EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

57,70-83

(1992)

Antiatherosclerotic Effects of Antioxidants Are Lesion-Specific When Evaluated in Hypercholesterolemic New Zealand White Rabbits M. A. BOCAN,' SANDRA BAK MUELLER, EDIE QUENBY BROWN, PAUL D. UHLENDORF, MICHELLE J. MAZUR, AND ROGER S. NEWTON

THOMAS

Department of Pharmacology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105 Received May 8, 1992, and in revised form July 27, 1992 Oxidative modification of LDL may represent an initiating event in the formation of monocyte-macrophage foam cells, a major cell present in fatty streaks and atherosclerotic fibrous plaques. Therefore, we studied the effect of such antioxidants as probucol (500 mg/kg) and vitamins E and C (500 mg/kg each) on the regression of induced iliac-femoral lesions and progression of naturally occurring thoracic aortic fatty streak lesions in hypercholesterolemic New Zealand White rabbits. Following an initial 9-week lesion induction phase, both therapies were evaluated for 8 weeks. Probucol lowered plasma cholesterol 47% while vitamins E and C had no effect on plasma cholesterol. Probucol decreased the cholesteryl ester (CE) content of the thoracic aorta by 31% without changing the thoracic aortic lesion coverage. Vitamins E and C decreased thoracic aortic CE content by 40% and lesion coverage by 46%. Neither probucol nor vitamins E and C altered the CE content, lesion size, or macrophagejlesion ratio of the iliac-femoral artery. Thus, we conclude that the effects of antioxidants are specific to the stage of atherosclerotic lesion development. Antioxidant therapy alters the progression and cholesteryl ester enrichment of diet-induced thoracic aortic fatty streaks but has no effect on the progression and/or regression of more complicated injury-induced iliac-femoral lesions. 8 1992Academic PRSS, IN.

INTRODUCTION Hypercholesterolemia or more specifically elevated plasma low density lipoprotein (LDL) cholesterol is an important risk factor for development and progression of atherosclerosis (Kannel et al., 1971; Keys, 1970). In addition to being present in plasma, LDL, identified by the presence of apolipoprotein B, has been observed in both normal and atherosclerotic human vessels using immunocytochemical techniques (Kao and Wissler, 1965; Knieriem et al., 1967; Yomantas et al., 1984; Hoff, 1983; Bocan et al., 1988). Lipid peroxides have also been found within atherosclerotic lesions (Glavind et al., 1952). Most recently, oxidatively modified LDL or such oxidation products as malondialdehyde-conjugated LDL or 4-hydroxynonenal-conjugated LDL have been localized to atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits (Boyd et al., 1989; Haberland et al., 1988; Palinski et al., 1989). Steinberg and colleagues (1989) have reported that oxidatively modified LDL may be important in the progression of atherosclerosis due to the observations that oxidized LDL is cytotoxic, chemotactic, and chemostatic. Monocyte-macrophages in an environment of oxidized LDL would avidly remove the LDL from the interstitium and generate macrophage foam cells, a major cell present within fatty streaks and fibrous plaques (Haust, 1981). Thus, one might hypothesize that prevention of the oxidative mod’ To whom reprint requests should be addressed. Fax: (313) 996-3135. 70 0014-4800/92 $5.00 Copyright 0 1992 by Academic Press, Inc. AU rights of reproduction in any fom reserved.

ANTIOXIDANTS

AND

ATHEROSCLEROSIS

71

i&cation of LDL by pharmacologic agents or naturally occurring antioxidants, i.e., vitamins E and C, would result in a retardation of atherosclerotic lesion development. Several studies investigating the antiatherosclerotic activity of antioxidants have been performed in both New Zealand white rabbits (Daugherty et al., 1989; Tawara et al., 1986; Stein et al., 1989; Carew et al., 1987) and Watanabe heritable hyperlipidemic (WHHL) rabbits (Daugherty et al., 1991; Kita et al., 1987). Butylated hydroxytoluene (BHT) at a dose of 1% in the diet, i.e., approximately 500 mg/kg, was fed to rabbits in a 1% cholesterol diet and was found to inhibit the development of thoracic aortic lesions by 68% as measured planimetrically (Bjorkhem et al., 1991). Brattsand (1975) has also shown in rabbits that the combination of vitamins E and -4 in a hypercholesterolemic diet blunted the cholesterol enrichment of the thoracic aorta. In contrast, Godfried and colleagues (1989) have reported that vitamin E alone potentiated atherosclerotic lesion development ,in balloon-denuded vessels despite receiving 2000 mg vitamin E/kg/day. The cholesterol-lowering agent, probucol, which has been reported to have antioxidant properties, has been shown to attenuate atherosclerotic lesion development (Daugherty et al., 1989; Tawara et al., 1986; Kita et al., 1987; Carew et al., 1987) and to have no effect on lesion formation (Stein et al., 1989). In the studies cited, all but Stein and co-workers (1989) administered the antioxidant therapy from the initiation of the study along with the hypercholesterolemic diet. In contrast to previous studies, the present report examines the effect of antioxidant therapy on lesion regression in a preestablished injury-induced iliacfemoral lesion and on lesion progression in the thoracic aorta. We have previously reported (Bocan et al., 1991a,b) that 8 weeks postinjury a fibrofoamy iliacfemoral lesion is observed while there is minimal lesion development in the thoracic aorta, i.e., 39% lesion coverage. Despite the absence of thoracic lesions, the animals are hypercholesterolemic for 9 weeks prior to initiation of antioxidant therapy. Thus, in the same group of animals one could assess the effect of therapy on both lesion regression and progression under conditions which may more closely mimic the human disease. MATERIALS Experimental

AND METHODS

Design

Male New Zealand White rabbits (Kuiper Farms, Gary, Indiana) weighing 1.Z 1.5 kg were meal-fed a chow diet (Purina, No. 5321) supplemented with 0.5% cholesterol (C), 3% peanut oil (PNO), and 3% coconut oil (CNO) between 0700 and 0900 h daily for a total of 17 weeks. The dietary regimen consisted of feeding 40 g for the first week, 50 g for the next 4 weeks, 60 g for another 4 weeks and 70 g for the final 8 weeks. After 1 week of diet initiation, a chronic endothelial injury was induced in the abdominal and femoral artery by surgically inserting a sterile indwelling 18-cm nylon monofilament with a diameter of 200 pm into the lumen of the right femoral artery. As reported previously (Bocan et al., 1991a,b), a tibrofoamy, lipid-rich iliac-femoral lesion had developed 8 weeks postinjury in 99% of the animals. The animals were randomized into groups of eight based on their plasma total cholesterol values which were determined in animals 24 h after their daily meal. The groups were chosen when the F-ratio determined by analysis of variance was

Antiatherosclerotic effects of antioxidants are lesion-specific when evaluated in hypercholesterolemic New Zealand white rabbits.

Oxidative modification of LDL may represent an initiating event in the formation of monocyte-macrophage foam cells, a major cell present in fatty stre...
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