholesteryl esters has been used as a surrogate marker of cholesterol absorption inhibition.18 Inhibition of cholesterol absorption by ezetimibe would be expected to result in reduced accumulation of hepatic free and esterified cholesterol. Long-term (6-month) administration of ezetimibe at doses of 4.65 to 5.93 mg/kg per day significantly reduced the accumulation of hepatic free cholesterol and esterified cholesterol in apoE-/- mice fed all 3 diets compared with untreated control mice (Figure 3, P<0.05). Reductions in cholesteryl esters ranged from 37% (cholesterol-free diet) to 66% (western diet), and reductions in hepatic free cholesterol ranged from 20% (cholesterol-free diet) to 43% (low-fat cholesterol diet, Figure 3A and 3B).
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Development and Progression of Atherosclerosis
Ezetimibe had dramatic inhibitory effects on the development and progression of atherosclerosis in the apoE-/- mice fed all 3 diets. The surface area of the aorta occupied with atherosclerotic lesions was determined in the cholesterol-fed groups. The aortic atherosclerotic lesion surface area of the entire aorta was reduced from 20.2% to 4.1% in the western diet group and from 24.1% to 7.0% in the low-fat cholesterol group by ezetimibe treatment (P<0.05). Atherosclerosis progression was most severe in the arch of the aorta in apoE-/- mice. In the western diet group, 44.5% of the aortic arch was occupied by atherosclerotic lesions. In the low-fat cholesterol group, 53.6% of the aortic arch had lesion involvement (Figure 4A and 4B). Ezetimibe treatment reduced aortic arch atherosclerosis by 87% and 81% in the western diet–fed and low-fat cholesterol–fed groups, respectively (Figure 4A and 4B). Atherosclerosis involvement of the thoracic aorta was reduced from 68% to 74%, and that of the abdominal aorta was reduced from 47% to 71% by ezetimibe in both cholesterol-fed apoE-/- groups (Figure 4A and 4B). The apoE-/- mice fed the cholesterol-free diet for 6 months did not develop extensive aortic atherosclerosis. Therefore, cross sections of the aorta from the aortic valves were evaluated. Ezetimibe treatment of the mice fed a cholesterol-free diet for 6 months caused a significant reduction of atherosclerosis at the base of the aorta, with an 89% reduction of intimal lesion area at 100 µm distal to the valves (Figure 5A). The proximal right carotid artery in apoE-/- mice rapidly develops atherosclerosis.16,23 The intimal lesion cross-sectional area of the carotid artery was reduced 91% to 97% by ezetimibe treatment from 0.098 to 0.003 mm2, from 0.142 to 0.004 mm2, and from 0.044 to 0.004 mm2 in the western diet, low-fat diet, and cholesterol-free diet groups, respectively (Figure 5B).
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Discussion |
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The protective role of apoE in the prevention of atherosclerosis occurs through diverse mechanisms of lipid transport and clearance, stimulation of cellular cholesterol efflux, and modulation of the immune response.12–15 In the apoE-/- mouse, increases in plasma cholesterol concentrations occur in response to diets that are either normal, high in fat, or cholesterol rich, resulting in rapid and severe atherosclerosis.13–17,23,24 The pathophysiology of the atherosclerosis in this model parallels that in humans with respect to vascular distribution, cellular components, and overall lipid content of the lesions.14–16,23
In the present study, it was confirmed that cholesterol absorption in apoE-/- mice is similar to that in wild-type mice.25 Ezetimibe significantly inhibited cholesterol absorption in wild-type and apoE-/- mice at 0.3 mg/kg per day and by >90% at 10 mg/kg per day. The fat and cholesterol content of the diet was previously found not to alter the activity of this class of compounds on cholesterol absorption.18 Therefore, cholesterol absorption inhibition by ezetimibe should have been similar among the 3 dietary conditions in the apoE-/- mice. Ezetimibe was previously found to be more potent in hamsters, rats, rabbits, dogs, and monkeys, with an ED50 of 0.5 µg/kg per day in rhesus monkeys.5–11 It has also been reported that this class of cholesterol absorption inhibitor specifically reduces the cholesterol content of postprandial chylomicrons in monkeys.9 ApoE is a ligand for the removal of chylomicron remnants and VLDL by the liver. In apoE-/- mice, chylomicron remnants and VLDL accumulate, and HDL levels are reduced.24,25 Chronic inhibition of cholesterol absorption in the apoE-/- mice by ezetimibe resulted in a dramatic reduction in the chylomicron/VLDL cholesterol levels in the western, low-fat cholesterol, and cholesterol-free dietary groups. The only source of intestinal cholesterol in the apoE-/- mice fed the cholesterol-free diet originates in the bile. Thus, ezetimibe specifically inhibited the absorption of biliary cholesterol, which resulted in an 87% reduction in chylomicron/VLDL cholesterol levels. The reduction in the chylomicron cholesterol levels by ezetimibe decreased the amount of exogenous cholesterol delivered to the liver and resulted in reduced hepatic cholesterol levels in apoE-/- mice fed all 3 diets. Ezetimibe reduced hepatic cholesteryl esters to a similar level in all 3 dietary groups, which may indicate that ezetimibe completely inhibited the intestinal absorption and delivery to the liver of dietary and biliary cholesterol. The significant reduction of hepatic cholesterol levels by ezetimibe treatment may have caused an upregulation of hepatic LDL receptors, which resulted in the reduction of LDL cholesterol levels in all the ezetimibe-treated groups.
