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ORIGINAL RESEARCH COMMUNICATIONS |
1 From the School of Medicine and Pharmacology, University of Western Australia, Royal Perth Hospital, Perth, Australia (EW, IBP, and KDC), and the Western Australian Institute for Medical Research, Perth, Australia (EW, IBP, and KDC).
2 Supported by a grant from the National Health and Medical Research Council of Australia (9937262) and through postgraduate research funding from the University of Western Australia (to EW). 3 Address reprint requests to KD Croft, School of Medicine and Pharmacology, University of Western Australia, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6847, Australia. E-mail: kcroft{at}cyllene.uwa.edu.au.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: This study examined stable biomarkers of lipid oxidative damage in relation to atherosclerotic disease progression in apolipoprotein E–deficient (Apoe-/-) mice and retardation of the disease by red wine polyphenols.
Design: One hundred male Apoe-/- mice and 50 male control (C57BL/6J) mice were given a high-fat, high-cholesterol diet for 20 wk. To examine the effect of the polyphenolic compounds on lesion development, 50 of the Apoe-/- mice were also given dealcoholized red wine for the duration of the study.
Results: Aortic lipid deposition was significantly greater in the Apoe-/- mice than in the control mice (P < 0.01). Plasma and aortic F2-isoprostanes did not differ between the treatment groups. Plasma concentrations of monocyte chemoattractant protein 1, which has been implicated in the development of atherosclerosis, were significantly higher in the Apoe-/- mice than in the control mice up to 16 wk (P < 0.05). Hydroxyeicosatetraenoic acid concentrations increased significantly over time in all groups (P < 0.05). Red wine polyphenols had no effect on markers of lipid peroxidation or monocyte chemoattractant protein 1 concentrations, but lipid deposition in the aorta at age 26 wk was significantly less in the mice given red wine than in those not given red wine.
Conclusion: These results suggest that lipid deposition is independent of lipid oxidation and that the protective action of red wine polyphenols is independent of any antioxidant action of these compounds.
Key Words: Hydroxyfatty acids • lipid peroxidation • F2-isoprostanes • red wine polyphenols • atherogenesis • apolipoprotein E
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most human intervention studies examining the effect of antioxidants on atherogenesis and its complications studied subjects with established atherosclerosis and used lipid-soluble 
-tocopherol as an antioxidant (13-15). These studies generally had negative results and called into question the oxidation hypothesis of atherosclerosis. Animal models, on the other hand, gave more support to the oxidation hypothesis, which allowed for the study of early disease processes and enabled the evaluation of interventions and treatments in the beginning stages of atherosclerosis. The apolipoprotein E–deficient (Apoe-/-) mouse develops lesions as early as 5 wk of age (16) and complex lesions, similar to the plaques observed in late-stage human atherosclerosis, by age 6 mo (17, 18). The Apoe-/- mouse model has been considered particularly useful for the study of the oxidation hypothesis of atherosclerosis. Palinski et al (19) showed that atherosclerotic plaques in Apoe-/- mice contained epitopes specific for oxidation products of LDL and that the plasma contained high concentrations of autoantibodies to malondialdehyde-lysine.
Markers of fatty acid peroxidation such as the F2-isoprostanes and hydroxyeicosatetraenoic acids (HETEs) were identified in late-stage human atherosclerotic plaque (9-11, 20). Many of these compounds also have biological activities that may be relevant to the progression of the atherosclerotic plaque (21-24), but it is difficult to determine their possible role in atherogenesis, given that most studies to date involved late-stage lesions. A recent study by Upston et al (12) examined the accumulation of lipid and protein oxidation products at different stages of human atherosclerosis and found a definite stage-dependent increase in lipid oxidation products, such as hydroperoxides and oxidized cholesterol compounds, during atherosclerosis progression.
The use of the Apoe-/- mouse model of atherosclerosis has shown inconsistent results for the effect of antioxidants on the progression of atherosclerosis (25-27). In addition, animal experiments using red wine polyphenols were shown to inhibit the development of atherosclerosis (28, 29) but not to reduce mature atherosclerosis (30). Whereas red wine polyphenols can reduce the ex vivo susceptibility of LDL to oxidation (28), their effect on in vivo markers of lipid peroxidation has not been investigated in an animal model.
