Abstract
Blueberries are rich in nutrients and (poly)phenols, popular with consumers, and a major agricultural crop with year-round availability supporting their use in food-based strategies to promote human health. Accumulating evidence indicates blueberry consumption has protective effects on cardiovascular health including vascular dysfunction (i.e., endothelial dysfunction and arterial stiffening). This narrative review synthesizes evidence on blueberries and vascular function and provides insight into underlying mechanisms with a focus on oxidative stress, inflammation, and gut microbiota. Evidence from animal studies supports beneficial impacts on vascular function. Human studies indicate acute and chronic blueberry consumption can improve endothelial function in healthy and at-risk populations and may modulate arterial stiffness, but that evidence is less certain. Results from cell, animal, and human studies suggest blueberry consumption improves vascular function through improving nitric oxide bioavailability, oxidative stress, and inflammation. Limited data in animals suggest the gut microbiome mediates beneficial effects of blueberries on vascular function; however, there is a paucity of studies evaluating the gut microbiome in humans. Translational evidence indicates anthocyanin metabolites mediate effects of blueberries on endothelial function, though this does not exclude potential synergistic and/or additive effects of other blueberry components. Further research is needed to establish the clinical efficacy of blueberries to improve vascular function in diverse human populations in a manner that provides mechanistic information. Translation of clinical research to the community/public should consider feasibility, social determinants of health, culture, community needs, assets, and desires, barriers, and drivers to consumption, among other factors to establish real-world impacts of blueberry consumption.
Keywords: Anthocyanins, Arterial stiffness, Blueberry, Endothelial function, Polyphenols, Cardiovascular disease
1. Introduction
Cardiovascular disease (CVD) is the leading cause of death in the United States, followed by cancer and COVID-19 in 2021 [1]. CVD is an umbrella term for disorders affecting the heart and blood vessels, including coronary heart disease, heart attack, stroke, and heart failure [2]. The underlying etiology of CVD is complex and multifactorial but involves nonmodifiable and modifiable risk factors such as age, menopausal status, hypertension (HTN), dyslipidemia, and diet/nutrition, with age being the primary risk factor [3]. Based on National Health and Nutrition Examination Survey 2015–2018 data, CVD prevalence in adults aged 20 years and older was estimated to be 49.2% and found to increase with age [4]. With aging, unfavorable structural and functional changes occur throughout the cardiovascular system, contributing to vascular dysfunction, which includes endothelial dysfunction and large elastic arterial stiffening [5]. Both endothelial dysfunction and arterial stiffness have been shown to be predictive of CVD and CVD-related events [6-8]. Two main underlying mechanisms of vascular dysfunction include oxidative stress and inflammation, whereas the gut microbiome has more recently emerged as a major contributing factor [9-11].
Epidemiological, animal, and clinical studies have shown that consuming (poly)phenol-rich foods may reduce CVD risk through improvements in vascular function [12-17]. (Poly)phenols are secondary plant metabolites necessary for plant health, serving as antioxidants and protecting against biotic and abiotic stressors in plant flowers, leaves, stems, and roots [18]. They are characterized by their phenolic structures and hydroxyl moieties and are classified as flavonoid and nonflavonoid compounds, with flavonoids having a 15-carbon skeleton consisting of 2 phenol rings and a heterocyclic ring, and with nonflavonoids containing a single phenol ring [18]. Flavonoid subclasses include anthocyanins (ACNs), flavan-3-ols, flavanones, flavones, flavonols, and isoflavones, and nonflavonoid subclasses include lignans, phenolic acids, and stilbenes. Following ingestion and depending on their chemical structure, bioaccessible (poly)phenols are absorbed and metabolized in the upper gastrointestinal (GI) tract and transported to the liver for further metabolism, whereas unabsorbed (poly)phenols pass to the large intestine for gut microbial metabolism followed by metabolite absorption and transport to the liver (Fig. 1). (Poly)phenols and their metabolites have been shown to exert physiological effects in animals and humans through various mechanisms including, but not limited to, attenuating oxidative stress and inflammation and modulation of the gut environment including the gut microbiota [19-21].
Fig. 1 –
Visual representation of blueberry (poly)phenol digestion, absorption, metabolism, and elimination that includes examples of (poly)phenol metabolites produced through phase I and II and gut microbial metabolism. Following ingestion of blueberries, their (poly)phenols travel through the gastrointestinal tract, where a minor amount is absorbed in the stomach following release from the food matrix and hydrolysis. Approximately 5% to 10% are absorbed in the small intestine following deconjugation reactions, and the remainder (~ 90%–95%) go to the large intestine for gut microbial and phase II metabolism. Following absorption and further metabolism in the liver, (poly)phenol metabolites enter systemic circulation for travel to target tissues and/or are eliminated through urine or feces. This figure was created with Biorender (https://www.biorender.com).
Blueberries are rich in nutrients and (poly)phenols, and epidemiological evidence supports that habitual blueberry consumption is protective against incident HTN and myocardial infarction, as well as other related cardiometabolic diseases and events such as type 2 diabetes and all-cause mortality [22-26], suggesting diverse cardiovascular and metabolic health benefits. This evidence is strengthened by human and animal studies with blueberries demonstrating protective effects on cardiovascular health, including vascular function [12,17]. Epidemiological, animal, and human data also suggest these health effects are due in large part to their (poly)phenols, particularly their ACN and flavonoid components [22,23]. Preclinical and clinical studies in animals suggest blueberries exert cardioprotective effects by improving oxidative stress and inflammation and modulation of the gut microbiota with blueberry consumption [17,27,28]. The aim of this narrative review is to synthesize the evidence on the impact of blueberries on vascular function, including endothelial function and arterial stiffness, in animals and humans. An additional aim is to provide insight into underlying mechanisms responsible for clinical effects in humans based on results from cell, animal, and human studies with a focus on oxidative stress, inflammation, and the gut microbiota. Background information on blueberries and vascular function is provided to give context to included studies evaluating the impact of blueberries and their (poly)phenols on vascular function.
2. Blueberry components and metabolism
Blueberries are one of the few fruits native to North America, and the United States is the world’s largest producer, with consumption significantly increasing during the past decade [29]. Consumption per capita has progressively increased and was estimated to exceed 1.79 pounds per person in 2017 and peaking in 2019 (based on most recently available data) [30]. Their popularity with consumers, year-round availability, and nutritional quality makes them ideal for use in food-based intervention strategies for preserving vascular function with aging, and thus reducing CVD risk. Indeed, blueberries are rich in nutrients including dietary fiber, vitamins C and K, and manganese [31], and nonnutrient bioactive compounds (i.e., (poly)phenols) such as flavonoid compounds like ACNs, proanthocyanidins, flavan-3-ols, and flavonols, and non-flavonoid compounds such as phenolic acids and pterostilbene (Table 1) [12,32-34].
Table 1 –
Nutrient contents of raw blueberries
| Amount per 100 g fresh weight | Amount per 1 cup/148 g fresh weight | |
|---|---|---|
| Nutrientsa | ||
| Energy (kcal) | 57 | 84 |
| Fat (g) | 0.31 | 0.46 |
| Total carbohydrates (g) | 14.6 | 21.6 |
| Dietary fiber (g) | 2.4 | 3.6 |
| Protein (g) | 0.7 | 1.0 |
| Vitamin C (mg) | 8.1 | 12.0 |
| Vitamin K (mcg) | 19.3 | 28.6 |
| Potassium (mg) | 86 | 127 |
| Magnesium (mg) | 6.2 | 9.2 |
| Phosphorus (mg) | 13 | 19 |
| Iron (mg) | 0.34 | 0.50 |
| Calcium (mg) | 12 | 18 |
| Manganese (mg) | 0.423 | 0.6 |
| Sodium (mg) | 1 | 1 |
| Zinc (mg) | 0.09 | 0.13 |
| Copper (mg) | 0.046 | 0.068 |
| (Poly)phenolsb | ||
| Total flavonoids (mg) | 173.79 | 257.21 |
| Anthocyanins (mg) | 133.99 | 198.31 |
| Phenolic acids (mg) | 136.48 | 201.99 |
| Flavonols (mg) | 38.69 | 57.26 |
| Flavan-3-ols (mg) | 1.11 | 1.64 |
Information obtained from FoodData Central, United States Department of Agriculture [173]. Information obtained did not specify the variety of blueberries but are believed to be highbush blueberries because of being fresh/raw and the location of origin provided.
Information obtained from Phenol-Explorer [174]. Information obtained and used was for highbush blueberries because there was limited information for wild/lowbush blueberries.
Although many of their beneficial health effects are largely attributed to their (poly)phenols (based on evidence), certain nutrients in blueberries may also protect against CVD through effects on vascular function. While nutrient concentrations do not equate to that found in dietary supplements, consumption of blueberries can contribute to meeting nutrient intake requirements important for normal physiological function and therefore cardiovascular health. For instance, a 1 cup/148 g serving of fresh blueberries provides a good source of vitamin C because it contains more than 10% of the Daily Value. Vitamin C supplementation has been widely studied and shown to protect against arterial stiffening, endothelial dysfunction, and high blood pressure [35,36]. Mechanisms for improved vascular health with vitamin C supplementation may include direct free-radical scavenging (i.e., reducing oxidative stress) and increased nitric oxide (NO) production/bioavailability [36]. Importantly, data from the Nurse’s Health Study (a large prospective study) indicated vitamin C intake (through supplements and food and ≥ 360 mg/day) was associated with a 27% lower risk for coronary heart disease in those taking vitamin C supplements [37]. However, vitamin C supplementation (and dietary supplementation with vitamins and minerals broadly) has not always been shown to translate to long-term CVD prevention, highlighting the benefit of consuming foods rich in a variety of nutrients and phytochemicals such as (poly)phenols that benefit health [38-40]. Vitamin K is another nutrient with cardiovascular benefits, and higher dietary intake and supplementation have been shown to have positive effects on vascular calcification, arterial stiffening, endothelial function, blood pressure, and other aspects of cardiovascular health [41]. Fresh blueberries provide an excellent source of vitamin K, with a 1 cup/148-g serving containing more than 20% of the Daily Value. They also provide an excellent source of the mineral manganese, which may have benefits for vascular health and CVD prevention because it is linked with lower inflammation and oxidative stress and improved antioxidant defense [42,43]. Lastly, dietary fiber is found in blueberries and has been shown to be a prebiotic (energy source) for gut microbiota, which then in turn can produce key metabolites (e.g., short chain fatty acids, (poly)phenol metabolites) beneficial for improving endothelial function and other aspects of cardiovascular health [44-46].
With respect to blueberry (poly)phenols, metabolites resulting from gut microbial metabolism and phase I and II metabolism (Fig. 1) are increasingly being linked to blueberries’ health effects rather than the parent compounds themselves, which have low bioavailability in the GI tract [12,47-49]. After (poly)phenol consumption, parent compounds travel to the stomach, where they are released from the food matrix, hydrolyzed, and a small amount of absorption may take place. Reports are mixed on the absorption of (poly)phenols in the stomach, but specific phenolic acids and ACNs (i.e., (poly)phenols found in blueberries) have been shown to undergo gastric absorption in humans and animals [50-54]. All remaining parent (poly)phenols travel to the small intestine, where some undergo deconjugation reactions such as deglycosylation and phase I and II metabolism in enterocytes after absorption, followed by metabolite release into portal circulation for transport to the liver for further phase II metabolism. Many (poly)phenols including ACNs remain intact until they reach the large intestine for gut microbial metabolism, colonocyte phase II metabolism, and absorption into portal circulation for transport to the liver [48,49]. Less than 1% of parent ACNs are found in urine after blueberry consumption, indicating low bioavailability [12,54]. Gut bacterial species involved in catabolism of (poly)phenols is not fully known, though several species have been identified that belong to abundant phyla in the human intestine including Bacillidota (Firmicutes), (Bacteriodota (Bacteroidetes), Actinomycetota (Actinobacteria), and Pseudomonadota (Proteobacteria). Gut microbiota can deconjugate glycosides, glucuronides, and organic acids to release aglycones, which then undergo further microbial transformations through ring and lactone fission, demethylation, dehydroxylation, decarboxylation, and other reactions. Examples of families and species involved in hydrolysis and enzymatic reactions of (poly)phenol compounds found in blueberries include Clostridiaceae (e.g., Clostridium sphenoides, C. saccharogumia), Lactobacillaceae (e.g., Lactobacillus acidophilus, L. plantarum), Bifidobacteriaceae (e.g., Bifidobacterium animalis, B. longum), Bacteroidaceae (e.g., Bacteroides fragilis, B. ovatus), Eubacteriaceae (e.g., Eubacterium cellulosolvens, E. ramulus), and Streptococcaceae (e.g., Lactococcus lactis, Lactococcus sp.). Following metabolite absorption and further metabolism in the liver, (poly)phenol metabolites enter systemic circulation for travel to target tissues and then elimination through urine or feces (Fig. 1) [12,48,49,55].
Because of this extensive and diverse metabolism, the pharmacokinetic profiles of (poly)phenols are varied. Specifically, blueberry (poly)phenol metabolites have been detected in plasma within the first couple of hours after ingestion (namely catechols, followed by benzoic acids, cinnamic acids, hippuric acids, and flavonols, among others), whereas gut-derived (poly)phenol metabolites (namely hippuric acids, followed by catechols and benzoic acids, among others) can peak at later points after acute and chronic blueberry consumption [54,56-58]. The detection of blueberry (poly)phenol metabolites in plasma is dependent on the amount of blueberries (and (poly)phenols) consumed and food matrix interactions, among other factors [54,57,59]. The contribution of blueberry (poly)phenols and their metabolites (individually, together, and in relation to other nutrient and nonnutrient components) to the beneficial effects of blueberries on vascular function is not fully understood but is an area of active investigation within the scientific community. Indeed, several studies have demonstrated acute blueberry consumption improves measures of vascular function, and that improvements are associated with increases in plasma (poly)phenol metabolites [56-58]. Few chronic intervention studies with blueberries have evaluated the relationship between improvements in vascular function and circulating (poly)phenol metabolites [28,58,60]. Those that have evaluated this relationship have not established a clear link [28,58,60], potentially because of the high turnover and excretion of (poly)phenol metabolites [61], background diet, and/or other factors.
3. Blueberries and vascular function evidence review methods
PubMed and Google Scholar were used to identify studies examining the impact of blueberry consumption on vascular function. Animal and human research studies were included in the search if the treatment was in the form of whole blueberries (i.e., fresh or frozen), blueberry powder, blueberry extract, and/or blueberry metabolites, and if outcome measures included endothelial function and/or arterial stiffness parameters. Cell culture research studies that assessed mechanisms associated with improvements in vascular function were also included in the search if they were conducted in vascular smooth muscle cells (VSMCs) or endothelial cells using any form of blueberry but were later screened for physiological relevance (i.e., whether they accounted for human digestion, absorption, and/or metabolism). For this, the search terms were as follows: blueberry cardiovascular, blueberry endothelial, blueberry endothelial dysfunction, blueberry endothelial function, blueberry flow-mediated dilation, blueberry reactive hyperemia index, blueberry pulse wave velocity, blueberry augmentation index, blueberry arterial stiffness, blueberry, blueberry human, blueberry human cardiovascular, blueberry vascular function, blueberry vascular dysfunction, blueberry smooth muscle cell, blueberry endothelial cell, blueberry gut microbiome, blueberry gut microbiome CVD, blueberries gut microbiome, blueberries pulse wave velocity, and blueberries gut microbiota pulse wave velocity. These terms were searched in PubMed using “AND” between terms as well.
Ten animal studies and 21 human studies assessing the impact of blueberry consumption on vascular function in vivo were included. There were 16 cell culture studies aiming to assess mechanisms associated with blueberry consumption and improved vascular function. However, a primary focus of the review is to gain insight into how blueberries improve vascular function in humans; therefore, only 8 cell studies that included methods to reflect human digestion, absorption, and/or metabolism were included. Specifically, included cell culture studies using blueberry (poly)phenol metabolites reported using physiologically relevant concentrations of (poly)phenol metabolites and/or parent compounds commonly found in human plasma after blueberry consumption [54,56,58,62]. To our knowledge, all cell, animal, and human studies to date that focused on blueberry consumption and vascular function have been accounted for in the article search and review.
