Vitamin E supplementation suppresses macrophage accumulation and endothelial cell expression of adhesion molecules in the aorta of hypercholesterolemic rabbits

Atherosclerosis 176 (2004) 265–272

Vitamin E supplementation suppresses macrophage accumulation and endothelial cell expression of adhesion molecules in the aorta of hypercholesterolemic rabbits Takuro Koga, Paul Kwan, Ligia Zubik, Clement Ameho, Donald Smith, Mohsen Meydani∗ Vascular Biology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA Received 2 September 2003; received in revised form 5 May 2004; accepted 17 May 2004

Abstract Suppression of cell adhesion molecule expression and macrophage accumulation by the endothelium is believed to play an important role in preventing the development of atherosclerosis. Earlier, we have shown that in vitro supplementation of human aortic endothelial cells with Vitamin E dose-dependently reduced expression of adhesion molecules and monocyte adhesion. Here, we report the in vivo down-regulation of endothelial cell adhesion molecules expression and macrophage accumulation in the aortas of hypercholesterolemic rabbits supplemented with Vitamin E. To this end, New Zealand White rabbits were fed a semi-purified diet containing 30 (control) or 1000 IU/kg Vitamin E. After 4 weeks, both groups’ diets were switched to an atherogenic diet (0.3% cholesterol, 9% hydrogenated coconut oil, and 1% corn oil) containing the respective levels of Vitamin E and fed for 2, 4, and 6 weeks. Vitamin E supplemented rabbits had significantly higher levels of Vitamin E in their plasma and aortas. Frozen aorta sections were fixed and stained by an avidin–biotin complex method using Rb2/3 and Rb1/9 monoclonal antibodies against rabbit ICAM-1 and VCAM-1, respectively, and with RAM-11 for macrophage and von Willebrand factor for endothelial cells, followed by staining with secondary antibodies and counterstaining and evaluation under the microscope. At 6 weeks on atherogenic diet treatment, a trend (P = 0.08) toward a lower score of ICAM-1 expression by endothelial cells was observed in the aorta of Vitamin E treated rabbits compared to the control. However, a decrease in the score of VCAM-1 expression by endothelial cells in Vitamin E treated rabbits did not reach to a statistical significance. At 4 and 6 weeks on atherogenic diet, Vitamin E supplementation also significantly (P = 0.003) inhibited the accumulation of macrophages in the aorta. These results support the concept that down-regulation of adhesion molecule expression and suppression of monocyte/macrophage activation by Vitamin E in vivo is one of the potential mechanisms by which Vitamin E may suppress the development of aortic lesions in a rabbit model of atherosclerosis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Vitamin E; Hypercholesterolemia; Adhesion molecule; ICAM-1; VCAM-1

1. Introduction Hypercholesterolemia is one of the most important risk factors for atherosclerosis and related occlusive vascular disease [1]. Hypercholesterolemia alters endothelial cell function and increases permeability to low density lipoprotein (LDL). Local adherence of circulating monocytes to the vas∗ Corresponding author. Tel.: +1 617 556 3126; fax: +1 617 556 3224. E-mail address: [email protected] (M. Meydani).

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.05.034

cular endothelial lining is one of the earliest morphological alterations in the arteries of animals after initiation of an atherogenic diet [2,3]. In animal studies, diet-induced hyperlipidemia up-regulated the expression of adhesion molecules and accumulation of monocytes resulting in foam cell formation [4,5]. Conflicting results regarding the effects of Vitamin E supplementation on atherosclerosis progression and cardiovascular disease (CVD) events have been reported from randomized controlled trials [6–15]. However, compelling evidence