The aims of the present study were to examine stable biomarkers of lipid oxidation, ie, F2-isoprostanes and HETEs, in relation to atherosclerotic disease progression in Apoe-/- mice and to ascertain whether antioxidant red wine polyphenols limit the extent of atherosclerosis or modify the formation of these fatty acid oxidation products.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and the eicosanoid standard 15-HETE were purchased from the Cayman Chemical Company (Ann Arbor, MI). All solvents used in the chromatography were of HPLC grade. The derivatizing reagents were N,O-bis-(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (99:1; Pierce Chemicals, Rockford, IL) and pentafluorobenzylbromide, N,N-diisopropylethylamine, and pyridine (all: Sigma Chemicals, St Louis). Preparation of the [18O2]15-HETE standard was similar to that described by Murphy and Clay (31). Briefly, H218O (Cambridge Isotopes, Andover, MA) was subjected to hydrogen chloride saturation by bubbling hydrogen chloride gas into the water solution. A 1-mol H218O/L solution was diluted with acetonitrile to 50:50 (by vol). The 15-HETE standard was first subjected to hydrogenation by using a rhodium on alumina catalyst (Sigma Chemicals) for 10 min. After removal of the catalyst and subsequent drying under nitrogen, the residue was dissolved in the acetonitrile:H218O solution (100 µL). The tube was purged with argon and heated for 12–15 h at 60 °C.
Animals
One hundred 4-wk-old male Apoe-/- mice that were homozygous for the disrupted Apoe gene and fifty 4-wk-old male C57BL/6J mice were obtained from the Animal Resource Centre (Canningvale, Australia). This study and all procedures used were approved by the Animal Ethics Committee of Royal Perth Hospital. Mice were housed in groups of 5 or 6 and immediately began to consume a nonpurified stock diet (Glenforrest Stockfeeds, Glenforrest, Australia) until 6 wk of age. At that time, all mice began to consume a high-fat, high-cholesterol diet containing 21.2% fat and 0.15% (by wt) cholesterol (Harlan Teklad diet TD88137; Glenforrest Stockfeeds). The Apoe-/- mice subsequently were randomly assigned to receive either dilute dealcoholized red wine (Cabernet Shiraz 1994; Hardys Nottage Hill, McLaren, Australia; n = 50) or water (n = 50). The C57BL/6J mice were all given water alone. The wine was dealcoholized by vacuum distillation as described previously (32); it contained 900 mg polyphenols/L. The dilute dealcoholized red wine was diluted 1:10 so that mice would consume 
0.6 mg polyphenol in their average daily fluid intake (which is equivalent to several glasses per day for a human). Both food and fluid intake were ad libitum. All fluids were changed each day.
Isolation of plasma and aortic tissue
Nonfasting mice from each group (n = 11–13) were studied at 11, 15, 21, and 26 wk of age. After the mice were anesthetized with the use of an intraperitoneal injection of Nembutal (Boehringer Mannheim, Mannheim, Germany), a blood sample was obtained via a heart puncture and collected into microfuge tubes containing 50 µL EDTA (1 g/10 mL in 0.9% saline). Samples were immediately centrifuged at 5000 x g for 30 s at room temperature, and the plasma was stored at -80 °C after the addition of 10 µL butylated hydroxytoluene (4 mg/mL). The abdominal and thoracic cavities were opened by ventral incision. The aorta from the iliac juncture to the top of the aortic arch was removed. All aortas were stripped of any external fatty deposits. Aortas for histopathologic analysis were cut longitudinally from the aortic arch down to the iliac juncture. Then the aortas were placed in phosphate-buffered saline (PBS) containing EDTA (0.38 mg/mL) and stored on ice for 3 h before being stained and fixed. After their removal and cleansing of extraneous fat, aortas for biochemical analysis were washed in PBS containing EDTA (0.38 mg/mL) and butylated hydroxytoluene, blotted dry, and immediately stored at -80 °C.
