Atheroprotective and plaque-stabilizing effects of enzymatically modified isoquercitrin in atherogenic apoE-deficient mice

Nutrition 25 (2009) 421– 427 www.nutritionjrnl.com

Basic nutritional investigation

Atheroprotective and plaque-stabilizing effects of enzymatically modified isoquercitrin in atherogenic apoE-deficient mice Koka Motoyama, M.D., Ph.D.a, Hidenori Koyama, M.D., Ph.D.a,*, Masamitsu Moriwaki, Ph.D.b, Kazuhiro Emurab, Shuji Okuyamab, Eisuke Sato, M.D., Ph.D.c, Masayasu Inoue, M.D., Ph.D.c, Atsushi Shioi, M.D., Ph.D.d, and Yoshiki Nishizawa, M.D., Ph.D.a a

Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan b Fundamental Research Division, San-Ei Gen F.F.I., Inc., Osaka, Japan c Department of Biochemistry and Molecular Pathology, Osaka City University Graduate School of Medicine, Osaka, Japan d Department of Cardiovascular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan Manuscript received November 15, 2007; accepted August 26, 2008.

Abstract

Objective: Enzymatically modified isoquercitrin (EMIQ), isoquercitrin with malto-oligosaccharides, has been recognized as “generally recognized as safe” by the Flavor and Extracts Manufacturers Association in the United States since 2003. The long-term antiatherogenic effect of EMIQ was examined using apolipoprotein E (apoE)– deficient atherogenic mice. Methods: Male apoE-deficient mice (6 wk old) were fed with a high-fat diet alone or a diet containing EMIQ for 14 wk. At 20 wk old, atherosclerotic lesions in the aorta and aortic sinus were measured by morphometry and histomorphometry. Results: In apoE-deficient mice, EMIQ did not significantly affect body weight, plasma total cholesterol, triacylglycerol, and high-density lipoprotein cholesterol throughout the experiment. EMIQ significantly suppressed the aortic atherosclerotic lesion area (control 8.8 ⫾ 3.5% versus EMIQ 4.4 ⫾ 1.5%, mean ⫾ SD, P ⫽ 0.022). Similarly, atherosclerotic plaque lesions in the aortic sinus were significantly reduced by EMIQ (control 37.7 ⫾ 3.6% versus EMIQ 30.2 ⫾ 2.0%, P ⫽ 0.010). Of note, the immunostained area for macrophage or 4-hydroxy-2-nonenal, a well-recognized marker of oxidative stress, at the plaque in the aortic sinus was markedly suppressed, whereas the area for collagen or smooth muscle cell were increased by EMIQ, suggesting a plaque-stabilizing effect of EMIQ. Conclusion: EMIQ has atheroprotective and plaque-stabilizing effects. © 2009 Elsevier Inc. All rights reserved.

Keywords:

Flavonoid; Atherosclerosis; Mice; Plaque; Quercetin

Introduction Oxidative stress, the imbalance between free radical production and antioxidant defenses, is associated with many cardiovascular diseases including atherosclerosis, hyperten-

This work was supported by a grant from the Japan Food Chemical Research Foundation to Y.N. and a grant from San-Ei Gen F.F.I., Inc., to H.K. and Y.N. * Corresponding author. Tel.: ⫹81-6-6645-3806; fax: ⫹81-6-66453808. E-mail address: [email protected] (H. Koyama). 0899-9007/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2008.08.013

sion, heart failure, stroke, and diabetes [1]. Reactive free radicals can cause oxidative modification of low-density lipoprotein (LDL), which is believed to be one of the key factors in the development of atherosclerosis [2]. There is compelling evidence in humans and experimental animals for oxidative stress in wide variety of vascular diseases and in risk factors for cardiovascular diseases. A review by Carr and Frei [3] revealed that in humans, a daily 90- to 100-mg intake of vitamin C appears to be effective for protection against the incidence of cardiovascular diseases. Surprisingly, however, stimuli that attenuate oxidative stress are not consistently beneficial in humans and experimental animals [4,5].