266

T. Koga et al. / Atherosclerosis 176 (2004) 265–272

from experimental in vitro studies and in vivo studies as well as from epidemiological studies support the oxidative hypothesis of atherosclerosis and the potential role of dietary antioxidants such as Vitamin E in reducing the risk of CVD, in particular when oxidative stress appears to be a contributing factor [12,16–24]. Vitamin E serves as a potent antioxidant in the LDL particle [25] and in the lipid bilayer of cell membranes [26] protecting them against oxidative damage. In addition, Vitamin E is incorporated into other components of the vascular system, including endothelial cells, smooth muscle cells, platelets, and immune cells, and has been shown to modulate a variety of inflammatory processes that are involved in atherogenesis [27]. In vitro studies have demonstrated that Vitamin E supplementation inhibited the production of proinflammatory cytokines by immune and endothelial cells and suppressed expression of adhesion molecules in endothelial cells and their adhesive interaction with immune cells [28–32]. However, the protective role of Vitamin E supplementation on the expression of adhesion molecules and the accumulation of macrophages prior to and during the development of hypercholesterolemia has not been investigated. Therefore, in this study, we examined the in vivo effect of Vitamin E on the modulation of adhesion molecule expression by the vascular endothelium and the accumulation of macrophages in the aortic walls of rabbits while they fed on an atherogenic diet.

2. Materials and methods 2.1. Animals and diets Thirty-three 3-month-old New Zealand white, Pasteurella-free rabbits weighing approximately 2.5 kg were purchased from Millbrook Farms (Amherst, MA). The animals were housed in the JM USDA-Human Nutrition Research Center on Aging at Tufts University (HNRCA) laboratory animal facility accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). Rabbits were housed individually in stainless steel cages in an air-conditioned room (23 ± 1 ◦ C and 55 ± 5% humidity) under a 12-h dark:12-h light cycle and were acclimated to a pelleted whole-grain commercial diet (Purina Mills Inc., Richmond, IN) for 1 week. Rabbits were randomly assigned to two dietary groups for 10 weeks. One group of rabbits (control group, N = 15) consumed a semi-purified diet (D30323, Research Diet Inc., New Brunswick, NJ) containing a normal level of Vitamin E (30 ppm) according to NRC requirements [33]. Another group (N = 15) received a semi-purified diet (D30339, Research Diet Inc.) containing 1000 ppm Vitamin E. After consumption of the respective diets for 4 weeks, the diets of the rabbits were switched to an atherogenic diet (D30324 and D30325, respectively, Research Diet Inc.) while the level of Vitamin E in the respective diets remained the same. The atherogenic diet contained 0.3% cholesterol and 9%

partially hydrogenated coconut oil (a source of C10–12 saturated fatty acid), and 1% corn oil (a source of essential and polyunsaturated fatty acids). The atherogenic diets were fed to rabbits from week 5 for 2, 4, and 6 weeks after which the animals were killed (at week 6, 8, and 10). Two rabbits were fed pelleted whole-grain regular commercial rabbit chow (Purina Mills Inc.) and killed after 8 h following an i.v. injection with 50 ␮g/kg lypopolysacchride (LPS) endotoxin (Sigma Chemical Co., St. Louis, MO), which induces adhesion molecule expression on the endothelium [4]. The aortas from these rabbits were used as a positive control. One rabbit, fed a control semi-purified basal diet, was killed after 4 weeks, and the aorta from this rabbit was used as a negative control. In a preliminary experiment, we found that lightly spraying semi-purified diet pellets with apple juice increased the palatability and normalized the food intake of rabbits that were accustomed to regular rabbit chow. Water was provided ad libitum. One hundred and 20 g of diet was provided to each rabbit every day. Food intake was recorded daily, and body weight was monitored weekly. The study was reviewed and approved by the institutional Animal Care and Use Committee in accordance with National Institute of Health guidelines. 2.2. Experimental protocol Blood was drawn from the marginal ear vein into EDTAcontaining tubes at 0, 2, 4, 6, 8, and 10 weeks. Five rabbits from the control and the supplemented groups were killed after 2, 4, or 6 weeks of feeding on the atherogenic diet. Thus, at the time of sacrifice, the Vitamin E supplemented rabbits had been treated with high levels of Vitamin E for 6, 8, or 10 weeks, respectively. Rabbits were sacrificed by intravenous injection of pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL, 50 mg/kg body weight) and xylazine (5 mg/kg body weight). The aorta was perfused with PBS through the left ventricle and the descending aorta was dissected and immersed in PBS, and the adventitia was removed. Five millimeters of segments of the thoracic aorta were obtained 1–3 mm above the aortic valve. The aortic rings were embedded in OTC, snap-frozen in liquid nitrogen, and stored at −80 ◦ C. Segments from the descending segments of thoracic aorta were also obtained, cleaned, and stored at −80 ◦ C for Vitamin E and cholesterol analysis. 2.3. Determination of Vitamin E and cholesterol in plasma and aortas Plasma and aorta Vitamin E was measured by HPLC [34]. Briefly, plasma Vitamin E was extracted with hexane and Tocol used as an internal standard. The extract was injected into the reverse phase HPLC column and detected with an electrochemical detector. For determination of Vitamin E levels in the aorta, 50 mg of aortic sample was homogenized in 4 mL cold PBS. The homogenate was extracted two times with 4 mL chloroform/methanol (2:1, v/v) containing 0.01%