Histologic analysis of mouse aortas
After a 3-h rinse in PBS, each aorta was placed in 2 mL of a formal sucrose solution, pH 7.4 (4 g paraformaldehyde, 5 g sucrose, 0.01 mg butylated hydroxytoluene, and 0.038 g EDTA in 100 mL double-distilled H2O). Samples were protected from light and left overnight. The next day, samples were rinsed in PBS containing EDTA for 6 h and then in 70% ethanol for 1 min. Aortic tissue was then immersed in a solution of Sudan IV (0.5 g Sudan, 35 mL ethanol, 50 mL acetone, and 15 mL double-distilled H2O; Sigma-Aldrich, St Louis) for 15 min. To remove excess stain, each aorta was rinsed several times with 100% ethanol. The extent of atherosclerosis was calculated from the ratio of lipid-rich, Sudan-stained tissue to total tissue area. This calculation was performed via image analysis using a microscope (Leitz, Wetlar, Germany) with a video attachment (Panasonic, Yokohama, Japan). By using VIDEOPRO 32 Color Image Analysis software (Leading Edge Pty Ltd, Adelaide, Australia), we calculated the lesion area as a percentage of the total tissue area. Samples were coded and analyzed blind.
Plasma cholesterol and monocyte chemoattractant protein 1
The cholesterol content of the mouse plasma samples was measured by using a commercially available cholesterol assay kit (Boehringer Mannheim). Mouse plasma monocyte chemoattractant protein 1 (MCP-1) concentrations were measured with the use of a commercially available kit (Pharmingen, San Diego).
Plasma F2-isoprostanes
Samples of plasma were thawed, and 200-µL aliquots were removed. To these aliquots, 400 µL double-distilled H2O and 1 mL 1 mol methanolic KOH/L were added. Samples were flushed with nitrogen, capped tightly, and incubated at 45 °C for 1 h. After the addition of the internal standard d4-8-iso-PGF2 (2 ng), samples were subjected to solid-phase extraction, HPLC purification, and quantitation with the use of gas chromatography–mass spectrometry as described previously (33).
Isolation of F2-isoprostanes from aortic tissue
Frozen tissue was thawed and weighed, cut into small pieces, and placed in 2 mL PBS. With the use of an Ultra Turax T8 blade homogenizer (IKA Labortechnik, Staufen, Germany), samples were homogenized for 1 min at the highest rotation speed. Ice-cold Folch solution (chloroform:methanol, 2:1 by vol) was added, and the samples were centrifuged at 1500 x g for 10 min at 4 °C. The bottom layer was removed and divided into 2 approximately equal volumes. One portion was stored at -80 °C to await the analysis of HETE content, and the other portion was dried under nitrogen and then reconstituted in 200 µL methanol before the addition of 1 mL aqueous 15% KOH and incubation at 45 °C for 1 h. After incubation, 5 mL 0.1 mol PBS/L (pH 4.0) and 2 ng d4-8-iso-PGF2 (stock 1 ng/10 µL) were added, and the entire solution was acidified to pH 3.0 with the use of 5 mol HCl/L. The samples were then applied to methanol-conditioned C18 Sep-Paks (Waters Corporation, Milford, MA) as described (34). The HPLC purification was omitted for the analysis of aorta F2-isoprostanes. Briefly, after C18 and silica solid-phase purification, samples were dried in a vacuum for 2 h and then derivatized and subjected to gas chromatography–mass spectrometry analysis as previously described (33).
Aortic fatty acid composition
Approximately 200 µL of the lipid extract obtained from the mouse aortas was placed in a glass boiling tube and dried under nitrogen. To this we added 2 mL 4% H2SO4 in methanol and 40 µL heptadecanoic acid as an internal standard (stock 1 mg/mL). Fatty acid methyl esters were analyzed by gas chromatography as described (34).
