Author + information
- Received December 6, 1996
- Revision received March 31, 1997
- Accepted April 17, 1997
- Published online August 1, 1997.
- Liying Chen, MDA,
- W.Herbert Haught, MDA,
- Baichun Yang, MD, PhDA,
- Tom G.P Saldeen, MD, PhD, FACCB,
- Sampath Parathasarathy, PhDC and
- Jawahar L Mehta, MD, PhD, FACCA,* ()
- ↵*Dr. Jawahar L. Mehta, Box 100277, University of Florida, Gainesville, Florida 32610.
Objectives. We sought to document the common mechanisms of the antiatherogenic effects of the cholesterol-lowering hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin, the dihydropyridine Ca2+blocker amlodipine and the antioxidant vitamin E.
Background. Vitamin E, HMG-CoA reductase inhibitors and Ca2+blockers each inhibit atherosclerosis in hypercholesterolemic animals.
Methods. New Zealand White rabbits were fed regular chow (Group A), chow with 1% cholesterol (Group B), 1% cholesterol diet plus lovastatin (Group C), 1% cholesterol diet plus vitamin E (Group D) or 1% cholesterol diet plus amlodipine (Group E) for 12 weeks. The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured as indexes of lipid peroxidation and antioxidant activity, respectively.
Results. Group A rabbits showed no atherosclerosis, whereas Group B rabbits had 17.4 ± 9.3% (mean ± SD) of the aorta covered with atherosclerosis, and Groups C, D and E rabbits had significantly less atherosclerosis. Plasma SOD activity was lower in Group B than in Group A (6.9 ± 1.1 vs. 12.8 ± 1.5 U/ml, p < 0.01) and was preserved in the groups given lovastatin, vitamin E or amlodipine with a high cholesterol diet. The serum MDA level was higher in Group B rabbits than Group A rabbits (12.1 ± 2.6 vs. 1.2 ± 0.1 nmol/ml, p < 0.01) and increased minimally in rabbits given lovastatin, vitamin E or amlodipine with a high cholesterol diet. In in vitro experiments, both lovastatin and amlodipine preserved SOD activity and reduced the oxidizability of low density lipoproteins by rabbit leukocytes.
Conclusions. This study suggests that a reduction in lipid peroxidation and preservation of SOD may be common mechanisms of antiatherosclerotic effects of lovastatin, vitamin E and amlodipine.
Elevated levels of total cholesterol and low density lipoproteins (LDL) in plasma are major risk factors for the development of atherosclerosis . Much evidence provides support for the concept that the oxidized form of LDL causes oxidant stress and increases intracellular Ca2+in the vessel wall, and represents the pathogenic element in hypercholesterolemia [2, 3]. Release of oxidant species from activated leukocytes, such as superoxide radical and hydroxyl radical, in principle, contributes to the oxidation of LDL . Thus, a strategy directed at the use of antioxidants such as vitamin E has been advocated to decrease the susceptibility of LDL to oxidation by interrupting free radical peroxidative chain reactions and to increase the resistance to atherosclerosis by protecting against endothelial dysfunction in cholesterol-fed animals [6, 7].
Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and Ca2+blockers have been shown to prevent or retard the progression of atherosclerosis [8–11]. The classic HMG-CoA reductase inhibitor, lovastatin, has been found to inhibit the oxidation of LDL in in vivo and in vitro studies . The calcium antagonists, including lacidipine and cinnarizine, have been reported to inhibit lipid peroxidation, probably by quenching several radical species [13, 14]. So far, the mechanism(s) by which HMG-CoA reductase inhibitors and Ca2+antagonists inhibit oxidation is unclear. It may be hypothesized that the antiatherogenic effect of these drugs may relate in part to their ability to limit oxidation of LDL. Notably, the direct effect of these agents on the endogenous antioxidant system, such as superoxide dismutase (SOD) , has not yet been well characterized.