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Flavonoids are polyphenolic compounds ubiquitously included in natural plants and are used currently for various kinds of foods and beverages as antioxidant additives [6]. Even in populations with a relatively high dietary intake of saturated fat, the consumption of red wine has been shown to be associated with a reduced cardiovascular mortality, which is known as the “French paradox” [7]. In several epidemiologic studies, there is an inverse association between intakes of flavonoids and mortality from coronary heart diseases [8 –12], although some controversies remain [12–14]. Accordingly, there have been numerous reports showing the protective effects of flavonoids on atherosclerosis in animal models [15–20]. Potential antiatherogenic mechanisms include inhibition of LDL oxidation [15,16], inhibition of platelet aggregation [16], upregulation of paraoxonase that reduce macrophage lipid peroxidation [16 – 21], inhibition of LDL phagocytosis in macrophages and inhibition of esterification of cholesterol [21], and improvement of antioxidative activity on arterial wall [18]. Among these flavonoids, quercetin, or quercetin 3-glucoside (isoquercitrin), is the major flavonol in the plant kingdom (especially in onion, broccoli, apple, and tea) and is found ubiquitously in the diet. Quercetin may be a powerful bioactive constituent of the human diet as a free radical scavenging agent and through interaction with various endogenous proteins (e.g., as an inhibitor of enzyme activity) [12]. Quercetin has been shown to reduce fatty streak formation and early stages of aortic atherosclerotic lesions in the hypercholesterolemic hamster [22], rabbit [23], apolipoprotein E (apoE)– deficient [15] or LDL receptor– deficient mice [24]. It is still not clear, however, if quercetin or isoquercitrin inhibits atherosclerosis in longer and in more advanced stages. Furthermore, the effects on plaque compositions are not entirely known. Enzymatically modified isoquercitrin (EMIQ), isoquercitrin with malto-oligosaccharides manufactured by trans-glycosylation with cyclodextrin glucanotransferase [25,26], is recognized worldwide as a safe additive [27] and has been included since 1996 in the List of Existing Food Additives in Japan and since 2003 in the list of Flavor and Extracts Manufacturers Association (FEMA) as “generally recognized as safe” (GRAS) in the United States. Recently, EMIQ was shown to have antihypertensive function in spontaneously hypertensive rats [28]. In the present study, the long-term antiatherogenic effect of EMIQ was examined using apoE-deficient mice. We found that EMIQ potently suppressed progression of atherosclerosis. We also found that EMIQ decreased the numbers of macrophages and increased collagen content in the plaque, suggesting its plaque-stabilizing effect.

Materials and methods Materials EMIQ was produced from rutin at San-Ei Gen F.F.I., Inc. (Osaka, Japan), as shown in Figure 1 [26]. In short, rutin

Fig. 1. Manufacturing process of EMIQ. EMIQ, enzymatically modified isoquercitrin.

was purified from enju through Sophora japonica L solvents and isoquercitrin was produced from the rutin by excluding rhamnose by ␣-L-rhamosidase. EMIQ is produced from isoquercitrin by the addition of several glucose molecules to the glucose branch by cyclodextrin glucanotransferase. EMIQ was analyzed by ultraviolet spectrophotometry and high-performance liquid chromatography and measured as rutin equivalent content because the molar absorbance coefficient of EMIQ was the same as that of rutin. Animals Male 6-wk-old apoE-deficient mice that were homozygous for the disrupted apoE gene were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and bred under a standard 12-h light cycle at 20°C. This study and all procedures used were approved by the animal ethics committee of Osaka City University. Mice were housed in groups of three or four and consumed a purified stock diet (Stockfeeds; Oriental Yeast Co., Ltd., Tokyo, Japan) for 4 d. At 6 wk of age, mice were randomly assigned to each group and started to be fed with a high-fat diet (47% as carbohydrate, 21% as fat, and 20% as protein; Nippon Formula Feed Mfg. Corp., Yokohama, Japan) under free access to food and water. Control mice were fed only with the high-fat diet, and the EMIQ group was fed with the diet containing 0.026% EMIQ. Food and fluid were changed three times weekly. Body weights were monitored throughout the study. At 4, 9, and 13 wk after the treatments, each mouse was housed in a metabolic cage and the consumption of the food and water was monitored. There were no significant differences in food consumption among the groups at each time point (data not shown). After 14 wk, mice were sacrificed under anesthesia with pentobarbital and processed as follows. Reproducibility of the results was checked by two sets of experiments.

