**Nutrition and Cardiovascular Health**

Special Issue Editors

**Paramjit S. Tappia Heather Blewett**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Special Issue Editors* Paramjit S. Tappia St. Boniface Hospital Canada

Heather Blewett Agriculture and Agri-Food Canada Canada

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: https://www.mdpi.com/ journal/ijms/special issues/nutrition cardiovascular health).

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## **Contents**

## **About the Special Issue Editors**

**Paramjit S. Tappia** received his B.Sc. (Honors) in Pharmacology from the University of Sunderland, U.K. in 1985 and a Ph.D. in Biochemistry from the University of Wolverhampton, U.K. in 1992. He received postdoctoral training in the Institute of Human Nutrition, University of Southampton, U.K. from 1992–1995. In 1996, he joined the Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Canada as a Research Associate. He is currently a Principal Investigator and Clinical Scientist in the Asper Clinical Research Institute, St. Boniface Hospital, Winnipeg, Canada. As a clinical trialist, Dr. Tappia has conducted several trials in the area of diabetes, heart failure, gut health, and cancer diagnostics, as well as nutrition and human health. To date, he has published 115 full-length papers in scientific journals and 28 book chapters in the area of cardiovascular sciences and nutrition with H-index of 34 (Google Scholar). He has co-edited 3 books in the area of human health and serves on the Editorial Boards of 3 international journals. Dr. Tappia also serves as the Editor of the CV Network, which is the official news bulletin of the International Academy of Cardiovascular Sciences; a global organization for the promotion of prevention of cardiovascular disease.

**Heather Blewett** (nee Hosea) completed her Ph.D. under the supervision of Dr. Carla Taylor in the Department of Human Nutritional Sciences at the University of Manitoba in 2006. Her Ph.D. thesis explored the effects of zinc deficiency on T-cell maturation and function in growing rats. She went on to work with Dr. Catherine Field at the University of Alberta (2006–2008). During this time, Dr. Blewett expanded both her nutritional and immunological training by studying the importance of amino acids and fatty acids for intestinal and immune defense against *E. Coli* using a novel piglet model. She also investigated the effect of a "natural" trans fat called vaccenic acid on the pro-inflammatory tendency of immune cells in a rat model of obesity. After leaving the University of Alberta, Dr. Blewett joined Peter Zahradka's lab at the Canadian Centre for Agri-Food Research in Health and Medicine (CCARM) to gain experience using clinical trials to test the efficacy of Canadian crops in the treatment of vascular disease (2009–2011). Dr. Blewett was hired into her dream job as a Research Scientist with the Government of Canada in the Department of Agriculture and Agri-food Canada in May 2011 and is located at the St. Boniface Research Centre in Winnipeg, Manitoba. She uses her expertise in human nutrition to perform the clinical trials that are needed to substantiate food health claims for Canadian crops including LDL cholesterol lowering, reduction in postprandial glucose response, and increased satiety. Her background in immunology also allows her to focus on the effect of Canadian food products on immune function in the context of chronic diseases (i.e., cardiovascular disease, diabetes, obesity).

## *Editorial* **Nutrition and Cardiovascular Health**

**Paramjit S. Tappia 1,\* and Heather Blewett 2**

Received: 23 March 2020; Accepted: 24 March 2020; Published: 26 March 2020

There is unequivocal experimental, epidemiological and clinical evidence demonstrating a correlation between diet and increased risk of cardiovascular disease (CVD). While nutritionally poor diets can have a significant negative impact on cardiovascular health, dietary interventions with specific nutrients and/or functional foods are considered cost-effective and efficient components of prevention strategies. It has been estimated that nutritional factors may be responsible for approximately 40% of all CVD [1]. Indeed, in one of the seminal studies conducted on modifiable risk factors and heart health (the INTERHEART study), >90% of all myocardial infarctions were attributed to preventable environmental factors with nutrition identified as one of the important determinants of CVD [2]. There is increasing public interest and scientific investigation into establishing dietary approaches that can be undertaken for the prevention and treatment of CVD. This Special Issue provides an insight into the influential role of nutrition and dietary habits on cardiovascular health and disease as well as the therapeutic and preventive potential of novel nutraceuticals and specific nutrients. Fourteen outstanding papers, from experts in the field, provide a broad range of contributions detailing various aspects of nutrition and cardiovascular health as well as highlighting possible mechanisms of beneficial action.

Casas et al. [3] have reviewed the role of overall nutrition, specific nutrients, foods and dietary practices in relation to cardioprotection and prevention of CVD. This review Furthermore describes some of the mechanisms in the cardioprotective properties of individual nutrients, foods and nutritional patterns.