4. Blueberries and endothelial (dys)function
4.1. Endothelial (dys)function overview and methods of assessment
The endothelium, once termed the “wallpaper” of the blood vessels, is a cell monolayer located in the tunica intima of the vasculature that acts as a barrier between blood and the vessel wall [63]. It controls vascular responses to stimuli such as hormones, neurotransmitters, and shear stress [63]. A healthy endothelium is defined by its ability to adequately maintain vascular tone (i.e., the balance between vasoconstriction and vasodilation) and to regulate inflammation, thrombosis, and other aspects of vascular health [64]. Endothelial dysfunction occurs when there is an imbalance, favoring a more vasoconstricted (decreased vasodilation), proinflammatory, prothrombotic, and pro-oxidant state [64,65], which is central to the initiation and progression of atherosclerosis, the cause of most CVDs [65].
The main hallmark or clinical manifestation of endothelial dysfunction is impaired endothelium-dependent dilation (i.e., vasodilation) resulting from the decreased production and/or bioavailability of the endothelium-derived vasodilatory molecule NO [5,66]. Stimuli such as shear stress causes production of NO from L-arginine through activation of endothelial NO synthase (eNOS) in the presence of essential co-factors such as tetrahydrobiopterin [66,67]. NO is then released into VSMCs to stimulate vasodilation for increased blood flow to peripheral tissues [66]. NO also has anti-inflammatory and antithrombotic properties [67]; therefore, reduced NO bioavailability can promote atherosclerotic CVD development. CVD risk factors such as advanced age, postmenopausal status, high blood pressure, and dyslipidemia can impair NO production and bioavailability and thus can promote endothelial dysfunction, atherosclerotic plaque accumulation, and ultimately increase CVD risk [3,5,9,67-72]. Specifically, vascular oxidative stress impairs NO production and bioavailability and is largely caused by excessive superoxide radical production resulting from increased NADPH oxidase (NOX) activity, mitochondrial dysfunction, and eNOS uncoupling (i.e., where eNOS produces superoxide instead of NO) [66,67]. Superoxide radicals reduce NO production and bioavailability through several mechanisms, including direct NO scavenging, thereby rendering it unavailable to function, and eNOS uncoupling [3,66,67]. Through a bidirectional relationship with oxidative stress, inflammation also contributes to endothelial dysfunction by inhibiting eNOS and increasing reactive oxygen species (ROS) production [3,9,73,74]. An additional mechanism responsible for impaired NO bioavailability is oxidative stress-driven dysregulation of arginine metabolism that depletes L-arginine and/or methylates it, leading to formation of methylarginines such as asymmetric dimethylarginine and NG-mono-methyl-L-arginine (L-NMMA), which are competitive inhibitors of eNOS and contributors to oxidative stress [75,76].
In animals, endothelial function is assessed by analyzing vasoconstriction and vasodilation using pressure myography ex vivo, which measures outer and lumen diameters and wall thickness of isolated and pressurized arteries [77]. Though each independent laboratory uses specific and varied techniques (e.g., choice of pharmacological agents used to assess endothelium-dependent mechanisms such as inhibition of enzyme activity, specific vessel type, or outcome units of measure), certain methods are more commonly used. In short, isolated vessels are placed on pressure myography prongs and are repressurized. Following phenylephrine (Phe)-induced contraction, increased concentrations of the vasodilator acetylcholine (Ach) are applied to the vessels to measure vessel diameter expansion over a specific period until maximum vasodilation occurs and is observed through an imaging microscope system [77]. Results obtained from studies and presented in this review are described as vasoconstriction, vasorelaxation, and vessel sensitivity.
In humans, there are several assessments of endothelium-dependent dilation, including noninvasive and invasive methods. For the purpose of this review, only two noninvasive methods used in published studies on blueberries and endothelial function in humans are included: ultrasound-assessed brachial artery flow-mediated dilation (FMD) and EndoPAT®-assessed peripheral arterial tonometry (PAT). Both methods assess blood flow in response to a hyperemic stimulus that is primarily NO-dependent (though other vasodilator pathways may contribute) and reflective of endothelial function, but each assessment has different features. FMD uses a high-resolution ultrasound to assess macrovascular function of conduit (e.g., brachial) arteries by measuring artery diameter change in response to increased blood flow after the release of arterial occlusion [78]. FMD is represented as the change in peak artery diameter in response to increased blood flow. Also, considering that shear stress (i.e., the stimulus for vasodilation) varies between individuals and populations, normalizing the FMD response to shear rate (FMD normalized to shear rate area under the curve, FMD/SRAUC) can provide better insight into endothelium-dependent dilation [79-81]. FMD is considered the gold standard for noninvasive assessment of endothelial function in humans because it correlates with coronary artery endothelial function and is highly predictive for CVD, but is highly operator-dependent, challenging, time-consuming to perform/analyze, and requires extensive operator training [82]. On the other hand, PAT uses the EndoPAT® device to measure microvascular endothelial function by assessing pulse wave amplitude through the fingertips in response to increased blood flow following the release of brachial artery occlusion, generating the reactive hyperemia index (RHI); each participant serves as their own control [78,83,84]. PAT has simpler procedures and operator independency but may not be directly comparable to FMD because each method assesses endothelial function in different vascular beds [78,85]. Though both have been shown to predict CVD and CVD events, FMD has been shown to be a more robust measure of endothelial function and a stronger predictor of CVD and CVD events [78,82,86]. Nonetheless, both methods have been shown to be reflective of endothelial function, validated, and predictive of CVD and related events, and, as such, are clinically relevant [7,78,83,84,87-91].
4.2. Effects of blueberries on endothelial (dys)function: animal studies
Ten studies examined the impact of blueberry consumption on endothelial function in animal models using pressure myography ex vivo (Table 2 ). Four studies were performed with healthy male rats. First, in thoracic aorta rings of male Sprague-Dawley (SD) rats fed a diet with 8% w/w freeze-dried wild (lowbush) blueberry powder (equivalent to 1.67 cups of fresh blueberries) for 13 weeks, a control diet for 13 weeks, or a control diet for 13 weeks followed by a diet with 8% w/w freeze-dried wild blueberry powder for 8 weeks (reverse diet), endothelium-dependent vasoconstriction to Phe decreased in both blueberry diet groups compared with the control group [92]. In another study with male SD rats, 8% w/w freeze-dried wild blueberry powder added to diets for 7 weeks decreased vasoconstriction (to Phe) and vessel sensitivity to Phe and Ach and increased Ach-induced vasorelaxation in thoracic aorta rings compared with the control group [93]. A study in male SD rats observed that vasoconstriction to Phe decreased in thoracic aorta rings after consuming a diet with 8% w/w freeze-dried wild blueberry powder for 7 weeks compared with a 7-week control diet and a 4-week wild blueberry diet [94]. The same study found that vessel sensitivity to Phe increased in thoracic aortas after the 4-week wild blueberry diet compared with control (with no effects on vasoconstriction), whereas a decrease in vessel sensitivity to Phe was found after 7 weeks of the wild blueberry diet compared with 7-week control and 4-week wild blueberry diets (potentially because of age-related impacts) [94]. Finally, a study assessed vasoconstriction and vasorelaxation in thoracic aorta rings of male Wistar rats fed 2% w/w freeze-dried highbush blueberry powder added to control chow or high-fat/high-cholesterol diets for 10 weeks. It showed decreased vasoconstriction (to Phe) in the blueberry-fed groups compared with control and high-fat/high-cholesterol diets, and increased vasorelaxation (to Ach) in the blueberry plus high-fat/high-cholesterol diet compared with the high-fat/high-cholesterol diet alone [95].
Table 2 –
Research studies examining the impact of blueberry consumption on endothelial function in animal models
| Reference and location |
Animal model |
Vascular measures |
Blueberry treatment |
Blueberry (poly)phenol concentrations |
Vascular results |
Possible mechanistic/pathway-related results |
Mechanism/ pathway conclusions |
|---|---|---|---|---|---|---|---|
| Norton et al. (2005) Maine, USA [92] |
Sprague-Dawley male rats (n = 30). | Vasoconstriction and vessel sensitivity in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w consumed for 13 wk. Three diet groups: control, WBB, and reverse (consumed control for 13 wk then switched to WBB diet for 8 wk). |
N/A | ↓ Vasoconstriction to Phe in WBB and reverse diet groups compared with control. No significant effects on other vascular measures. |
Diets did not have effects on vasoconstriction nor vessel sensitvity in endothelium-denuded rings. | WBB improves vascular function through the endothelium. |
| Kalea et al. (2009) Maine, USA [93] |
Sprague-Dawley male rats (n = 20). | Vasoconstriction, vasorelaxation, and vessel sensitivity in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 7 wk. Two diet groups: control and WBB. |
N/A | ↓ Vasoconstriction to Phe in WBB diet group compared with control. ↑ Vasorelaxation to Ach after WBB diet compared to control. ↓ Vessel sensitivity to Phe and Ach in WBB group compared to control. |
↑ Vasoconstriction in both groups after L-NMMA administration compared with no L-NMMA, with WBB group being higher than control. ↓ Vasorelaxation in both groups after L-NMMA administration compared with no L-NMMA, with WBB group having less vasorelaxation than control. ↓ Vessel sensitivity to Ach in WBB group after L-NMMA as well as MFA administration compared to control group after administration(s). |
WBB improves vascular function through the eNOS/NO pathway. |
| Kalea et al. (2010) Maine, USA [96] |
Spontaneously hypertensive male rats (n = 20). | Vasoconstriction, vasorelaxation, and vessel sensitivity in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 7 wk. Two diet groups: control and WBB. |
N/A | ↑ Vasorelaxation to Ach in WBB group compared with control without impacting maximum vasorelaxation force. ↑ Vessel sensitivity to Ach in WBB group compared with control. No significant effects on other vascular measures. |
↑ Vasoconstriction in WBB + L-NMMA compared to control + L-NMMA, as well as compared with WBB and control with no L-NMMA. ↓ Vasoconstriction in both groups with MFA compared to no MFA, and WBB + MFA had ↓ vasoconstriction compared with control + MFA. ↓ Vasorelaxation in both groups after L-NMMA administration compared to no L-NMMA, with WBB + L-NMMA group having greater vasorelaxation than control + L-NMMA. ↑ Vasorelaxation in both groups with MFA administration, with control + MFA group having greater vasorelaxation than WBB + MFA. ↓ Vessel sensitivity to Phe in WBB + L-NMMA as well as WBB + MFA compared with control + L-NMMA groups. ↑ Vessel sensitivity to Ach in WBB + L-NMMA compared with control + L-NMMA. ↓ Vessel sensitivity to Ach in WBB + MFA compared with control + MFA. |
WBB improves vascular function through the eNOS/NO and COX pathways. |
| Kristo et al. (2010) Maine, USA [97] |
Spontaneously hypertensive male rats (n = 40). | Vasoconstriction, vasorelaxation, and vessel sensitivity in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 8 wk. Two diet groups: control and WBB. |
21 ACNs detected in WBB powder. Peonidin-3-glucoside and malvidin-3-galactoside most abundant making up ~13% of total ACN content (1.6 ± 0.2 mg/100 mg) |
↓ Vasoconstriction to Phe in WBB group compared with control. ↑ Vasorelaxation in WBB group at lower Ach doses but ↓ at higher Ach doses compared with control. No significant effects on other vascular measures. |
↑ Vasoconstriction in both groups after L-NMMA administration compared to no L-NMMA, but no difference between groups. ↓ Vasoconstriction in both groups after MFA administration compared to no MFA with ↓ vasoconstriction in WBB + MFA compared with control + MFA. ↓ Vasorelaxation and vessel sensitivity in both groups after L-NMMA compared to no L-NMMA, with WBB + L-NMMA having greater vasorelaxation compared with control + L-NMMA and BB group vessel sensitivity lower than control with L-NMMA. ↑ Vasorelaxation in WBB + MFA compared to control + MFA. ↓ Vessel sensitivity in control + MFA compared to control without MFA. ↑ Vessel sensitivity in WBB + MFA compared to control + MFA. |
WBB improves vascular function through the COX pathway. |
| Del Bo et al. (2012) Maine, USA [94] |
Sprague-Dawley male rats (n = 40). | Vasoconstriction and vessel sensitivity in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 4 or 7 wk. Four diet groups: control 4-wk, WBB 4-wk, control 7-wk, and WBB 7-wk. |
21 ACNs detected in blueberry powder. Peonidin-3-glucoside and malvidin-3-galactoside most abundant of total ACNs (1.6 ± 0.2 mg/100 mg), with about 20.7 mg/d of ACNs consumed in WBB groups. | ↓ Vasoconstriction to Phe for WBB 7-wk diet compared with 7-wk control and 4-wk WBB diets. ↑ Vessel sensitivity to Phe for WBB 4-wk diet compared with 4-wk control diet. ↓ Vessel sensitivity to Phe for WBB 7-wk diet compared with 7-wk control and 4-wk WBB diets. |
↑ Vasoconstriction in all groups after L-NMMA administration compared to controls without L-NMMA. ↑ Vasoconstriction for WBB 7-wk diet with L-NMMA compared to 7-wk control with L-NMMA, WBB 7-wk without L-NMMA, and WBB 4-wk with L-NMMA. ↓ Vessel sensitivity in both 4-wk diet groups after L-NMMA compared to no L-NMMA. ↑ Vessel sensitivity in both 7-wk diets after L-NMMA compared to no L-NMMA 7-wk diets, but ↓ compared to their 4-wk diet counterparts after L-NMMA. |
WBB improves vascular function through eNOS/NO pathway. |
| Kristo et al. (2013) Maine, USA [98] |
Spontaneously hypertensive male rats (n = 20). | Vasoconstriction and vessel sensitivity in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 8 wk. Two diet groups: control and WBB. |