K. Motoyama et al. / Nutrition 25 (2009) 421– 427

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Table 1 Body weight and lipid concentration in apolipoprotein E– deficient mice* Experiment 1

Body weight (g) 6 wk 20 wk TC (mg/dL) 6 wk 20 wk TG (mg/dL) 6 wk 20 wk HDL-C (mg/dL) 6 wk 20 wk

Experiment 2

Control (n ⫽ 10)

EMIQ (n ⫽ 10)

P

Control (n ⫽ 8)

EMIQ (n ⫽ 7)

P

22.8 ⫾ 2.0 30.7 ⫾ 1.9

22.8 ⫾ 2.0 29.6 ⫾ 2.2

NS NS

23.3 ⫾ 2.1 28.5 ⫾ 3.8

23.0 ⫾ 1.4 29.4 ⫾ 2.3

NS NS

626 ⫾ 149 1405 ⫾ 253

677 ⫾ 253 1399 ⫾ 282

NS NS

689 ⫾ 110 1522 ⫾ 465

683 ⫾ 98 1228 ⫾ 182

NS NS

237 ⫾ 68 105 ⫾ 43

218 ⫾ 47 103 ⫾ 38

NS NS

233 ⫾ 42 64 ⫾ 27

254 ⫾ 69 54 ⫾ 30

NS NS

34 ⫾ 8 22 ⫾ 5

39 ⫾ 8 18 ⫾ 5

NS NS

26 ⫾ 10 15 ⫾ 7

27 ⫾ 5 16 ⫾ 9

NS NS

EMIQ, enzymatically modified isoquercitrin; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triacylglycerol * Male 6-wk-old apolipoprotein E– deficient mice were fed with a high-fat diet for 14 wk. Two independent sets of experiments were performed to check reproducibility. Control mice were fed with a high-fat diet alone and the EMIQ group was fed with a diet containing 0.026% EMIQ. Values are means ⫾ SDs.

Plasma lipid analyses Blood was drawn from the tail vein after overnight fasting, and sera were obtained by centrifugation. Total cholesterol, high-density lipoprotein cholesterol, and triacylglycerol concentrations were measured by regular enzymatic assays that were adapted to a Fuji-DryChem3500 multianalyzer system (Fujifilm Medical Co., Ltd., Tokyo, Japan). Quantification of atherosclerotic lesions At 20 wk old, mice were anesthetized by pentobarbital, blood was taken, and perfused with saline followed by 2% formaldehyde. The heart and entire aorta were rapidly dissected out and postfixed by immersion in 4% paraformaldehyde at room temperature. For evaluation of atherosclerotic lesions on the aorta (experiments 1 and 2), thoracic and abdominal parts of the aorta were opened longitudinally and pinned down on a board. Atherosclerotic lesions were stained by oil red O, photographed, and analyzed by image analysis software (IPAP, Sumitomokagaku Technoservice Inc., Osaka, Japan). For histological analyses of plaque lesions in the aortic sinus (experiment 2), the top half of the hearts was obtained under stereoscopic observation and fixed by immersion in paraformaldehyde. Specimens were routinely processed for paraffin embedding. Cross sectioning started within the heart and worked in the direction toward the aorta. Once the aortic sinus was identified, every consecutive 5-␮m-thick section throughout the aortic root area (300 ␮m, 60 sections per mouse) was taken for analysis. Every 12 sections (total of 5 sections) were stained with Elastica Van Gieson, and the intimal plaque area was examined for histomorphometric analyses using a computerized image analyzer (Lumina Vision 2.20, Mitani Corporation, Tokyo, Japan) attached to a microcamera and microscope. Each plaque lesion area is normalized by the area of aortic sinus, and the average of five specimens was