T. Koga et al. / Atherosclerosis 176 (2004) 265–272

BHT. The combined chloroform layers were evaporated under nitrogen gas and the residue was dissolved in 500 ␮L of isopropanol. A portion of the extract was used to analyze Vitamin E by HPLC and a portion was used for cholesterol analysis, which was measured enzymatically [35] using automated COBAS FARA analyzer (Roche Diagnostic System, Nutley, NJ). 2.4. Antibodies The following antibodies were used for immunohistochemical analysis: Rb2/3 and Rb1/9, monoclonal antibodies against rabbit ICAM-1 and VCAM-1, respectively (generous gifts from Dr. M.I. Cybulsky, University of Toronto); RAM-11, a monoclonal antibody against rabbit macrophages (Dako, Carpinteria, CA). The preservation of aortic endothelial cells in the frozen section was evaluated by localization of von Willebrand factor (vWF), a constitutively expressed endothelial cell marker, using polyclonal goat anti-human antibody (Incstar Co., Stillwater, MN), which cross-reacts with rabbit vWF. 2.5. Immunohistochemistry Serial 5-␮m-thick frozen sections from rabbit aortas were adhered to glass slides (Superfrost Plus, Eric Scientific Co, Portsmoth, NH) and fixed in acetone at −20 ◦ C for 5 min. Sections were incubated with Rb2/3 (1:50 dilution), Rb 1/9 (1:50 dilution), RAM-11 (1:1000 dilution), or vWF (1:5000 dilution) for 1.5 h. After washing, specific biotinylated secondary antibodies (biotinylated horse anti-mouse IgG for Rb2/3, Rb1/9, RAM-11, and biotinylated horse anti-goat IgG for vWF) were applied, followed by avidin–biotin peroxidase complexes (ABC Elite kit, Vector Labs, Burlingame, CA). Antibody binding was visualized with diaminobenzidine tetrahydrochloride. Sections were counterstained with Gill’s hematoxylin. Positive control for Rb2/3, Rb1/9, and RAM-11 were from aorta sections of rabbits that were treated with LPS endotoxin. To provide a quantitative measure of adhesion molecule expression and macrophage accumulation, the extent of positive staining of sections was evaluated under the microscope according to the 5-grade evaluation. The thick and complete circle to patchy and spotty staining of endothelial cells in the lumen of the aorta for ICAM-1 and VCAM-1 and the prevalence of macrophages stained as spots in the aortic ring were used to score the lesions. The 6-grade scoring scales are presented in Table 1. The sections were evaluated and scored by three individuals who were blind to the animal grouping. 2.6. Statistical analysis Data were analyzed by using the SYSTAT statistical package (version 9.0; SPSS Inc, Chicago). The overall treatment effect was determined by analysis of variance followed by

267

Table 1 Grading scale for scoring expression of ICAM-1, VCAM-1 and prevalence of macrophages in the aortic rings of rabbits Grade

ICAM-1/VCAM-1

Macrophage

5 4 3 2 1 0

Thick complete stained ring Thin complete stained ring Thick irregular patches Thin irregular patches Spotty staining Negative

10 spots or more 8 spots 6 spots 4 spots 2 spots Negative

Fisher’s least-significant-difference post hoc test. Significance was set at P < 0.05.