The objective of the present study was to document the antiatherogenic effects of the cholesterol-lowering HMG-CoA reductase inhibitor lovastatin, the dihydropyridine Ca2+blocker amlodipine and the well known antioxidant vitamin E. We used the hypercholesterolemic rabbit as a model for experimental atherosclerosis. The effects of these agents on lipid peroxidation and on the endogenous antioxidant system were assessed in these rabbits. To further study the antioxidant mechanism(s), the effects of lovastatin and amlodipine on LDL oxidation mediated by CuSO4or leukocytes were also investigated. In addition, we examined if a relation exists between the extent of atherosclerosis and lipid peroxidation and endogenous antioxidant defense.
This study was approved by the University of Florida and complied with all local, state and federal guidelines.
Lovastatin was obtained from Merck and Co. Amlodipine besylate was obtained from Pfizer Laboratories. Vitamin E (dl-alpha-tocopherol acetate in a base of vegetable oil) was obtained from Nature’s Bounty. All other chemicals were purchased from Sigma Chemical Co.
1.2 Feeding protocol.
New Zealand White rabbits (all males, starting age 4 to 6 weeks) were randomly assigned to one of the five dietary groups for 12 weeks: Group A = regular chow (n = 4); Group B = regular chow with 1% cholesterol (n = 6); Group C = high cholesterol diet plus lovastatin, 2 mg/kg body weight per day (n = 4); Group D = high cholesterol diet plus vitamin E, 0.04 g/kg per day (n = 4); Group E = high cholesterol diet plus amlodipine, 2 mg/kg per day (n = 5).
These diets were prepared by mixing lovastatin, amlodipine and vitamin E directly into regular chow containing a high cholesterol diet. The complete consumption of chow was checked daily to assure that the rabbits had completely consumed their food (∼100 g).
1.3 Blood and aortic tissue collections.
Blood was collected from an ear vein before the dietary feeding was started. Blood was collected again at 12 weeks of dietary intervention from the carotid artery before the rabbits were killed by administration of sodium pentobarbital (100 mg/kg) into the marginal ear vein. The aorta was isolated along its entire length, removed and washed in saline for measurement of the area of sudanophilia as index of atherosclerosis. Blood was used for measurement of serum cholesterol, malondialdehyde (MDA) and vitamin E levels and plasma or leukocyte SOD activity.
1.4 Quantification of area of atherosclerosis.
After fixation, the lumen surface of the aorta from all the rabbits was stained with Sudan III/IV solution (prepared by dissolving 1 g of Sudan III in 100 ml of 70% ethanol and 0.1 g of Sudan IV in 100 ml of 70% ethanol/acetone and mixing Sudan III and IV). After washing with water for 5 min, the total and fatty streak surface areas were quantified by use of computerized planimetry and recorded. The area of atherosclerosis was expressed as percent total aortic area.
1.5 Determination of serum MDA, cholesterol and vitamin E levels.
Malondialdehyde in serum was determined by a modification of the method of Ohkawa et al. . The assay mixture consisted of 0.1 ml of the serum, 0.4 ml of 0.9% NaCl, 0.5 ml of 3% sodium dodecylsulfate, 3 ml of thiobarbituric acid reagent (containing equal parts of 0.8% aqueous thiobarbituric acid and acetic acid) and was heated for 75 min at 95°C. Thereafter, the mixture was added to 1 ml of cold 0.9% NaCl and cooled in tap water and extracted by adding 5 ml of n-butanol. After centrifugation at 3,000 rpm for 15 min, the butanol phase was assayed spectrophotometrically at 532 nm. Amounts of 0, 20, 40, 60 and 80 nmol of tetramethoxypropane served as the external standard and were assayed in the previously described fashion. Serum MDA content was expressed as nmol/ml. Serum cholesterol was estimated by use of a routine automated system. Plasma vitamin E (alpha-, beta- and gamma-tocopherol) was measured by high performance liquid chromatography with fluorescence detection, as described earlier .
