There is increasing evidence that the oxidative modification of low-density lipoprotein (LDL) is involved in the progression of atherosclerosis.1-3 Oxidized LDL (OxLDL) and its lipid constituents impair endothelial production of nitric oxide and induce the endothelial expression of chemoattractants for monocytes, leukocyte adhesion molecules, and smooth-muscle growth factors, which may be involved in atherogenesis. However, the study of the causal linkage between the LDL oxidation in vivo and the progression of atherosclerosis using antioxidants has been hampered by the lack of a specific assay for functional OxLDL in plasma.
Lectin-like OxLDL receptor-1 (LOX-1) is a receptor for OxLDL mainly expressed in endothelial cells.4 Beside OxLDL, LOX-1 can phagocytose activated platelets5 and aged/apoptotic cells,6 suggesting potential pathologic and physiologic functions. Expression of LOX-1 can be upregulated by inflammatory cytokines,7,8 fluid shear stress,9 and its ligand OxLDL,10 which have been implicated in the atherogenesis. Furthermore, LOX-1 was found to be expressed in arterial endothelial cells in human atherosclerotic lesions,11 suggesting that OxLDL uptake through this receptor may be involved in endothelial activation or dysfunction in atherogenesis or both. In 2000 the expression of LOX-1 was found to be upregulated in Watanabe heritable hyperlipidemic (WHHL) rabbit aortas compared with normal rabbit aortas.12 LOX-1 was also identified in the endothelium of nonlesion areas of WHHL rabbit aortas, suggesting the involvement of LOX-1 in the initial stage of atherosclerosis.
More recently, a novel sandwich enzyme immunoassay for LOX-1-ligand, using a recombinant soluble form of LOX-1 and anti-apoB antibody, has been developed to detect the circulating modified LDL via the mechanisms of specific binding to LOX-1.13 They demonstrated the accumulation of LOX-1-ligand in plasma and in the atherosclerotic lesions of WHHL rabbits. In the present study, to examine the causal linkage between oxidation of LDL in vivo and the progression of atherosclerosis, we investigated the effects of antioxidants including probucol, vitamin E, and fluvastatin, an HMG-CoA (hydroxy-3-methylglutaryl coenzyme A) reductase inhibitor with antioxidative property, on plasma levels of LOX-1-ligand during the progression of atherosclerosis in WHHL rabbits.
Fluvastatin was donated by the Department of Pharmacology, Novartis Pharma (Ibaraki, Japan). Anti-rabbit apoprotein B (apoB) immunoglobulin Y (IgY) was a kind gift from Dr. Hiroyuki Itabe (Showa University). All other chemicals used in this study were standard high-purity materials obtained from commercial sources.
These experiments were reviewed and approved by the Committee on Ethics of Animal Experiments, Tanabe Seiyaku Co, Ltd.
WHHL and Japanese White (JW) rabbits (male, 2 months of age) were obtained from Oriental Yeast (Tokyo, Japan). After a 1-week acclimation to the animal facility (temperature; 21 ± 2°C, humidity; 55 ± 5%, illumination time 7:00 to 19:00) with food and water available ad libitum, rabbits were exposed to dietary treatment. In Experiment 1, rabbits were assigned to 4 treatment groups: JW rabbits fed regular diet (LRC4: oriental Yeast) (as healthy control), WHHL rabbits fed regular diet (disease control group), WHHL rabbits fed diet supplemented with 0.03% of fluvastatin (fluvastatin group), and WHHL rabbits fed diet supplemented with 0.5% of vitamin E (vitamin E group). In Experiment 2, rabbits were assigned to 3 treatment groups: JW, WHHL, and WHHL rabbits fed a diet supplemented with 1% of probucol (probucol group). Rabbits were fed 100 g of chow per day for 6 months. Diets, with or without drug supplementation, were prepared by Oriental Yeast.