determined to represent the atherosclerotic plaque lesion area in each animal. For evaluation of collagen content, the sections were stained with Masson’s trichrome. For immunohistologic analysis of macrophages or vascular smooth muscle cells, the sections were stained with rat Mac-3 antibody (clone M3/84, BD Biosciences, Japan) or with mouse anti–␣-smooth muscle actin antibody (clone 1A4 from Sigma, St. Louis, MO, USA) counterstained with Harris’s hematoxylin stain. 4-Hydroxy-2nonenal (4-HNE), a major lipid peroxidation-derived aldehyde, within human atheromatous lesions was stained with mouse anti– 4-HNE monoclonal antibody (clone 4-HNE–modulated keyhole limpet hemocyanin from the Japan Institute for the Control of Aging, Nikken SEIL Corporation). The sections were visualized by a Vectastain ABC kit (Vector Laboratories) with 3,3=diaminobenzidine tetrahydrochloride as the substrate. The stained area was morphometrically analyzed by a computerized image analyzer and normalized by the total area of the aortic sinus. Statistical analysis All measurements are expressed as mean ⫾ standard deviation (SD). Differences were considered statistically significant at P ⬍ 0.05. Differences between means were compared by using Student’s t test using Stat View V software (SAS Institute, Cary, NC, USA).

Results EMIQ does not affect body weight and lipid profile in apoE-deficient mice In apoE-deficient mice, treatment with EMIQ for 14 wk did not affect the food intake of mice as evaluated every week. As presented in Table 1, EMIQ did not significantly

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Fig. 2. Morphometric evaluation of aortic lesions of apolipoprotein E– deficient mice fed with a high-fat diet for 14 wk. In experiments 1 (A) and 2 (B,C), control mice were fed with a high-fat diet alone. EMIQ-treated mice were fed with the diet containing 0.026% EMIQ. In both experiments, atherosclerotic lesion area at the thoracic and abdominal aorta were measured by lipid deposition stained with oil red O. (C) Atherosclerotic lesion area at cross sections of the aortic sinus was analyzed by Elastica Van Gieson staining (experiment 2) and measured as intimal plaque area, which was normalized by the area of the aortic sinus. Arrows indicate plaque lesions. Bars ⫽ 500 ␮m. Values are means ⫾ SDs. EMIQ, enzymatically modified isoquercitrin.

affect body weights at 20 wk old. Furthermore, plasma total cholesterol, triacylglycerol, and high-density lipoprotein cholesterol were not significantly different between groups at 20 wk old. EMIQ inhibits progression of atherosclerotic lesions in apoE-deficient mice We next examined the effects of quercetin and EMIQ on the progression of atherosclerosis in hypercholesterolemic apoE-deficient mice. As shown in Figure 2A (experiment 1), EMIQ significantly suppressed the area of aortic atherosclerotic lesions by about 38% (control 9.5 ⫾ 4.1% versus EMIQ 5.9 ⫾ 2.5%, P ⫽ 0.037). In a separate experiment (experiment 2; Fig. 2B), EMIQ reproducibly and significantly suppressed the area of aortic atherosclerotic lesions by about 50% (control 8.8 ⫾ 3.5% versus EMIQ 4.4 ⫾ 1.5%, P ⫽ 0.022). In experiment 2, the plaque area in the aortic sinus was also examined and was also significantly inhibited by EMIQ (control 37.7 ⫾ 3.6% versus EMIQ 30.2 ⫾ 2.0%, P ⫽ 0.010; Fig. 2C). EMIQ suppresses accumulation of lipid peroxidation product and alters the composition in atherosclerotic plaque in apoE-deficient mice The formation of lipid peroxidation products in atherosclerotic lesions is a common characteristic associated with oxidative stress. 4-HNE is one of the lipid peroxidation products and is known to have potent cytotoxicity [29]. Thus, we next examined the effect of EMIQ on a 4-HNE– immunostained area in the plaque area of the aortic sinus. As shown in Figure 3A, EMIQ potently suppressed the 4-HNE–positive area compared with control (control 14.2 ⫾ 5.8% versus EMIQ 7.5 ⫾ 2.9%, P ⫽ 0.033). Thus, EMIQ markedly suppressed accumulation of the lipid peroxidation