3. Results In this study, before inducing of hypercholesterolemia with the atherogenic diet, rabbits were fed either a control diet or a diet supplemented with Vitamin E for 4 weeks. Rabbits that consumed either the control or Vitamin E supplemented diet had similar daily food intakes and growth rate as determined by body weight (data not shown). Concentration of plasma cholesterol was the same for both groups of rabbits up to the 4th week and significantly increased after introducing the atherogenic diet (Fig. 1A). In Vitamin E supplemented rabbits, the plasma concentration of Vitamin E (␣-tocopherol) was significantly higher than control rabbits at 2 and 4 weeks. Following the introduction of the atherogenic diet after the 4th week, because of the increase in plasma cholesterol levels, the plasma level of Vitamin E was further increased (Fig. 1B), and it was significantly higher when the levels were adjusted per mg of plasma cholesterol (Fig. 1C). Vitamin E supplementation also increased the levels of Vitamin E in the aorta (Fig. 2A). However, the levels of cholesterol in the aortic tissue were the same in both groups (Fig. 2B). Endothelial ICAM-1 and VCAM-1 expression and macrophage accumulation was analyzed immunohistochemically in the ascending aortas of rabbits. Sections treated with appropriate negative control serums did not show any staining. ICAM-1 but not VCAM-1 was constitutively expressed by the endothelial cells of aortas of the negative-control rabbits. The score of ICAM-1 and VCAM-1 expression in the aortas of the negative control rabbit was 3 and 0, respectively. In positive control rabbits (LPS injected), the expression of ICAM-1 and VCAM-1 was prominent, while staining for macrophages was absent. At 6 weeks of feeding the rabbits an atherogenic diet supplemented with Vitamin E showed a trend (P = 0.08) toward a lower score of ICAM-1 expression by the endothelial cells in the aortas compared to control rabbits. (Figs. 3 and 6A and B). However, down-regulation of VCAM-1 expression by Vitamin E supplementation in the endothelial cells of the aortas of rabbits fed the atherogenic diet did not reach a statistical significance (P = 0.1) (Figs. 4 and 6C and D).

268

T. Koga et al. / Atherosclerosis 176 (2004) 265–272

Fig. 2. Concentration of Vitamin E (A) and cholesterol (B) in aorta of Vitamin E supplemented (open bars) and control (solid bars) rabbits. Values are mean ± S E. ∗∗ P < 0.001 compared to control.

dietary Vitamin E suppressed the upregulation of adhesion molecule expression by endothelial cells and the accumulation of macrophages in the aortas of rabbits fed the atherogenic diet.

4. Discussion

Fig. 1. Plasma concentrations of total cholesterol (A), Vitamin E (B), and ratio of Vitamin E per mg of cholesterol (C) in Vitamin E supplemented (open square) and control rabbits (solid square). Values are mean ± S.E. ∗∗ P < 0.001 compared to control.

At 4 and 6 weeks on the atherogenic diet treatment, the accumulation of macrophages in aortic walls was significantly lower (P = 0.003) in the aortas of rabbits supplemented with Vitamin E compared to those fed an atherogenic diet containing control levels of Vitamin E (Figs. 5 and 6E and F). The aortas of all the rabbits that consumed the atherogenic diet with control levels of Vitamin E for 6 weeks contained areas of thickened intima with foam cell lesions, whereas such changes were not present in the aorta of Vitamin E supplemented rabbits. Thus, the supplementation of rabbits with

