Prevention of Atherosclerosis by Berries â the ... - ACS Publications

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Cite This: J. Agric. Food Chem. 2018, 66, 9172−9188

Prevention of Atherosclerosis by Berries: The Case of Blueberries Xianli Wu,*,† Thomas T. Y. Wang,‡ Ronald L. Prior,§ and Pamela R. Pehrsson† Nutrient Data Laboratory and ‡Diet, Genomics and Immunology Laboratory, USDA−ARS Beltsville Human Nutrition Research Center, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States § Department of Food Science, University of Arkansas, Fayetteville, Arkansas 72704, United States

J. Agric. Food Chem. 2018.66:9172-9188. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/11/18. For personal use only.

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ABSTRACT: Berry consumption has been associated with cardiovascular disease prevention in recent years. Atherosclerosis is one of the major causes of cardiovascular diseases. However, research on the prevention of atherosclerosis through consuming individual whole berries, specifically direct evidence, remains scarce. Therefore, further elucidating the role that berries play in the prevention of atherosclerosis is warranted. In this perspective, blueberries were selected to articulate research strategies for studying atheroprotective effects of berries. Studies from human subjects and various animal models are summarized. The mechanisms by which blueberries may act, through reducing oxidative stress, decreasing inflammation, improving endothelial dysfunction, regulating cholesterol accumulation and trafficking, along with potentially influencing gut microbiota, are also discussed. Blueberries contain high levels of polyphenolic compounds, which were widely indicated as major bioactive compounds. Nonetheless, the metabolites/catabolites after blueberry consumption, such as simple phenolic acids, rather than original compounds in berries, may be the actual in vivo bioactive compounds. Future research should focus on obtaining more direct evidence, preferably in humans, understanding of the mechanisms of action at the molecular level, and identifying bioactive compounds as well as which compounds act synergistically to convey health benefits. The research strategy discussed here may also be applied to the studies of other fruits and berries. KEYWORDS: blueberries, atherosclerosis, oxidative stress, inflammation, endothelial function, cholesterol, bioactive compounds, gut microbiota

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INTRODUCTION

were found to contain an extremely high level of polyphenols.13,14 Growing evidence shows berries and/or berry polyphenols may prevent CVD through various mechanisms.15−19 However, research on the prevention of atherosclerosis by individual whole berries, especially the direct evidence of molecular mechanisms and in vivo bioactive compounds, is still scarce. A majority of the existing studies are based on in vitro and animal studies without disease outcomes.13 In addition, although collectively referred to as berries, it is important to point out that each individual berry fruit could be very different from the others in terms of their nutrient and/or phytochemical profiles, which may further determine their potential beneficial or even detrimental effects in the human body and the underlying mechanisms. Thus far, most research on the prevention of atherosclerosis by individual berries has focused on several commonly consumed berries, such as pomegranates,20 blueberries,21 strawberries,22 and açai ́ (Euterpe oleracea Mart.).23 They all showed preventive effects and, to some extent, shared similar mechanisms, such as antioxidant and anti-inflammatory activities. Blueberries are the focus of this perspective to articulate research strategies for studying atheroprotective effects of berries. The aim is to summarize the current knowledge on the preventive effects of blueberries on atherosclerosis and possible molecular mechanisms. The potential in vivo bioactive compounds, mainly polyphenols and their in vivo catabolites,

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous plaques in the large arteries, which leads to hardening and narrowing of the arteries. It is the major cause of heart attacks, strokes, and peripheral vascular disease, which together are known as “cardiovascular diseases (CVDs)”. CVDs are the number one cause of death globally, with more people dying annually from CVDs than from any other causes. An estimated 17.3 million people died from CVDs in 2008, representing 30% of all global deaths.1 CVDs are projected to remain the number one leading cause of death in the foreseeable future.2 CVD is a complex condition resulting from the interplay between genetic and environmental factors. Among the modifiable environmental factors, diet and lifestyle are largely responsible for increased risk of CVDs at population levels in recent years.3 Diet plays an important role in the development and prevention of CVDs.4 On the other hand, certain diet models, such as the Mediterranean diet, have shown promising preventive effects against CVDs.5,6 The traditional Mediterranean diet is characterized by a high intake of fruit and vegetables. Numerous epidemiological studies and experimental studies have linked a diet rich in fruits and vegetables to the lower risk of CVDs.7−9 Fruits and vegetables contain various nutrients and nonnutrient phytochemicals. Nutrients such as dietary fiber, vitamins, and minerals could be beneficial. However, the evidence accumulated in recent years have suggested that certain phytochemicals, polyphenolic compounds in particular, may be largely responsible for their cardioprotective effects.10−12 Among all fruits and vegetables, dark color berries © 2018 American Chemical Society

Received: Revised: Accepted: Published: 9172

June 19, 2018 August 9, 2018 August 9, 2018 August 9, 2018 DOI: 10.1021/acs.jafc.8b03201 J. Agric. Food Chem. 2018, 66, 9172−9188

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Journal of Agricultural and Food Chemistry

Figure 1. Representative chemical structures of five major groups of polyphenolic compounds in blueberries.

provinces westward to Quebec in Canada and southward to Michigan and West Virginia in the U.S. The berries of V. angustifolium are smaller than cultivated highbush blueberries. In the U.S., most of the production of blueberries comes from high- and lowbush types.24 Consumption of blueberries has increased rapidly in the past decade, largely driven by health claims. Currently, blueberry is among the most commonly consumed berries in the U.S. In addition to being consumed as raw fruits, blueberries are also used in jellies, jams, and pies, baked into muffins, and are an ingredient of many other snacks and delicacies. Blueberry juice is considered a fast growing category of juice in consumer stores. According to a comparison of commonly consumed polyphenol-rich beverages in the U.S., blueberry juice was ranked in the top 4 after pomegranate juice, red wine, and concord grape juice.25 Major Nutrients in Blueberries. In general, fruits and berries are major contributors of vitamins, dietary fiber, and certain minerals in the human diet.26 According to National Nutrient Database for Standard Reference Legacy Release,27 raw blueberries (nutrient profiles combining high- and lowbush blueberries) provide 7.5 g of sugar (3.7 g of glucose and 3.7 g of fructose), 1.8 g of dietary fiber, 7.3 mg of vitamin C [12% daily value (DV)], 14.5 μg of vitamin K (36% DV), and 0.25 mg of manganese (25% DV) per serving size (1/2 cup or 75 g). Major Polyphenolic Compounds in Blueberries. Anthocyanins. Anthocyanins are water-soluble plant secondary metabolites responsible for the blue, purple, and red colors of many plant tissues. They occur primarily as glycosides of their respective aglycone anthocyanidin chromophores (Figure 1). Different blueberry cultivars were found to contain 12−27 anthocyanins, which included five or six of the most common aglycones: cyanidin, delphinidin, peonidin, petunidin,

are further discussed. In addition, a general research strategy in conducting similar studies with other berries or dietary factors is also discussed. PubMed and Google Scholar were selected as primary search engines for the literature review. The following keywords were used in different combinations: blueberries, blueberry, atherosclerosis, cardiovascular disease, oxidative stress, inflammation, polyphenol, anthocyanin, phenolic acid, endothelial function, gut microbiota, and cholesterol. From a nutritional standpoint and considering the whole food approach, only the studies using whole blueberries, either fresh or dried blueberry powders, and 100% blueberry juice were included. No in vitro studies, unless those using physiologically relevant compounds/mixture as test materials, were included as a result of the lack of biological relevance. Because the majority of the studies on atheroprotective effects of blueberries have been focused on high- or lowbush blueberries, the efforts are restricted on the data from these two main species. To avoid confusion, studies with no specification of the blueberry species were excluded.