Total phenolics (gallic acid equivalents) were identified at 4.4% w/w, with chlorogenic acid most abundant (510 mg/100 g). 1.5% w/w ACNs concentration, with malvidin 3-galactoside and peonidin 3-glucoside making up 13% of ACNs content (1.6 ± 0.2 mg/100 mg). | ↓ Vasoconstriction to Phe in WBB group compared with control. No significant effects on other vascular measures. |
↑ Vasoconstriction in both groups after ODQ administration compared with no ODQ, with ↑ vasoconstriction in WBB + ODQ compared with control + ODQ. ↓ Vasoconstriction in both groups after TCP administration compared with no TCP but did not change association of WBB response curve to control. ↓ Vasoconstriction in WBB + TCP compared with control + TCP. ↓ Vasoconstriction in WBB + dazoxiben compared with control + dazoxiben; however, TXA2 was not associated with WBB improving vascular function because ↓ vasoconstriction in WBB group was seen compared with control regardless of inhibitor administration. ↑ Plasma cGMP and 6kPGF1α in WBB group compared with control. |
WBB improves vascular function through NO-sGC-cGMP signaling pathway. |
| Rodriguez-Mateos et al. (2013) Tokushima, Japan and Reading, England, UK [95] |
Wistar male rats (n = 32). | Vasoconstriction and vasorelaxation in aorta rings. | Freeze-dried highbush BB powder added to diets at 2% w/w for 10 wk. Four diet groups: control chow, control chow + BB, high-fat, and high-fat + BB. |
Flavonoids (mg/100 g diet) in 2% blueberry diets: ACNs 11.80, procyanidins 8.46, procyanidin monomers 0·63, and procyanidin dimers 1.78. Total flavonoids measured were 20.26 mg/100 g diet. | ↓ Vasoconstriction to Phe in control chow + BB group compared with control chow and high-fat group. ↓ Vasoconstriction to Phe in high-fat + BB group compared with control chow and high-fat group. ↑ Vasorelaxation to Ach in high-fat + BB group compared with high-fat group. |
Total flavonoid intake was ~3.85 mg of total flavonoids. ↓ SBP at 8 and 10 wk in control chow + BB group compared with control chow. ↓ SBP in high-fat + BB group at 10 wk compared with high-fat control. |
Highbush BB improves vascular function through the endothelium. |
| Vendrame et al. (2014) Maine, USA [105] |
Obese Zucker male rats (n = 72). | Vasoconstriction, vasorelaxation, and vessel sensitivity in aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 8 wk. Four diet groups: lean, lean + WBB, obese, obese + WBB. |
Total ACNs was 1.5% w/w, with peonidin-3-glucoside and malvidin-3-galactoside most abundant. | ↑ Vasoconstriction and ↓ vasorelaxation in obese + WBB group compared with obese control. ↑ Vasorelaxation in lean + WBB group compared with lean control. ↑ Vessel sensitivty to Ach in lean + WBB and obese control compared with lean control. ↓ Vessel sensitivty to Ach in obese + WBB compared with obese control. |
↑ Vasoconstriction in obese and lean control groups after MFA or L-NMMA administration compared with controls without inhibitor, with obese + WBB + MFA ↑ vasoconstrcition compard with obese control + MFA and a further ↑ in vasoconstrcition in obese + WBB + L-NMMA to the level of lean control. ↓ Vasorelaxation in obese + WBB + L-NMMA compared with obese + L-NMMA control. ↑ Vessel sensitivity to Phe in both obese and lean + L-NMMA and MFA compared with obese and lean without inhibitor. ↑ Vessel sesnistivity in obese + WBB + L-NMMA compared with obese + WBB and obese control + L-NMMA. ↑ Vessel sensitivity in obese + WBB + MFA and obese control + MFA compared with obese + WBB and obese control, respectively. ↓ Plasma NO in obese + WBB compared with obese control. ↑ Plasma NO in lean + WBB and obese control compared with lean control. ↑ Aortic effluent 6kPGF1α in obese + WBB compared with obese control. ↑ iNOS aorta gene expression in obese control and ↓ in lean + WBB compared with lean control. ↓ iNOS aorta gene expression in obese + WBB compared with obese control. ↑ COX-2 aorta gene expression in obese control compared with lean control. ↓ COX-2 aorta gene expression in obese + WBB group compared with obese control. |
WBB improves vascular function through COX and eNOS/NO pathways, with eNOS/NO pathway seeming to be greater. |
| Klimis-Zacas et al. (2016) Maine, USA [99] |
Obese Zucker male rats (n = 72). | Vasoconstriction and vasorelaxation in thoracic aorta rings. | Freeze-dried WBB powder added to diets at 8% w/w for 8 wk. Four diet groups: lean control, lean WBB, obese control, and obese WBB. |
21 different ACNs were identified in WBB powder: Peonidin-3-glucoside and malvidin-3-galactoside most abundant of total ACNs, and total ACNs content of WBB powder was 1.5% w/w. | ↓ Vasoconstriction to Phe in obese control compared with lean control. ↑ Vasoconstriction to Phe in obese WBB compared with obese control. ↑ Vasorelaxation to Ach in lean control compared with obese control. |
↓ IL-6, TNF-α, and CRP in obese WBB group compared with obese control. ↓ NO in obese WBB group compared with obese control. ↑ NO in lean WBB groups compared with lean control. ↑ 6kPGF1α in obese WBB group compared with obese control. ↓ Aorta gene expression of iNOS in obese and lean WBB group compared with their controls. ↓ Aorta gene expression of COX-2 in obese WBB group compared with control. |
WBB improves vascular function through eNOS/NO and COX pathways. |
| Petersen et al. (2022) Utah, USA [27] |
Diabetic db/db male mice (n = 45). | Vasorelaxation of mesenteric arteries. | Freeze-dried WBB powder added to diets at 3.8% w/w for 10 wk. Three diet groups: control non-diabetic mice, control diabetic mice, and diabetic mice + WBB. |
N/A | ↑ Endothelium-dependent vasorelaxation to Ach in WBB group compared with diabetic control. | ↓ ICAM-1 and VCAM-1 and monocyte binding to vessel in WBB group compared with diabetic control. ↓ Serum inflammatory chemokines, MCP1/JF and KC, in WBB group compared with diabetic control. ↓ Monocyte binding and KC secretion in mouse arterial endothelial cells incubated with serum from WBB group compared with diabetic control. ↓ NOX4 and IκkB expression in arterial endothelial cells and aortic vessel in WBB group compared with diabetic control. Gut microbes significantly differed between all groups. Trends in microbiota alpha diversity indices (Shannon and Simpson) in WBB group compared with diabetic control. Beta diversity significantly different at the phylum and genius level between all groups. WBB supplementation significantly increased Actinobacteria abundance compared with diabetic control. WBB supplementation significantly increased Adlercreutzia, Bifidobacterium, and Dorea compared with diabetic control. WBB supplementation showed changes in predicted pathways in diabetic mice compared with diabetic control. |
WBB improves endothelium-dependent vascular function through reduced NOX4 activity (and likely reduced oxidative stress), and potentially through reduced inflammation and changes in the microbiome. |
Abbreviations: Ach, acetylcholine (vasodilator); 6kPGF1α, 6-keto prostaglandin F1; ACN, anthocyanin; BB, blueberry; CRP, C-reactive protein; COX-2, cyclooxygenase 2; cGMP, cyclic guanosine monophosphate; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; IκkB, inhibitor kappa B kinase; ICAM-1, intercellular adhesion molecule 1; IL-6, interleukin-6; L-NMMA, L-NG-monomethyl-arginine (eNOS inhibitor); MFA, mefenamic acid (COX inhibitor); MCP-1, monocyte chemoattractant protein-1; N/A, not available; NO, nitric oxide; NOX4, NADPH oxidase 4; ODQ, 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (sGC inhibitor); Phe, L-phenylephrine (vasoconstrictor); PDE5, phosphodiesterase-5; Phe, phenylephrine; PGI2, prostaglandin I2; SBP, systolic blood pressure; TXA2, thromboxane A2, TCP, tranylcypromine (PGI2 synthase inhibitor); TNF-α, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion molecule 1; WBB, wild blueberry.
Three studies were performed with a male rat model of HTN. One study observed an increase in vasorelaxation and vessel sensitivity to Ach in thoracic aorta rings from male spontaneously hypertensive rats following 7 weeks of consuming a diet with 8% w/w freeze-dried wild blueberry powder compared with the control group [96]. In another study, Phe-induced vasoconstriction decreased, whereas Ach-induced vasorelaxation increased in thoracic aorta rings from male spontaneously hypertensive rats following consumption of a diet with 8% w/w freeze-dried wild blueberry powder for 8 weeks compared to control [97]. A later study examined 8% w/w freeze-dried wild blueberry powder added to the diets of male spontaneously hypertensive rats for 8 weeks and found that Phe-induced vasoconstriction decreased after wild blueberry consumption compared with control [98].
Three studies were performed with male animal models of obesity and diabetes. Evidence indicates endothelium-dependent vasodilator responses are exaggerated, whereas vasoconstrictor responses are impaired in obese Zucker rats, likely because of a compensatory adaptation to preserve blood flow in the presence of oxidative stress and inflammation [99-104]. The first study found that male obese Zucker rats fed a diet with 8% w/w freeze-dried wild blueberry powder for 8 weeks had partial restoration of Phe-induced vasoconstriction compared with obese Zucker rats on a control diet. Lean Zucker rats had no improvements [105]. The researchers also found that vasorelaxation (to Ach) increased in lean rats consuming 8% w/w freeze-dried wild blueberry powder added to the diets for 8 weeks compared with the lean control diet, whereas there was no change in blueberry-fed obese rats compared with control diet-fed obese rats [105]. Similarly, an additional study using male obese and lean Zucker rats found obese rats had impaired vasoconstrictor responses to Phe and exaggerated vasorelaxation responses to Ach compared with lean rats and found that vasoconstriction (but not vasorelaxation) was partially restored with the addition of 8% w/w freeze-dried wild blueberry powder added to diets for 8 weeks. Vasorelaxation to Ach was increased in only lean blueberry-fed rats [99]. Lastly, male obese diabetic db/db mice were found to have impaired endothelium-dependent vasorelaxation to Ach relative to control obese mice that was restored by 3.8% w/w freeze-dried wild blueberry powder added to diets for 10 weeks [27].
Overall, the findings from these animal studies suggest that blueberry consumption improves endothelial function through regulation of vascular tone, though differential effects were observed across studies that may be dependent on the animal model and disease state, animal age, and/or the blueberry intervention (dose and length). Although many of the studies used a high dose of blueberry powder (i.e., 8%), studies using 2% and 3.8% added to diets demonstrated beneficial effects, suggesting lower effective doses that are reasonable for daily human consumption are feasible.
4.3. Effects of blueberries on endothelial (dys)function: human studies
4.3.1. Acute blueberry consumption
Nine studies examined the impact of acute blueberry consumption (i.e., single dose) on endothelial function in diverse human populations through assessment of RHI and FMD (Table 3 ). For reference, studies have indicated that an 11 g dose of freeze-dried blueberry powder equates to 100 g fresh wild blueberries, whereas 22 to 26 g equates to 1 cup of fresh highbush blueberries (148–150 g) [28,56,58,106]. A serving size of fresh blueberries is 1 cup (148 g). Five studies evaluated acute effects of blueberries on endothelial function in healthy men. The first study observed no effects on RHI 1 hour after consumption of 300 g of whole highbush blueberries partially thawed into a jelly-like mixture, or a control jelly consisting of 20 g of gelatin plus added sugars made to match the consistency and nutrient composition of the blueberry treatment [107]. Another study investigated the time-course and dose-dependent acute effects of freeze-dried wild blueberry powder mixed with water on FMD in healthy males in 2 different randomized controlled trials [56]. Results from the time-course trial showed that 34 g and 57 g of freeze-dried blueberry powder increased FMD at 1 hour (2.4% and 2.2% unit increase from baseline, respectively) and 2 hours (1.5% and 1.5% unit increase from baseline, respectively) after consumption compared with baseline and placebo consumption. FMD after consumption of 80 g of freeze-dried wild blueberry powder was increased compared with baseline (2.4% unit increase) and placebo at 1 hour and compared with placebo (but not baseline) at 2 hours [56]. The 34-g dose increased FMD 6 hours after consumption compared with baseline (1.2% unit increase) and placebo, whereas the 57-g dose increased FMD compared with placebo (but not baseline) [56]. Next, the dose-dependent study showed that 14-, 28-, and 34-g doses of freeze-dried wild blueberry powder dose-dependently increased FMD 1 hour after consumption and plateaued (~ 2.2% unit increase) thereafter, with no additional benefits observed for 57-g and 80-g doses [56]. Another study conducted in healthy males examined the effects of 34 g of freeze-dried wild blueberry powder in a baked product (processed) or as a drink in water (unprocessed) on FMD [57]. They found that FMD significantly increased at 1, 2, and 6 hours after consumption of the baked product containing the wild blueberry powder compared with the control baked product, with maximal increases (2.6% unit increase) at 2 hours after consumption [57]. The same findings were seen for the unprocessed wild blueberry drink; however, maximal increases in FMD were seen at 1 instead of 2 hours, indicating that food matrix might play a role in determining the acute effects of blueberry consumption on endothelial function in healthy males, likely because of differences in (poly)phenol metabolite bioavailability [57]. Furthermore, these results suggest that food processing with wild blueberry powder may not reduce its beneficial effects on endothelial function. Finally, a more recent study examined FMD in healthy men after consumption of 11 g of freeze-dried wild blueberry powder mixed in water compared with a control drink, control drink + fiber, control drink + vitamins/minerals, and pure ACN drink to understand the possible role of ACNs as major bioactives in blueberry [58]. They found that FMD increased at 2 and 6 hours after wild blueberry powder (~ 2.5% and ~ 2.3% unit increase compared with control, respectively) and ACN drink (~ 2.2% and ~ 2.2% unit increase compared with control) consumption compared with the control drink, with no significant differences between the 2 drinks, and no effects on FMD for the other treatments [58]. These results indicate that 11 g of wild blueberry powder (150 mg of ACNs) is the lowest dose needed to acutely improve endothelial function in healthy men, and that effects are largely the result of ACN contents.