1.6 Determination of SOD activity.
Total SOD activity in plasma was measured spectrophotometrically by monitoring the SOD-inhibitable autoxidation of pyrogallol, as described by Marklund and Marklund . The reaction mixture (4.5 ml) contained 0.2 mmol/liter of pyrogallol, 1 mmol/liter of diethylenetriamine pentaacetic acid, 50 mmol/liter of Tris-cacodylic acid buffer (pH 8.2) and 4 μg of catalase. The reaction was carried out at 25°C. The rate of increase in absorbance at 420 nm was recorded. One unit of enzyme activity is defined as 50% inhibition of pyrogallol autoxidation under the assay conditions. The SOD activity in plasma is expressed as units per milliliter.
1.7 Preparation of leukocytes.
Rabbit leukocytes were isolated using the method described by Chand et al. . Briefly, about 30 ml of blood from the carotid artery was collected in 3.8% sodium citrate and mixed with 7.5 ml of 6% dextran in isotonic saline and allowed to stand for 30 min at 37°C. The leukocyte-rich upper layer was transferred to another tube containing 2.25 ml of 0.1 mol/liter of EDTA and centrifuged at 1,000 rpm at 4°C. The leukocytes were washed with Tris buffer (composition in mmol/liter: Tris, 25; NaCl, 120; KCl, 5; bovine albumin, 0.03%; and dextrose, 0.1%) with or without 0.6 mmol/liter of CaCl2and 1 mmol/liter of MgCl2at 4°C.
1.8 Lovastatin, amlodipine and LDL oxidation.
To investigate the effect of lovastatin on copper- or leukocyte-mediated LDL oxidation, LDL (50 μg/ml) was incubated with CuSO4(10 μmol/liter) or phorbol 12-myristate 13-acetate (PMA, 100 ng/ml)–stimulated rabbit leukocytes (106cells/ml) in 2 ml of Tyrode’s buffer (composition in mmol/liter: NaCl, 137; KCl, 2.7; MgCl2, 1.0; CaCl2, 1.0; NaH2PO4, 0.35; NaHCO3, 11.9; and glucose, 5.5 at pH 6.5) with or without different concentrations of lovastatin (5 and 10 μmol/liter) or amlodipine (50 and 100 μg/ml) at 37°C. After 1 h of incubation, leukocytes were centrifuged and used to measure SOD activity with the method described earlier. The kinetics of LDL oxidation in the aliquots of supernate were determined by monitoring the change in absorbance (234 nm) on a Perkin-Elmer lambda 5 UV-spectrophotometer. Absorbance was recorded every hour for 6 h. From the kinetic absorbance profile of each experiment, the maximal rate of oxidation was calculated from the slope of the absorbance curve during the propagation phase, expressed as nmol dienes/min per mg LDL protein. Superoxide dismutase activity in leukocytes treated as described previously was measured and expressed as U/2 × 106cells.
All data are presented as mean value ± SD. Comparison among different groups was made by analysis of variance followed by the Student-Newman-Keuls test. Correlation between MDA and SOD values and extent of sudanophilia was made by stepwise linear regression analysis using the Statview version 4.2 program.
2.1 Body weight and feed intake.
There was no significant difference in body weight among the different groups of rabbits. No difference in food intake was observed, as all rabbits completely finished their rations every day.
2.2 Atheromatous fatty streak formation.
Representative examples of the areas of atherosclerosis are presented in Fig. 1. Group A rabbits fed regular chow for 12 weeks showed no visible atherosclerosis lesions in all the aortas. In Group B rabbits, fatty streaks covered 17.4 ± 9.3% of the aortic area. However, the extent of atherosclerosis was significantly decreased in rabbits fed a high cholesterol diet with either lovastatin, vitamin E or amlodipine (extent of atherosclerosis as percent aortic area 5.3 ± 0.7%, 9.7 ± 1.7% and 10.2 ± 4.2%, respectively, all p < 0.05 vs. Group B).