Blood samples were collected at 0, 1, 3, and 5 months of drug administration from the marginal ear artery using a heparinized syringe. At 6 months of drug administration, blood was collected from the abdominal aorta of anesthetized rabbits immediately before they were sacrificed. Plasma was separated by low-speed centrifugation. Plasma cholesterol, triglyceride, and phospholipid levels were enzymatically determined by using Cholestezyme V555 (Eiken Chemical Co, Tokyo, Japan), Triglyzyme (Eiken), and Phospholipid C test Wako (Wako Pure Chemicals, Osaka, Japan), respectively.
Quantification of Aortic Atherosclerosis
After the final collection of blood, rabbits were sacrificed and the proximal part of the aorta including the arch was isolated. After removal of surrounding tissue, the aorta was opened by longitudinal incision and photographed. The images of aortas were captured with an image scanner (Scanjet 4C/T, Hewlett Packard) and the lesion areas covering the aortic surface were picked up on the digital images using a computer-aided manipulator (image-operating computer program, Macscope®, Mitani Corporation, Fukui, Japan). The extent of atherosclerosis was expressed as a percentage of the aortic surface area covered by lesions. For measurement of the cholesterol content, the aorta was homogenized in Dalbecco's phosphate-buffered saline (PBS). The lipids were extracted by chloroform/methanol (2:1), and the chloroform fraction was evaporated. The residue was solubilized with isopropanol, and total cholesterol was determined enzymatically. The protein concentration of tissue homogenate was measured by the bicinchoninic acid method.
Preparation of Lipoproteins
LDL (1.019 < d < 1.063) was isolated from rabbit plasma by sequential ultracentrifugation (Beckman XL-90, CA, USA), as described previously. The protein concentration was measured by the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA.) with bovine serum albumin (BSA) as a standard. For the preparation of cupric ion-oxidized LDL (Cu-OxLDL) for the standard of enzyme immunoassay, rabbit LDL was oxidatively modified by CuSO4, as described previously.13 Oxidation was verified as enhanced mobility by use of agarose gel electrophoresis. The value of relative electrophoretic mobility for Cu-OxLDL compared with native LDL was 2.6.
Measurement of LOX-1-Ligand Levels
Plasma LOX-1-ligand levels were determined by a sandwich enzyme-linked immunosorbent assay (ELISA)-like method, as described,13 with minor modification. A fusion protein of extracellular domain of bovine LOX-1 and Fc region of human IgG (LOX-Fc) was prepared as described previously.6 LOX-Fc or human IgG (5 μg/mL in PBS) was immobilized on 96-well plate (Maxisorb, Nunc) by incubating for 24 hours at 4°C. The plates were washed with PBS and blocked with 25% Block Ace (Dainippon Pharmaceutical Co, Ltd, Osaka, Japan) in PBS by overnight incubation at 4°C. The plates were washed with PBS, and plasma samples or the standard rabbit OxLDL, which were diluted in PBS containing 20% newborn calf serum (Gibco BRL), were applied to the wells. After incubation for 24 hours at 4°C, the plates were washed with PBS and applied with antirabbit apo B IgY (diluted 1:1000 in PBS containing 1% BSA). After 2 hours of incubation at room temperature, the plates were washed with PBS and incubated with alkaline phosphatase (AP)-labeled anti-IgY IgG (Jackson Immunoresearch Laboratory, diluted 1:2000 in PBS containing 1% BSA) for 1 hour at room temperature. The plates were washed with PBS, and AP activity was measured using p-nitrophenyl phosphate as a substrate. Calibration was performed with copper ion-oxidized rabbit LDL. The specific binding to LOX-Fc was determined by the subtraction of the AP activity obtained with human IgG from that with LOX-Fc.
All results are expressed as mean ± SEM. Data in plasma parameters were analyzed by repeated-measures analysis of variance (ANOVA). Statistical comparisons of data between the WHHL group and drug-treated groups in the assessment of lesion area and cholesterol contents were carried out by Student's unpaired t-test or one-way ANOVA followed by Tukey-Kramer's test for multiple comparison with Bonferroni adjustment in alpha level. For all comparisons, the probability less than 5% was considered to be statistically significant.