Table 3 –
Human clinical trials examining the impact of acute blueberry consumption on vascular function
| Reference and location |
Study design | Participant baseline characteristics |
Vascular measures |
Blueberry treatment |
Blueberry (poly)phenol concentrations |
Vascular results | Other related results |
|---|---|---|---|---|---|---|---|
| Del Bo et al. (2013) Milan, Italy [107] |
Randomized, crossover, controlled trial (blinding not specified). | Healthy males (n = 10). aAge: 20.8 ± 1.6 y; BMI 22.5 ± 2.1 kg/m2; SBP 119.5 ± 8.8 mmHg; DBP 76.5 ± 6.2 mmHg. aBB group: SBP 119.5 ± 8.8 mmHg; DBP 76.5 ± 6.2 mmHg; RHI 1.96 ± 0.39. a Control group: SBP 122.5 ± 10.4 mmHg; DBP 76.3 ± 4.8 mmHg; RHI 1.94 ± 0.30. |
RHI. Measures taken at baseline and 1 h after treatment consumption. |
300 g whole highbush BB partially thawed into a gelatinous mixture. Control jelly consisted of 20 g food grade gelatin + sugars (~27.1 g total sugars, 16.4 g fructose, 10.7 g glucose, and food colorant) to match BB, which was mixed in 200 mL hot water and cooled to solidify to match BB consistency. Each treatment period separated by 10-d washout. |
727 mg total phenolic acids, 348.3 mg ACNs, and 90.3 mg chlorogenic acid. | No significant effects on vascular measures. | ↓ H2O2-induced DNA damage in participant’s PBMCs at 1 h, not 2 or 24 h, after BB consumption compared with control. |
| Rodriguez-Mateos et al. (2013) England, UK [56] |
Randomized, double-blind, crossover, controlled trial. Time-course trial. |
Healthy males (n = 10). bAge 27 ± 1.3 y; BMI 25 ± 0.8 kg/m2; SBP 123 ± 2.3 mmHg; DBP 71 ± 2.1 mmHg; FMD 7.1 ± 0.1%. b34 g WBB group: FMD 7.0 ± 0.2%; PWV 5.9 ± 0.2 m/s; AIx 2.3 ± 3.9%; DVP-SI 6.0 ± 0.4 m/s; DVP-RI 67 ± 4%. b57 g WBB group: FMD 7.2 ± 0.5%; PWV 5.9 ± 0.2 m/s; AIx 2.3 ± 3.4%; DVP-SI 6.2 ± 0.4 m/s; DVP-RI 78 ± 2%. b80 g WBB group: FMD 7.0 ± 0.3%; PWV 5.8 ± 0.2 m/s; AIx 1.4 ± 3.8%; DVP-SI 6.1 ± 0.2 m/s; DVP-RI 70 ± 5%. bControl group: FMD 7.1 ± 0.3%; PWV 5.9 ± 0.4 m/s; AIx 3.1 ± 5.9%; DVP-SI 6.4 ± 0.2 m/s; DVP-RI 69 ± 7%. |
FMD, PWV, AIx, DVP-SI, and DVP-RI. Measures taken at baseline and 2, 4, and 6 h after treatment consumption. FMD was also measured 1 h after treatment consumption. |
34, 57, and 80 g of freeze-dried WBB powder mixed with 500 mL of low-nitrate water. Placebo drink was matched to the 57 g blueberry drink for sugar (15 g fructose and 13 g glucose) and vitamin C (6.8 mg) Washout period not specified. |
34 g WBB powder contains 766 mg total (poly)phenols, 310 mg ACNs, 137 mg proanthocyanidins, and 273 mg chlorogenic acid. 57 g WBB powder contains 1278 mg total (poly)phenols, 517 mg ACNs, 228 mg proanthocyanidins, and 455 mg chlorogenic acid. 80 g WBB powder contains 1791 mg total (poly)phenols, 724 mg ACNs, 320 mg proanthocyanidins, and 637 mg chlorogenic acid. |
↑ FMD 1 h after WBB consumption (all doses) compared with baseline and placebo. ↑ FMD 2 h after 34 g and 57 g WBB consumption compared with baseline and placebo, whereas ↑ FMD at 2 h for 80-g dose only compared with placebo. ↑ FMD 6 h after WBB consumption for 34 g dose compared with baseline and placebo, whereas ↑ FMD at 6 h for 54-g dose compared only with placebo. No significant effects on other vascular measures. |
↑ Plasma benzoic acid at baseline and 2 h after 34 g WBB consumption compared with placebo. ↑ In 6 plasma (poly)phenol metabolites at increase 1-2 h after 34 g WBB consumption and later increase of 8 metabolites 6 h after 34 g WBB consumption. ↓ Neutrophil NOX activity 1-2 and 6 h after 34 g WBB consumption. Δ Plasma vanillic acid, hippuric acid, and homovanillic acid predicted ↓ in NOX activity. Significant correlation between Δ FMD and NOX activity after 34 g WBB consumption. |
| Rodriguez-Mateos et al. (2013) England, UK [56] |
Randomized, double-blind, crossover, 6-arm controlled trial. Dose-dependent trial. |
Healthy males (n = 11). bAge 27 ± 1.0 y; BMI 22 ± 0.9 kg/m2; SBP 120 ± 1.7 mmHg; DBP 65 ± 1.8 mmHg; FMD 6.3 ± 0.2%. |
FMD. Measures taken at baseline and 1 h after treatment consumption. |
14, 28, 34, 57, and 80 g freeze-dried WBB powder mixed with 500 mL of low-nitrate water. Placebo drink was matched to 57 g WBB drink for sugar (15 g fructose and 13 g glucose) and vitamin C (6.8 mg). Washout period not specified. |
14 g WBB powder contained 319 mg total (poly)phenols, 129 mg ACNs, 57 mg proanthocyanidins, and 114 mg chlorogenic acid. 28 g WBB powder contained 639 mg total (poly)phenols, 258 mg ACNs, 114 mg proanthocyanidins, and 228 mg chlorogenic acid. 34 g WBB powder contained 766 mg total (poly)phenols, 310 mg ACNs, 137 mg proanthocyanidins, and 273 mg chlorogenic acid. 57 g WBB powder contained 1278 mg total (poly)phenols, 517 mg ACNs, 228 mg procyanidins, and 455 mg chlorogenic acid. 80 g WBB powder contained 1791 mg total (poly)phenols, 724 mg ACNs, 320 mg procyanidins, and 637 mg chlorogenic acid. |
↑ FMD at 1 h after WBB consumption up to 34 g WBB and plateaued thereafter for other doses. | No other significant findings. |
| Rodriguez-Mateos et al. (2014) England, UK [57] |
Randomized, crossover, controlled trial. | Healthy males (n = 10). bAge 27 ± 1 y; BMI 25 ± 0.8; SBP 124 ± 2.6 mmHg; DBP 74 ± 2.5 mmHg; FMD 7.0 ± 0.1%. |
FMD. Measures taken at baseline, and 1, 2, 4, and 6 h after treatment consumption. |
Baked product using 34 g freeze-dried WBB powder or 34 g unprocessed freeze-dried WBB powder drink or matched control baked product (bun). Each bun (60 g: 40 g dough and 20 g filling) consisted of the following (% w/w): strong white flour (46.4), freeze-dried WBB powder (12.4), eggs (5.9), butter (5.9), yeast (1.1), salt (0.5), skimmed milk powder (4.6), sugar (7.7), and water. The filling consisted of the following: freeze-dried WBB powder (31.8), sugar (22.2), salt (0.1), evaporated milk (12.6), corn flour (4.6), and eggs (28.7). The control bun was voided of WBB powder and was not specified how the ingredients were adjusted. For the bun interventions, participants consumed 3 buns. Each treatment period separated by 1-week washout. |
Baked WBB product contained 196 ± 7.7 mg ACNs, 140 ± 7.4 procyanidins, and 221 ± 10 mg chlorogenic acid. Unprocessed WBB drink contained 339 ± 6.1 mg ACNs, 111 ± 4.1 procyanidins, and 179 ± 1 mg chlorogenic acid. |
↑ FMD 1, 2, and 6 h after baked WBB product consumption compared with control bun with maximum increase at 2 h. ↑ FMD 1, 2, and 6 h after unprocessed WBB drink consumption compared with control bun with max increase at 1 h (instead of 2 h seen with baked product). |
At 1-2 h after bun consumption, plasma vanillic and ferulic acids predicted ↑ FMD, whereas vanillic and benzoic acids predicted ↑ FMD after unprocessed WBB drink consumption. At 4-6 h after unprocessed WBB drink consumption, plasma hippuric, hydroxy hippuric, and homovanillic acids predicted ↑ FMD after 4-6 h unprocessed WBB drink consumption. Compared with control bun: At 1-2 h, ↑ in 4 metabolites after baked WBB product and ↑ in 6 metabolites after unprocessed BB drink consumption. At 6 h, ↑ in 4 metabolites after baked WBB product and increase in 8 metabolites after unprocessed WBB drink consumption. Compared with unprocessed WBB drink: ↑ Cmax of plasma 3-hydroxyphenylacetic acid after baked WBB product consumption and ↓ in plasma benzoic and hippuric acids. ↓ AUC 0-6 h plasma benzoic, hippuric, salicylic, and sinapic acids and ↑ AUC 0-6 h plasma ferulic, hydroxy hippuric, and 3-hydroxyphenylacetic acids after baked WBB product consumption. |
| Del Bo et al. (2014) Milan, Italy [108] |
Randomized crossover, 3-arm, controlled trial (blinding not specified). | Healthy male smokers, defined as ~15 cigarettes/d (n = 16). bAge 23.6 ± 0.7 y; BMI 23.0 ± 0.5 kg/m2; SBP 116.0 ± 1.7 mmHg; DBP 76.1 ± 2.1 mmHg; RHI 2.23 ± 0.07; F-RHI 0.65 ± 0.07; dAIx −8.6 ± 2.0%; dAIX @ 75 bpm −18.4 ± 2.2%. |
RHI, F-RHI, dAIx, and dAIx @ 75 bpm. Measures were taken at baseline and 20 min after treatment consumption. |
300 g whole highbush BB + smoking, control treatment (300 mL water with 16 g fructose, and 11 g glucose to match BB sugar content) + smoking, or smoking alone with no BB or control treatment. Each treatment period separated by 1-week washout. |
726.6 mg total phenolic acids, 348.3 mg ACNs, and 90.3 mg chlorogenic acid. | ↓ RHI, F-RHI, dAIx, and dAIx @ 75 bpm after smoking. Highbush BB consumption counteracted impairments in RHI and F-RHI. No significant effects on other vascular measures. |
BB significantly counteracted ↑ SBP from smoking. |
| Del Bo et al. (2017) Milan, Italy [109] |
Randomized, crossover, 2-arm controlled pilot trial (blinding not specified). | Nonsmoking men with peripheral arterial dysfunction, defined as RHI ≤ 1.67 (n = 12). bAge 24.2 ± 1.2 y; BMI 22.5 ± 1.2 kg/m2; SBP 116.9 ± 3.2 mmHg; DBP 75.3 ± 2.9 mmHg; RHI 1.41 ± 0.07; dAIx −14.6 ± 2.7%; dAIX @ 75 bpm −20.0 ± 5.8%. |
RHI, dAIx, and dAIx @ 75 bpm. Measures were taken at baseline and 120 min after treatment consumption. |
300 g whole highbush BB or control treatment (300 mL water with 16.4 g fructose and 10.6 g glucose to match BB sugar content). Each treatment period separated by 1-week washout. |
856 mg total phenolic acids, 309 mg ACNs, and 30 mg chlorogenic acid. | ↑ RHI after highbush BB consumption in nonsmokers compared with control. No significant effects on other vascular measures. |
No other significant findings. |
| Del Bo et al. (2017) Milan, Italy [109] |
Randomized, crossover, 3-arm controlled pilot trial (blinding not specified). | Smoking men with peripheral arterial dysfunction, defined as RHI ≤ 1.67 (n = 12). bAge 24.5 ± 1.9 y; BMI 22.9 ± 1.1 kg/m2; SBP 118.2 ± 2.9 mmHg; DBP 75.7 ± 2.7 mmHg; RHI 1.47 ± 0.05; dAIx −12.7 ± 2.5%; dAIx @ 75 bpm −18.2 ± 5.0%. |
RHI, dAIx, and dAIx @ 75 bpm. Measures were taken at baseline and 120 min after treatment consumption. |
300 g whole highbush BB + smoking, control treatment (300 mL water with 16.4 g fructose and 10.6 g glucose to match BB sugar content) + smoking, or smoking alone with no BB or control treatment. Each treatment period separated by 1-week washout. |
856 mg total phenolic acids, 309 mg ACNs, and 30 mg chlorogenic acid. | ↑ RHI in highbush BB + smoking and control treatment + smoking groups compared with smoking alone. No significant effects on other vascular measures. |
No other significant findings. |
| Dodd et al. (2019) Berkshire, UK [125] |
Randomized, crossover, controlled trial (blinding not specified). | Older adults with cognitive decline (n = 18). aAge 68.72 ± 3.30 y; BMI 25.89 ± 4.46 kg/m2; SBP 135.02 ± 7.34 mmHg; DBP 78.55 ± 3.38 mmHg. |
DVP (secondary outcome). Measures taken at baseline and 1 h after treatment consumption. |
Freeze-dried highbush BB powder (~ 30 g) and control powder mixed (19.92 g total sugars, 9.94 g fructose, 9.76 g glucose, 0.22 g sucrose, 1 g citric acid, and 19 mg vitamin C) in 300 mL semi-skimmed milk. Washout period not specified. |
507.79 mg ACNs and 71.03 mg epicatechin oligomers. | No significant effects on vascular measures. | BB treatment trended toward attenuation of ↑ SBP after control treatment. |
| Rodriguez-Mateos et al. (2019) England, UK [58] |
Randomized, double-blind, crossover, 5-arm controlled trial. | Healthy males (n = 5). aAge 23 ± 3 y; BMI 24 ± 3 kg/m2; SBP 124 ± 11 mmHg; DBP 71 ± 9 mmHg. |
FMD. Measures taken at baseline and 1, 2, and 6 h after treatment consumption. |
5 different drinks consisting of: (1) 11 g freeze-dried WBB powder drink mixed with 500 mL of low-nitrate water, (2) control drink (3.5 g fructose), (3) control drink + fiber (3.5 g fructose and 5 g dietary fiber), (4) control drink + minerals and vitamins (3.5 g fructose, 5 g dietary fiber, 75 mg potassium, 50 IU total beta carotene, 12.5 mg vitamin C, 20 mg calcium, 0.63 mg iron, 1.88 IU vitamin E, 0.18 mg vitamin B1, 0.1 mg vitamin B2, 0.25 mg vitamin B6, 15.6 mg phosphorus, 12.5 mg magnesium, 0.63 mg zinc, 2.25 mg manganese, and 2.5 mg niacin), and (5) 160 mg pure ACNs drink. Each treatment period separated by 1-week washout. | WBB drink: 150 mg ACNs and 64 mg chlorogenic acid. ACNs drink: 160 mg ACNs. |
↑ FMD at 2 and 6 h after WBB and ACNs drink consumption compared with control drink. | Plasma ferulic acid-4-O-sulfate, isoferulic acid-3-O-β-D-glucuronide, dihydroferulic acid, dihydroferulic acid 4-O-β-D-glucuronide, chlorogenic acid, vanillic acid, homovanillic acid, protocatechuic acid, syringic acid, 4-hydroxybenozic acid, 4-methylgallic acid-3-O-sulfate, 1-methylpyrogallol-O-sulfate, 4-hydroxyphenyl acetic acid, and quercetin 3-O–β-D-glucuronide all significantly correlated to 2-h FMD in WBB group. |
| Curtis et al. (2022) Anglia, UK [106] |
Randomized, double-blind, parallel arm, controlled trial. | Adults with metabolic syndrome (n = 45). aAge 63.2 ± 8.8 y; aBMI 31.5 ± 2.9 kg/m2; cSBP 136 (133–140) mmHg; cDBP 82.9 (81.0–84.8) mmHg; cFMD 2.1 (1.6–2.7)%; cAIx @ 75 bpm 35.7 (33.7–37.7)%; cPWV 11.0 (10.5–11.4) m/s. |
FMD, AIx @ 75 bpm, and cfPWV. Measures were taken at baseline, and then at 3, 6, and 24 h after consumption. |
26 g freeze-dried highbush BB or 26 g placebo-matched powder (dextrose, maltodextrin, and fructose (31% glucose, 30% fructose, and 0% sucrose), which were produced as purple powder, and BB aromatics and artificial coloring). Powder added to 500 g energy-dense emulsion meal and 50 g water into opaque shaker bottle and consumed by participant within 15 min. |
879 mg total phenolic acids and 364 mg ACNs. | No significant effects on vascular measures. | ↑ Serum and urine ACNs metabolite concentrations 24 h after consumption, with greater ↑ serum concentrations at 6 and 24 h, and greater ↑ urinary concentrations at 6–24 h compared with placebo. ↑ Postprandial plasma glucose 1 h and ↓ postprandial plasma glucose and insulin in BB group 3 h compared with placebo. ↑ HDL at 90 min and 6 h in BB group compared with placebo. ↑ apo-A, XL-HDLP, and L-HDLP at 6 h as well as XL-HDLP at 3 h in BB compared with placebo. ↓ TC at 24 h in BB compared with placebo. |
Abbreviations: ACNs, anthocyanins; AIx, augmentation index; AIx @ 75 bpm, augmentation index normalized to 75 beats/minute; Apo-A1, apolipoprotein A-1; AUC, area under the curve; baPWV, brachial-ankle pulse wave velocity; BB, blueberry; BMI, body mass index; BP, blood pressure; cfPWV, carotid-femoral pulse wave velocity; Cmax, maximum concentration; dAIx, digital augmentation index; dAIx @ 75 bpm, digital augmentation index @ 75 beats/minute; DBP, diastolic blood pressure; DVP-RI, digital volume pulse reflection index; DVP-SI, digital volume pulse stiffness index; FMD, flow-mediated dilation; F-RHI, Framingham reactive hyperemia index; HDL-C, high-density lipoprotein-cholesterol; NO, nitric oxide; PBMC, peripheral blood mononuclear cells; PWA, pulse wave analysis; PWV, pulse wave velocity; RHI, reactive hyperemia index; SBP, systolic blood pressure; XL/L-HDLP, extra-large/large high-density lipoprotein particles; TC, total cholesterol; WBB, wild blueberry.
Data are presented as mean ± SD.
Data are presented as mean ± SEM.
Data are presented as mean (ranges).
Three studies examined acute consumption of 300 g of whole fresh highbush blueberries on endothelial function via RHI in smoking and nonsmoking men [108,109] and found that RHI decreased in healthy men after smoking and highbush blueberry consumption counteracted these impairments 20 minutes after consumption [108]. There was also an increase in RHI 120 minutes after consumption of whole highbush blueberries in nonsmokiaung men with peripheral arterial dysfunction (defined as RHI ≤ 1.67) compared with control treatment [109]. However, in smoking men with peripheral arterial dysfunction, RHI increased 120 minutes after whole highbush blueberry and control treatment consumption compared with smoking alone [109]. The results of these studies suggest that blueberry consumption may attenuate smoking-induced impairments in endothelial function in healthy men and men with peripheral arterial dysfunction. Lastly, the impact of 26 g of freeze-dried highbush blueberry added into a 500-g energy-dense emulsion meal + 50 g of water on FMD in adults with metabolic syndrome was evaluated at baseline (0 hours) and 3, 6, and 24 hours after consumption, and no effects were observed, potentially because of impacts of the meal [106]. Collectively, these studies strongly suggest that acute blueberry consumption improves endothelial function in healthy men and smoking and nonsmoking men with and without peripheral arterial dysfunction. They also suggest that the beneficial impacts of blueberries may be reduced when consumed with a high-fat meal. Biphasic time-dependent improvements in endothelial function have been observed 1 to 2 and 6 hours after blueberry consumption that were paralleled by and correlated with increases in circulating (poly)phenol metabolites, suggesting blueberry (poly)phenol metabolites mediate improvements [56]. It is difficult to determine the exact source of blueberry (poly)phenol metabolites because they are not specific to blueberries and can be formed from different parent compounds and through different processes (e.g., phase II metabolism, gut microbial metabolism, degradation) (Fig. 1). Evidence suggests that (poly)phenol metabolites peaking 1 to 2 hours following blueberry consumption reflect upper GI tract absorption and metabolism, whereas the later response reflects gut microbial metabolism in the lower GI tract. Regarding differential responses observed across studies, factors determining responses cannot be established based on the current evidence. It is possible that compositional differences in blueberry treatments impacted responses but they appear to play a minor role. For example, incorporation of freeze-dried wild blueberry powder into a baked product changed the (poly)phenol composition compared with unprocessed freeze-dried wild blueberry powder. These changes had differential impacts on plasma (poly)phenol metabolite concentrations and the timing of the early FMD peak (i.e., blueberry powder in a drink peaked at 1 hour whereas blueberry powder in a baked product peaked at 2 hours). However, both treatments led to FMD increases at 1, 2, and 6 hours after blueberry consumption, suggesting that the food processing impacts on the food matrix do not have major effects on endothelial function responses. Rather, differential responses observed across studies may be related to differences in methodology, including endothelial function assessment and populations studied. Further research is needed in diverse populations to establish these acute effects and their generalizability to other healthy sexes and genders, races and ethnicities, and individuals and populations with chronic diseases and/or risk factors.