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BLUEBERRIES AND MAJOR NUTRIENTS AND POLYPHENOLS American Blueberries. The blueberry of the genus Vaccinium is a native American species. Blueberries may be cultivated or picked from semi-wild or wild bushes. In North America, the predominant cultivated species is Vaccinium corymbosum (highbush blueberry), which grow all over the United States. Hybrids of this with other Vaccinium species adapted to southern U.S. climates are known collectively as southern highbush blueberries, of which Vaccinium ashei (southern rabbiteye blueberry) is the most common species. Vaccinium angustifolium is commonly known as “wild” or lowbush blueberry. It is a species found from the Atlantic 9173

DOI: 10.1021/acs.jafc.8b03201 J. Agric. Food Chem. 2018, 66, 9172−9188

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Figure 2. Illustration of the early events in the pathogenesis of atherosclerosis. The initial event in atherogenesis is the increased transport of LDL across endothelium into intima, where it is subjected to oxidative modifications to form ox-LDL. This infiltration occurs preferentially at sites where the endothelium becomes dysfunctional. Upregulation of adhesion molecules leads to the attachment and rolling of immune cells (e.g., monocytes) on the endothelial wall. After adhesion to the endothelial surface, monocytes undergo direct migration into the artery wall mediated by chemokines, such as MCP-1. Once resident in the arterial intima, these recruited monocytes, upon exposure to comitogenic mediators, such as M-CSF, differentiate into macrophages. Macrophages are able to take up and degrade ox-LDL through overexpressed scavenger receptors, such as SR-A and CD36. Meanwhile, accumulated cholesterol can be removed from macrophages by cholesterol efflux through various ATP-binding membrane cassette transporters (e.g., ABCA1). Increasing cellular cholesterol leads to the formation of lipid-laden foam cells, the most significant early hallmark of atherosclerosis. Foam cells could produce more proinflammatory cytokines and ROS oxidative stress, which could further accelerate the atherosclerosis process.

stilbenes, pterostilbene and piceatannol, were found in rabbiteye and highbush blueberries, respectively36 (Figure 1).

and malvidin. The lowbush blueberry was found to contain 27 anthocyanins, and the highbush blueberry contained 25 anthocyanins, whereas only 12 anthocyanins were detected in the rabbiteye blueberry.28−30 Proanthocyanidins. Proanthocyanidins represent a group of condensed flavan-3-ols. (+)-Catechin and (−)-epicatechin are the basic monomeric units of this group for blueberry (Figure 1). Proanthocyanidins are usually divided into A and B types. For the B type, the monomeric units are mainly linked through C4 → C8 or sometimes C4 → C6 bonds. Only B-type proanthocyanidins were found in blueberries.31 Phenolic Acids. Phenolic acids are another group of important phenolic compounds in blueberries. They share a phenolic ring and an organic carboxylic acid group (Figure 1). The chlorogenic acid is the principle phenolic acid in all of the most common cultivated Vaccinium species, and indeed, it is the most abundant single phytochemical in blueberries. Chlorogenic acid makes up over 99% of total phenolic acids in lowbush blueberries and 74.38% of total phenolic acids in highbush blueberries.32 Other Flavonoids. Except for anthocyanins and flavan-3-ols, flavonols and their glycosides were found in blueberries. There are three types of aglycones reported, namely, kaempferol, quercetin, and myricetin33 (Figure 1). The only differences of these compounds are the numbers of hydroxyl groups on ring B. The sugar chains of flavonol glycosides usually include glucose, galactose, rhamnose, arabinose, and rutinose.33−35 Stilbenes. Resveratrol, pterosilbene, and piceatannol are naturally occurring compounds considered as derivatives of stilbenes, which is an ethane double bond substituted with a phenyl group on both carbon atoms of the bond. Resveratrol, which was found in considerable quantities in grapes, was detected in all three blueberry varieties (highbush, lowbush, and rabbiteye blueberries) at a low content. The other two

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EARLY EVENTS IN THE PATHOGENESIS OF ATHEROSCLEROSIS AND STRATEGIES OF PRIMARY PREVENTION BY DIETARY FACTORS Atherosclerosis is a multifactorial disease with different stages of development. Before the preventive effects of blueberries are discussed, it is helpful to provide a brief overview on the early events of the initiation and progression of atherosclerosis. Atherosclerosis is primarily a disease of the intima of arteries, the innermost layer of blood vessels that lies between the endothelial cells and the smooth muscle cells of the media. Atherosclerosis was traditionally viewed as a set of lipid disorders. However, it is now widely recognized as an inflammatory disease,37,38 and oxidative stress plays a key role in the initiation and propagation of the disease.39,40 The initial event in atherogenesis is the increased transport of lowdensity lipoprotein (LDL) across endothelium into intima, where it is subjected to oxidative modifications to form highly oxidized and aggregated LDL, referred to as oxidized LDL (oxLDL). This infiltration occurs preferentially at sites with disturbed flow dynamics, where the endothelium becomes dysfunctional at the cellular and molecular level. Activated by the disturbed flow, the endothelium shows enhanced platelet, increased endothelial permeability to LDL, and increased gene expression of adhesion molecules and chemotactic factors. Upregulation of adhesion molecules, such as intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, leads to the attachment and rolling of immune cells (e.g., monocyte and T lymphocyte) on the endothelial wall. After adhesion to the endothelial surface, monocytes undergo direct migration into the artery wall mediated by chemokines, such as monocyte 9174


9175

fresh highbush blueberries, 300 g one serving

randomized, controlled, crossover trial, separated by 1 week washout period randomized, controlled, crossover trial, separated by 1 week washout period randomized controlled trial

young male subjects

male smokers

young male smokers and non-smokers

randomized, crossover trial

300 g of fresh highbush blueberries, single dose

a single serving of 300 g of fresh-frozen highbush blueberries

fresh highbush blueberries, 300 g one serving

25 g of freeze-dried wild blueberry powder in a drink, 6 weeks

randomized, crossover trial

randomized controlled trial

obese males and females with metabolic syndrome male volunteers with cardiovascular risk factor young male smokers and non-smokers

yogurt- and skim-milk-based smoothie with 22.5 g of freeze-dried highbush blueberry powder for 6 weeks yogurt- and skim-milk-based smoothie with 22.5 g of freeze-dried highbush blueberry powder for 6 weeks 50 g of freeze-dried highbush blueberries, 8 weeks

randomized, double-blind, placebo-controlled trial


100% wild blueberry juice, 240 mL, 7 day treatment separated by an 8 day washout period

randomized, placebo-controlled, crossover trial

adults with metabolic syndrome

freeze-dried highbush blueberry powder, 22 g for 8 weeks


treatment freeze-dried highbush blueberry powder, 22 g for 8 weeks

study design


human subject

healthy postmenopausal women with pre- and stage 1 hypertension healthy postmenopausal women with pre- and stage 1 hypertension men and women adults with increased risk for type 2 diabetes adults with metabolic syndrome

outcome/disease marker

reduced H2O2-induced DNA damage in cell model but no effects on endogenous DNA damage, peripheral function, and NO levels

blueberry consumption counteracted the impairment of the RHI induced by smoking and Framingham reactive hyperemia and increase of systolic blood pressure after smoking