WHHL rabbits at 2 months of age were enrolled in the experiment and age-matched Japanese white (JW) rabbits were used as healthy control. Two experiments were carried out: Experiment 1 for the effects of fluvastatin and vitamin E and Experiment 2 for probucol. All drugs were supplemented with normal chow and administered for 6 months. There was no difference in the body weight gain among all groups throughout the experimental period (data not shown).
In Experiment 1, total plasma cholesterol was higher in WHHL rabbits than in JW rabbits (898.7 ± 34.7 mg/dL compared with 41.7 ± 1.6 mg/dL) at 2 months of age, and this was sustained throughout the experimental period (Fig. 1). Treatment with vitamin E did not influence total cholesterol, triglyceride, and phospholipid levels. Fluvastatin significantly reduced plasma total cholesterol (P < 0.01) and phospholipid (P < 0.01) levels but had no effect on triglyceride levels.
LOX-1-ligand levels were more than 5 times higher in WHHL rabbits than in JW rabbits (450.5 ± 119.0 compared with 75.7 ± 7.9 ng/mL Cu-OxLDL equivalent) as early as 2 months of age and sustained throughout the experimental period (Fig. 2). Both vitamin E and fluvastatin significantly (P < 0.01) reduced LOX-1-ligand levels in contrast to their effects on lipid levels. As for fluvastatin, the extent of reduction in LOX-1-ligand levels appeared to be more prominent than in the case of total cholesterol.
Rabbits were sacrificed at 8 months of age and their proximal aortas containing the arch were assessed for lesion area and cholesterol content (Fig. 3). In JW rabbits, no fatty streak formation was detectable, whereas lesions were present in all the WHHL rabbits studied. The lesion area of proximal aorta covered 68.3 ± 3.6% of the total vessel surface. The percentages of plaque area in groups fed fluvastatin and vitamin E were 44.3 ± 4.7% and 49.9 ± 4.3%, respectively, which were significantly less severe than that of the WHHL group (P < 0.01 and P < 0.05, respectively). In the WHHL group, total cholesterol content of aortic arches was 0.77 ± 0.06 mg/mg protein, whereas vessel segments from vitamin E and fluvastatin-fed rabbits exhibited lowered values of 0.65 ± 0.06 and 0.56 ± 0.05 mg/mg protein (P < 0.05), respectively.
These results suggest that the antioxidative effects of these agents suppressed the development of atherosclerosis by reducing oxidative modification of LDL. To test this hypothesis, we investigated the effect of another antioxidant, probucol.
In Experiment 2, probucol had little effect on plasma total cholesterol, triglyceride, and phospholipid levels (Fig. 4). As expected, in the probucol-treated group, LOX-1-ligand was maintained to lower levels than in the WHHL group from as early as 1 month of treatment to the end of the experiment (P < 0.05, Fig. 5). Six months of administration of probucol resulted in the significant reduction in both lesion area development (P < 0.05) and cholesterol accumulation (P < 0.01) of aortic arches (Fig. 6).
A causal role of OxLDL in the progression of atherosclerosis was first suggested by experiments showing that OxLDL induced foam cell formation and endothelial activation or dysfunction, or both, and was further supported by studies of antioxidant inhibition of atherogenesis demonstrated in several different animal models.14 Several markers, such as isoprostanes and 8-hydroxy-2′-deoxyguanine, have been used to assess the state of oxidative stress in vivo. However, the correlation between the LDL oxidation in vivo and the progression of atherosclerosis has been confused by the lack of a specific assay for functionally active OxLDL in circulating plasma. Although ex vivo oxidation of purified LDL has been commonly used to assess in vivo efficacy of antioxidants, it has been argued that different in vitro oxidizing conditions can give different results. Antibodies against OxLDL have been used to investigate the relationship between in vivo oxidation reaction and various pathologic conditions.15-18 In the present study, we made use of the newly developed ELISA-like method using the recombinant soluble form of LOX-1 and anti-apoB antibody to evaluate the levels of LOX-1-ligand in plasma.13 LOX-1 can bind oxidized LDL but weakly or hardly binds acetylated and native LDL.19 We detected LOX-1-ligand only in LDL fraction (1.019 < d < 1.063) but not in other lipoprotein fractions (data not shown). Thus, LOX-1-ligand, which we measured in this study, is considered to be oxidatively modified LDL. Further studies are needed to clarify the biochemical nature of circulating LOX-1-ligand in plasma.