4.3.2. Short-term and chronic blueberry consumption
Eight studies examined the impact of short-term and chronic blueberry consumption (i.e., more than one dose for more than one day) on endothelial function in humans through assessment of RHI or FMD (Table 4 ). First, two studies were performed in healthy men. In the first study, FMD increased after 1 week (~ 1% unit increase compared with baseline) of consuming 22 g of freeze-dried wild blueberry powder mixed in water, with further improvements observed at 2 weeks (almost a 2% unit increase compared with baseline) but plateaued thereafter up to 28 days [58]. In a different subset of healthy men, similar increases in FMD were observed 2 hours following acute (1.5% unit increase compared with baseline) and 28 days following chronic (2.3% unit increase compared with baseline) consumption of 22 g/day of freeze-dried wild blueberry powder compared with placebo powder, whereas no acute on chronic improvements in FMD were observed on day 28 (i.e., after consuming a single dose of 22 g of freeze-dried wild blueberry powder following 28 days of daily wild blueberry powder consumption) [58].
Table 4 –
Human clinical trials examining the impact of short-term and chronic blueberry consumption on vascular function
| Reference and location |
Study design |
Participant baseline characteristics |
Vascular measures |
Blueberry treatment |
Blueberry (poly)phenolic concentrations |
Vascular results |
Other related results |
|---|---|---|---|---|---|---|---|
| Riso et al. (2013) Milan, Italy [110] |
Randomized, crossover, controlled trial (blinding not specified). | Healthy males with at least one CVD risk factor (n = 18). aAge 47.8 ± 9.7 y; BMI 24.8 ± 2.6 kg/m2; SBP 121 ± 16 mmHg, and DBP 79.4 ± 8.7 mmHg; RHI 1.84 ± 0.46; F-RHI 0.32 ± 0.27; AIx 5.22 ± 18.5; AIx @ 75 bpm 0.0 ± 17.4. |
RHI, F-RHI, AIx, and AIx @ 75 bpm. Measures taken at baseline and after 6 weeks of treatment consumption. |
25 g freeze-dried WBB powder or placebo powder containing similar sensory characteristics as WBB powder (7.5 g fructose, 7 g glucose, 0.5 g citric acid, and 0.03 g BB flavor) mixed with 250 mL of water and consumed daily for 6 wks. Each treatment period separated by 6-wk washout. |
WBB treatment contained 375 mg ACNs and 127.5 mg chlorogenic acid. | No significant effects on vascular measures. | ↓ Endogenous oxidized DNA bases and H2O2-induced DNA damage in blood mononuclear cells in WBB group compared with placebo. |
| McAnulty et al. (2014) North Carolina, USA [127] |
Randomized, double-blind, parallel arm, controlled trial. | Sedentary men and postmenopausal women (n = 25). aBB group (n = 13): age 46.15 ± 11.92 y; BMI 27.8 ± 5.46 kg/m2; SBP 117.23 ± 7.85 mmHg; DBP 74.61 ± 11.46 mmHg; cfPWV 8.4 ± 1.1 m/s2; AIx 18.91 ± 11 m/s2. aPlacebo group (n = 12): age 39.92 ± 13.38 y; BMI 24.23 ± 3.44 kg/m2; SBP 113.53 ± 10.39 mmHg; DBP 70.15 ± 8.8 mmHg; cfPWV 8.8 ± 1.9 m/s2; AIx 23.2 ± 7.79 m/s2. |
cfPWV and AIx. Measures taken at baseline and after 6 wks of treatment consumption. | 19 g Tifblue/Rubel BB powder or 19 g placebo powder (blend of maltodextrin, fructose, BB flavoring, coloring, citric acid, silica) consumed 2x/d (38 g/d total) each night with evening meal for 6 wk. | N/A | ↓ AIx in BB group compared to placebo. No significant effects on other vascular measures. |
↓ Aortic systolic pressure in BB group compared with placebo. ↓ DBP in prehypertensive participants. ↑ NK cells in BB group compared with placebo. |
| Johnson et al. (2015) Florida, USA [128] |
Randomized, double-blind, placebo-controlled, parallel arm, controlled trial. | Postmenopausal women with elevated BP or stage 1 HTN (n = 40). aBB group (n = 20): age 59.7 ± 4.58 y; BMI 30.1 ± 5.94 kg/m2; SBP 138 ± 14 mmHg; DBP 80 ± 7 mmHg; baPWV 1498 ± 179 cm/s; cfPWV 1234 ± 201 cm/s. 2 Placebo group (n = 20): age 57.3 ± 4.76 y; BMI 32.7 ± 6.54 kg/m2; SBP 138 ± 15 mmHg; DBP 78 ± 8 mmHg; baPWV 1,464 ± 194 cm/s; cfPWV 1,233 ± 194 cm/s. |
baPWV and cfPWV. Measures taken at baseline, 4 wk, and 8 wk. |
11 g freeze-dried highbush BB powder or 11 g calorie-matched placebo powder mixed (0.02 g fat, 20.82 g total carbohydrates consisting of maltodextrin and fructose, 0.17 g protein, natural BB flavoring, artificial color, citric acid, and silica dioxide) in 1 cup (240 mL) of water consumed 2x/d (22 g/d total) for 8 wk. | BB treatment contained 844.58 mg phenolics and 469.48 mg ACNs. | ↓ baPWV in BB group compared with baseline and placebo. No significant effects on other vascular measures. |
↓ SBP and DBP in BB group compared with baseline and placebo. ↑ plasma NO in BB group compared with baseline. |
| Stull et al. (2015) Louisiana, USA [112] |
Randomized, double-blind, parallel arm, controlled trial. | Adults with metabolic syndrome (n = 44). bBB group (n = 23): age 55 ± 2 y; BMI 35.2 ± 0.8 kg/m2; SBP 125.7 ± 2.2 mmHg; DBP 82.7 ± 1.9 mmHg; RHI 1.94 ± 0.11 (n = 22 for RHI). bPlacebo group (n = 21): age 59 ± 2 y; BMI 42.8 ± 1.6 kg/m2; SBP 125.0 ± 3.2 mmHg; DBP 77.5 ± 1.9 mmHg; RHI 2.34 ± 0.16 (n = 18 for RHI). |
RHI. Measures were taken at baseline and after 6 wks of treatment consumption. |
45 g freeze-dried highbush BB powder or placebo-matched powder (51 g carbohydrates, 4.5 g fiber, 7.9 g protein, 1.0 g fat, 0.6 g saturated fat, 1146 mg sodium, 807.3 IU vitamin A, 0.04 mg iron, and 287.5 mg calcium) added in a smoothie containing 12 oz yogurt and skim milk consumed 2x/d (22.5 g) for 6 wk. | BB treatment contained 773.6 mg phenolics and 290.3 mg ACNs. | ↑ RHI in BB group compared with control. | No other significant results. |
| Stote et al. (2017) New York, USA [111] |
Randomized, crossover, single-blind, controlled trial. | Adults at risk for type 2 diabetes (n = 19). aAge 53 ± 6.3 y; BMI 31.4 ± 2.9 kg/m2; SBP 126.6 ± 14.0 mmHg; DBP 81.7 ± 8.7 mmHg. |
RHI. Measures were taken at baseline and after 7 d of treatment consumption. |
120 mL WBB juice and 120 mL placebo drink (27.5 g total sugars, 13 g glucose, 14.5 g fructose, artificial BB flavoring, and tartaric acid) containing similar characteristics to BB but without (poly)phenols consumed 2x/d (240 mL) for 7 d. Each treatment period separated by 8-d washout. |
WBB treatment contained 2138 mg phenolics, and 314 mg ACNs. | No significant effects on vascular measure. | Trend ↓ SBP after WBB treatment. ↑ Plasma nitrate and nitrite concentrations after WBB treatment compared with placebo. |
| Curtis et al. (2019) Anglia, UK [113] |
Randomized, double-blind, parallel arm, controlled trial. | Adults with metabolic syndrome (n = 115). aBB group (13 g) (n = 37): age 62.6 ± 7.2 y; BMI 31.2 ± 2.6 kg/m2; 3SBP 134–138 mmHg; cDBP 80.3–83.0 mmHg. 1BB group (26 g) (n = 39): age 63.0 ± 5.9 y; BMI 31.3 ± 3.4 kg/m2; cSBP 134-138 mmHg; cDBP 79.6–82.2 mmHg. aPlacebo group (n = 39): age 62.9 ± 8.1 y; BMI 31.1 ± 3.0 kg/m2; cSBP 134–138 mmHg; cDBP 79.9–82.5 mmHg. |
FMD, AIx, and cfPWV. Measures taken at baseline and after 6 mo of treatment consumption. |
3 interventions: (1) 26 g freeze-dried highbush BB, (2) 13 g freeze-dried BB and 13 g placebo-matched powder, and (3) 26 g placebo-matched powder consumed as drink/smoothie, or added to cereals/yogurts, banana toast, or to salads as a dressing 1x/d for 6 mo. Placebo powder contained dextrose, maltodextrin, and fructose (31% glucose, 30% fructose, and 0% sucrose) which were produced as purple powder, and BB aromatics and artificial coloring. | 26 g BB treatment contained 364 mg ACNs and 879 mg phenolics. 13 g BB treatment contained 182 mg ACNs and 439.5 mg phenolics. |
↑ FMD after 26 g BB consumption compared to 13 g BB dose and placebo. ↓ AIx after 26 g BB consumption compared with 13 g dose and placebo. No significant effects on other vascular measures. |
↑ Mean plasma cGMP concentrations after 26 g BB treatment compared with placebo. ↑ Serum and 24-h urine ACNS concentrations after BB treatment in a dose-dependent manner. ↑ HDL-C, ApoA1, and HDL-P in non-statin users after 26 g BB consumption compared with placebo. |
| Rodriguez-Mateos et al. (2019) England, UK [58] |
Uncontrolled, single-arm pilot study (blinding not specified). | Healthy males (n = 5). 2Age 24 ± 2 y; BMI 24 ± 3 kg/m2; SBP 122 ± 7 mmHg; DBP 73 ± 8 mmHg. |
FMD. Measures taken at baseline and 7, 14, 21, and 28 d after treatment consumption. |
11 g WBB powder consumed 2x/d (22 g total) mixed with 500 mL water for 28 d. | WBB treatment contained 150 mg ACNs, 49 mg flavanol monomers, 31 mg flavanol oligomers, and 64 mg chlorogenic acid. | ↑ FMD after 1 wk of WBB treatment with further improvement at 2 wk and plateaued thereafter. | No other related outcomes measured. |
| Rodriguez-Mateos et al. (2019) England, UK [58] |
Randomized, double-blind, parallel 2-arm, acute-on-chronic, controlled trial. | Healthy males (n = 40). aAge 33 ± 6 y; BMI 24 ± 3 kg/m2; SBP 128 ± 10 mmHg; DBP 75 ± 9 mmHg. |
FMD, cfPWV, and AIx. Measures taken at baseline and after 28 d of treatment consumption. For the acute-on-chronic study, measures were taken at 0 and 2 h after blueberry consumption on day 28 of intervention. |
11 g WBB powder or placebo-matched powder (3.5 g fructose) consumed 2x/d (22 g total) mixed with 500 mL water for 28 d. | WBB treatment contained 150 mg ACNs, 49 mg flavanol monomers, 31 mg flavanol oligomers, and 64 mg chlorogenic acid. | ↑ FMD after 28 d of WBB treatment compared with placebo. No further improvement in FMD after acute consumption of WBB treatment on 28th day. No significant effects on other vascular measures. |
↓ 24-h SBP in WBB group compared with placebo. 21 plasma (poly)phenol metabolites significantly correlated to FMD increase after WBB consumption. 357 genes upregulated and 251 genes downregulated in PBMCs after 28 d of WBB treatment. 3 miRNA differentially expressed in PBMCs after 28 d of WBB treatment. |
| Wang et al. (2022) England, UK [126] |
Randomized, crossover, controlled trial. | Healthy adults (n = 37). aAge 25.86 ± 6.81; BMI 23.15 ± 3.12. bWhole BB group (n = 37): SBP 108.727 ± 1.411 mmHg; DBP 64.059 ± 1.440 mmHg; cfPWV 7.630 ± 0.153 m/s. bBB powder group (n = 37): SBP 110.278 ± 1.711 mmHg; DBP 64.333 ± 1.482 mmHg; cfPWV 7.993 ± 0.258 m/s. bPlacebo group (n = 37): SBP 109.314 ± 1.691 mmHg; DBP 63.676 ± 1.395 mmHg; cfPWV 8.265 ± 0.209 m/s. |
cfPWV. Measures taken at baseline and after 1 wk of treatment consumption. |
160 g fresh whole highbush BB, 20 g freeze-dried BB powder, and control capsule containing plant-derived fiber microcrystalline cellulose (1.0 g total carbohydrates) consumed 1x/d for 1 wk. Each treatment period separated by 1-wk washout. |
Fresh highbush BB contained 220.48 mg/d total (poly)phenols. Freeze-dried BB powder contained 288.43 mg/d total (poly)phenols. |
No significant effects on vascular measures. | No other significant results. |
| Woolf et al. (2023) Colorado, USA [28] |
Randomized, double-blind, parallel arm, controlled trial. | Postmenopausal women with elevated BP or stage 1-HTN (n = 43). bBB group (n = 22): age 60 ± 1 y; BMI 27.6 ± 1.0 kg/m2; SBP 130 ± 1 mmHg; DBP 80 ± 1 mmHg; FMD 3.84 ± 1.02%; cfPWV 7.9 ± 0.2 m/s; AIx 36 ± 1%; AIx @ 75 bpm 29 ± 2%. 1Placebo group (n = 21): age 57.3 ± 4.76 y; BMI 27.8 ± 61.1 kg/m2; SBP 128 ± 1 mmHg; DBP 82 ± 1 mmHg; FMD 4.69 ± 0.55%; cfPWV 7.4 ± 0.2 m/s; AIx 37 ± 1%; AIx @ 75 bpm 31 ± 1%. |
FMD, FMD/SRAUC, cfPWV, AIx, and AIx @ 75 bpm. Measures taken at baseline and after 12 wk of treatment consumption. |
11 g freeze-dried highbush BB powder or 11 g placebo powder matched for calories (<1 g fat, 21 g total carbohydrates that consisted of dextrose, maltodextrin, and fructose, <1 g protein, and artificial coloring and flavoring) mixed in 1 cup (240 mL) of water consumed 2x/d (22 g/d total) for 12 wk. | BB treatment contained 766 mg total (poly)phenols and 224 mg ACNs. | ↑ FMD/SRAUC after 12 wk of BB consumption and the Δ from baseline to 12 wk in FMD/SRAUC ↑ compared with placebo group. FMD statistically nonsignificant but a clinically significant unit increase of 1.34% after 12 wk of BB consumption compared with baseline. No significant effects on other vascular measures. |
Supraphysiologic ascorbic acid infusion acutely ↑ FMD/SRAUC in both groups at baseline but significant differences went away at 12 wk in the BB group while trending differences were still present for placebo group at 12 wk, and the difference between ascorbic acid and saline FMD/SRAUC ↓ at 12 wk compared with baseline in the BB group but not for the placebo group. ↑ Sum of plasma (poly)phenol metabolites in BB group compared with baseline and placebo at 4, 8, and 12 wk. Δ from baseline to 4 and 12 wk in sum of (poly)phenol metabolites ↑ in BB group compared with placebo. ↑ Plasma hippuric acid, 3-hydroxyhippuric, vanillic acid-4-sulfate, protocatechuic acid, and 2,5-dihydroxybenzoic acid in BB group. Δ in 8 plasma (poly)phenol metabolites from baseline to 12 wk derived from myricetin, phenyl propanoic acid, benzoic acid, cinnamic acid, and phenylacetic acid were correlated with Δ FMD/SRAUC from baseline to 12 wk in BB group but not placebo. 1 plasma (poly)phenol metabolite at 12 wk derived from benzoic acid was correlated to Δ in FMD/SRAUC from baseline to 12 wk in BB group. Δ p47phox from baseline to 12 wk was negatively correlated Δ FMD/SRAUC from baseline to 12 wk, and p-eNOS at 12 weeks was positively correlated to Δ in FMD/SRAUC from baseline to 12 wk in BB group. |
| Wood et al. (2023) England, UK [60] |
Randomized, double-blind, parallel arm, controlled trial. | Healthy older adults aged 65–80 y (n = 61). aBB group (n = 32): age 69.44 ± 3.48 y; BMI 24.57 ± 2.7 kg/m2; SBP 128.52 ± 11.63 mmHg; DBP 81.05 ± 7.86 mmHg; FMD 3.62 ± 1.53%; cfPWV 7.94 ± 3.07 m/s; AIx @ 75 bpm 27.8 ± 7.11%. aPlacebo group (n = 29): age 70.76 ± 3.81 y; BMI 23.16 ± 2.59 kg/m2; SBP 128.36 ± 10.01 mmHg; DBP 79.59 ± 5.59 mmHg; FMD 4.11 ± 1.14%; cfPWV 8.47 ± 2.35 m/s; AIx @ 75 bpm 29.4 ± 11.0%. |
FMD, cfPWV, and AIx @ 75 bpm. Measures taken at baseline and after 12 wk of treatment consumption. |
26 g freeze-dried WBB powder or 26 g placebo-matched powder (17.6 g total carbohydrates, 9.23 g fructose, 8.32 g glucose, 5.17 g dietary fiber, 4.16 g insoluble fiber, 1.01 g soluble fiber, and 90 vitamin C) mixed in water consumed 1x/d for 12 wk. | WBB treatment contained 302 mg ACNs and 202.1 mg chlorogenic acid. | ↑ FMD after 12 wk of WBB compared with placebo. No significant effects on other vascular measures. |
↓ 24-h SBP in WBB group compared with placebo at 12 wk. ↑ Total 24-h urinary (poly)phenol excretion in the WBB group compared with placebo at 12 wk. ↑ Plasma pyrogallol-O-sulfate, 2 methylpyrogallol-O-sulfate, 4-methylcatechol-O-sulfate, isoferulic acid, and 4-methylcatechol acid in WBB compared with placebo at 12 wk. ↓ Plasma phenylacetic acid and vanillic acid in plasma from WBB group compared with placebo group at 12 wk. Δ in 6 plasma (poly)phenol metabolites derived from cinnamic acid, benzoic acid, phenylacetic acid, and hippuric acid from baseline to 12 wk were correlated to Δ in FMD from baseline to 12 wk. |
Abbreviations: ACNs, anthocyanins; AIx, augmentation index; AIx @ 75 bpm, AIx normalized to 75 bpm; ApoA1, apolipoprotein A1; baPWV, brachial-ankle pulse wave velocity; BB, blueberry; BMI, body mass index; BP, blood pressure; cfPWV, carotid-femoral pulse wave velocity; crPWV, carotid-radial pulse wave velocity; DBP, diastolic blood pressure; FMD, flow-mediated dilation; F-RHI, Framingham-reactive hyperemia index; HDL, high-density lipoprotein particle concentration; HDL-C, high-density lipoprotein cholesterol; H2O2, hydrogen peroxide; HTN, hypertension; N/A, not available; NO, nitric oxide; p47phox, NADPH oxidase/p47; p-eNOS, phosphorylated endothelial nitric oxide synthase; PWV, pulse wave velocity; RHI, reactive hyperemia index; SBP, systolic blood pressure; WBB, wild blueberry.