RHI was higher and after blueberry treatment for non-smokers; acute cigarette smoke significantly increased blood pressure and heart rate; blueberry and control treatment significantly improved peripheral arterial function; and no difference between the two treatments no significant effect of treatment was observed for markers of oxidative stress and antioxidant defense

reduced both endogenous oxidized DNA bases and H2O2-induced DNA damage and no effects on endothelial function and inflammatory markers

systolic and diastolic blood pressures decreased, plasma ox-LDL and MDA decreased, and no effects on serum glucose concentration and lipid profiles

the RHI was improved significantly but no changes for blood pressure and insulin sensitivity

a trend for lowering systolic blood pressure, increased serum concentrations of nitrates and nitrites, and no changes in glucose, insulin, insulin sensitivity, triglycerides, inflammatory markers, adhesion molecules, oxidative stress, endothelial function, or blood pressure blueberry treatment decreased superoxide and total ROS in whole blood and monocytes, decreased monocyte gene expression of TNF-α, IL-6, TLR4, reduced serum GMCSF, and increased myeloid dendritic cell

8-OHdG levels were significantly lower in the blueberry group compared to the control group at 4 weeks, and other biomarkers of oxidative stress, inflammation, and antioxidant defense were not affected by blueberry consumption

systolic blood pressure and diastolic blood pressure and brachial-ankle pulse wave velocity were significantly lower than baseline levels, and nitric oxide levels were greater in the blueberry powder

Table 1. Evidence of Atheroprotective Effects of Blueberries from Human Studies conclusion

blueberries improved oxidative stress and endothelial dysfunction caused by smoking blueberries improved cell antioxidant defense

single blueberry portion did not modulate markers of oxidative stress and antioxidant

single-dose blueberry consumption may not render protection on endothelial function to smokers

blueberries increased the resistance to oxidative damages

blueberries reduced blood pressure and markers of oxidative stress

blueberries improved endothelial function but did not improve blood pressure

blueberry juice promoted cardioprotective effects by improving systolic blood pressure, possibly through nitric oxide production blueberries exerted immunomodulatory effects and attenuate oxidative stress and inflammation

blueberries provided modest protection against oxidative DNA damage in a high-risk population

blueberries lowered blood pressure and arterial stiffness, which may be due to increased nitric oxide production

reference

Del Bò et al.57

Del Bò et al.52

Del Bò et al.56

Del Bò et al.55

Riso et al.48

Basu et al.51

Stull et al.54

Nair et al.47

Stote et al.53

Johnson et al.46

Johnson et al.50

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dated.21,24 In this section, atheroprotective effects of blueberries from human or animal studies are summarized. Evidence from Human Studies. Human studies specifically on the cardioprotective effects of blueberries are emerging. Nonetheless, there is still no direct evidence in human subjects. Published studies primarily focused on certain CVD risk factors/biomarkers or in vivo antioxidant and antiinflammatory effects (Table 1). The human subjects were usually selected who had a high cardiovascular risk, such as hypertension, metabolic syndrome (MetS), diabetes, or smokers. With regard to the experimental design, most studies were randomized controlled trials. Of the 11 studies summarized, 9 studies tested the effects of highbush blueberries and only 2 used wild blueberries. In vivo antioxidant and anti-inflammatory effects of blueberries were demonstrated in several recent human studies. Consumption of freeze-dried highbush blueberries for 4 weeks was found to reduce 8-hydroxy-2′-deoxyguanosine (8-OHdG), a blood biomarker of oxidative DNA damage, in postmenopausal women with pre- and stage 1 hypertension.46 In another study, adults with metabolic syndrome consumed freeze-dried highbush blueberry in a smoothie for 6 weeks. Blueberry treatment decreased superoxide and total reactive oxygen species (ROS) in whole blood and monocytes. The monocyte gene expressions of tumor necrosis factor α (TNF-α), interleukin (IL)-6, toll-like receptor 4 (TLR4), and serum granulocyte−macrophage colony-stimulating factor (GMCSF) were also significantly reduced in the blueberry treatment group compared to the placebo.47 In male volunteers with cardiovascular risk factor, wild blueberry drink intake for 6 weeks significantly reduced the levels of endogenously oxidized DNA bases and the levels of H2O2induced DNA damage.48 Hypertension is a risk factor for the development of atherosclerosis.49 Consuming 22 g of freeze-dried blueberries for 8 weeks was shown to lower systolic blood pressure and diastolic blood pressure in healthy postmenopausal women with pre- and stage 1 hypertension.50 Similar results were also observed in another study, in which both systolic and diastolic blood pressures decreased in obese men and women with MetS after consuming 50 g of freeze-dried blueberries daily for 8 weeks.51 Smoking significantly increases the systolic blood pressure. Blueberry treatment (300 g of fresh blueberries in a single dose) was found to counteract the increased systolic blood pressure in healthy male smokers.52 However, the hypotensive effects of blueberries were not observed in another two studies.53,54 One interesting difference between these two studies and the three studies mentioned above is that none of these two studies used fresh or freeze-dried blueberries. One of them used 100% wild blueberry juice, and the other one used a yogurt- and skim-milk-based smoothie with freeze-dried blueberry powder. Blueberry consumption has also been shown to reduce brachial-ankle pulse wave velocity in healthy postmenopausal women with pre- and stage 1 hypertension,50 and decreased plasma ox-LDL in obese men and women with MetS.51 Blueberries have also been shown to alter a number of markers related to oxidative stress, antioxidant defense, chronic inflammation, and endothelial dysfunction. Nevertheless, these should be considered as mechanisms rather than evidence and, therefore, will be discussed in a later section. It is worth pointing out that of the 11 studies summarized, 4 studies are acute single-dose studies.52,55−57 Results from these

