Atherogenesis is a chronic inflammatory process that involves a complex interplay between circulating cellular and blood elements and the cells of the arterial wall.¹,² While many factors are involved, the “oxidation hypothesis” has been a central focus in the investigation of the pathogenesis of the atherosclerotic process, suggesting that the oxidative modification of low-density lipoprotein (LDL), or other lipoproteins, is central, if not obligatory for the atherogenic process.³,⁴

Oxidation of LDL is a lipid peroxidation process resulting in formation of a wide range of biologically active products, including peroxides and malondialdehyde. Oxidation of LDL results in the generation of aldehydes that substitute lysine residues in the apoprotein B-100 moiety of LDL and causes apolipoprotein B-100 fragmentation. As a result, the particle loses its affinity for the LDL receptor and binds avidly to scavenger receptors on macrophages, resulting in foam cell formation.⁵,⁶

Advances in the management of hypercholesterolemia are primarily due to the development of the 3-hydroxy-3-methylglutaryl coenzyme (HMG-Co A) reductase inhibitors, ie, the statins.⁷ Recently, many statins have been used for their overall antihypercholesterolemic action in patients with atherosclerosis. They are presumed to work by upregulating the LDL receptor, thereby lowering LDL cholesterol in particular. In addition to lowering cholesterol levels, statins seem to act on the formation and progression of atherosclerotic plaque through alternative mechanisms involved in progression of the disease.⁸ The various statins differ in their pharmacokinetics, therapeutic efficacy, drug interactions, dosage, and administration, and therefore they differ in their potential advantages and disadvantages. Reduction of lipid peroxidation may be one of the important features explaining the favorable effect of statins in preventing or reducing atherosclerosis. Even though some investigators have shown the antioxidant potential of statins in vitro,⁸ in vitro investigations may not be the best approach to evaluate the effects of these agents because many other relevant events may be taking place in vivo.

In this study, we examined lipid peroxidation products, ie, thiobarbituric acid reactive substances (TBARS) and LDL-conjugated diene, formed as a result of oxidative stress generated in a hypercholesterolemic atherosclerosis model in rabbits. The antioxidant activity of defensive enzymes, including superoxide dismutase, catalase, and paraoxonase, was also investigated to determine oxidative status. The effects of atorvastatin, a potent HMG-Co A reductase inhibitor, with the longest half-life of its group, was evaluated for its influence on lipid profiles, lipid peroxidation, and the antioxidant system, as well as its impact on paraoxonase activity. In addition, atherosclerotic plaque formation and the outcome of atorvastatin treatment was assessed histopathologically.

Even though atherosclerosis is a complex, progressive, and multifactorial disease, nowadays there is no doubt that elevated plasma cholesterol levels play a dominant role in its pathogenesis. HMG-CoA reductase inhibitors, ie, statins, are widely prescribed in many countries to lower cholesterol levels in patients at risk.¹⁴ In this study, we investigated the effect of atorvastatin on the oxidant–antioxidant status of LDL cholesterol and erythrocytes, as well as in arcus aortic tissue.

In the animal model used, an atherogenic diet severely increased cholesterol levels (Table 1). Plasma total cholesterol and LDL cholesterol levels reached about 20 times the initial values, consistent with the results of another study reported by Bustos et al.¹⁵ The increase in triglyceride levels may have been associated with the addition of butter to the atherogenic diet.

Besides being a potent HMG-CoA reductase inhibitor, atorvastatin also has the longest half-life among the available statins, which contributes to its high efficacy. In view of this and conflicting reports about the antioxidant characteristics of the drug, atorvastatin was chosen from the available statins for this study. The dose of atorvastatin used (5 mg/kg/day) was decided on the basis of previous experiments using 1, 2.5, and 10 mg/kg/day.¹⁵

It must be emphasized that, in contrast with other studies, we did not start atorvastatin treatment at the beginning of the experiment, but started it after 7 weeks of the atherogenic diet, in order to mimic conditions of human atherosclerosis, where statins are used in most cases after atherogenesis is established. Most studies in humans are performed after the onset of clinical signs when the disease is well advanced. In this respect, animal models are more convenient to use because they allow study of the initiation and progression of the preclinical stages of atherosclerosis in a more controlled setting within a shorter period of time. As expected, atorvastatin achieved a 25% decrease in LDL cholesterol levels, consistent with previous studies.¹⁵–¹⁷

LDL levels in the atherogenic diet group were higher than in the control group in spite of treatment with atorvastatin, probably due to the excessive amount of cholesterol added to the diet. However, this gave us the opportunity to examine and compare the beneficial effects of atorvastatin additional to lowering of cholesterol levels.