2.3 Serum MDA, cholesterol and vitamin E levels and plasma SOD activity.
The mean serum MDA level was increased ∼10-fold in Group B rabbits compared with Group A rabbits (12.1 ± 2.6 vs. 1.2 ± 0.1 nmol/ml, p < 0.01). The increase in serum MDA was significantly (p < 0.05) reduced by supplementation of a cholesterol-rich diet with lovastatin, vitamin E or amlodipine (Table 1).
The average baseline serum cholesterol level in Group A rabbits was 60 ± 17 mg/dl. Serum cholesterol levels were significantly greater in rabbits fed high cholesterol, whether or not supplemented with lovastatin, vitamin E or amlodipine (Table 1).
The baseline serum vitamin E level was 1.96 ± 0.19 μg/ml in Group A and remained unchanged with feeding of regular chow. Serum vitamin E levels were significantly increased in all rabbits fed a high cholesterol diet. As expected, a high cholesterol diet supplemented with vitamin E caused a further increase (27.2 ± 7.7 vs. 8.4 ± 0.01 μg/ml in Group B, p < 0.01) (Table 1).
Plasma SOD activity was markedly decreased in rabbits fed a high cholesterol diet (6.9 ± 1.1 vs. 12.8 ± 1.5 U/ml plasma in Group A, p < 0.01). Treatment with lovastatin, vitamin E or amlodipine increased and preserved SOD activity despite intake of a high cholesterol diet (Table 1).
2.4 Effect of lovastatin or amlodipine on oxidation of LDL and leukocyte SOD activity.
Copper sulfate (CuSO4) or leukocyte-induced oxidation was quantitated by continuously monitoring the conjugated diene formation, and the oxidizability of LDL was expressed as the maximal rate of oxidation. Treatment with 5 μmol/liter of lovastatin significantly decreased the maximal rate of oxidation induced by leukocytes from 2.2 ± 0.1 to 1.2 ± 0.3 nmol dienes/min per mg LDL protein, and 10 μmol/liter of lovastatin totally blocked the initiation of LDL oxidation induced by leukocytes. Amlodipine also decreased the oxidizability of LDL mediated by leukocytes, but to a lesser extent. Results of representative experiments are shown in Fig. 2and Fig. 3, and the results from four experiments are summarized in Table 2. Neither lovastatin nor amlodipine affected CuSO4(10 μmol/liter)–induced oxidation of LDL (data not shown).
Superoxide dismutase activity was significantly preserved by both lovastatin and amlodipine in PMA-stimulated leukocytes. Interestingly, SOD activity was preserved in the presence of LDL, and it increased further in the presence of lovastatin or amlodipine in PMA-stimulated leukocytes. The results are summarized in Table 2.
2.5 Extent of atherosclerosis, serum MDA level and plasma SOD activity.
There was a linear relation between the extent of atherosclerosis and serum MDA level (y = 3.143x − 7.4224, r = 0.866, n = 23). There was an insignificant inverse relation between plasma SOD activity and the extent of atherosclerosis. A similar absence of relation was found between plasma SOD and serum MDA levels.
3.1 Lipid peroxidation, antioxidant activity and atherosclerosis.
The causative role of increased cholesterol level and lipid peroxidation in the development of atherosclerosis , as well as in the antiatherogenic effects of HMG-CoA reductase inhibitors, Ca2+blockers and antioxidants, has been well documented [5–11]. This study, for the first time, provides definitive evidence that the extent of atherosclerosis is proportional to the extent of lipid peroxidation. In support of this concept, we found a positive correlation between the extent of aortic atherosclerosis and plasma MDA values. We also found that lovastatin, amlodipine and vitamin E each protected against lipid peroxidation and preserved the endogenous antioxidant enzyme SOD in hyperlipidemic rabbits. In addition, we demonstrated the antioxidant activity of both lovastatin and amlodipine, by their inhibitory effects on the oxidation of LDL in in vitro experiments.