chemoattractant protein 1 (MCP-1). Once resident in the arterial intima, these recruited monocytes, upon exposure to comitogenic mediators, such as macrophage colony-stimulating factor (M-CSF), differentiate into macrophages. Macrophages are able to take up and degrade ox-LDL through overexpressed scavenger receptors, including type A scavenger receptor (SRA), a cluster of differentiation 36 (CD36), and lectin-like oxLDL receptor (LOX-1).41 Unlike the classical low-density lipoprotein receptor (LDLR), which is downregulated by increasing cellular cholesterol levels, the ability of scavenger receptors to take up modified LDL is not inhibited by increasing cellular cholesterol. This leads to the appearance of macrophage-derived foam cells, whose cytoplasm is swollen with lipid droplets. Lipid-laden foam cell formation is considered as the most significant early hallmark of atherosclerosis, which further leads to the formation of the fatty streak, the earliest visible lesion of atherosclerosis42 (Figure 2). The development of atherosclerosis is a life-long process, beginning in early childhood.43 Multiple risk factors contribute to the development of atherosclerosis, and they can be classified as modifiable and non-modifiable risk factors. Modifiable risk factors include hyperglycemia, type 2 diabetes, hyperlipidemia [specifically high LDL and very low-density lipoprotein or low high-density lipoprotein (HDL) or a high LDL/HDL ratio], cigarette smoking, sedentary lifestyle, hypertension, and vitamin B6 deficiency. Non-modifiable risk factors include increasing age, gender (males are at a higher risk), and genetics (familial hypercholesteremia). What these risk factors all have in common is that they all promote inflammation and oxidative stress, key components of the development and progression of atherosclerosis.44 There are a number of effective strategies for the prevention of atherosclerosis. These can be done through changes in lifestyle or via pharmacological treatment. For primary prevention of atherosclerosis in early life, lifestyle modifications, mainly nutritional intervention without pharmacological treatment, would be the optimal strategy.45 On the basis of the understanding of pathogenesis of atherosclerosis, the principal strategies for primary prevention of atherosclerosis by dietary factors may include (1) lowering LDL by reducing cholesterol intake and improving in vivo cholesterol homeostasis, (2) reducing LDL oxidation through decreasing LDL subendothelial retention, lowering systematic oxidative stress, and inhibiting oxidative enzymes, (3) enhancing LDL or cholesterol efflux from intima or enhancing LDL/cholesterol degradation, leading to reduced lipid accumulation, (4) slowing inflammatory processes on multiple targets, including inhibiting expression of adhesion molecules, chemotactic proteins, and other cytokines or chemokines, (5) maintaining normal endothelial functions mainly through regulation of normal nitric oxide (NO) production and bioavailability, and (6) reducing other risk factors associated with atherosclerosis, including hypertension, smoking, insulin resistance, and obesity.

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ATHEROPREVENTIVE EFFECTS OF BLUEBERRIES: EVIDENCE FROM HUMAN AND ANIMAL STUDIES Blueberries have been implicated in preventing CVDs for many years. However, only recently were their atheroprotective effects demonstrated in animal and human studies and the underlying mechanism and bioactive components eluci9176


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this group were performed in the obese Zucker rat (OZR) model, which is considered as a valid experimental model for human MetS. 77 MetS is a multiplex risk factor for atherosclerotic cardiovascular disease.59 It was characterized by a cluster of risk factors, including central obesity, dyslipidemia, insulin resistance, glucose intolerance, hypertension, pro-oxidative, prothrombotic, and pro-inflammatory status. Their results in the OZR model showed antioxidant and anti-inflammatory effects of blueberries, including downregulating inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) pathways,73,76 reducing MCP-1, pro-inflammatory cytokines (TNF-α and IL-6), and C-reactive protein (CRP), and increasing adiponectin.75,76,78 They also found that blueberries improved lipid profiles and modulated the expression of key enzymes (e.g., fatty acid synthase) and transcription factors of lipid metabolism [peroxisome proliferator-activated receptor (PPAR)α, PPARγ, and SREBP1].72 In another study,74 they found that blueberries also normalized some markers related to glucose metabolism, including lowering plasma glycated hemoglobin HbA1c, RBP4, and resistin concentrations. Animal studies conducted by other research groups in several other models also supported the beneficial effects of blueberries related to cardiovascular diseases. Kalt et al.79 reported that, in male castrated pigs, blueberries beneficially affected plasma lipid status by decreasing total, LDL, and HDL cholesterol. In male spontaneously hypertensive stroke-prone rats (SHRSP), blueberries were found to slow the development of hypertension, at least partly, as a result of the antioxidant effects implicated by prevention of renal oxidative damage.80 In the same animal model, Elks et al.81 showed blueberries lowered mean arterial and systolic pressure. Coban et al.82 suggested in their study conducted in male Dankin Hartley guinea pigs that blueberries displayed anti-atherogenic effects by antioxidant and antilipidemic actions, which were shown to attenuate the increased cholesterol peroxidation markers induced by a high-cholesterol diet. Ahmet and colleagues published three studies on blueberries in preventing myocardial infarction and possible underlying mechanisms.83 They found that blueberries resulted in 22% less myocardial infarction than the control diet and increased myocardial tolerance to ischemic damage. The possible mechanisms were related to attenuating necro−apoptosis, inflammation, and oxidative stress. In another study, male Wistar rats fed 2% blueberries showed significant reductions in systolic blood pressure of 11 and 14% at weeks 8 and 10, respectively, relative to rats fed the control diet. Systolic blood pressure was reduced by 14% in rats fed with the high-fat diet plus 2% blueberry diet at week 10 compared to those on the high-fat diet. Furthermore, aortas harvested from blueberry-fed animals exhibited significantly reduced contractile responses (to Lphenylephrine) compared to those fed the control chow or high-fat diets. These suggested that blueberries may lower blood pressure and improve endothelial dysfunction induced by a high-fat- and high-cholesterol-containing diet.84

studies, although given the similar amount of blueberries (∼300 g of fresh blueberries), only showed marginal to no protective effects. These may indicate the possible accumulated effects of blueberries. The exact effects and underlying mechanisms warrant further investigations. Evidence from Animal Studies. Most animal studies published in the last 10 years on the atheroprotective effects of blueberries were conducted primarily on rodent models and focused on different aspects of their protective effects (Table 2). Of these animal studies, only one study actually examined the disease outcome (atherosclerotic lesions).58 The rest of them either focused on specific risk factors/biomarker or antioxidant and anti-inflammatory effects. Notably, with recognition of obesity and MetS as emerging risk factors,59,60 some of the most recent animal studies targeted the effects of blueberries against MetS and associated risk factors. The animal studies that provided direct evidence showing that blueberries could cause plaque in arteries to regress was published by Wu et al.58 In this paper, the apolipoprotein-Edeficient (apoE−/−) mouse model was used. This model has been widely used in cardiovascular research.61,62 ApoE deficiency in mice leads to the development of atherosclerotic lesions, resembling those in humans.63 The mice were fed either the control diet or control diet supplemented with 1% freeze-dried wild blueberries for 20 weeks. The plaques were measured at two sites on the aorta (arteries leading from the heart). The results showed that the lesion areas were 39 and 58% smaller in the mice fed blueberries compared to those fed the control diet. Notably, a series of 13 animal studies published by KlimisZacas and her colleagues in past the decade significantly advanced our knowledge on cardioprotective effects of wild blueberries.64−76 These studies were conducted in three different animal models and focused on different aspects of protective effects and/or risk factors. In their studies performed on weanling male Sprague Dawley (SD) rats, they found that (1) wild blueberries affect the vascular smooth muscle contractile machinery by suppressing the α1-adrenergic receptor agonist-mediated contraction, which may have implications in blood pressure regulation,71 (2) wild blueberries caused structural alterations in rat aortic tissue glycosaminoglycans (GAGs), which may affect cellular signal transduction pathways and the biological function of GAG molecules within the vascular environment,67 (3) dietary blueberries suppress the α1-adrenergic agonist-induced vasoconstriction via a NO-mediated pathway, suggesting that blueberries preserved NO bioavailability in the vasculature under basal and stimulated levels,65 and (4) blueberries positively affected vascular smooth muscle contractility and sensitivity, but these effects were evident only after 7 weeks of blueberry consumption, indicating the duration of exposure to the diet is crucial for wild blueberries and their bioactive components to exert their beneficial effects.64 Another animal model that this group used was the spontaneously hypertensive rat (SHR), which was used to investigate the effects of blueberries on vasoconstriction and vasorelaxation. They found that blueberries modified NO pathways of vasomotor control and improved the vascular tone,66 affecting the endotheliumdependent vasorelaxation by modulating the production/ activity of cyclooxygenase (COX)-derived products,69 enhancing the protective features of GAG molecules associated with endothelial dysfunction,70 and benefiting key signaling steps of NO and COX pathways.68 Several recent studies reported by