Erythrocytes are a very appropriate medium via which cholesterol can exert a pro-oxidant action due to the potential for free radical generation. Therefore, in this study, oxidant status was studied in erythrocytes¹⁸,¹⁹ as well as in LDL fractions. Our results show that an atherogenic diet caused a prominent increase in superoxide dismutase and catalase antioxidant activity and also in TBARS levels (Table 2). The increase in superoxide dismutase activity represents an important physiological adaptation counteracting the increase in oxidative stress.¹⁹ The enhanced levels of TBARS are also an indicator of the oxidant stress induced by hypercholesterolemia. In this study, the increase in TBARS levels in spite of enhanced superoxide dismutase activity suggests that the elevation in superoxide dismutase activity was inadequate for complete prevention of lipid peroxidation in the hypercholesterolemic group. The significant decrease in superoxide dismutase activity in erythrocytes and changes in TBARS levels after 8 weeks of atorvastatin treatment, indicate the antioxidant effect of the drug, in support of previous studies.²⁰,²¹

Wassmann et al²² reported increased catalase protein expression and enzyme activity following upregulation of catalase mRNA, with no significant alteration in mRNA expression of superoxide dismutase in vascular smooth muscle cells in vitro. In the same study, while atorvastatin did not have any significant effect on vascular mRNA expression of superoxide dismutase, vascular catalase mRNA expression was increased in the aortic tissue of hypertensive rats treated with atorvastatin in comparison with controls. In the present study, the changes in superoxide dismutase and catalase activity coincide with the in vivo results of the study reported by Wassmann et al. The more distinctive changes in superoxide dismutase activity than in catalase activity may be due to the different animal models used.

Atorvastatin and its metabolites formed in in vivo circumstances may prevent enhancement of the hydroxyl and peroxyl radicals generated by hypercholesterolemia, probably via their radical scavenging, metal-chelating capacity, and the attenuating effect on output of superoxide anions.

In this study, LDL TBARS and LDL-conjugated diene levels were measured at baseline and after induction of oxidation using Cu²⁺ to evaluate the oxidation status of LDL. The significant increase in LDL TBARS and LDL-conjugated diene levels in the hypercholesterolemic rabbits represents the effect of hypercholesterolemia on the oxidative modification of LDL, probably via increased susceptibility to oxidation.²

The results of this study demonstrate the success of atorvastatin therapy in alleviating the production of LDL TBARS and LDL-conjugated diene. The oxidatively modified lipids and their degradation products are believed to have proinflammatory, immunogenic, and cytotoxic properties which contribute to both the initiation and progression of atherosclerotic lesions. It is reported that products of oxidatively modified LDL contribute to recruitment of monocytes and T cells, directly or indirectly, via induction of chemokines and endothelial cell adhesion molecules.² The present study supports this hypothesis, with histopathological examination showing atherosclerotic plaque formation and nearly 50% luminal narrowing (Figures 1A and 1B) in aortic tissue from the nontreated atherogenic diet group.

There is no doubt that statins have a positive effect on regulation of total cholesterol and LDL cholesterol levels. Some authors have suggested that inhibition of LDL oxidation by statins may be independent of any lipid-lowering effects.²³ According to earlier studies, statins were assumed to reduce susceptibility of LDL to in vitro oxidation.²⁴ It has been suggested that the inhibitory effect of statins on LDL oxidation may result from enhanced removal from plasma of “aged” LDL, which is more prone to oxidation than newly synthesized LDL. This effect could be due to statin-induced stimulation of LDL receptor activity in liver cells and to the inhibition of very low-density LDL (VLDL) and LDL production in the liver.²⁵ However, a few studies have reported that, with the exception of fluvastatin, none of the parent forms of the hypolipidemic drugs studied demonstrated a direct inhibitory antioxidant effect on LDL oxidation.²⁶

Singh et al reported that atorvastatin therapy had a dose-response effect with regard to decreasing oxidized LDL. They also reported other pleiotropic effects of the drug, evidenced by a significant decrease in related inflammatory markers, including high sensitivity C-reactive protein, matrix metalloproteinase-9, and NF-κB activity, especially at high doses, ie, 80 mg/day.²⁷ Aviram et al reported that atorvastatin did not affect oxidation of LDL in vitro, even at high concentrations.²⁰ However, low pharmacological concentrations of specific hydroxylated metabolites of the drug demonstrated a very potent inhibitory effect on LDL, VLDL, and HDL oxidation, both in metal ion-dependent and metal ion-independent systems. The molecular structure of atorvastatin hydroxymetabolites enables them to act as electron donors and, hence, as potent antioxidants.²⁰ Therefore, it may be proposed that even if the parent drug does not show antioxidant properties, its metabolites may have antioxidant activity in vivo. This may be the reason for the conflicting data generated by in vivo and ex vivo studies.