product, which could be associated with an inhibition of atherosclerotic progression. The cellular composition and other phenotypic features of an atheromatous plaque determine its plaque size and its stability. To further understand the underlying mechanisms related to EMIQ suppression of atherosclerotic progression, histologic analyses of the plaque in the aortic sinus were performed. Interestingly, macrophage accumulation as determined by Mac-3 immunostaining was profoundly suppressed in the EMIQ-treated group compared with that of the control group (control 13.3 ⫾ 4.4% versus EMIQ 7.0 ⫾ 2.1%, P ⫽ 0.005; Fig. 3B). In contrast to macrophage accumulation being related to plaque instability, numbers of differentiated vascular smooth muscle cells and collagen content have recently been shown to be associated with plaque stabilization [30,31]. To examine the possibility that EMIQ stabilizes plaque, smooth muscle cell accumulation and collagen content were examined by ␣-smooth muscle actin immunostaining and Masson’s trichrome staining. The ␣-smooth muscle actin–immunostained area was significantly increased in EMIQ-treated mice (control 1.0 ⫾ 0.4% versus EMIQ 1.8 ⫾ 0.3%, mean ⫾ SD, P ⫽ 0.011), suggesting that the numbers of smooth muscle cells were increased by EMIQ (Fig. 3C). Collagen content in EMIQ-treated mice was also increased compared with that of control mice (control 11.5 ⫾ 3.0% versus EMIQ 16.2 ⫾ 2.8%, P ⫽ 0.027; Fig. 3D). Discussion In the present study, we showed, to our knowledge for the first time, that EMIQ, a flavonol recently included in the FEMA GRAS, has long-term potent antiatherogenic and plaque-stabilizing properties. Antiatherogenic properties of quercetin or quercetinglucoside were examined using several animal models. Hayek et al. [15] first evaluated the effect of quercetin on apoE-

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Fig. 3. Immunohistologic evaluation of atherosclerotic lesions of apolipoprotein E– deficient mice fed with a high-fat diet for 14 wk. Control mice were fed with a high-fat diet alone. EMIQ-treated mice were fed with a high-fat diet with 0.026% EMIQ. The histologically stained area was studied at cross sections of aortic valves. (A) 4-HNE, (B) Mac-3, (C) ␣-SMA, (D) collagen stained by Masson’s trichrome. Stained area was normalized by the area of the aortic sinus. Values are means ⫾ SDs. Arrows indicate specific staining. Red bar ⫽ 500 ␮m, black bar ⫽ 50 ␮m. ␣-SMA, ␣-smooth muscle actin; EMIQ, enzymatically modified isoquercitrin; 4-HNE, 4-hydroxy-2-nonenal.

deficient atherogenic mice. They found that 6 wk of quercetin treatment reduced the area covered with atherosclerotic plaque by 46%. More recently, treatment with isoquercitrin for 8 wk was shown to suppress atherosclerotic lesion area in LDLreceptor–null mice [24]. Antiatherogenic effects of quercetin were also confirmed in hamster and rabbit atherosclerotic models [22,23]. These findings, however, focused on the early phase of the atherogenic process, particularly fatty streak formation. In the present study using apoE-deficient atherogenic mice fed with a high-fat diet, we evaluated, to our knowledge for the first time, the effects of EMIQ, an enzymatically modified quercetin-glucoside, on the more advanced phase of atherosclerosis and found that EMIQ profoundly suppressed formation of advanced atherosclerotic lesions at the aortic surface and at the aortic sinus. Moreover, we carefully evaluated the composition of the plaques and found that EMIQ markedly suppressed accumulation of macrophages and enhanced the numbers of smooth muscle cells and accumulation of collagens. Thus, EMIQ not only inhibits progression of atherosclerosis but also could stabilize the plaque.