Ezetimibe treatment for 6 months resulted in a nearly complete inhibition of the development of atherosclerosis in apoE-/- mice fed a western, low-fat cholesterol, or a cholesterol-free diet. In ezetimibe-treated mice, the aortic surface area involved with lesions was <10%, and the cross-sectional area of the carotid artery atherosclerotic lesions was reduced by >90%. With such a dramatic reduction in atherosclerosis, it was not possible to determine the effect of ezetimibe on the cellular and extracellular composition of the atherosclerotic lesions or to correlate lipoprotein changes to the extent of atherosclerosis. The only lesions present in the ezetimibe-treated groups were small fatty streaks. The inhibition of atherogenesis by specifically inhibiting cholesterol absorption with ezetimibe in this chylomicron remnant model of atherosclerosis may have occurred primarily by reducing the remnant and lower density lipoprotein levels. Clinical and preclinical studies have reported results supporting the concept that chylomicron remnants and remnant-like particle cholesterol levels may significantly increase the risk of atherosclerosis and coronary heart disease.26–29 Alternatively, some of the antiatherogenic activity of ezetimibe may have occurred through increasing the low HDL cholesterol levels found in the apoE-/- mice.24,25 HDL cholesterol was increased >2-fold in the cholesterol-fed apoE-/- mice and by 65% in the mice fed the cholesterol-free diet by ezetimibe treatment. It has been well documented that HDL cholesterol levels are negatively correlated with coronary heart disease in humans.30 ApoE-/- mice that overexpress human apoA-1 and have increased HDL cholesterol levels develop less atherosclerosis.31 In addition, apoE-/- mice fed a western diet and treated with troglitazone were reported to have an increased HDL cholesterol level and a reduction in atherosclerosis.32 Ezetimibe has not been found to significantly increase HDL cholesterol levels in other animal models, and the mechanism by which it occurs in apoE-/- mice is not known.9–11,33
Previous preclinical studies with ezetimibe have demonstrated significant antihypercholesterolemic activity in rodents, rabbits, dogs, and monkeys when given alone or in combination with HMG-CoA reductase inhibitors.5–11 The present studies extend these findings by establishing that ezetimibe inhibits the development of atherosclerosis in apoE-/- mice. As a single agent, the effect of ezetimibe on plasma cholesterol has been investigated extensively in studies using normocholesterolemic and hypercholesterolemic animal models. In cholesterol-fed animals, including hamsters, rats, dogs, rabbits, and rhesus monkeys, ezetimibe effectively lowered levels of plasma and hepatic cholesterol.5–11 In a hamster model of combined hyperlipidemia induced by moderate-cholesterol/high-fat diets, ezetimibe normalized hypercholesterolemia and hypertriglyceridemia and reduced the accumulation of hepatic cholesterol.33 In animals with preestablished hypercholesterolemia, including rabbits and monkeys, ezetimibe normalized or reversed hypercholesterolemia primarily through normalization of LDL levels, a finding directly applicable to patients with hypercholesterolemia.9,11 In contrast to the effect of ezetimibe in apoE-/- mice, in hamsters and dogs fed cholesterol-free diets, ezetimibe alone produced rather modest reductions in plasma and hepatic cholesterol levels.10,11,33 In these studies, ezetimibe reduced the delivery of biliary cholesterol from the intestine to the liver, producing a corresponding reduction of hepatic cholesterol stores and subsequent upregulation of hepatic HMG-CoA reductase activity.10,11,18 The upregulation of hepatic cholesterol synthesis resulted in a normalization of circulating cholesterol levels. Lipid-lowering agents that target synthesis of hepatic cholesterol, such as the statins, may effectively counter the upregulation of cholesterol synthesis in response to ezetimibe. In normocholesterolemic dogs, ezetimibe at doses as low as 0.007 mg/kg per day in combination with lovastatin, pravastatin, fluvastatin, simvastatin, or atorvastatin was found to cause significant synergistic reductions in plasma cholesterol levels.10,11 Significant reductions of rabbit aortic atherosclerosis were also observed with the use of ezetimibe alone or in combination with a statin.11
Ezetimibe is rapidly progressing through human clinical trials. In hypercholesterolemic patients, ezetimibe at 10 mg/d significantly reduced LDL cholesterol levels by 
18% and increased HDL cholesterol levels.34 Coadministering ezetimibe at 10 mg/d with simvastatin at 10 or 20 mg/d, lovastatin at 20 or 40 mg/d, or atorvastatin at 10 mg/d resulted in a 16% to 18% additive reduction in LDL cholesterol levels compared with statin monotherapy in hypercholesterolemic humans.35–38 Pharmacological strategies for lipid management that rely on the diversity and synergy among the mechanisms involved in cholesterol production, transport, and balance would be expected to favorably impact plasma cholesterol levels.
Ezetimibe, the first of a new class of lipid-lowering agents for patients with hypercholesterolemia, selectively inhibits cholesterol absorption by preventing its uptake at the level of the intestinal wall. Ezetimibe localizes in the intestine and undergoes enterohepatic recirculation, thus delivering the drug repeatedly to its intestinal site of action.7,8 Results from the present study, which used a well-established animal model of atherosclerosis, demonstrated that ezetimibe inhibited cholesterol absorption, decreased total plasma cholesterol largely through chylomicron remnant and VLDL reductions, increased HDL levels, and inhibited the progression of atherosclerosis in apoE-/- mice under high-fat western, low-fat cholesterol, and cholesterol-free dietary conditions. Ezetimibe may inhibit atherogenesis clinically in hypercholesterolemic individuals that, despite the diversity of available therapies, cannot reach target LDL cholesterol concentrations to reduce their risk of coronary heart disease.
Received July 24, 2001; accepted September 26, 2001.
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