Data are presented as mean ± SD.
Data are presented as mean ± SEM.
Data are presented as ranges.
Six studies were performed in populations with cardiometabolic risk factors. One study examined the effects of consuming 25 g/day freeze-dried wild blueberry powder mixed in water for 6 weeks on RHI in heathy men with at least one CVD risk factor but observed no statistically significant effects [110]. Another study in adults at risk for type 2 diabetes found that 240 mL of wild blueberry juice consumption for 7 days did not lead to statistically significantly improvements in RHI [111]. However, another study in adults with metabolic syndrome observed an increase in RHI (0.32-unit increase) compared with control following consumption of 45 g/day of freeze-dried highbush blueberry powder mixed in a yogurt smoothie (12 oz yogurt and skim milk) for 6 weeks [112]. Similarly, in adults with metabolic syndrome, 26 g/day of freeze-dried highbush blueberry powder consumed as drink/smoothie or added to cereals, yogurts, banana toast, or as a salad dressing for 6 months improved FMD (1.45% unit increase compared with baseline) compared with the 13-g dose of freeze-dried highbush blueberry powder and the 26 g dose of placebo powder [113]. In postmenopausal women with above-normal blood pressure, daily consumption of 22 g/day of freeze-dried highbush blueberry powder mixed in water for 12 weeks improved FMD/SRAUC by 96% compared with baseline and placebo [28]. Furthermore, there was a clinically (but not statistically) significant 1.34% unit increase in FMD not normalized to shear rate after 12 weeks of blueberry consumption and no change in the placebo group [28]. Finally, healthy older adults had increased FMD following 12 weeks of consuming 26 g/day freeze-dried wild blueberry powder mixed in water compared with placebo [60].
In conclusion, these studies indicate that short-term and chronic blueberry consumption can improve endothelial function in healthy adults and those with CVD risk factors. In particular, evidence suggests daily chronic consumption of at least 22 g/day of freeze-dried blueberry powder for at least 1 week and up to 6 months can improve endothelial function in healthy men and adults with major CVD risk factors (i.e., metabolic syndrome, postmenopausal women with above-normal blood pressure). Whether the different doses and intervention durations can be generalized beyond each study is not known and requires further investigation. It is possible that higher doses are needed to observe improvements in endothelial function in chronic intervention periods of shorter duration (e.g., 1–6 weeks) for populations with CVD and type 2 diabetes risk factors. Dose-dependent and time-course studies in diverse human populations (e.g., sex, gender, race, ethnicity, disease risk or clinical disease) are needed to further establish the effects of blueberry consumption on endothelial function.
5. Blueberries and arterial stiffness
5.1. Arterial stiffness overview and methods of assessment
Arterial stiffness occurs due to functional and structural changes in the tunica media and is characterized by the loss of arterial elasticity and compliance [3,64]. With each heartbeat, a pulse wave is generated that travels down the aorta and arterial tree buffering arteries for dilation [64]. Healthy elastic arteries distend during systole and recoil during diastole to facilitate continuous blood flow throughout the body to maintain proper blood pressure and flow [64]. However, changes to the arterial wall seen with advancing age and other CVD risk factors, including oxidative stress, inflammation, and reduced NO bioavailability, result in increased collagen deposition (i.e., fibrosis), elastin fragmentation, and advanced glycation end-product formation, which interferes with arterial distensibility and promotes arterial stiffening [3,6]. Other factors, including sympathetic nervous system activity and the renin-angiotensin-aldosterone system, may also contribute [114]. With this, arteries are less able to buffer the pulse wave generated by the heart, leading to an early arrival of the reflection wave to the aorta and resulting in increased systolic blood pressure and an unchanged (or lower) diastolic blood pressure (i.e., isolated systolic HTN), and is considered a robust predictor of CVD risk [6].
Clinically, arterial stiffness can be assessed noninvasively at various locations along the vascular tree, and several assessments exist. For the purpose of this review, only three noninvasive methods used in published studies that assessed the impact of blueberries on arterial stiffness are included: pulse wave velocity (PWV), augmentation index (AIx), and digital volume pulse (DVP). PWV is the speed of arterial pressure wave that is propagated throughout the arterial tree and is commonly used to assess arterial stiffness because of its feasibility to measure, high reproducibility, and ability to predict CVD events [3,6,115]. Using a Doppler ultrasound or an applanation tonometer, PWV is calculated as the distance traveled and pulse transit time between two arterial sites [6,115]. A higher PWV value is indicative of arterial stiffness because pulse waves travel faster down the arterial trees in less compliant vessels. The carotid to femoral (cfPWV) arterial segment, also referred to aortic PWV (aPWV), is reflective of aortic stiffness and considered to be the gold standard [3,115,116]. Brachial to ankle PWV (baPWV) is also used to assess arterial stiffness but is reflective of both central and peripheral arterial stiffness. Nonetheless, both assessments have been shown to be independent predictors of CVD events [115-119]. PWV is affordable and validated, but there is possible error associated with distance estimation, measurement in individuals with excess body fat or obesity only allows for regional assessment, and misrepresentation in the acquisition signal [120].
Another noninvasive but more indirect measure of arterial stiffness is AIx, which is the proportion of central pulse pressure caused by arterial wave reflection and is derived during pulse wave analysis assessments. AIx has been shown to predict CVD mortality [121]. Pulse wave analysis, and thus, AIx can be assessed through applanation tonometry, echo tracking, or volumetric displacement using a blood pressure cuff [3]. Its advantages include its noninvasive and simplistic attributes in addition to its validity and reproducibility [3]. However, certain physiological factors such as heart rate, sex, blood pressure, height, and endothelial function can affect the outcome value, which is why it is recommended that AIx also be normalized to heart rate (AIx @t 75 beats/min, AIx @ 75 bpm) and used in conjunction with PWV to assess arterial stiffness [3]. Finally, DVP assesses pulse waveforms through measuring absorption of infrared light across the finger (i.e., photoplethysmography) [56,122,123]. DVP is validated, reproducible with low intra-observer variation, and comparable to PWV [124].
5.2. Effects of blueberries on arterial stiffness: animal studies
No published studies that evaluated the impact of blueberry consumption on arterial stiffness in animals were identified. This is a major gap in the knowledge that would benefit from further investigation because it may provide insight into the impact of blueberries on arterial stiffness in humans, as well as underlying mechanisms that could support interventions aimed at enhanced efficacy in humans. Mechanisms contributing to endothelial function and arterial stiffness overlap [114] and therefore blueberries likely modulate arterial stiffness in a similar manner; however, this requires evaluation.
5.3. Effects of blueberries on arterial stiffness: human studies
5.3.1. Acute blueberry consumption
Six studies examined the effects of acute blueberry consumption (≤ 24 hours) on arterial stiffness (Table 3). In healthy men consuming 34 g, 57 g, and 80 g of freeze-dried wild blueberry powder mixed in water, there were no effects on AIx or DVP at 2-, 4-, or 6-hour post-treatment consumption [56]. Three studies analyzed digital AIx (dAIx) and dAIx at 75 beats/min (dAIx @ 75 bpm) 20 and 120 minutes after consuming 300 g of whole highbush blueberries in healthy male smokers, and nonsmoking and smoking males with peripheral arterial dysfunction (RHI ≤ 1.67) but saw no effects [108,109]. Furthermore, in older adults with cognitive decline, no effects were seen on DVP 1 hour after consuming ~ 30 g of freeze-dried highbush blueberry powder mixed in a semi-skimmed milk [125]. Lastly, there was no effect on AIx @ 75 bpm or cfPWV in adults with metabolic syndrome 3, 6, or 24 hours after consuming 26 g of freeze-dried highbush blueberry powder added to 500 g of energy-dense emulsion meal + 50 g of water [106]. This supports that structural vascular changes occur over time and not acutely.
5.3.2. Chronic blueberry consumption
Eight studies examined chronic blueberry consumption (> 24 h) on arterial stiffness in diverse human populations (Table 4). Two studies were performed in healthy individuals. In one study, daily consumption of 11 g of freeze-dried wild blueberry powder mixed in water for 28 days did not improve AIx or cfPWV in healthy men [58]. In a different study, consuming 160 g of fresh whole highbush blueberries or 20 g of freeze-dried blueberry powder daily for 1 week did not improve cfPWV in healthy adults [126].
Six studies were performed in adults at risk for CVD. In a study in men with at least one CVD risk factor, no effects were observed on AIx or AIx @ 75 bpm after consuming 25 g of freeze-dried wild blueberry powder mixed in water daily for 6 weeks [110]. In another study with sedentary men and postmenopausal women, AIx improved after consuming 36 g of Tifblue/Rubel blueberry powder daily at evening meals for 6 weeks compared with placebo, with no effects on cfPWV [127]. Similarly, a study in postmenopausal women with above-normal blood pressure found that daily consumption of 22 g of freeze-dried highbush blueberry powder mixed in water for 8 weeks decreased baPWV, but not cfPWV, compared with baseline and placebo [128]. Furthermore, a study evaluating daily consumption of 26 g of freeze-dried highbush blueberry powder consumed as a drink/smoothie or added to cereals, yogurts, banana toast, or as a salad dressing for 6 months improved AIx, but not cfPWV, in adults with metabolic syndrome compared with those who consumed 13 g of freeze-dried highbush blueberry powder and placebo powder [113]. A study with postmenopausal women with above-normal blood pressure found that daily consumption of 22 g of freeze-dried highbush blueberry mixed in water for 12 weeks had no effects on cfPWV, AIx, or AIx at 75 beats/min [28]. Finally, healthy older adults had no changes in cfPWV or AIx at 75 beats/min following 12 weeks of consuming 26 g/d of freeze-dried wild blueberry powder mixed in water [60].
These findings suggests that chronic blueberry consumption may improve arterial stiffness assessed as AIx and baPWV in sedentary men and postmenopausal women, as well as postmenopausal women with above-normal blood pressure, respectively [127,128]. Limited studies have assessed chronic blueberry consumption on cfPWV, but existing research has not demonstrated benefits regardless of dose and length of intervention. It is possible that peripheral arteries are more responsive to dietary interventions such as blueberries, and/or that intervention lengths have not allowed for sufficiently capturing structural improvements in the aorta. Indeed, age-related impacts on central/aortic arterial stiffness have been shown to be more pronounced than impacts on peripheral arterial stiffness, potentially because of structural differences or differential remodeling [129]. Further research is needed to understand the effects of blueberry consumption on arterial stiffness.
6. Potential mechanisms by which blueberries modulate vascular function
6.1. Overview of mechanisms contributing to vascular (dys)function
The endothelium is responsible for producing NO, a critical vasodilatory molecule, through activation of eNOS by stimuli such as shear stress or Ach [130,131]. When the substrate L-arginine is available, eNOS produces NO, which can diffuse out of endothelial cells and into VSMCs, resulting in activation of soluble guanylyl cyclase, and thus, formation of cyclic guanylyl monophosphate (cGMP) and initiating vasodilation [66,130]. Vasodilation also occurs through activation of cyclooxygenase (COX), which metabolizes arachidonic acid to yield prostaglandin I2 (PGI2, also known as prostacyclin). PGI2 can diffuse out of endothelial cells and into VSMCs, triggering activation of cyclic adenosine monophosphate thus causing vasodilation [66,130]. COX is also responsible for vasoconstriction, but through the production of vasoconstrictors such as thromboxane instead of vasodilators and under different conditions [132]. Both NO- and COX-related pathways are activated through increased intracellular calcium.
Current evidence indicates that impaired endothelium-dependent dilation (resulting from endothelial dysfunction) is primarily caused by impairments in NO production and/or bioavailability secondary to excessive oxidative stress and inflammation [9,10]. The underlying mechanisms contributing to endothelial dysfunction are complex and not fully understood, and this review focuses on mechanisms related to oxidative stress, inflammation, and the gut microbiota. Regarding vascular oxidative stress, the main source of reactive oxygen/nitrogen species is NOX [133], whereas mitochondrial metabolism and dysfunction and xanthine oxidase 1 are also contributors [73,134]. With aging and the presence of other CVD risk factors such as HTN, dyslipidemia, and metabolic syndrome, increased reactive oxygen/nitrogen species production without adequate antioxidant defense (e.g., from decreased or insufficient antioxidant enzyme activity including superoxide dismutase and heme oxygenase-1 [HO-1]) promotes increased vascular oxidative stress [9,73,135,136]. This can lead to eNOS uncoupling through oxidation of its essential cofactor tetrahydrobiopterin, rendering eNOS unable to produce NO and instead producing superoxide radicals, further promoting vascular oxidative stress. ROS also reacts directly with NO, resulting in decreased NO bioavailability and increased formation of peroxynitrite (i.e., reactive nitrogen species), which increases vascular dysfunction [66]. ROS can also stimulate the release of pro-inflammatory molecules (e.g., C-reactive protein [CRP], tumor necrosis factor alpha [TNF-α], COX-2), which can decrease NO production and bioavailability and enhance vasoconstrictor molecule production [9,67,73,74,130,132,137]. Inflammation also stimulates ROS production, perpetuating the vicious cycle of oxidative stress and inflammation contributing to endothelial dysfunction [3,9,73]. Monocyte adhesion to endothelial cells also leads to vascular inflammation and atherogenesis and occurs with endothelial dysfunction [9,138,139]. Likewise, oxidative stress and inflammation contribute to arterial stiffening through endothelium-related mechanisms mentioned previously, including reduced NO bioavailability [6,140]. ROS also promote immune cells to release matrix metalloproteinases responsible for uncoiled collagen and elastin degradation in the vessel wall, leading to vascular fibrosis and VSMC proliferation and resulting in structural changes [140].