Atherosclerosis, which is a chronic inflammatory disease of arteries, is characterized by a thickening of the vascular wall and an infiltration of macrophages and lymphocytes. In animals with diet-induced or genetically determined hyperlipidemia, the earliest morphological changes in arteries include focal adherence of mononuclear leukocytes to the endothelium and accumulation of monocyte-derived foam cells in the intima [36,37]. Oxidative stress and the production of proinflammatory cytokines are believed to be prerequisites for the activation of the endothelium to induce chemokines and adhesion molecules to attract and adhere to monocytes at the early stage of fatty streak formation. A high level of LDL and its oxidation contributes to the recruitment of circulating monocytes into the arterial intima [38]. In vivo, free radicals generated by endothelial cells and activated macrophages oxidize the LDL particles [39] making

T. Koga et al. / Atherosclerosis 176 (2004) 265–272

Fig. 3. Effect of Vitamin E supplementation on aortic endothelial cells expression of ICAM-1. Frozen rabbit aorta sections were incubated with monoclonal antibody against rabbit ICAM-1, followed by applying biotinylated secondary antibody and avidin–biotin peroxidase complexes and counterstained with Gilles hematoxylin. Sections were scored by three individuals under the microscope according to the 5-grade scaling evaluation. The open bars represent the ICAM-1 expression in the aorta of Vitamin E supplemented rabbits fed atherogenic diet. The hatched bars represent the ICAM-1 expression in the aorta of control rabbits fed atherogenic diet. The solid bar represents the ICAM-1 expression in the aorta of chow-fed rabbit injected with LPS as a positive control. Values are mean ± S.E. ∗ P = 0.08 compared to Vitamin E supplemented rabbits.

them chemotactic to attract monocytes and to elicit inflammatory responses by increasing adhesion molecule expression directly and/or through production of cytokines such as IL-1␤ [40–42]. As previously reported [4], after 6 weeks,

Fig. 4. Effect of Vitamin E supplementation on aortic endothelial cells expression of VCAM-1. Frozen rabbit aorta sections were incubated with monoclonal antibody against rabbit ICAM-1, followed by applying biotinylated secondary antibody and avidin–biotin peroxidase complexes and counterstained with Gilles hematoxylin. Sections were scored by three individuals under the microscope according to the 5-grade scaling evaluation. The open bars represent the VCAM-1 expression in the aorta of Vitamin E supplemented rabbits fed atherogenic diet. The hatched bars represent the VCAM1 expression in the aorta of control rabbits fed atherogenic diet. The solid bar represents the VCAM-1 expression in the aorta of chow-fed rabbit injected with LPS as a positive control. Values are mean ± S.E.

269

Fig. 5. Effect of Vitamin E supplementation on aortic macrophages. Frozen rabbit aorta sections were incubated with RAM-11 monoclonal antibody and vWF followed by applying biotinylated secondary antibody and avidin–biotin peroxidase complexes and counterstained with Gilles hematoxylin. Sections were scored by three individuals under the microscope according to the 5-grade scaling evaluation. The open bars represent the macrophage expression in the aorta of Vitamin E supplemented rabbits fed atherogenic diet. The hatched bars represent the macrophage expression in the aorta of control rabbits fed atherogenic diet. Values are mean ± S.E. ∗∗ P = 0.003 compared to Vitamin E supplemented rabbits.

the atherogenic diet treatment induced hypercholesterolemia in rabbits, resulting in an enhancement of ICAM-1 and VCAM-1 expression and an accumulation of macrophages in the aortas. Thus, the atherogenic diet used in this study was effective in inducing atherosclerosis in this animal model. Dietary antioxidants including Vitamin E, in addition to protecting LDL from oxidation, may increase the antioxidant capacity of vascular and immune cells and reduce oxidative stress and the inflammatory response associated with hypercholesterolemia. We reported that IL-1␤ and chemokines production by activated endothelial cells in culture was decreased with Vitamin E supplementation [31,32]. Devaraj et al. [43] reported that Vitamin E supplementation in humans reduced the secretion of IL-1␤ by monocytes. We and others have also demonstrated that Vitamin E prevented the upregulation of adhesion molecule expression by endothelial cells and reduced leukocyte adhesion to endothelial cells in culture when activated with cytokines, native LDL, and oxidized LDL [29,31,32,44,45]. Fruebis et al. [46] reported that Vitamin E and probucol significantly reduced the upregulation of VCAM-1 mRNA and protein expression in the vascular walls of rabbits during cholesterol-induced atherosclerosis. In the present study, we investigated the supplementation of rabbits with Vitamin E prior to, and during the progressive increase of hypercholesterolemia on the suppression of ICAM-1 and VCAM-1 and the accumulation of macrophages in the aortas. In concurrence with an earlier report [47], plasma and arterial Vitamin E levels were significantly higher in the Vitamin E supplemented rabbits compared to control rabbits in this study. Thus, supplementation with Vitamin E certainly