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MECHANISMS OF ATHEROPROTECTIVE EFFECTS OF BLUEBERRIES Blueberries have long been known to contain high levels of polyphenols 24 and appeared to show extremely high antioxidant capacities accessed by various in vitro assays.85,86 However, more recent studies suggested that the mechanisms of atheroprotective effects of blueberries may go beyond 9177


diet/dose

wild blueberry freeze-dried powder (8%, w/w) for 8 weeks wild blueberry freeze-dried powder (8%, w/w) for 8 weeks

wild blueberry freeze-dried powder (8%, w/w) for 8 weeks

wild blueberry freeze-dried powder (8%, w/w) for 4 or 7 weeks wild blueberry freeze-dried powder (8%, w/w) for 8 weeks


wild blueberry freeze-dried powder (8%, w/w) for 8 weeks wild blueberry freeze-dried powder (8%, w/w) for 4 or 7 weeks

OZR and LZR

OZR and LZR

OZR and LZR

9178

OZR and LZR

male SHRs

SD rat

OZR and LZR

OZR and LZR


wild blueberries freeze-dried powder (1%, w/w) for 20 weeks wild blueberry freeze-dried powder (8%, w/w) for 7 weeks wild blueberry freeze-dried powder (8%, w/w) for 7 weeks wild blueberry freeze-dried powder (8%, w/w) for 7 weeks wild blueberry freeze-dried powder (8%, w/w) for 7 weeks

weanling male SD rats

weanling male SD rats

young SHRs

adult SHRs

SHRs

apoE-deficient mouse

animal model

4 weeks of blueberry feeding only increased pD2 in the absence of L-NMMA; feeding blueberries significantly attenuated contraction in response to L-Phe and resulted in lower pD2; and 7 weeks of having the greater response inhibited NOS-induced increase in the constrictor response

blueberry consumption decreased TNF-α, IL-6, and CRP in plasma and downregulated TNF-α, IL-6, and NF-κB in the liver and abdominal adipose tissue of OZR, and blueberry diet also attenuated plasma NO, increased aortic effluent PGI2 concentration, and downregulated iNOS and COX-2 expression in the OZR aorta plasma lipid concentrations were significantly lower following blueberry consumption; in blueberry-fed rat group, PPARα and PPARγ in the OZR was increased in the abdominal adipose tissue (AAT), while that of SREBP-1 was decreased in the liver and AAT; and the expression of fatty acid synthase was significantly decreased in both the liver and AAT, and that of ABCA1 was increased in the AAT following blueberry consumption plasma glycated hemoglobin HbA1c, RBP4, and resistin concentrations were significantly lower in OZR following the blueberry diet; resistin expression was downregulated in the liver of both OZR and LZRs; and RBP4 expression was downregulated in the abdominal adipose tissue of both OZR and LZR blueberry diet partially restored Phe-induced constrictor responses and attenuated Ach-induced relaxant responses in OZR; plasma nitric oxide was significantly attenuated with the blueberry feeding; PGI2 in the aortic effluent concentration significantly increased; and blueberreis downregulated iNOS and COX-2 expression blueberry consumption resulted in decreased plasma concentrations of TNF-α, IL-6, and CRP and increased adiponectin concentration; expression of IL-6, TNF-α, and NF-κB was downregulated in both the liver and the abdominal adipose tissue, while CRP expression was downregulated only in the liver; and in the abdominal adipose tissue, similar trends were also observed in LZR following blueberry treatment blueberries were involved in primary aspects of GAG metabolism, such as population redistribution and sulfation in the SHR aorta

blueberry diet increased maximal constrictor force and adiponectin and decreased MCP-1 and IL-8 in perivascular adipose tissue

blueberries diminished vasoconstrictor response to Phe in aortic rings; the blueberry group showed greater response in increasing the constrictor response caused by inhibition of NO synthase and in the participation of the NO pathway in endothelium-dependent vasorelaxation induced by Ach; and the vessel sensitivity of aortic rings to the vasoconstrictor and vasodilator was significantly reduced in rats fed blueberries blueberries led to a 13% higher amount of total GAGs in aortas as a result of a higher content of GalAGs and an overall lower concentration of oversulfated disaccharides in both HS and GalAG populations in the aortas of the blueberry-fed group

the vasoconstriction elicited by phenylephrine was reduced in the blueberry group, attributed to the NO pathway, and acetylcholine-induced vasorelaxation in the blueberry group was possibly mediated through the COX but not the NO pathway blueberries in the diet affects the endothelium-dependent vasorelaxation by modulating the production/activity of COX-derived products in the young SHR aorta

blueberries decreased phenylephrine-mediated vasoconstriction in SHR aortic rings under basal conditions by enhancing NO−cGMP signaling without a significant involvement of the COX pathway

the mean lesion area for apoE−/− mice fed blueberries was reduced by 39% in the aorta sinus and 58% in the descending aorta compared to control-diet-fed mice

key finding/result

Table 2. Evidence of Atheroprotective Effects of Blueberries from Animal Studies conclusion

blueberries enhanced the protective features of GAG molecules associated with endothelial dysfunction and vascular pathology blueberries positively affected vascular smooth muscle contractility and sensitivity, but these effects were evident only after 7 weeks of blueberry consumption

blueberries exerted an overall anti-inflammatory effect in the OZR, an animal model of the metabolic syndrome

blueberries normalized some markers related to glucose metabolism in the OZR model of metabolic syndrome blueberries improved endothelial function of OZR, which was related to its antioxidant and anti-inflammatory actions

blueberries improved lipid profiles and modulate the expression of key enzymes and transcription factors of lipid metabolism in severely dyslipidaemic rats

blueberries improved endothelial function and exerted anti-inflammatory effects

blueberries altered rat aortic structural tissue GAGs, which may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment blueberries attenuated local inflammation in perivascular adipose tissue

blueberries modified major pathways of vasomotor control and improved the vascular tone with endothelial dysfunction blueberries affected the endothelium-dependent vasorelaxation by modulating alternative metabolic pathway(s) blueberries affected NO metabolic pathways in the aorta at basal and stimulated levels