The scientific debate continues on how HDL exerts its protective effect against atherosclerosis. Until recently, the focus of attention was on the role of HDL in reverse cholesterol transport. The human serum paraoxonase 1 enzyme might be one of the ways in which HDL exerts its antiatherogenic action.²⁸,²⁹ Serum paraoxonase 1 is a 45 kDa glycoprotein which is tightly bound to HDL sub-fractions containing apolipoprotein A-I and apolipoprotein J (clusterin).³⁰ Paraoxonase 1 hydrolyzes aromatic carboxylic acid esters, organophosphates, carbamates, and lactones, and acts to protect LDL against oxidative modification, thereby exhibiting anti-atherogenic and anti-inflammatory effects.²⁹ This proposal is based on the studies of Aviram and Mackness who showed that in vitro supplementation of human HDL or whole serum with purified paraoxonase 1 significantly inhibited Cu²⁺-induced lipoprotein oxidation in a concentration-dependent manner.²⁸,³¹,³² The results of the present study show that an atherogenic diet results in a sharp decrease in paraoxonase activity, leading to loss of another guardian against oxidation and atherosclerosis. The inadequate paraoxonase activity seen in the atherogenic diet groups in this study probably contributes to the increased levels of oxidation markers (TBARS, oxidized LDL, LDL-conjugated dienes). The low paraoxonase activity observed in the atherogenic diet groups is consistent with the results of other research.³²

Loss of antioxidant paraoxonase activity from HDL or repressed expression of the enzyme may explain the decrease in paraoxonase activity seen with an atherogenic diet. In this study, although paraoxonase activity was studied, paraoxonase mass or expression was not assayed, so more detailed information will have to come from further studies.

Leitinger et al reported that an atherogenic diet resulted in the formation of oxidized phospholipids in livers of mice rendered genetically susceptible to diet-induced atherosclerosis.³⁴ These oxidized phospholipids are believed to induce an acute-phase response which results in production of proinflammatory HDL susceptible to atherosclerosis.³⁴ LDL-derived oxidized phospholipids were found to induce interleukin-6 in hepatocytes and to repress paraoxonase mRNA levels. In humans, paraoxonase 1 activity and serum HDL susceptibility to oxidation are reported to be inversely related in several groups of patients with increased risk for atherosclerosis, such as those with hypercholesterolemia, noninsulin-dependent diabetes, or vascular disease.³²,³⁵,³⁶

Superoxide dismutase and catalase activity and malondialdehyde levels in aortic tissue gave us the chance to compare and evaluate oxidant status in aortic tissues from different groups (Figure 3). The results obtained coincide with the findings for LDL fractions and erythrocytes. Superoxide dismutase activity in the aortic tissue from the atherogenic diet group was significantly higher than in Group 1 controls. Superoxide dismutase activity in Group 4 rabbits receiving statin treatment with an atherogenic diet was as low as in Group 1 controls. Catalase activity in the same tissues did not show any statistically significant difference between groups, even though there was a slight increase of activity in the atherogenic diet group. These results can be explained as a collaborative interplay for defense against the rise of free oxygen radicals.¹⁸ Lipid peroxidation and aortic antioxidant defense mechanisms take action before the formation of gross lipid infiltration in the vessels of animals receiving an atherogenic diet, and some antioxidant mechanisms may act more prominently than others during this interplay.³⁷ Other studies have also demonstrated significantly increased enzymatic activity and protein expression of superoxide dismutase in macrophage-rich atherosclerotic lesions in accordance with our data.³⁸,³⁹ The vessel wall being 100-fold richer than the other tissues for extracellular superoxide dismutase activity may also be the reason why the changes in the superoxide dismutase activity both in hypercholesterolemia and atorvastatin treatment were more expressive than the catalase activity observed in the aortic tissue.⁴⁰ Malondialdehyde levels showed a two-fold increase in the atherogenic diet group in comparison with controls, and atorvastatin therapy lowered the levels of malondialdehyde to a point even lower than the control group. The aortic tissue probably was the best medium for atorvastatin to show its antioxidant and antiatherosclerotic effects, which are clearly shown by the oxidant status markers and histopathological data (Figure 4). The reason for this is probably because atherosclerosis is a complex and miscellaneous disease, and atorvastatin not only has a favorable influence on oxidation and lipid profiles, but also exhibits pleiotropic effects on inflammation, macrophage accumulation, and even the calcification phases the atherosclerotic plaque goes through.²⁷,⁴¹–⁴⁴ Reflecting the summation of all these effects of atorvastatin, aortic tissue was the best medium through which to observe the outcome of treatment.

Even though animal studies are very valuable, some mechanisms may differ from those in humans, and this may be one of the limitations of this study. Another limitation was that the animals did not undergo angiography. However, the histological data obtained at the end of the experiment could not have been obtained in human patients, and that is an advantage of the present study and similar studies carried out using animal models.

The benefit of atorvastatin therapy certainly appears to exceed the cholesterol-lowering effects of the statins, and this study provides evidence that atorvastatin has an anti-atherogenic effect in a primary prevention setting, not just by decreasing lipid levels, but also by suppressing oxidation of LDL. The beneficial effect of atorvastatin on oxidative status seems to be one of its most important benefits, ie, a preventative effect on plaque formation.

Despite the discussion on the widespread use of statins and at what point their use is essential, the present study shows that atorvastatin has a dramatic anti-atherosclerotic effect, and part of this effect seems to be due to the anti-oxidant features of the parent drug and/or its metabolites, favoring inhibition of LDL oxidation in addition to its other pleiotropic effects. We believe comprehending the entire spectrum of benefits associated with atorvastatin therapy would allow better therapeutic application and foster the early and appropriate use of a statin.

Disclosure

The authors report no conflicts of interest in this work.