The presence of lipid peroxides in tissue and cell lipids has long been assumed to be a sign of atherosclerosis . Despite the presence of potent antioxidant defenses in plasma, there are detectable levels of lipid peroxides in plasma in atherosclerosis [21–24]. The present study extended these previous observations. We found that the increased lipid peroxidation was associated with decreased activity of the endogenous antioxidant enzyme SOD, but there was no direct correlation between the degree of lipid peroxidation and loss of antioxidant activity. Furthermore, there was no significant correlation between SOD activity and the extent of atherosclerosis. These observations, nonetheless, provide evidence that increased oxidative stress or decreased antioxidant activity, or both, are major mechanism(s) involved in the pathogenesis of atherosclerosis . These data may also be taken to affirm the view that plasma lipid peroxidation is a consequence of the atherosclerosis process.
The weak correlation between plasma SOD activity and atherosclerosis may reflect the presence of multiple antioxidants, of which SOD is only one component. The levels of different antioxidants may vary during atherogenesis. Superoxide dismutase activity, perhaps the most important antioxidant enzyme , fell in our study, whereas the levels of vitamin E, the most abundant naturally occurring lipid-soluble antioxidant, increased in all rabbits fed a high cholesterol diet. The precise mechanism of increased levels of vitamin E in plasma in these rabbits is not clear; however, it has been reported that LDL isolated from plasma contains considerable amounts of antioxidants, such as vitamin E and carotenoids . Because vitamin E is predominantly associated with LDL, its increased levels in hypercholesterolemic animals may simply reflect elevated plasma lipid levels. The pathophysiologic significance of this phenomenon remains unclear. It may be speculated that the increased vitamin E level in plasma may affect SOD measurement, resulting in an increased level of the latter. However, we found that SOD activity was significantly decreased in Group B rabbits, whereas the plasma vitamin E level was increased. We therefore believe that a high vitamin E level in the blood is unlikely to be the sole basis of preservation of antioxidant activity in Group C, D and E rabbits.
3.2 Calcium blockers, statins and lipid peroxidation.
The proximity of the endothelium to circulating leukocytes makes it an important early target for leukocyte adherence . The activation of leukocytes can damage the vascular tissues by release of free radicals and proteolytic enzymes. Superoxide radicals from activated leukocytes adherent to the blood vessels have been recognized as an initiating factor in the oxidation of LDL . Thus, lipid peroxidation is evident at all stages of atherosclerosis, especially in early atherosclerotic lesions . It has been suggested that antioxidants exert their greatest beneficial effect on early lesions . Epidemiologic evidence and clinical trials have suggested that vitamin E may slow the atherogenic process and prevent nonfatal myocardial infarction in patients with coronary artery disease [29, 30]. Similar to vitamin E, clinical trials and angiographic studies have demonstrated that statins and Ca2+blockers significantly decrease the frequency of cardiac events and the number of new atherosclerotic lesions [31–33]. However, so far it is uncertain as to how statins and Ca2+blockers have an effect similar to that of vitamin E. In the present study, supplementation with vitamin E was found to further increase plasma vitamin E levels and decrease lipid peroxidation by increasing endogenous SOD activity in rabbits fed a high cholesterol diet. Importantly, lovastatin and amlodipine also significantly decreased plasma lipid peroxides and preserved plasma SOD activity. Our in vitro study also shows that SOD activity in PMA-activated leukocytes is significantly increased by both lovastatin and amlodipine, indicating that the antiatherogenic effect of these three agents may be, at least in part, mediated through a common antioxidant pathway.