It is not clear at present how EMIQ exerts antiatherogenic effects. The effects of quercetin or isoquercitrin on lipid metabolism are dependent on the animal models and experimental conditions (duration of the treatment, dosage of quercetin or isoquercitrin). In our present study, EMIQ failed to decrease serum cholesterol or triacylglycerol levels in accordance with the effect of quercetin using apoE-deficient mice [15]. In contrast, in a rabbit or hamster model fed with a high-fat diet, even lower dosages of quercetin for suppression of atherosclerosis is associated with suppressed serum cholesterol or triacylglycerol levels [22,23]. In LDL-receptor– deficient mice fed with an atherogenic diet, isoquercitrin, but not quercetin, significantly suppressed serum cholesterol levels [24]. In our experiment, serum cholesterol level increased, whereas serum triacylglycerol decreased with high-fat diet feeding, and Jiang et al. [32] reported an increase in serum triacylglycerol during treatment. This could be due to an alteration of very low density lipoprotein (VLDL) composition by different components in a highfat diet. Alteration of VLDL components has been shown in mice treated with fish oil [33]. It appears unlikely that EMIQ

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markedly changed the components of VLDL, a major lipoprotein in apoE-knockout mouse. The atheroprotective effect of EMIQ may be attributed to its antioxidative property, because quercetin and isoquercitrin have been shown to markedly inhibit LDL oxidation in vitro [6 –15]. Quercetin and related compounds have been shown to bind to the surface of LDL particles by the formation of an ether bond, which limits the access by oxidants and their attack on the surface [15]. In apoE- or LDL receptor– deficient mice in vivo, quercetin or isoquercitrin retarded the onset of plasma LDL oxidation as evidenced by the prolongation of the lag phase for conjugated diener formation [15–24]. However, these effects are not necessarily associated with the inhibition of the serum levels of the endproduct of LDL oxidation. In our present study, EMIQ failed to inhibit levels of the serum endproduct of LDL oxidation as measured by thiobarbituric acid–reactive substances (data not shown). EMIQ may directly suppress oxidative activity in the arterial wall, as shown for green tea– derived catechin or isoquercitrin [18–31,34]. Indeed, the abundance of 4-HNE, a major lipid peroxidation-derived aldehyde, was significantly suppressed in mice treated with EMIQ. However, because the localization of 4-HNE in the lesions was closely associated with that of the macrophages, this result may simply be attributed to the gross inhibition of atherosclerotic progression. Beyond the traditional view of atherosclerosis as an accumulation of oxidized lipids in the arterial wall, activation of arterial endothelial cells appear to play a key role in the initiation and progression of the disease [35,36]. Recently, Lotito and Frei [37] showed that 5,7-dihydroxyl substitution of a flavonoid A-ring and 2,3-double bond and 4-keto group of the C-ring were the main structural requirements for inhibition of adhesion molecule expression, which is essential for leukocyte– endothelial interactions and leukocyte emigration to the subendothelium. Thus, flavonols such as quercetin, isoquercitrin, and EMIQ could suppress adhesion molecules in endothelial cells, which may result in suppression of the initiation and progression of atherosclerosis. In the present study, we showed, to our knowledge for the first time, that EMIQ modified the composition of plaque toward stabilization, increased smooth muscle cells and collagen content, and decreased macrophage accumulation. Several lines of clinical evidence suggest that an acute coronary syndrome, such as acute myocardial infarction and unstable angina pectoris, is mainly due to vessel lumen occlusion by a thrombus formed at the contact of a disrupted atherosclerotic plaque (unstable plaque) [38]. Pathologic studies have shown that vulnerable or unstable plaques are rich in inflammatory cells and exhibit a substantial loss in smooth muscle cell and collagen content [30,31]. Moreover, such plaques show a significant increase in apoptotic cell death leading to the formation of a highly thrombogenic lipid core [39,40]. Thus, EMIQ may be beneficial for the prevention of

plaque rupture and potentially be useful in patients with a high risk for an acute coronary syndrome. In summary, using a well-validated mouse model of human-like atherosclerosis, we showed that EMIQ has strong inhibitory effects against atherosclerotic plaque development, progression, and instability. With the safety data of EMIQ as in the FEMA GRAS, further studies are warranted to accelerate the development of EMIQ as a potential therapeutic tool to fight against the occurrence of cardiovascular events.

Acknowledgments Gratitude is expressed to Yuriko Matsuo, an assistant staff member at our laboratory, for data collection and intellectual input.

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