blueberries benefited key signaling steps of endothelial function

blueberries prevented atherosclerosis in apoE−/− mouse model

reference

Del Bò et al.64

Kristo et al.70

Vendrame et al.78

Vendrame et al.73

Vendrame et al.74

Vendrame et al.72

Klimis-Zacas et al.76

Vendrame et al.75

Kalea et al.67

Kalea et al.65

Kalea et al.66

Kristo et al.69

Kristo et al.68

Wu et al.51

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Kalt et al.79

Rodriguez-Mateos et al.84

blueberries resulted in a decrease in total, LDL, and HDL cholesterol in pigs fed a high-plant-based diet, with 2% showing the greatest reduction, and the lipid-modulating effect of blueberries was attenuated in a low-plant-based diet

blueberries lowered blood pressure and improved endothelial dysfunction induced by a high-fat- and high-cholesterol-containing diet blueberries affected plasma lipid status, which may function synergistically blueberry feeding led to significant reduction in systolic blood pressure, reduced contractile responses, and aorta relaxation was significantly greater in response to acetylcholine

male SHRSP and male Wistar rats (normotensive controls) male Wistar rats

male castrated pigs

Shaughnessy et al.80 blueberries slowed the development of hypertension in a rat model

antioxidant activities.21,24,51 Studies conducted in animals, human subjects, and cell culture models suggested that blueberries affected multiple events that related to the occurrence and progression of atherosclerosis, which included counteracting oxidative stress by improving serum antioxidant status, inhibiting inflammation, improving endothelial dysfunction, and regulating cholesterol accumulation and trafficking. In this section, the possible mechanisms of atheroprotective effects of blueberries were discussed primarily on the basis of human and animals studies, along with the cell culture studies, in which the in vivo bioactive compounds were used. Reducing Oxidative Stress. LDL oxidation is a key step in the early stages of atherosclerosis. Ox-LDL initiates a series of inflammatory events leading to the recruitment of leukocytes, including monocyte and T lymphocyte, into intima and differentiation of monocyte into macrophages.87,88 In addition to being taken up rapidly by macrophages to form foam cells, ox-LDL was shown to be atherogenic through other mechanisms.89 Early studies showed blueberries inhibited human LDL and liposome oxidation in cell cultures and significantly reduced lipid hydroperoxides in human subjects.90−92 Many recent studies assessed the effects of blueberries in reducing lipid peroxidation by measuring specific biomarkers. It was found that blueberries reduced the concentration of a biomarker of lipid peroxidation, F2-isoprostane, in the livers of blueberry-fed apoE−/− mice.58 Specific fatty acid oxidation products, including hydroxyoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acids (HETEs), were also lower in the thioglycollate-elicited peritoneal macrophages of blueberry-fed apoE−/− mice.93 Another end product of lipid peroxidation, malondialdehyde (MDA), was found to be increased in highcholesterol-fed guinea pigs but was significantly reduced with blueberry intervention.82 Similar results were also observed in human trials. In obese men and women, the decreases of plasma ox-LDL, serum MDA, and hydroxynonenal concentrations were found to be greater in the blueberry group than in the control group.51 However, the mechanisms of reducing lipid peroxidation by blueberries are still not clear. One possible mechanism was that they boosted antioxidant enzymes. For instance, blueberries did not change the overall antioxidant status in serum but increased expression and activities of some major antioxidant enzymes in aorta, such as superoxide dismutase (SOD)1, SOD2, glutathione reductase (GSR), thioredoxin reductase 1, and paraoxonase 1.58 Boosting the cell or serum/plasma antioxidant status has been implicated as a possible preventative means to reduce the development of cardiovascular disease. For adults with MetS, treatment with blueberries (22.5 g of freeze-dried highbush blueberries) markedly decreased superoxide and total ROS in whole blood and monocytes compared to the placebo.47 A single blinded crossover study was performed in a group of eight middle-aged male subjects.94 Subjects consumed a highfat meal, and a control supplement followed 1 week later by the same high-fat meal supplemented with 100.0 g of freeze-dried lowbush blueberry powder. Serum antioxidant status was determined using the oxygen radical absorbance capacity assay and the total antioxidant status assay. Significant increases in serum antioxidant status above the controls were observed at 1 and 4 h post-consumption of the high-fat meal. It was concluded that the consumption of wild blueberries was associated with a diet-induced increase in the in vivo serum antioxidant status. Changes in plasma antioxidant capacity

highbush blueberry freeze-dried powder (2%, w/w) for 10 weeks highbush blueberry powder was added to diets at concentrations of 0, 1, 2 and 4% (w/w) in trial 1 and in 1.5% (w/w) in trial 2

Norton et al.71 weanling male SD rats

reference

blueberries may have implications in blood pressure regulation

blueberry feeding affected the vascular smooth muscle contractile machinery by suppressing the α1-adrenergic receptor agonist-mediated contraction, and their mechanism of action seems to be accomplished through an endothelium-dependent pathway in SHRSP consuming blueberries, systolic blood pressure was 19% lower at week 4 and 30% lower at week 6, relative to SHRSP on the control, and blueberry feeding had no effect on urinary excretion of F2-isoprostanes but had reduced markers of renal oxidative stress

blueberries displayed anti-atherogenic effects that may be related to their antioxidant and antilipidemic actions

conclusion key finding/result

high-cholesterol diet increased cholesterol and peroxidation markers in aorta, liver, or serum, and blueberries attenuated these atherosclerotic changes

highbush blueberries (8%, w/w) in control or high-cholesterol diet (1.25% cholesterol) wild blueberry freeze-dried powder (8%, w/w) for 13 weeks wild blueberry freeze-dried powder (3%, w/w) for 8 weeks

diet/dose animal model

male Dankin Hartley guinea pigs

Table 2. continued

Coban et al.82


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Figure 3. Blueberries reduced LPS-induced pro-inflammatory cytokines (e.g., TNF-α and IL-6) by inhibiting the phosphorylation of IκB and NFκB p65 of the NF-κB pathway and p38 and JNK of the MAPK pathway.

Mediating Inflammatory Factors. Inflammation plays a central role in the pathogenesis to atherosclerosis.100 Early development of atherosclerosis often begins with inflammatory changes in the endothelium, which can express the adhesion molecules, such as VCAM-1 and ICAM-1. Adhesion molecules attract monocytes, which then migrate through the endothelial layer under the influence of various pro-inflammatory chemoattractants. Once within the arterial intima, the monocytes continue to undergo inflammatory changes, transform into macrophages, engulf lipids, and become foam cells. T lymphocytes also migrate into the intima, where they release pro-inflammatory cytokines that amplify the inflammatory activity. Through these inflammatory processes, the initial lesion of atherosclerosis, the fatty streak, is formed.101,102 Despite the numerous reports suggesting the anti-inflammatory effects of berries or berry bioactive compounds,13,17,103,104 the human studies showing the effects of consuming whole blueberries in reducing chronic inflammation are still limited and controversial. In a double-blind, randomized, and placebocontrolled study, adults with MetS received 22.5 g of freezedried blueberry powder or placebo in a smoothie twice daily for 6 weeks. Blueberry treatment was found to decrease monocyte gene expression of TNF-α, IL-6, and TLR4 and reduce serum GM-CSF when compared to the placebo treatment.47 However, in three other studies, after human subjects were given comparable amounts of blueberries for similar or shorter durations, no changes of inflammatory biomarkers (including TNF-α, IL-6, and CRP) were observed.46,48,53 Anti-inflammatory effects of blueberries were shown in several animal studies. After obese or lean Zucker rats (LZR) were fed a control or an 8% wild blueberry diet for 8 weeks from age 8 to 16 weeks, plasma concentrations of TNF-α, IL-6, and CRP were significantly lower and adiponectin was significantly higher in both obese and lean rats fed blueberries compared to those fed the respective control diet. In addition,