270

T. Koga et al. / Atherosclerosis 176 (2004) 265–272

Fig. 6. Frozen rabbit aorta sections were incubated with monoclonal antibody against rabbit ICAM-1(Rb2/3), VCAM-1(Rb1/9) and macrophage (RAM-11) followed by applying biotinylated secondary antibody and avidin–biotin peroxidase complexes and counterstained with Gilles hematoxylin. Representative photomicrographs of aortic sections from rabbits fed atherogenic diet without Vitamin E supplement (A, C, E) and rabbits fed atherogenic diet supplemented with Vitamin E (B, D, F) are shown. Aorta sections from rabbits fed atherogenic diet showed an intense staining of ICAM-1 (A) and VCAM-1 (C) on the intima and presence of foam cells/macrophages in the media (E). In contrast, aorta sections from rabbits supplemented with Vitamin E and treated with atherogenic diet showed relatively little ICAM-1 staining (B) and staining was absent for VCAM-1 (D), and macrophages (E).

increased the levels of Vitamin E in the aorta, where it protected the aorta from the oxidative stress of diet-induced hypercholesterolemia. Our results demonstrate that Vitamin E at the dose of a 1000 IU/kg diet tended to suppress expression of adhesion molecules such as ICAM-1 and VCAM-1 by the endothelium of rabbit aortas; also, this level of Vitamin E suppressed the accumulation of macrophages in aortic walls, when rabbits were pre-supplemented with Vitamin E for 4 weeks prior to inducing hypercholesterolemia, and they continued to receive Vitamin E supplementation during the 6 weeks of feeding on the atherogenic diet. Taken together, the results of our study and the evidence from other studies support the concept that down-regulation of adhesion molecule

expression and suppression of macrophage accumulation by Vitamin E are the potential mechanisms by which dietary and supplemental Vitamin E may prevent and reduce the risk of atherosclerosis as it was demonstrated in this rabbit model of atherosclerosis.

Acknowledgments This material is based upon work supported by the US Department of Agriculture agreement no. 58-1950-9-001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do

T. Koga et al. / Atherosclerosis 176 (2004) 265–272

not necessary reflect the view of the US Department of Agriculture. Dr. Takuro Koga was a Visiting Scientist at Vascular Biology Laboratory and was supported by Noda Institute for Scientific Research, Japan. The authors thank Hong Wang for her technical assistance and Stephanie Marco for her assistance in the preparation of the manuscript.