Analysis of HETEs in mouse aortas
The stored lipid extract obtained from the homogenized mouse aortas was thawed; 10 ng of the internal standard, [18O2]15-HETE, was added, and then rhodium on alumina (5 mg) was added. The solution was purged with hydrogen, sealed, and agitated for 10 min. The catalyst was removed by centrifugation, and the supernatant was dried under nitrogen. The residue was reconstituted in 200 µL ethanol, 1 mL aqueous 15% KOH was added, and the mixture was incubated at 45 °C for 1 h. We added 5 mL of 0.1 mol PBS/L (pH 4.0) and adjusted the solution to pH 3.0 by using 1 mol HCl/L. Samples were applied to C18 solid-phase cartridges (Sep-Pak, Waters Corporation) and washed with 5 mL double-distilled H2O and 10 mL acetonitrile:water (15:85, by vol), and the HETEs were eluted by using 5 mL hexane:ethyl acetate:2-propanol (30:65:5, by vol). The eluate was dried under nitrogen, and the residue was derivatized to the pentafluorobenzoate by using 40 µL pentafluorobenzylbromide (10% solution in acetonitrile) and 20 µL N,N-diisopropylethylamine (10% solution in acetonitrile), incubated at room temperature for 30 min, and then dried under nitrogen. N,O-bis-(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (20 µL) and pyridine (10 µL) were then added to the residue, the mixture was incubated at 45 °C for 20 min, and solvents were evaporated with the use of nitrogen. The derivatized HETEs were reconstituted in 25 µL isooctane and analyzed by using negative ion chemical ionization gas chromatography–mass spectrometry. Chromatography was performed by using an HP-5MS column (30 m x 0.25 mm x 0.25 µm; Hewlett-Packard, Palo Alto, CA) with helium as the carrier gas and methane as the reagent gas at an ion source pressure of 1.8 torr. The initial column temperature of 160 °C was held for 1 min, after which the temperature was programmed to rise to 300 °C at a rate of 15 °C/min and then was maintained at 300 °C for 5 min. The mass spectrometer was operated in selective ion monitoring mode, and HETEs were detected by monitoring the peak corresponding to their carboxylate anion (M-181)- [mass-to-charge ratio (m/z): 399] and the corresponding peak of the [18O2]15-HETE standard (m/z: 403).
Statistical analysis
Statistical analysis was performed with the use of SPSS software (version 11.5; SPSS Inc, Chicago). Results are reported as means ± SEMs. Differences between mouse groups over time were analyzed by using generalized linear modeling to determine the significance of time main effects, treatment main effects, and treatment x time interactions. When there was a significant interaction, subgroup analysis was carried out at each time point by using analysis of variance with Bonferroni adjustment for post hoc multiple comparisons of group means. Pratico et al (10) previously reported a decrease of 
100% in both lesion area and plasma and tissue isoprostanes in this mouse model after vitamin E treatment (n = 8–11 animals per group). With plasma sampled from 11–13 animals per group at each time point, our study had >95% power to detect a 50% decrease in plasma isoprostanes, and with aortic tissue sampled from 6–8 animals per group at each time point, it had 50% power to detect a 100% decrease in aortic isoprostanes and HETEs. Statistical significance was accepted at a value of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histologic assessment of aortic lesions
Atherosclerotic lesion development as the percentage of the total area that had become lipid lesion is shown for each mouse group at 4 time points (Figure 1
). The amount of lipid deposition remained relatively low and did not differ significantly from baseline in the C57BL/6J mouse group. In contrast, both groups of Apoe-/- mice tended to show increased lesion development over the 6-mo period. The percentage of lipid deposition was significantly higher in the 26-wk-old Apoe-/- mice receiving water alone than that in those given red wine polyphenols (P < 0.02). The amount of lipid deposition was significantly higher in both Apoe-/- mouse groups than in the control C57BL/6J mouse group (P < 0.05).
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. There was no significant difference between the cholesterol concentrations in Apoe-/- mice receiving red wine polyphenol treatment and those in Apoe-/- mice not receiving treatment. Even with consumption of a high-fat, high-cholesterol diet, the cholesterol concentrations in the C57BL/6J mice remained relatively lower and constant over the 6-mo time course (average: 110 ± 14 mg/dL).
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). However, by 21 wk of age, there was no significant difference between the 3 groups. There was also no significant effect of red wine polyphenols on MCP-1 concentrations (Table 1).
Plasma and aortic F2-isoprostanes
The plasma F2-isoprostane concentrations for each group from 11 to 26 wk are shown in Table 1. The concentration of F2-isoprostanes remained relatively constant over the 6-mo study period for each of the mouse treatment groups, and there was no significant difference between groups over time. F2-isoprostanes in mouse aortas also showed no significant difference between any of the groups over time, whether expressed in nanograms per gram of tissue weight (as shown in Table 1) or corrected for tissue arachidonic acid concentrations.