The enhanced expression of LOX-1 has been demonstrated in human atherosclerotic lesions11 and WHHL rabbit aortas.12 The highly inducible nature of LOX-1 expression under atherosclerosis-related conditions has been demonstrated in vitro.7-10 Furthermore, LOX-1- mediated induction of adhesion molecule expression and metalloproteinase expression in endothelial cells by OxLDL have been reported.20,21 Taken together, LOX-1 is strongly suggested to have pathophysiologic roles in atherogenic processes, and thus LOX-1-ligand should have potential roles in these processes.
It was suggested that LDL in WHHL rabbit plasma undergo mild oxidative modification.22 The present study shows that LOX-1-ligand levels were elevated in WHHL rabbits as early as 2 months of age, when most of the lesions were in the early stages, suggesting that oxidant stress is elevated in the early stages of disease progression. Because LOX-1 is accumulated in the proatherogenic aortas and in the initial atherogenic lesions,12 LOX-1 and its ligands should be involved in initiating and promoting the vicious cycles of atherogenic processes. In this study, the LOX-1-ligand level of the WHHL control group in Experiment 1 was higher than that in Experiment 2; correspondingly the atherosclerotic lesion of the WHHL group in Experiment 1 was more severe than that in Experiment 2. Additionally, the LOX-1-ligand level in the vitamin E group in Experiment 1 was similar to the level in the WHHL control group in Experiment 2; corresponding lesions were also similar in severity. These results also support the pathologic role of LOX-1-ligand in the progression of atherosclerosis. Further biochemical characterization of circulating LOX-1-ligand may provide a means of specifically antagonizing the action of LOX-1-ligand that would show a direct link between the increased level of LOX-1-ligand and the progression of atherosclerotic lesion.
We investigated the effects of antioxidants, vitamin E, and probucol. Supplementation of these agents reduced the evolution of atherosclerosis with little effects on plasma lipid levels. These observations were in agreement with previous reports.23,24 In both vitamin E- and probucol-treated animals, plasma LOX-1-ligand was maintained to lower levels than in control WHHL rabbits. Although we have not assessed the effect on LOX-1 in this study, it is probable that the expression of LOX-1 is also reduced by these antioxidants via the reduction of LOX-1-ligand because OxLDL induces LOX-1 expression.10 Therefore, it is suggested that the antioxidative effects of these agents suppressed the development of atherosclerosis by reducing oxidative modification of LDL.
Fluvastatin, the first totally synthesized HMG-CoA reductase inhibitor, is widely used as a cholesterol-lowering drug. Some in vitro studies showed that fluvastatin and its major metabolites have antioxidative effects on LDL oxidation and superoxide radical scavenging activity ascribable to its unique chemical structure.25-28 In this study, supplementation of fluvastatin to the diet significantly reduced both LOX-1-ligand and total cholesterol levels. The extent of reduction in plasma total cholesterol levels was less prominent than in the case of LOX-1-ligand levels. Although the extent of the contribution of the cholesterol-lowering effect of fluvastatin is unclear, present results show that the reduction in atherosclerotic areas observed in the fluvastatin-treated group may be attributable, at least partly, to the reducing effect on LOX-1-ligand level of this compound as a result of its antioxidative property. It also has been reported that fluvastatin exhibits antioxidative effects in clinical29 and animal30 studies. These characteristics may contribute to the beneficial effects of fluvastatin in the prevention of cardiovascular diseases.
In conclusion, the present study provides evidence that oxidatively modified LDL is elevated in vivo from the early stages of atherogenesis, is of functional importance in the progression of the disease, and can be suppressed by antioxidant treatment. Further studies are needed to clarify the biochemical nature, functional activity, and pathologic significance of circulating LOX-1-ligand.
The authors would like to thank Hiroyuki Itabe (Showa university) for providing Anti-rabbit apoprotein B IgY. This study was supported by the grant from the National Institute of Biomedical Innovation.
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