(AOC) following consumption of a single meal of different berries or fruits, including lowbush blueberry, were studied in five clinical trials with 6−10 subjects per experiment.95 Consumption of blueberries in two studies increased the value of hydrophilic AOC. This study also demonstrated that consumption of blueberries was associated with increased plasma AOC in the postprandial state and consumption of an energy source of macronutrients containing no antioxidants was associated with a decline in plasma AOC. However, in the apoE−/− model, 1% blueberry feeding for 20 weeks did not change the total antioxidant capacity measured by the same assay,58 suggesting that dose response studies are needed to understand the in vivo antioxidant activity of blueberries. In addition, recent evidence has revealed that the antioxidant activities of dietary polyphenols may be achieved through regulating redox-sensitive signaling pathways, such as the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2).96 Nrf2 is referred to as the “master regulator” of the antioxidant response, regulating the expression of multiple antioxidant enzymes, including glutathione peroxidase (GPx), glutathione S-transferase, heme oxygenase-1 (HO-1), catalase, NAD(P)H:quinone oxidoreductase 1 (NQO1).97,98 In a recent study,99 three healthy individuals were recruited to consume single-strength cold-pressed blueberry juice (5 mL/ kg of body weight). The profiles of bioavailable plasma polyphenol metabolites following intake of blueberry juice were analyzed. The effects of three physiologically relevant mixtures of blueberry-derived phenolic acids was further investigated for their ability to induce Nrf2 nuclear translocation and downstream gene expression in human endothelial cells. The results indicated that, at the concentrations observed in plasma, blueberry phenolic acids could induce low-level production of Nrf2-regulated antioxidant response proteins HO-1 and glutamate−cysteine ligase modifier subunit (GCLM) in human endothelial cells exposed to 2.5 μM H2O2. 9180


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Figure 4. Blueberries downregulated scavenger receptors SR-A and CD36 and reduced oxidized fatty acids. Blueberries increased expression and protein levels of ABCA1. The potential mechanisms were related to downregulating PPARγ.

expression of IL-6, TNF-α, and nuclear factor κB (NF-κB) was downregulated in both the liver and abdominal adipose tissue, while CRP expression was downregulated in only the liver.78 In another study conducted by the same group with the same experimental design using the same model, wild blueberry consumption attenuated local inflammation in the perivascular adipose tissue (PVAT) of OZR. Following the blueberry diet, PVAT concentrations of IL-8 were significantly lower and adiponectin concentrations were significantly increased in both OZR and LZR.75 In another study, Xie et al.105 showed that, after apoE−/− mice were fed a control or control diet formulated to contain 1% wild blueberries for 5 weeks, TNFα and IL-6 were lower in serum and TNF-α expression in aorta was downregulated with blueberry feeding. The protein level and mRNA expression of TNF-α and IL-6 were significantly lower in the peritoneal macrophages from mice fed blueberries without or with lipopolysaccharide (LPS) or ox-LDL stimulation. Furthermore, RAW264.7 macrophages were treated with polyphenol-enriched extracts made from the sera of rats fed control (SEC) or the sera of rats fed control containing 10% blueberries (SEB). SEB significantly inhibited LPS-induced mRNA expression and protein levels of TNF-α and IL-6. SEB inhibited the phosphorylation of IκB, NF-κB p65, mitogen-activated protein kinase (MAPK) p38, and c-Jun N-terminal kinase (JNK), suggesting that blueberries affected two major chronic inflammation-related signaling pathways: NF-κB and MAPK pathways106 (Figure 3). Improving Endothelial Function. As the major regulator of vascular homeostasis, the endothelium exerts a number of vasoprotective effects.107 Endothelial dysfunction, including impaired vasomotor disturbance, abnormal coagulation, and increased vascular proliferation, is an early sign of atherosclerosis.108 It is closely associated with the development of atherosclerosis and precedes the clinical manifestations of atherosclerosis.109,110 Many non-invasive techniques were developed to assess endothelial function. The peripheral arterial tonometry reactive hyperemia index (RHI), an index of the endothelial-dependent flow-mediated dilation, was shown to be more sensitive to metabolic risk factors.111 RHI was improved significantly more

in adults with MetS consuming the blueberries versus the placebo group.54 A single serving of 300 g of fresh blueberries was found to counteract the impairment of RHI induced by smoking in male smokers.52 However, similar effects were not observed in another study.48 After male volunteers with cardiovascular risk factor consumed 25 g of blueberries for 6 weeks, no significant changes were found in RHI before and after blueberry consumption or between blueberry and control groups. Oxidative stress and proinflammatory cytokines are considered as major pathophysiological stimuli of endothelial dysfunction in atherogenesis.112 Blueberries were found to improve the endothelial function by reducing the oxidative stress and chronic inflammation. In the OZR model, blueberries were shown to improve endothelial function by increasing prostaglandin I2 (PGI2) and downregulating iNOS and COX-2 expression in the OZR aorta.73,76 In human aortic endothelial cells, lipotoxicity-induced endothelial dysfunction was attenuated by blueberry metabolites at the concentrations known to circulate in humans. The possible explanation for this finding is that blueberry metabolites suppress NOXmediated ROS production, which increases bioavailable NO.113 In addition, blueberries were also observed to improve vascular tone by decreasing phenylephrine-mediated vasoconstriction in SHR aortic rings in both adult SHRs68,69 and weanling male SD rats.65 Regulating Cholesterol Accumulation and Trafficking. Cholesterol-laden macrophage foam cells are the primary component of the fatty streak, the earliest atherosclerotic lesion.41 Data from murine models of atherosclerosis have demonstrated a significant role of the two major scavenger receptors, SR-A and CD36, in atherosclerotic foam cell development and vascular lesion initiation.114,115 CD36 and SR-A are the principal receptors responsible for the binding and uptake of modified LDL in macrophage. Together they account for 75−90% of the uptake and degradation of acetylated and ox-LDL.116 Reverse cholesterol transport (RCT) is a pathway by which excessive cholesterol is transported from the vessel wall to the liver for excretion, thus preventing cholesterol accumulation. A critical part of 9181


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Figure 5. Seven simple phenolic acids that were identified from sera of rats after fed blueberries. These seven compounds were either new or their concentrations were much higher in sera of rats fed blueberries than those fed the control diet. Thus, these seven compounds are considered as catabolites of blueberry constituents.

Figure 6. Molecular mechanisms of atheroprotective effects of blueberries.