Aortic HETEs
A typical selected ion chromatogram of a mouse aortic extract is shown in Figure 2, with monitoring of the ions 399 and 403, which represent the major fragmented ions of the HETEs (m/z: 399; top panel) and the [18O2]15-HETE internal standard (m/z: 403; bottom panel). With the use of authentic standards, 15-HETE was identified as the peak detected at 12.90 min, whereas the peak detected at 12.75 min was found to have the same elution time as authentic standards of 8-, 9-, 11-, and 12-HETE, which co-elute under these conditions. The concentrations of 15-HETE and all other HETEs were quantitated against the standard O18–15-HETE. The aortic concentrations of 15-HETE in each mouse group over time are shown in Figure 3. The differences between the 3 groups were not significant, but there was a significant increase in the concentrations over time in all groups (P < 0.001).
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) show the same time-dependent significant increase as that observed for 15-HETE. There was no significant difference between the groups according to treatment or genotype. The same trend in HETE formation was observed whether the concentrations were expressed in nanograms per gram of tissue weight (Table 1) or corrected for aortic arachidonic acid concentrations as in Figure 3.
Aortic tissue fatty acid composition
The distribution of major fatty acids in aortic tissue for each group at 26 wk of age is shown in Table 2. The 2 Apoe-/- mouse groups had greater amounts of all fatty acids, which was expected, given the greater amount of lipid deposition in these groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Apoe-/- mouse has been widely used to study the processes involved in atherosclerosis, but few studies examined the formation of oxidized fatty acid in relation to disease progression. A comprehensive study by Letters et al (35) examined the changes to lipids and antioxidants in Apoe-/- mice fed a high-fat diet. They showed that progression of atherosclerosis was associated with increased aortic lipid peroxidation, as assessed by the concentrations of cholesterol ester hydroperoxides. However, values were not compared with those in normolipidemic control mice. We have noted time-dependent increases in the major oxidation products of arachidonic acid (HETEs) in both normolipidemic control C57BL/6J mice and Apoe-/- mice. Moreover, there was no significant difference in the concentrations of aortic HETEs in the control C57BL/6J mice and in the Apoe-/- mice.
There were no changes in the concentrations of F2-isoprostanes, expressed per substrate fatty acid, in plasma or aortic tissue over a 6-mo period in either Apoe-/- mice or C57BL/6J mice. Aortic concentrations of total F2-isoprostanes in our animals were similar to those previously reported in hyperlipidemic mice (36). Pratico et al (25) reported that concentrations of a particular isoprostane isomer (iPF2-VI) were greater in Apoe-/- mice than in C57BL/6J mice after 18 wk of age. Whereas the mice in that study were fed a normal diet rather than a high-fat diet, there is little evidence that fat feeding influences isoprostanes (37). It is therefore not clear why F2-isoprostane concentrations did not differ between control and Apoe-/- mice in our study. F2-isoprostanes are regarded as one of the best available markers of in vivo lipid peroxidation. However, they are subject to metabolism via ß-oxidation pathways (38, 39), and there is still conjecture as to whether they represent systemic or localized oxidative stress (40). Hydroxyfatty acids such as the HETEs have been reported to be present in human atherosclerotic plaque in concentrations that are many times higher than those of F2-isoprostanes (20). HETEs have not previously been detected or quantitated in Apoe-/- mice. In this study, we show that relatively high concentrations of 15-HETE and of other HETEs are present in the aortas of mice fed a high-fat, high-cholesterol diet. The concentrations of 15-HETE and other HETEs in aortic tissue were 36-fold and 600-fold, respectively, those of the F2-isoprostanes. Whereas HETEs are among the major products resulting from the reduction of lipid hydroperoxides (primary products of arachidonic acid oxidation), it is also possible that a contribution from lipoxygenase activity may be present in early lesions (41). Our analytic methods cannot distinguish the stereospecific products, but we previously showed by analysis of the chirality of HETEs in advanced human lesions that most HETEs are formed via the nonenzymatic pathway (11). There is also little direct evidence for the involvement of 15-lipoxygenase in human atherosclerotic lesions (42). HETEs were observed to increase significantly in a time-dependent manner in both Apoe-/- and control C57BL/6J mice. The increasing concentrations of HETEs may therefore reflect increased oxidative stress associated with aging (43, 44).