Accumulating evidence from epidemiological, animal, and human clinical studies indicate the gut microbiome is an important mediator of vascular function and CVD risk [11,141-143]. For instance, suppression of the gut microbiome with a broad-spectrum antibiotic cocktail in mice improved age-related vascular dysfunction (i.e., endothelial function and arterial stiffness), and vascular oxidative stress and inflammation [144]. Also, transplantation of an obesity-associated human gut microbiota into germ-free mice led to impaired endothelium-dependent dilation and increased arterial stiffness [145]. Though what is considered to be a “healthy” gut microbiome is complex and not completely understood, it is commonly accepted that higher microbial diversity, higher abundance of certain health-promoting bacteria such as Lactobacillus and Faecalibacterium prausnitzii, high proportion of Bacteriodota (Bacteroidetes) relative to Bacillidota (Firmicutes) phyla, resilience against external pressures, and higher abundance of bacteria that can metabolize complex carbohydrates has been shown in healthy individuals and those meeting the dietary guidelines [11,146,147]. The gut microbiome is responsible for digesting and metabolizing food components such as (poly)phenols and fiber, regulating the immune system, maintaining intestinal homeostasis, protecting against diseases, and producing certain nutrients and bioavailable metabolites important for human health, among numerous other functions [11,141,148,149]. Gut microbial dysbiosis is a compositional change away from what is considered a “healthy” gut microbiome including the loss of beneficial bacteria, expansion of pathobionts, and loss of diversity [11]. Gut microbial dysbiosis is linked with vascular dysfunction because of various mechanisms including, but not limited to, impaired intestinal barrier function resulting in entry of lipopolysaccharide (Gram-negative bacterial wall component) into circulation and Toll-like receptor signaling that increases production of pro-inflammatory cytokines, free radicals, and trimethylamine N-oxide (microbial metabolite of choline), which can promote inflammation, platelet reactivity, and thrombosis [11,141-143,150].
Diet and nutrition are important determinants of oxidative stress, inflammation, and gut microbial composition and function. A typical Western diet (e.g., high saturated-fat, high-refined carbohydrates, low fruits and vegetables) is linked to gut microbial dysbiosis, oxidative stress, inflammation, arterial stiffness, and endothelial dysfunction [11,151,152]. Conversely, in vitro studies with human gut microbiomes, in vivo animal studies, and clinical trials have reported (poly)phenols to beneficially modulate the gut microbiota through increasing growth of bacterial families linked to health such as Bifidobacteriaceae and Lactobacillaceae, increasing specific health-promoting bacteria such as Akkermansia, Prevotella, and Bacteroides, inhibiting the growth of pathogenic bacteria such as Escherichia coli and Helicobacter pylori, and reducing the Bacillidota/Bacteriodota (Firmicutes/Bacteroidetes) ratio, which may contribute to decreased CVD risk [153].
6.2. NO-related mechanisms for improvements in vascular (dys)function with blueberries
6.2.1. Evidence from cell culture studies
One study evaluated the impact of blueberry consumption on biomarkers related to NO. They demonstrated that treatment of human aortic endothelial cells (HAECs) with palmitate suppressed insulin-stimulated NO production but was restored by treatment with physiological levels of blueberry (poly)phenol metabolites (i.e., 700 nM of benzoic acid-4-sulfate, 75 nM of isovanillic acid-3-sulfate, 75 nM of vanillic acid-sulfate, 3 μM of hydroxy hippuric acid, and 5 μM of hippuric acid) [154]. No other physiologically relevant cell culture studies that examined blueberry (poly)phenols and/or their metabolites on mechanistic pathways related to NO were identified except for those evaluating impacts on oxidative stress and inflammation, which are described in a later section. This highlights a gap in the scientific knowledge, and future studies should aim to consider physiological relevance when designing studies that account for digestion, absorption, and metabolism.
6.2.2. Evidence from animal studies
Ten studies evaluated NO-related mechanisms associated with improvements in vascular function with blueberry consumption in animals (Table 2). With respect to endothelial function, studies have demonstrated that blueberries mediate beneficial effects through endothelium-dependent mechanisms [92,95]. In SD, hypertensive, and obese Zucker male rats, vasoconstriction (induced through eNOS inhibition with L-NMMA) was restored following blueberry supplementation compared with respective controls without inhibitors [93,94,96,97,105], whereas vasorelaxation was lower following blueberry consumption (also induced through eNOS inhibition with L-NMMA) compared with the controls [93,96,97,105], indicating improvements in vascular function were mediated through the eNOS/NO pathway. To support that improved vascular function with blueberry consumption is mediated through the eNOS pathway, male spontaneously hypertensive rats were found to have increased vasoconstriction (induced through guanylyl cyclase inhibition) and plasma cGMP following wild blueberry supplementation compared with the control [98]. Furthermore, decreased plasma NO metabolite concentrations were observed in obese male rats after wild blueberry consumption compared with obese controls, whereas increased NO metabolite concentrations were observed in lean rats after wild blueberry consumption compared with lean controls, suggesting improved NO bioavailability [99,105]. Interestingly, overproduction of NO coupled with low NO bioavailability has been observed in pro-inflammatory and oxidative stress–driven conditions including obesity and metabolic syndrome, likely from adaptive compensation, suggesting that a reduction in NO could be beneficial in certain populations [104,105]. Furthermore, aortic gene expression of eNOS and inducible NOS (iNOS) and plasma NO concentrations were increased in obese Zucker rats compared with lean Zucker rats, and blueberry-fed obese Zucker rats had decreased iNOS gene expression (but not eNOS) compared with obese controls and increased NO concentrations compared with control lean Zucker rats [99,105]. Overall, these studies support that improvements in endothelial function are mediated through the eNOS/NO pathway.
6.2.3. Evidence from human studies
Seven studies evaluated the impact of blueberry consumption on biomarkers related to NO. Of the human clinical trials that found improved vascular function after blueberry consumption, one found significantly increased NO metabolite concentrations in postmenopausal women with above-normal blood pressure following 8 weeks of daily consumption of 22 g of freeze-dried highbush blueberry powder mixed in water [128]. Similarly, another study found significantly increased plasma NO metabolite concentrations after consuming 240 mL of wild blueberry juice for 7 days in adults at risk for type 2 diabetes, even though RHI did not significantly improve [111]. Lastly, plasma cGMP concentrations were increased after consuming 26 g/day of freeze-dried highbush blueberry powder for 6 months in adults with metabolic syndrome, while NO metabolite concentrations were unchanged [113]. Finally, four studies measured NO metabolites but found no statistical differences in NO metabolite concentrations after blueberry consumption [28,107,110,126], though one study observed an increase in endothelium/NO-dependent dilation as indicated by an improvement in FMD/SRAUC but no change in endothelium-dependent dilation to oral sublingual nitroglycerin [28]. Together, these findings suggest that improvements in endothelial function in humans may be through the eNOS/NO pathway. However, further research is needed to understand the extent to which improvements in endothelial function are mediated by NO and the underlying mechanisms related to NO production/bioavailability, particularly in diverse populations that include healthy individuals and those at risk for and/or have chronic diseases that influence the eNOS/NO pathway. Assessment of NO status accurately is complex because of its short half-life and high reactivity; thus, a single sampling of blood may not reflect tissue levels. Current methods assess products of NO signaling, which may over- or underestimate NO concentrations rather than provide an accurate representation of bioavailability. More recently, erythrocyte 5-α-coordinated heme nitrosyl-hemoglobin has emerged as a biomarker of NO bioavailability and NO-dependent endothelial function because of its relationship with clinical endothelial function and the formation of NO in vivo [155]. The effects of blueberries on NO production and/or bioavailability, particularly as it relates to measures of endothelial function in humans, should be evaluated in future randomized controlled trials to better understand mechanisms. Studies could apply multiple assessments of NO in biological samples coupled with functional assessments (e.g., assessment of endothelium-dependent and endothelium-independent dilation).
6.3. Effects of blueberries on vascular oxidative stress
6.3.1. Evidence from cell culture studies
Two cell culture studies examined the effects of physiologically relevant levels of blueberry (poly)phenols and/or their metabolites commonly found in human plasma after blueberry consumption on endothelial cell–derived oxidative stress. One study observed that treatment of HAECs with palmitate increased NOX4 mRNA expression and ROS production but was attenuated by the addition of synthetic blueberry (poly)phenol metabolites (i.e., 700 nM of benzoic acid-4-sulfate, 75 nM of isovanillic acid-3-sulfate, 75 nM of vanillic acid-sulfate, 3 μM of hydroxy hippuric acid, and 5 μM of hippuric acid) [154]. In a different study, the impact of three different blueberry (poly)phenol metabolite mixtures on antioxidant defense (i.e., nuclear factor erythroid 2-related factor [Nrf2] and cellular HO-1 protein expression) in human umbilical venous endothelial cells (HUVECs) treated with sublethal dosages of hydrogen peroxide (H2O2) was evaluated [156]. For that study, (poly)phenol metabolites were first measured over a 24-hour period following consumption of a single dose of blueberry juice by three healthy humans and found that 12 metabolites were increased compared with baseline and served as the basis for the metabolite mixtures used: (1) Early mixture/metabolites found in plasma with a time to maximum ≤2 hours, including 50 nM 2-hydroxyhippuric acid, 60 nM 4-hydroxyhippuric acid, 450 nM syringic acid, 15 nM protocatechuic acid, 600 nM vanillic acid, 60 nM trans-ferulic acid, and 40 nM p-coumaric acid, totaling 1275 nM metabolites; (2) Late mixture/metabolites found in plasma with a time to maximum ≥4 hours including 30 nM protocatechuic acid, 100 nM gentisic acid, 1000 nM vanillic acid, 60 nM trans-ferulic acid, 50 nM p-coumaric acid, 2000 nM dihydroferulic acid, 200 nM dihydrocaffeic acid, 850 nM dihydro-m-coumaric acid, and 300 nM homovanillic acid, totaling 4590 nM metabolites; (3) Whole mixture/metabolites containing all of the early and late mixtures including 50 nM 2-hydroxyhippuric acid, 70 nM 4-hydroxyhippuric acid, 450 nM syringic acid, 25 nM protocatechuic acid, 100 nM gentisic acid, 1000 nM vanillic acid, 60 nM trans-ferulic acid, 50 nM p-coumaric acid, 2000 nM dihydroferulic acid, 200 nM dihydrocaffeic acid, 850 nM dihydro-m-coumaric acid, and 300 nM homovanillic acid, totaling 5155 nM metabolites [156]. Results showed that pretreatment with the whole mixture of blueberry (poly)phenol metabolites upregulated Nrf2-regulated antioxidant response proteins including HO-1 and the glutamate-cysteine ligase modifier subunit in HUVECs exposed to 2.5 μM of H2O2 compared with H2O2 control [156]. These studies suggest that blueberries may reduce endothelial cell–derived oxidative stress through modulation of ROS-producing and antioxidant defense enzymes.
6.3.2. Evidence from animal studies
One study examined blueberry consumption on oxidative stress as related to vascular function and showed that adding 3.8% w/w freeze-dried blueberry powder to the diets of diabetic db/db male mice for 10 weeks reduced NOX4 mRNA expression in vascular endothelial cells and aortic vessel compared with the diabetic control group (Table 2) [27]. No other studies in animals that evaluated vascular oxidative stress were identified, but future studies could support understanding oxidative stress–related mechanisms contributing to improvements in vascular function with blueberry consumption.
6.3.3. Evidence from human studies
Nine studies evaluated the effects of blueberry consumption on oxidative stress as related to vascular function. Acute consumption of 300 g of whole highbush blueberries was found to decrease H2O2-induced DNA damage in peripheral blood mononuclear cells in healthy men 1 hour after blueberry consumption compared with controls [107]. Another study observed decreased endogenous oxidized DNA bases and H2O2-induced DNA damage in peripheral blood mononuclear cells from healthy males with at least one CVD risk factor after consuming 25 g/d of freeze-dried wild blueberry powder for 6 weeks [110]. In a different study, decreases in neutrophil NOX activity were found 1 to 2 and 6 hours after consumption of 34 g of freeze-dried wild blueberry powder in healthy males that were directly associated with improvements in FMD and predicted by plasma (poly)phenol metabolites (vanillic acid, hippuric acid, and homovanillic acid) [56]. A study in postmenopausal women with above-normal blood pressure found that 4 weeks of consuming 22 g/d of freeze-dried highbush blueberry powder led to improvements in 8-hydroxy-2′-deoxyguanosine, which is indicative of DNA damage, though levels increased back to baseline at 8 weeks (follow-up study to Johnson et al.), whereas other circulating biomarkers of oxidative stress and antioxidant defense were not affected by blueberry consumption [157]. In adults with metabolic syndrome, a study found that 6 weeks of consuming 45 g/d of freeze-dried highbush blueberry powder found decreased whole blood superoxide radicals and monocyte ROS (follow-up study to Stull et al.) [158]. Finally, a recent study found that FMD/SRAUC increased after acute infusion of a supraphysiological dose of the antioxidant ascorbic acid (vitamin C) in postmenopausal women with above-normal blood pressure at baseline (indicating oxidative stress–mediated suppression of endothelial function), but not after 12 weeks of daily consumption of 22 g of freeze-dried highbush blueberry powder (whereas differences stayed trending for the control group) [28]. Additionally, the blueberry group had a reduction in the response to ascorbic acid from baseline to 12 weeks, whereas the placebo group had no change, indicating that improvements in endothelial function were mediated by reductions in oxidative stress [28]. Three other studies also measured biomarkers of oxidative stress after blueberry consumption in relation to vascular function but did not find improvements [110,111,127]. Other human studies have evaluated biomarkers of inflammation and oxidative stress, but were not performed in the context of vascular function and were not included but have been evaluated through a systematic review that indicated results are mixed because of various factors [159]. Nonetheless, circulating biomarkers do not necessarily reflect vascular oxidative stress, highlighting the need to better understand the impact of blueberries on vascular oxidative relating to vascular function, as well as the mechanisms involved.
