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* Department of Pathology/Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27157 and Bell Institute of Health and Nutrition, General Mills Company, Minneapolis, MN, 55440
2 To whom correspondence should be addressed. E-mail: madams{at}wfubmc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vegetables • atherosclerosis • inflammation • mice • lipoproteins
The 2005 Report of the Dietary Guidelines Advisory Committee to the Department of Health and Human Services and the United States Department of Agriculture concluded that increased fruit and vegetable intake is associated with a reduced risk of some cardiovascular diseases (1). In agreement with this conclusion, a review of prospective observational studies (2) found that the relative risk of cardiovascular disease in individuals with the highest intake of fruits and vegetables was 0.82 (95% CI = 0.76–0.89) compared with those with the lowest intake. Furthermore, a meta-analysis that combined the results of 11 prospective cohort studies found that individuals in the 90th percentile of fruit or vegetable intake (
5 servings/d) had a risk of myocardial infarction that was 15% lower than those in the 10th percentile of intake (3). In the largest and longest study to date, the dietary habits of 110,000 men and women were followed for 14 y. In this cohort, a higher average daily intake of fruits and vegetables was associated with a lower risk of developing coronary heart disease. Compared with those in the lowest category of fruit and vegetable intake (<1.5 servings/d), those who consumed a mean of 
8 servings/d were 20% less likely to experience myocardial infarction (4). However, 2 other recent observational studies found no relation between the intake of fruits and vegetables and coronary risk (5,6).
The relation between fruit and vegetable intake and risk factors for cardiovascular disease has also been investigated. In the Dietary Approaches to Stop Hypertension study (7), increased consumption of fruits and vegetables was associated with a reduction in blood pressure. The Women's Health Initiative Trial assessed cardiovascular disease outcome following behavior modification to increase fruit and vegetable intake along with a reduction of total fat intake (8). Although there were no significant reductions in the risk of coronary heart disease, stroke, or overall cardiovascular disease, the intake of fruits and vegetables had increased by only 1.1 servings/d in this cohort of postmenopausal women, who were followed for 8.1 y. Improvement in plasma lipoprotein profiles associated with increased fruit and vegetable consumption may contribute to a reduction in cardiovascular risk. In the Family Heart Study, 4466 subjects consumed a mean of 3 servings of fruits and vegetables/d. Men and women with the highest daily consumption (>4 servings/d) had modestly lower (<10%) levels of LDL cholesterol than those with a lower consumption (9). A reduction of this magnitude may contribute to the reduced coronary risk associated with increased vegetable consumption.
In an extensive review of literature aimed at identifying dietary strategies for prevention of coronary heart disease, Hu and Willett (10) identified a "diet high in fruits, vegetables, nuts, and whole grains" as 1 of 3 effective strategies. Relative to this finding, these authors also concluded that areas that remain unsettled include the effects of individual phytochemicals, antioxidant vitamins, and minerals contained in these food items. Nonetheless, despite compelling evidence supporting the health benefits of increasing vegetable consumption, the intake of vegetables by Americans remains low, with a mean consumption of 3.2 servings/d for people >2 y. Furthermore, 40% of this intake is contributed by starchy vegetables (mostly fried potatoes). In contrast, the 2005 Dietary Guidelines (1) recommends 5 servings of vegetables/d based on a 500 kJ intake.
The underlying cause of coronary heart disease is the development of atherosclerotic plaques in coronary arteries. Most myocardial infarctions are caused by a plaque rupture, the formation of an occlusive thrombus, and the loss of blood flow to the myocardium. Although a few studies have investigated the effects of single vegetables on cholesterol metabolism and antioxidant status (11–13), or oxidative stress and inflammatory activity in the cardiovascular system (14) of rodents, to our knowledge, the effect of increased vegetable consumption on the development or progression of atherosclerosis has not been addressed. Therefore, our goal was to determine the effect of a diet rich in a mixture of the 5 most commonly consumed green and yellow vegetables in the United States on the development of atherosclerosis in a mouse model. Concentrations of serum amyloid A (SAA), an acute-phase protein used as a nonspecific index of inflammatory activity in mice, were also assessed.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The control diet was a vegetable-free semipurified diet (Table 1). The vegetable diet (Table 1) was 30% (w/w) an equal-parts mixture of freeze-dried broccoli, peas, green beans, corn and carrots (Table 2) (Green Giant frozen vegetables, General Mills, Minneapolis, MN). The vegetables selected represent 5 of the top 10 vegetables consumed based on frequency of intake in the total U.S. population and collectively represent nearly 40% of all vegetable intake, excluding potatoes (18). To balance nutrient and fiber content between the diets, protein, carbohydrate, fat and fiber constituents of the control diet were reduced by the amount of the corresponding nutrient contained in the vegetable mix (Table 1). Each diet contained the same amount of energy with 20% of kJ as protein, 16% as fat, 64% as carbohydrate, and 10% as fiber (w/w). Body weights were determined at 4-wk intervals.
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Atherosclerosis and plasma lipoproteins. Plasma lipoproteins were separated by HPLC (19) and aliquots of isolated lipoprotein fractions were used for enzymatic determination of cholesterol (20).