References [1] Ross R, Harker L. Hyperlipidemia and atherosclerosis: chronic hyperlipidemia initiates and maintain lesions by endothelial cell desquamation and lipid accumulation. Science 1976;193:1094–100. [2] Gerrity RG, Naito HK, Richardson M, Schwartz CJ. Dietary induced atherogenesis in swine morphology of the intima in prelesion stages. Am J Pathol 1979;95:775–92. [3] Faggiotto A, Ross R. Studies of hypercholesterolemia in the nonhuman primate II fatty streak conversion to fibrous plaque. Arteriosclerosis 1984;4:341–56. [4] Li H, Cybulsky MI, Gimbrone MA, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Artherioscler Thromb 1993;13:197–204. [5] Truskey GA, Herrmann RA, Kait J, Barber KM. Focal increases in vascular cell adhesion molecule-1 and intimal macrophages at atherosclerosis-susceptible sites in rabbit aorta after short-term cholesterol feeding. Artherioscler Thromb Vasc Biol 1999;19:393–401. [6] Rapola JM, Virtamo J, Ripatti S, et al. Randomised trial of [alpha]tocopherol and [beta]-carotene supplements on incidence of major coronary events in men with previous myocardial infarction. Lancet 1997;349:1715–20. [7] Virtamo J, Rapola JM, Ripatti S, et al. Effect of vitamin E and beta carotene on the incidence of primary nonfatal myocardial infarction and fatal coronary heart disease. Arch Intern Med 1998;158:668–75. [8] Stephens NG, Parsons A, Schofield PM, et al. Randomized, controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 1996;347:781–6. [9] GISSI-Prevenzione Investigators. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet 1999;354:447–55. [10] The Heart Outcomes Prevention Evaluation Study. Vitamin E supplementation and cardiovascular events in high-risk patients. N Engl J Med 1999;342:154–60. [11] Investigators. THOPES. Vitamin E supplementation and cardiovascular events in high-risk patients. N Engl J Med 2000;342:154–60. [12] Boaz M, Smetana S, Weinstein T, et al. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): randomised placebo-controlled trial. Lancet 2000;356:1213–8. [13] Blot W, Li J-Y, Taylor PR, et al. Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence and disease-specific mortality in the general population. J Natl Cancer Inst 1993;85:1483–92. [14] Salonen JT, Nyyssonen K, Salonen R, et al. Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study: a randomized trial of the effect of vitamins E and C on 3-year progression of carotid atherosclerosis. J Intern Med 2000;248:377–86. [15] Brown BG, Zhao XQ, Chait A, et al. Simvastatin and niacin antioxidant vitamins, or the combination for the prevention of coronary disease. N Engl J Med 2001;345:1583–92. [16] Steinberg D, Witztum JL. Is the oxidative modification hypothesis relevant to human atherosclerosis? Do the antioxidant trials conducted to date refute the hypothesis? Circulation 2002;105:2107–11.