6.4. Effects of blueberries on vascular inflammation
6.4.1. Evidence from cell culture studies
Five cell culture studies examined the effects of physiologically relevant blueberry (poly)phenol metabolites commonly found in human plasma after blueberry consumption on endothelial-derived inflammation. In one study, HUVECs were incubated with blueberry (poly)phenol metabolites including gallic acid, syringic acid, and protocatechuic acid (among other parent ACNs and phenolic acids), and then monocytes (THP-1) were added with TNF-α [160]. Results showed that 0.01 to 10 μg/mL of gallic acid reduced monocyte adhesion compared with HUVECs treated with TNF-α alone, whereas 0.01 to 10 μg/mL of syringic acid and 0.1 μg/mL of protocatechuic acid (independently) increased adhesion [160]. Another study showed that incubation of a blueberry (poly)phenol metabolite mixture (i.e., 700 nM benzoic acid-4-sulfate, 75 nM isovanillic acid-3-sulfate, 75 nM vanillic acid-sulfate, 3 μM hydroxy hippuric acid, and 5 μM hippuric acid) attenuated palmitate-induced increases in relative mRNA expression of interleukin 8 (IL-8), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IκBα, as well as monocyte binding to HAECs [154]. In type 2 diabetic HAECs, a study showed that monocyte (THP-1) adhesion and relative mRNA expressions of IL-8, VCAM-1, and E-selectin were significantly increased compared with healthy HAECs, but reduced with the addition of a blueberry (poly)phenol metabolite mixture (i.e., 700 nM benzoic acid-4-sulfate, 75 nM isovanillic acid-3-sulfate, 75 nM vanillic acid-sulfate, 3 μM hydroxy hippuric acid, and 5 μM hippuric acid) [161] . In a different study, monocyte (THP-1) adhesion to healthy HUVECs was stimulated with TNF-α followed by incubation with 1 and 10 μg/mL protocatechuic acid, 1 and 10 μg/mL gallic acid, and 0.01 μg/mL malvidin-3-glucoside, which independently decreased monocyte adhesion to HUVECs. Furthermore, in the same study, soluble E-selectin in supernatant from HUVECs with TNF-α was decreased after incubation of the following compounds and concentrations: (1) 0.01 μg/mL, 0.1 μg/mL, and 1 μg/mL of malvidin-3-glucoside; (2) 10 μg/mL cy-3-glc; (3) 1 μg/mL protocatechuic acid; and (4) 1 μg/mL and 10 μg/mL gallic acid [162]. Lastly, three different blueberry phenolic acid mixtures at physiological levels reflecting early (0–4 hours), late (4–24 hours), or whole (0–24 hours) circulating metabolites after blueberry juice consumption (previously reported in Tang et al., 2018) were used to assess the effects of blueberry (poly)phenol metabolites on monocyte binding and inflammation in TNF-α induced dysfunctional HUVECs [163]. Results showed that 4 hours of pretreatment with the late mixture, but not the early or whole mixtures, decreased monocyte adhesion to HUVECs without affecting endothelial cell surface expression of adhesion molecules [163]. Interestingly, they also observed that exclusion of syringic acid from early and whole mixtures led to reduced monocyte adhesion, whereas treatment with syringic acid alone had no effects on monocyte adhesion [163]. Overall, these studies suggest that blueberry (poly)phenol metabolites exert anti-inflammatory effects in the vasculature that can attenuate processes involved in atherosclerosis.
6.4.2. Evidence from animal studies
Seven studies were identified that examined blueberry consumption on inflammation as related to vascular function in animals (Table 2). Two studies in male spontaneously hypertensive rats found that inhibition of COX with mefenamic acid ex vivo with thoracic aortic rings decreased vasoconstriction after wild blueberry diet consumption compared with control [96,97], whereas another study observed a decrease in vasoconstriction with inhibition of PGI2 synthase after wild blueberry diet consumption compared with control [98]. Furthermore, male spontaneously hypertensive and obese Zucker rats were found to have increased plasma 6kPGF1α (a COX pathway intermediate) following blueberry diet consumption in several studies, demonstrating improvements in endothelial function [98,99,105]. Lastly, aortic COX-2 gene and iNOS gene expression decreased in obese Zucker male rats after wild blueberry diet consumption compared with control [99,105]. Furthermore, blueberry diet consumption in obese male Zucker rats decreased plasma concentrations of interleukin-6 (IL-6), TNF-α, CRP, and adiponectin as well as increased PGI2 concentrations in aortic effluent compared with the obese control [99]. Finally, a study found that blueberry powder added to the diets of diabetic db/db male mice for 10 weeks decreased serum inflammatory markers (monocyte chemoattractant protein-1 [MCP-1]/JF, KC/IL-8), decreased aortic gene expression of Iκκβ, KC/IL-8, MCP-1/JE, ICAM-1, and VCAM-1, and decreased carotid endothelial cell gene expression of Iκκβ, KC/IL-8, MCP-1/JE, and VCAM-1 compared with control [27]. Additionally, blueberry (poly)phenol-rich serum from db/db mice that consumed wild blueberries suppressed glucose and palmitate-induced monocyte binding to, and KC secretion from, mouse aortic endothelial cells compared with the db/db control [27]. Together, these studies suggest that blueberries may improve vascular function through attenuation of inflammation in HTN, obesity, metabolic syndrome, and diabetes.
6.4.3. Evidence from human studies
Six studies evaluated the effects of blueberry consumption on inflammation as related to vascular function. In postmenopausal women with above-normal blood pressure, 4 weeks of consuming 22 g/d freeze-dried highbush blueberry powder improved the DNA damage product 8-hydroxy-2′-deoxyguanosine but circulating biomarkers of inflammation were unaffected (follow-up study to Johnson et al.) [157]. A study performed in adults with metabolic syndrome found that 6 weeks of consuming 45 g/d freeze-dried highbush blueberry powder found decreased monocyte gene expression of TNF-α, IL-6, Toll-like receptor 4, as well as serum granulocyte macrophage colony-stimulating factor and increased myeloid dendritic cells (follow-up study to Stull et al.) [158]. A study performed in healthy men found that 28 days of consuming 22 g/d of freeze-dried wild blueberry powder led to differential expression of numerous genes in peripheral blood mononuclear cells that are involved in immune regulation, inflammation, and cell adhesion, among other processes [58]. Lastly, three studies also measured biomarkers of inflammation (e.g., VCAM-1, IL-6, CRP, TNF-α) after blueberry consumption in relation to vascular function but did not find any statistically significant changes in such biomarkers [28,110,111].
6.5. Effects of blueberries on the gut microbiome
6.5.1. Evidence from cell culture studies
No relevant studies using cell culture models related to the gut microbiome and vascular function were identified. In vitro fermentation has allowed researchers to investigate the effects of blueberries on the gut microbiome and their potential impacts on human health. Though a limited number of studies have been performed, existing data from studies using whole blueberry, fermented blueberry pomace, and blueberry (poly)phenol extracts and fractions suggest that blueberries beneficially modulate the gut microbiota such as increasing Lactobacillus, Bifidobacterium, Ruminococcus, Akkermansia, and other microbes, increasing alpha diversity, and protecting fecal microbial richness and against infections [164-168]. Whether such effects translate to beneficial effects on the vasculature remains unknown.
6.5.2. Evidence from animal studies
Only one study in animal models investigates the effects of blueberries on the gut microbiome and vascular health were identified. In diabetic db/db male mice fed 3.8% w/w freeze-dried wild blueberry powder for 10 weeks, a study observed trends in changes of microbial alpha diversity indices (Shannon and Simpson) for the blueberry group compared with the db/db control, and beta diversity was significantly different at the phylum and genius level between groups [27]. The blueberry supplementation also significantly increased Actinobacteria, Adlercreutzia, Bifidobacterium, and Dorea abundance compared with db/db control [27].
6.5.3. Evidence from human studies
Studies in humans investigating the effects of blueberries on the gut microbiome in relation to vascular function are limited because only one study was identified. In older adults, consumption of 26 g/day of freeze-dried wild blueberry powder for 12 weeks did not significantly alter alpha or beta diversity of the gut microbiome; however, alpha diversity increased from baseline in the whole cohort (i.e., blueberry and placebo groups combined) that appeared to be driven by the blueberry group. They observed an increased abundance of Ruminiclostridium 9 and Ruminiclostridium 5 following blueberry consumption, a positive correlation between Cocoprocus and Family XIII AD03011 with FMD, and a negative correlation between Parabacteroides and Ruminococus UCG.003 with FMD [60]. Increases in bacteria observed were generally butyrate producers, and butyrate has been demonstrated to exert protective effects on cardiovascular health including attenuation of endothelial dysfunction through NOX-associated ROS production and NO-mediated mechanisms [169,170]. Though no major effects of blueberry consumption on the gut microbiome were observed, their overall study results suggest that improvements in endothelial function may be mediated by the gut microbiome [60]. Other studies have investigated the effects of blueberries on the composition of the gut microbiome but not in the context of vascular function. Briefly, studies in healthy adult men and young/old women indicate that chronic blueberry consumption for 6 weeks increase Bifidobacterium spp. and health-associated taxa [168,171,172]. These data suggest that blueberry consumption may modulate the gut microbiota, but it is unclear as to the extent to which the gut microbiome mediates improvements in vascular function and is a major gap in the field.
7. Conclusions
Blueberries are rich in nutrients and (poly)phenols, with demonstrated protective effects on cardiovascular health. They are a widely consumed fruit in the United States, a major agricultural crop with year-round availability, and have high nutritional quality and acceptability by consumers, making them ideal for use in food-based strategies for promoting human health including cardiovascular health. Blueberries and their (poly)phenol metabolites have been shown to improve measures of vascular function in animals and humans. Evidence from animal studies support that blueberry consumption promotes vasorelaxation and reduces vasoconstriction. Evidence from randomized controlled trials support that blueberry consumption can enhance endothelial function acutely in men (healthy men and smokers), and chronically in healthy young and older adults, adults with metabolic syndrome, and postmenopausal women with above-normal blood pressure. The totality of evidence suggests that blueberries improve endothelial function to a greater extent in those with increased CVD risk factors, but also that dosing may need to be tailored to the extent of CVD risk and disease progression. Regarding arterial stiffness, the data are less conclusive because no studies assessing arterial stiffness have been performed in animals and limited randomized controlled trials have been performed in humans. However, existing data suggest chronic blueberry consumption for at least 6 weeks may improve certain measures of arterial stiffness, whereas a longer duration may be needed to observe reductions in aortic arterial stiffness. Overall, the data suggest that strongly support that blueberry consumption improves endothelial function in high-CVD risk populations and is mediated in part through reductions in oxidative stress and inflammation, whereas the role of the gut microbiome is less clear (Fig. 2).
Fig. 2 –
Graphical schematic of the impact of blueberry consumption on endothelial function and arterial stiffness (i.e., vascular dysfunction) and potential major mechanisms based on evidence from cell, animal, and human studies. Vascular dysfunction occurs with aging and is accelerated by chronic disease risk factors and contributes to cardiovascular disease (CVD). Evidence indicates major mediators include oxidative stress, inflammation, and the gut microbiome. Preclinical and clinical evidence supports that blueberries improve vascular function, particularly endothelial function. The evidence supporting effects on arterial stiffness is less conclusive. Effects are mediated through modulation of oxidative stress and inflammation, and potentially the gut microbiome. This figure was created with Biorender (https://www.biorender.com).
It is difficult to determine which compounds present in blueberries contribute to their beneficial impacts on vascular function because many of their nutrients and (poly)phenols benefit cardiovascular health. Translational research studies with animals and humans have been performed to investigate this and findings strongly suggest that ACN metabolites mediate beneficial impacts [58]. In those studies, a clinical trial with healthy adult males demonstrated that purified ACNs improved FMD to the same extent as whole blueberry powder containing the same ACN content with maximal FMD increases of 1.3% units and 1.1% units at 1 and 6 hours, respectively, whereas control drinks containing equivalent amounts of fiber, vitamins, and minerals had no effects on FMD. They also found that plasma ACN metabolites correlated with improvements in FMD, and that injection of those same metabolites into mice improved FMD. These findings need to be extended to other populations, including those at increased risk for CVD. Additionally, recognizing the beneficial impacts of ACN metabolites does not exclude the possibility that other (poly)phenols and nutrients present in blueberries exert additive and/or synergistic protective effects on vascular function or other benefits to cardiovascular health. Indeed, preclinical evidence suggests that numerous (poly)phenol metabolites mediate the beneficial effects of blueberries on vascular function; therefore, a reductionist approach for defining specific compounds and their bioactivities is likely to be unsuccessful. The mechanisms by which blueberries, their nutrients, (poly)phenols, and/or polyphenol metabolites lead to improvements in endothelial function is not fully understood but potential mechanisms have been identified (Fig. 3). Results from cell, animal, and human studies suggest that blueberry consumption improves NO bioavailability, likely because of circulating (poly)phenol metabolites and their impact on oxidative stress, thus leading to changes in vascular function. Animal studies suggest a link between the gut microbiota and blueberry-induced improvements in vascular function, though this was not supported in a recent human study. Studies in animals and cells suggest that mechanisms may also include reductions in inflammation and increased antioxidant defense. An area of investigation worth pursuing is dysregulated L-arginine metabolism, which is driven by and drives oxidative stress and impaired NO bioavailability, and whether blueberry consumption beneficially impacts vascular function through attenuation of L-arginine depletion or methylarginine (e.g., asymmetric dimethylarginine, L-NMMA) formation.
Fig. 3 –
Potential mechanisms by which blueberries, their components, and/or resulting metabolites improve endothelial function based on evidence from cell, animal, and human studies. Evidence indicates blueberries may improve endothelial function through antioxidant defense (i.e., Nrf2/Keap1/ARE), reduced free radical production/oxidative stress (i.e., NADPH oxidase), reduced inflammation (i.e., iNOS, NF-κB, COX-2), endothelial cell nitric oxide production (i.e., eNOS), and gut microbiota modulation. This figure was created with Biorender (https://www.biorender.com). Abbreviations: COX-2, cyclooxygenase-2; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor kappa B; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; Nrf2/Keap1/ARE, nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1/antioxidant response element.
Based on the findings discussed in this narrative review, gaps identified, and expert opinion, we believe further research is needed (Fig. 4) to establish the dose-dependent and time-course clinical efficacy of blueberries alone and in combination with other foods, compounds, and in dietary patterns to improve endothelial function and arterial stiffness in diverse human populations. Additionally, research aimed at understanding determinants and mechanisms of improvements in vascular function is needed, particularly those that are translational and/or reverse translational in nature and can evaluate factors and mechanisms responsible for clinical responses in humans. Finally, translation of clinical research to the community/public in a manner that considers feasibility, social determinants of health, culture, community needs, assets, and desires, barriers and drivers to consumption, among other factors while evaluating efficacy is needed to establish real-world impacts.
Fig. 4 –
Knowledge gaps and suggestions for future research with blueberries related to cardiovascular health in humans. Major knowledge gaps related to blueberry consumption in humans were identified include dose-dependent and time-course clinical efficacy, underlying mechanisms and factors contributing to efficacy, and translation to clinical/medical, community, and public health settings. This figure was created with Biorender (https://www.biorender.com). Abbreviations: CVD, cardiovascular disease; FMT, fecal microbiota transplant; RCTs, randomized controlled trials.
Acknowledgment
The authors have no acknowledgments to declare.
Sources of Support
This work was supported by the USDA National Institute of Food and Agriculture [grant no. 2020-67017-30833/project accession no. 1021875] (principal investigator: S.A.J.). E.K.W. is currently supported by a NIDDK T32 grant (T32 DK64584-20) at Pennington Biomedical Research Center.
Abbreviations:
- Ach
acetylcholine
- ACNs
anthocyanins
- AIx
augmentation index
- AIx @ 75 bpm
augmentation index normalized to 75 beats/minute
- aPWV
aortic pulse wave velocity
- baPWV
brachial to ankle pulse wave velocity
- cfPWV
carotid to femoral pulse wave velocity
- cGMP
cyclic guanylyl monophosphate
- COX
cyclooxygenase
- CRP
C-reactive protein
- CVD
cardiovascular disease
- dAIx
digital augmentation index
- dAIx @ 75 bpm
digital augmentation index at 75 beats/min
- DVP
digital volume pulse
- eNOS
endothelial nitric oxide synthase
- FMD
flow-mediated dilation
- FMD/SRAUC
flow-mediated dilation normalized to shear rate area under the curve
- GI
gastrointestinal
- H2O2
hydrogen peroxide
- HAECs
human aortic endothelial cells
- HO-1
heme oxygenase-1
- HTN
hypertension
- HUVECs
human umbilical venous endothelial cells
- ICAM-1
intercellular adhesion molecule-1
- IL-6
interleukin 6
- IL-8
interleukin-8
- iNOS
inducible nitric oxide synthase
- L-NMMA
NG-mono-methyl-L-arginine
- MCP-1
monocyte chemoattractant protein-1
- NO
nitric oxide
- NOX
NADPH oxidase
- Nrf2
nuclear factor erythroid 2-related factor
- PAT
peripheral arterial tonometry
- Phe
phenylephrine
- PGI2
prostaglandin I2
- PWV
pulse wave velocity
- RHI
reactive hyperemia index
- ROS
reactive oxygen species
- SD
Sprague-Dawley
- THP-1
human monocytic cells
- TNF-α
tumor necrosis factor alpha
- VCAM-1
vascular cell adhesion molecule-1
- VSMC
vascular smooth muscle cells
Footnotes
Author Declarations
The authors declare no conflicts of interest. For full transparency, S.A.J. has received grant support from the US Highbush Blueberry Council and the North American Wild Blueberry Association but did not influence or support the development of this publication.
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