RCT is cholesterol efflux, by which accumulated cholesterol is removed from macrophages in the intima of the vessel wall by ATP-binding membrane cassette transporter A1 (ABCA1), ATP-binding membrane cassette transporter G1 (ABCG1), HDL, scavenger receptor class B type I (SR-BI), or other mechanisms, including passive diffusion.117,118 Xie et al.93 showed that, after apoE−/− mice were fed the control or control formulated to contain 1% wild blueberries for 20 weeks, gene expression and protein levels of scavenger receptors CD36 and SR-A in aorta and thioglycollate-elicited peritoneal macrophages were lower in blueberry-fed mice. In addition, in the same study, apoE−/− mice were fed the control or blueberry diet for 5 weeks. Gene expression and protein levels of CD36 and SR-A were found to be lower in peritoneal macrophages of blueberry-fed mice, and fewer ox-LDLinduced foam cells were formed in comparison to those from mice fed the control. The potential mechanism was suggested as partly downregulating PPARγ. In another study also conducted by Xie et al.,106 in vivo catabolites following wild blueberry consumption in rats increased expression and protein levels of ABCA1, suggesting the ability of blueberries in facilitating cholesterol efflux and reducing cholesterol accumulation in macrophages (Figure 4). Alteration of gene expressions related to the lipid metabolism in adipose tissue

and liver through blueberry consumption was also observed in the animal model. Following wild blueberry consumption, the expression of the transcription factors PPARα and PPARγ in the OZR was increased in the adipose tissue, while that of SREBP-1 was decreased in the liver and adipose tissue. The expression of fatty acid synthase was significantly decreased in both the liver and adipose tissue, while the expression of ABCA1 was increased in the adipose tissue.72 Influencing Gut Microbiota. Recent studies have linked gut microbiota to atherosclerosis. Disruption of the gut microbiota (dysbiosis) can lead to a variety of different diseases, including atherosclerosis.119 As described in a recent review, gut microbiota may affect atherosclerosis through three pathways: (1) local or distant infections might cause a harmful inflammatory response that aggravates plaque development or triggers plaque rupture; (2) metabolism of cholesterol and lipids by gut microbiota can affect the development of atherosclerotic plaques; and (3) diet and specific components that are metabolized by gut microbiota can have various effects on atherosclerosis.120 Foods have a direct or an indirect impact on all three pathways. For example, metabolites of the dietary lipid phosphatidylcholine−choline, trimethylamine N-oxide, and betaine by gut microbiota were found to promote atherosclerosis.121 Gut microbiota was thus proposed as a 9182


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Journal of Agricultural and Food Chemistry novel cardiovascular therapeutic target.122 Certain berries, such as lingonberries, have been reported to reduce atherosclerosis in apoE−/− mice by altering gut microbiota.123 Blueberries or blueberry polyphenols have been shown to influence the gut microbiota. In a recent study, highbush blueberries were found to alter microbiota composition changes in male Wistar rats caused by feeding a high-fat diet.124 Highbush blueberries were also found to alter the composition and metabolism of the cecal microbiota and colon morphology in mdr1a−/− mice.125 In another study, a lowbush blueberry-enriched diet resulted in a significant reduction in the relative abundance of the genera Lactobacillus and Enterococcus and a significant increase in the relative abundance of the phylum Actinobacteria, the order Actinomycetales, and several novel genera under the families Bifidobacteriaceae and Coriobacteriaceae.126 In human subjects, Bifidobacterium spp. significantly increased following consumption of a lowbush blueberry drink for 6 weeks, suggesting that lowbush blueberries positively modulated the composition of the intestinal microbiota.127 However, there have been no studies that directly linked the consumption of blueberries to prevention of atherosclerosis through altering gut microbiota. More studies are warranted in this exciting new area of research. In summary, blueberries may prevent atherosclerosis through multiple mechanisms (Figure 6). These mechanisms are likely inter-related. For example, blueberries reduced the levels of lipid oxidation product HODEs, which could further alleviate the inflammatory effects caused by HODEs.128 Several key molecular pathways, such as NF-κB, may underpin these mechanisms. Future studies should focus more on the integration of these mechanisms to obtain a clearer understanding of all of the components that blueberries contain that provide their claimed atheroprotective effects.

are various phenolic acids and their derivatives, such as benzoic acids (C6−C1), phenylacetic acids (C6−C2), and phenylpropionic acids (C6−C3).142 Many of these catabolites are efficiently absorbed in the colon, appear in the blood, and enter target tissues. As a result, some of these compounds can reach a considerable concentration in vivo. Because microbial catabolites may be present at many sites of the body in a higher concentration than the parent compounds, it is proposed that at least a part of the biological activities ascribed to berry polyphenols is due to their colonic catabolites. In one recent study, lipotoxicity-induced endothelial dysfunction was attenuated by blueberry metabolites (hydroxyhippuric acid, hippuric acid, benzoic acid-4-sulfate, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate) at the concentrations in human circulation but not by parent anthocyanins in the berries.113 This indicated that, instead of original compounds in berries, their metabolites/catabolites may be actual in vivo bioactive compounds. Seven simple phenolic acids (7PA), which appeared to be colonic catabolites of polyphenols, were identified in the serum of rats fed the blueberry-containing diet143 (Figure 5). After murine macrophage cell line RAW 264.7 was treated with the same concentrations in rat serum, 7PA were found to inhibit LPS-induced mRNA expression and protein levels of pro-inflammatory cytokine TNF-α and IL-6 by reducing MAPK JNK, p38, and extracellular signalregulated kinase (ERK)1/2 phosphorylation. After treatment with 7PA for 2 weeks, mRNA expression and protein levels of scavenger receptor CD36 were decreased, whereas SR-A remained unchanged. Foam cell formation induced by oxLDL and ox-LDL binding to macrophages was also inhibited by 7PA. In addition, 7PA increased expression and protein levels of ABCA1. These results indicated that certain phenolic acids are potential in vivo atheroprotective compounds following blueberry consumption in the rodent model. Nevertheless, because blueberries contain many phytochemicals, other yet to be identified bioactive compounds may also be important in preventing atherosclerosis in this model and, possibly, in humans.

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IN VITRO VERSUS IN VIVO ATHEROPROTECTIVE CONSTITUENTS IN BLUEBERRIES Identifying bioactive compounds in foods is crucial to understand their health effects and, thus, has been an active and important area in nutritional and food research.129 The primary challenge in this area is the number and diversity of compounds in a given food. Absorption, metabolism, excretion, catabolism, and biotransformation by gut microbiota are all key factors to link the in vivo biological activity and the bioactive compounds in foods. To add another layer of complexity, compounds in foods may also work synergistically.130 Blueberries contains high levels of polyphenols, including anthocyanins, proanthocyanidins, phenolic acids, and other phenolic compounds.131−134 Polyphenols are widely considered as major bioactive compounds in blueberries responsible for protective effects against vascular diseases, which have been shown in numerous in vitro studies.35,135,136 The absorption and metabolism of polyphenols are key factors in determining their biological activities.137 Studies have clearly shown that the major polyphenols in blueberries, including anthocyanins and proanthocyanidins, were poorly absorbed.138,139 For example, only a very small proportion (

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