Lovastatin, an HMG-CoA reductase inhibitor, is known for its potent LDL cholesterol-lowering effect [8, 9]. Interestingly, in the present study, lovastatin had no significant effect on plasma cholesterol levels, perhaps because the dose used in the present study may have been too low and the cholesterol load too high, and yet it reduced the extent of aortic atherosclerosis. Similar to our study, Saitoh et al. examined the effect of fenofibrate in hyperlipidemic rabbits and found an antiatheromatous effect independent of the hypolipidemic effect of this agent .
The clinically relevant dose of lovastatin used in our study might act as an antioxidant in this atherosclerotic model and exert an antiatherosclerotic effect. In our in vitro studies, we observed that lovastatin at a high concentration abolished PMA-activated leukocyte-induced oxidation of LDL. This antioxidant effect of lovastatin was associated with an increase in SOD activity in rabbit leukocytes. The lovastatin molecule, per se, does not contain an antioxidant center. However, by inhibiting the isoprenoid reaction during the activation of NADPH oxidase, lovastatin may affect the generation of oxygen radicals . Indeed, lovastatin and a related compound, simvastatin, have been reported to inhibit LDL oxidation induced by activated macrophages by reducing superoxide production [12, 37].
It is noteworthy that SOD activity in PMA-activated rabbit leukocytes was also preserved in the presence of LDL. This effect of LDL may be related to its large amounts of antioxidant components such as vitamin E, which can act as the scavengers of free radicals . In addition, LDL serves as a target of superoxide anion in this system .
Like lovastatin, treatment of rabbits with amlodipine also resulted in a marked increase in antioxidant activity. Amlodipine is a long-acting dihydropyridine Ca2+antagonist with vascular selectivity. In vitro studies have suggested that the dihydropyridine Ca2+antagonists act as antioxidants by directly quenching several radicals species [13, 39]. The antioxidant activity of these Ca2+antagonists probably contributes to their Ca2+blocking effect . Our in vitro studies also demonstrate that amlodipine reduces the oxidizability of LDL rabbit leukocytes. The studies provide strong evidence that the antioxidant activity of amlodipine, both in vivo and in vitro, may relate to the antiatherosclerotic effect of this agent.
Neither lovastatin nor amlodipine had any significant effect on CuSO4-induced LDL oxidation in vitro, but both agents reduced leukocyte-induced oxidation of LDL. Notably, the leukocyte-induced oxidation of LDL depends on the ability of myeloperoxidase to generate free radicals that oxidize LDL. In fact, this model of oxidizability of LDL is similar to the in vivo conditions, because the concentration of free copper or iron, the most effective ions in the oxidation of LDL, is extremely low in the body. Our observations of the antioxidant effect of lovastatin or amlodipine appear to be related to their inhibitory effect on free radical generation from leukocytes and other tissues.
We have shown an association between increased cholesterol levels and lipid peroxidation in the development of atherosclerosis, and we have documented the antiatherogenic effects of lovastatin, amlodipine and vitamin E in an atherosclerotic rabbit model. We have also demonstrated the antioxidant effect of lovastatin and amlodipine both in vivo and in vitro, and their antioxidant effects were comparable to the reference antioxidant, vitamin E. This study provides evidence that the extent of atherosclerosis is proportional to the extent of lipid peroxidation and that oxidative stress is increased in the atherosclerotic process. The antioxidant effect of lovastatin, amlodipine and vitamin E may be a common mechanism underlying their antiatherosclerotic effect.
☆ This study was supported by a grant-in-aid from the American Heart Association, Florida Affiliate, St. Petersburg; a Research Fellowship to Dr. Haught from the American Heart Association, Florida Affiliate, St. Petersburg; a Merit Review Award from the Department of Veterans Affairs, Washington, D.C.; and a Research Development Award from the Division of Sponsored Research, University of Florida, Gainesville, Florida.
- copper sulfate
- hydroxymethylglutaryl coenzyme A
- low density lipoprotein
- phorbol 12-myristate 13-acetate
- superoxide dismutase
- Received December 6, 1996.
- Revision received March 31, 1997.
- Accepted April 17, 1997.
- The American College of Cardiology
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