The significant increases in the isoprostane iPF2-VI in young mice observed by Pratico et al (25) preceded the development of atherosclerosis, which suggested that lipid peroxidation may have functional relevance to atherogenesis. Whether lipid peroxidation initiates the early atherosclerotic lesion or coincides with the disease remains a matter of some conjecture. A recent study examining the formation of lipid and protein oxidation products in human atherosclerosis development found that the accumulation of nonoxidized lipid precedes that of oxidized lipid in human aortic lesions (12). In the hyperlipidemic rabbit, atherogenesis has been dissociated from the accumulation of lipid hydroperoxides in aortic tissue (45), and, in a cholesterol-fed rabbit model, aortic hydroxyoctadecadienoic acids and oxysterols did not reflect tissue severity of disease (46). Witting et al (47) treated Apoe-/- mice with a lipid-soluble antioxidant, probucol, and noted a dissociation between inhibition of disease and inhibition of aortic lipoprotein lipid peroxidation. In our study, lipid deposition was independent of lipid peroxidation, as measured by the formation of fatty acid oxidation products. Whereas the concentrations of fatty acid oxidation products were similar in all groups, it was only the Apoe-/- mice that had significant lipid deposition. Therefore, oxidative processes did not appear to contribute to the uptake of lipid into the arterial wall. Our results do not rule out the possibility that fatty acid oxidation products may play an important secondary role in atherosclerosis development. In agreement with our observations, the study by Stocker and O'Halloran (48), using a different approach, showed that red wine polyphenols decrease atherosclerosis without inhibiting lipoprotein oxidation in the vessel wall.
Some epidemiologic studies suggested a protective influence of red wine in reducing the risk of coronary artery disease (49, 50). Red wine polyphenols protect LDL from oxidation in vitro. However, this has not been a consistent finding: the acute consumption of red wine or dealcoholized red wine showed variable effects on the ex vivo oxidation of LDL in humans (51-53). The results of studies involving animal models of atherosclerosis and red wine intervention also are inconclusive (28, 30, 54, 55). Previous studies investigating the action of red wine on atherogenesis in the Apoe-/- mouse did not examine specific markers of oxidative stress (28, 30).
Hayek et al (28) observed less progression of atherosclerosis in Apoe-/- mice after the consumption of red wine, and they attributed this change to the reduced susceptibility of LDL to ex vivo oxidation and aggregation. Other studies investigating the development of atherosclerosis in this model observed increased expression of MCP-1, especially in early development of the disease (56, 57). Feng et al (58) showed that red wine inhibited MCP-1 expression in rabbits fed a high-fat, high-cholesterol diet. In the present study, we also observed that, in the initial stages of disease progression, the 2 Apoe-/- mouse groups had significantly higher concentrations of plasma MCP-1 than did the control mice, although the red wine polyphenols had no effect on plasma MCP-1 concentrations in the Apoe-/- mice. In addition, MCP-1 concentrations increased with age in the control C57BL/6J mice despite the absence of an increase in atherosclerosis, which raises the question of the significance of plasma MCP-1 to disease progression in this model. It is also possible that there may be alternative actions for red wine polyphenols, such as the inhibition of smooth muscle cell proliferation (59) or the regulation of adhesion molecules (60-62), which may influence atherosclerotic progression.
In conclusion, we showed a time-dependent increase in the formation of arachidonic acid oxidation products, HETEs, in Apoe-/- mice and control C57BL/6J mice fed the same high-fat, high-cholesterol diet. It is interesting that we found increased aortic lipid deposition only in the Apoe-/- mice, which suggested that uptake of lipid into the arterial wall was not dependent on lipid peroxidation. The lack of effect of red wine polyphenols on biomarkers of oxidative stress in Apoe-/- mice suggests that the ability of polyphenols to reduce aortic lipid deposition may be independent of the inhibition of lipid peroxidation in this animal model.
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ACKNOWLEDGMENTS |
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EW was responsible for data collection and analysis and for drafting the manuscript. IBP was responsible for study design, statistical analysis, and intellectual input into the manuscript. KDC was responsible for study design, project supervision, and intellectual input into the manuscript. None of the authors had a conflict of interest.
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), and Apoe-/- mice given water only (
). n = 4–5 animals at each time point. There was a significant treatment x time interaction by generalized linear modeling, P < 0.01. Groups with different letters at 26 wk were significantly different, P < 0.02 (ANOVA with Bonferroni-adjusted post hoc comparison).