271

[17] Pratico D, Tangirala RK, Rader DJ, Rokach J, FitzGerald GA. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat Med 1998;4:1189–92. [18] Crawford RS, Kirk E, Rosenfeld ME, LeBoeuf RC, Chait A. Dietary antioxidants inhibit development of fatty streak lesions in the LDL receptor-deficient mouse. Artheriosclar Thromb Vasc Biol 1998;18:1506–13. [19] Gey KF, Puska P, Jordan P, Moser UK. Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in crosscultural epidemiology. Am J Clin Nutr 1991;53:326S–34S. [20] Gaziano JM, Manson J, Buring JE, Hennekens CH. Dietary antioxidants and cardiovascular disease. Ann NY Acad Sci 1992;669:249–59. [21] Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993;328:1444–9. [22] Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 1993;328:1450–6. [23] Morrow JD, Frei B, Longmire AW, et al. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers Smoking as a cause of oxidative damage. N Engl J Med 1995;332:1198–203. [24] Fang JC, Kinlay S, Beltrame J, et al. Effect of vitamins C and E on progression of transplant-associated arteriosclerosis: a randomised trial. Lancet 2002;359:1108–13. [25] Noguchi N, Niki E. Dynamics of vitamin E action against LDL oxidation. Free Radic Res 1998;28:561–72. [26] Kaneko T, Nakano S, Matsuo M. Protective effect of vitamin E on linoleic acid hydroperoxide-induced injury to human endothelial cells. Lipids 1991;26:345–8. [27] Meydani M. Vitamin E and atherosclerosis: beyond prevention of LDL oxidation. J Nutr 2001;131:366S–8S. [28] Cannon JG, Meydani SN, Fielding RA, et al. Acute phase response in exercise. II. Associations between vitamin E, cytokines, and muscle proteolysis. Am J Physiol 1991;260:R1235–40. [29] Faruqi R, de la Motte C, DiCorelto PE. a-Tocopherol inhibits against induced monocytic cell adhesion to cultured human endothelial cells. J Clin Invest 1994;94:592–600. [30] Devaraj S, Li D, Jialal I. The effects of alpha tocopherol supplementation on monocyte function: decreased lipid oxidation, interleukin 1b secretion, and monocyte adhesion to endothelium. J Clin Invest 1996;98:756–63. [31] Martin A, Foxall T, Blumberg JB, Meydani M. Vitamin E inhibits low density lipoprotein-induced adhesion of monocytes to human aortic endothelial cells in vitro. Arterioscler Thromb Vasc Biol 1997;17:429–36. [32] Wu D, Koga T, Martin KR, Meydani M. Effect of vitamin E on human aortic endothelial cell production of chemokines and adhesion to monocytes. Atherosclerosis 1999;147:297–307. [33] Board of Agriculture NRC Nutrient Requirements of Rabbits. 2nd ed. Washington, DC: National Academy of Science; 1977. p. 30. [34] Meydani M, Evans WJ, Handelman G, et al. Protective effect of vitamin E on exercise-induced oxidative damage in young and older adults. Am J Physiol 1993;33:R992–8. [35] McNamara JR, Schaefer EJ. Automated enzymatic standardized lipid analyses for plasma and lipoprotein fractions. Clin Chem Acta 1987;166:1–8. [36] Klurfeld DM. Identification of foam cells in human atherosclerotic lesions as macrophages using monoclonal antibodies. Arch Pathol Lab Med 1985;109:445–9. [37] Rogers KA, Hoover RL, Castellott JJJ, Robinson JM, Karnovsky MJ. Dietary cholesterol-induced changed in macrophage characteristics relationship to atherosclerosis. Am J Pathol 1986;125: 284–91. [38] Napoli C, D’Armiento FP, Mancini FP, et al. Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia intimal accumulation of low density lipoprotein

272

[39]

[40]

[41] [42]

[43]

T. Koga et al. / Atherosclerosis 176 (2004) 265–272 and its oxidation precede monocyte recruitment into early atherosclerotic lesions. J Clin Invest 1997;100:2680–90. Carr AC, McCall MR, Frei B. Oxidation of LDL by myeloperoxidase and reactive nitrogen species Reaction pathways and antioxidant protection. Artherioscler Thromb Vasc Biol 2000;20:1716–23. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone JMA. Interleukin-1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J Clin Invest 1985;76:2003–11. Faruqi RM, DoCorleto PE. Mechanisms of monocyte recruitment and accumulation. Br Heart J 1993;69:S19–29. Berliner JA, Navab M, Fogelman AM, et al. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 1995;91:2488–96. Devaraj S, Jialal I. Alpha-tocopherol decreases interleukin-1beta release from activate human monocytes by inhibition of 5-

[44]

[45]

[46]

[47]

lipoxygenase. Arterioscler Thromb Vasc Biol 1999;19:1125– 33. Yoshikawa T, Yoshida N, Manabe H, Terasawa Y, Takemura T, Kondo M. a-Tocopherol protects against expression of adhesion molecules on neutrophils and endothelial cells. Biofactors 1998;7:15–9. Yoshida N, Yoshikawa T, Manabe H, et al. Vitamin E protects against polymorphonuclear leukocyte-dependent adhesion to endothelial cells. J Leukocyte Biol 1999;65:757–63. Fruebis J, Silvestre M, Shelton D, Napoli C, Palinski W. Inhibition of VCAM-1 expression in the arterial wall is shared by structurally different antioxidants that reduce early atherosclerosis in NZW rabbits. J Lipid Res 1999;40:1958–66. Keaney JFJ, Gaziano MJ, Xu A, et al. Dietary antioxidants preserve endothelium-dependent vessel relaxation in cholesterol-fed rabbits. Proc Natl Acad Sci USA 1993;90:11880–4.