The analysis for aortic free and esterified cholesterol content was conducted as described previously (21). The aorta was placed on the platform of a dissecting microscope and the adventitia was carefully and completely dissected away from the intima/media and removed. The intima/media was then placed in 3 mL of chloroform:methanol (2:1, v:v) containing 5 
-cholestane as an internal standard, and the lipids were extracted. The lipid extract was separated by filtration and extracts were dried under N2 at 60°C and then dissolved in hexane. Analysis of free and total cholesterol was performed with 2 injections per sample on a DB 17 (0.53 mm i.d. x 15 m x 1 µm) gas-liquid chromatography column at 250°C and installed in a Hewlett Packard 5890 gas chromatograph equipped with a Hewlett Packard 7673A automatic injector and an on-column injection and a flame ionization detector. Cholesteryl ester was calculated as the difference between free and total cholesterol, as measured before and after saponification and reextraction of the nonsaponifiable sterol into hexane. The delipidated tissue protein was then digested and dissolved in 1 mol/L NaOH and total protein determined (22). We used µg esterified cholesterol/mg protein as the primary measure of atherosclerosis extent.
SAA. SAA concentrations were measured using enzyme-linked immunosorbent assay (Biosource International).
Data analysis. Data sets that were not normally distributed underwent logarithmic transformation before analysis. Pairwise comparisons were made using unpaired Student's t test. Simple associations between variables were assessed using Pearson's product moment correlation. Multiple linear regression was used to assess the relation among plasma lipoproteins, body weight, and atherosclerosis. Differences with P < 0.05 were considered significant. Values are presented as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weight. Body weights were lower (P < 0.05) at weeks 8, 12, and 16 in mice fed the vegetable-rich diet (data not shown). The difference between groups was greatest at week 16 when body weights were 7% lower.
Regression analyses. Multiple linear regression was used to determine the extent to which differences in body weight and plasma lipoproteins predicted atherosclerosis extent. Each variable was used alone and in multiple combinations. In all cases adjusted means were minimally different from actual means and a main effect of diet persisted (P < 0.01). The single best model included all variables. In this model, adjusted mean atherosclerosis extent of the vegetable-rich diet group was 29% (P < 0.01) (Fig 1) lower than the control group.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma total cholesterol, VLDL + ILDL cholesterol, and to a lesser extent LDL cholesterol, were reduced in mice fed the vegetable-rich diet. The mechanism by which these reductions were achieved remains uncertain. However, in a multiple regression model, neither body weight nor lipoprotein variables contributed substantially to the observed reduction in atherosclerosis extent. This suggests that factors other than plasma lipoproteins may be involved. The 37% reduction in SAA (an acute phase protein and nonspecific marker of inflammatory activity in mice) indicates that vegetable consumption inhibited inflammatory activity. In this regard, it is well known that atherosclerosis progression is intimately linked with pro-oxidant and proinflammatory activity in the arterial intima. Watzl et al. (23) assessed the effect of carotenoid-rich fruit and vegetables on indices of immune function and plasma C-reactive protein (an acute phase protein frequently used as a nonspecific index of inflammatory activity in human beings) concentrations in 64 healthy men. Men consuming 8 servings of fruits and vegetables/d for 8 wk had a reduction in plasma C-reactive protein compared with men consuming 2 servings/d. In another study (14), the consumption of 200 mg of broccoli sprouts/d for 14 wk resulted in decreased oxidative stress and inflammation in the heart and major arteries of spontaneously hypertensive stroke-prone rats. Aortic endothelial function was also improved in these rats. Glucoraphanin, a precursor of the phase-2 protein inducer, sulforaphane, which is found in broccoli sprouts, was implicated in mediating these effects. It has also been found that while the consumption of a diet containing 15 or 20% lyophilized carrots, or 20% lettuce, results in a reduction in plasma and hepatic concentrations of cholesterol and triglycerides and increased fecal neutral and total sterol excretion, it also results in an improvement in antioxidant status of the plasma of mice or rats fed atherogenic diets (11–13). Taken together, these results further support the idea that increased vegetable consumption inhibits atherosclerosis progression through antioxidant and anti-inflammatory pathways. Although the constituents of green or yellow vegetables responsible for these effects are not completely clear, there are numerous candidates. Among these are phase-2 protein inducers, such as sulforaphane. Furthermore, carotenoids, vitamin C, vitamin E, and selenium are all potent antioxidants. In addition, substantial evidence indicates that polyphenols found ubiquitously in plants possess antioxidant and anti-inflammatory properties (reviewed in 24). These and other constituents may act additively or synergistically to inhibit the initiation and progression of atherosclerosis.
As with all animal models of human disease, the LDL receptor –/–, apolipoprotein B transgenic mouse has limitations as a model of atherosclerosis. Notably, with rare exceptions, human beings have LDL receptors whereas these mice do not. The cardiovascular effects of increased vegetable intake should be studied further in other models and human beings. However, the usefulness of this mouse model in atherosclerosis research is derived from the fact that it overproduces atherogenic lipoproteins and has a limited capacity to catabolize them. These same metabolic factors are responsible for most types of hyperlipoproteinemia seen in human beings. Elevations in plasma LDL are frequently the result of a marked reduction in expression of LDL or other lipoprotein receptors (and consequently reduced catabolism of LDL) or overproduction of apoB-containing lipoproteins. Therefore, this model and these results may have particular applicability to individuals with this metabolic pattern and its related increase in coronary risk.
Although the pathway(s) involved remain unclear, we conclude that the consumption of a diet containing 30% green and yellow vegetables results in a substantial inhibition of atherosclerosis progression in a mouse model of atherosclerosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 16 March 2006. Initial review completed 22 March 2006. Revision accepted 7 April 2006.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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