This Special Issue contains two reviews on the effects of long-chain polyunsaturated omega-3 fatty acids on CVD. Innes and Calder [4] provide a review of the literature on the use of marine derived omega-3 (n-3) fatty acids (eicosapentaenoic acid and docosahexaenoic acid) with a focus on primary and secondary prevention of CVD, along with a discussion of the potential mechanisms for their effects. Goel et al. [5] discuss the inconsistencies regarding the cardioprotective effects of fish and fish oils. Although many experimental studies and some clinical trials have documented the benefits of fish oil supplementation in decreasing the incidence and progression of atherosclerosis, myocardial infarction (MI), heart failure (HF), arrhythmias and stroke, recent large-scale clinical studies have failed to demonstrate any benefit on cardiovascular outcomes and mortality. This is an area of investigation that needs some refinement in order to fully understand the beneficial effects of fish oils and n-3 polyunsaturated fatty acids in general. Indeed, we have Furthermore reported inconsistencies in outcomes of CVD management in some trials with n-3 fatty acids [6,7].

Micronutrient deficiency is present in HF and is associated with adverse clinical signs and symptoms. Indeed, a pathophysiological role as well as prognostic value has been ascribed to micronutrient deficiency. In this regard, Cvetinovic et al. [8] review evidence that demonstrate a correction/normalization of micronutrient status is linked to a concomitant improvement in physical performance and quality of life in HF. The Special Issue Furthermore includes articles on specific micronutrients. Globally, vitamin D deficiency is highly prevalent and has been linked to many non-communicable diseases, but the role of vitamin D in the pathogenesis of HF remains to be defined. Roffe-Vazquez [9] discuss the molecular mechanisms involved in inflammatory processes, remodeling, fibrosis and atherosclerosis in humans due to a deficiency in vitamin D, through in vitro and animal experimentation. Furthermore, by employing human cardiac microvascular endothelial cells and a rat model of isoproterenol-induced fibrosis, Lai et al. [10] specifically report that vitamin D may, in fact, reduce the development of fibrosis. Although more research is required, vitamin D could be part of a prevention strategy for individuals at risk of HF.

The role of magnesium (Mg) deficiency as a risk factor of arterial hypertension is not completely known. Kostov and Halecheva [11] describe the many benefits of Mg and discuss the role of Mg deficiency in increasing the risk of atherosclerosis, endothelial dysfunction and arterial stiffness, which all contribute to hypertension. Thus, healthy dietary practices that include the recommended amounts of Mg may constitute a nutritional approach for normal blood pressure.

Nutraceuticals and functional foods have established efficacy for the treatment and/or prevention of adverse human health. Among these natural health products is American Ginseng. Parikh et al. [12] describe the cardioprotective effects of American Ginseng root extract (GBE) using a rat model of congestive heart failure due to MI induced by coronary artery ligation. Although treatment with GBE did not improve cardiac remodeling and function, attenuation of oxidative stress and TNFα was seen. The reduction of TNFα to below baseline levels was suggestive of the use of GBE as a prophylactic or as a preventive adjunct for cardiovascular disease. Jakovljevic et al. [13] have examined the potential health benefits of Aronia melanocarpa extract (SAE) in a rat model of metabolic syndrome (MetS). SAE was found to lower blood pressure and improve cardiac function. It is noteworthy that SAE improved glucose tolerance, liver damage and reduced oxidative stress. From the evidence provided, nutritional supplementation with SAE may potentially exert cardioprotective effects in patients with MetS. Cocaine is a potent stimulant drug that disrupts the electrical signals of the heart, increases blood pressure, heart rate, and the occurrence of heart-related fatal events including sudden cardiac death. In the review by Kim and Park [14] the cellular and molecular mechanisms of cocaine on the cardiovascular system are described. While there is unequivocal information on the adverse acute effects of cocaine on the heart (electrophysiological abnormalities, arrhythmia, and acute MI), the data on the chronic effects of cocaine on the vascular system (coronary artery disease (CAD) and/or subclinical atherosclerosis) are less clear and inconsistent. However, interestingly, chronic effects of cocaine are more likely in individuals with higher CAD risk and with deleterious health choices and behaviour.

Extracellular matrix (ECM) remodeling and fibrosis are key players in HF. Although some experimental studies have reported that nutraceuticals can diminish cardiac fibrosis, clinical data are conflicting. Jahan et al. [15] detail the molecular mechanisms involved in the control of fibroblast activation. Specifically, the role of Zeb2 transcription factor which could serve as an effective target for the attenuation/prevention of cardiac fibrosis is discussed and the potential of specific nutraceuticals described. Lim et al. [16] describe the current understanding of α-Klotho, a protein with anti-aging properties, as a potential therapeutic against age-associated vascular abnormalities. From the evidence presented in this review, it is conceivable that Klotho-based interventional trials could be initiated and yield important data for the effective clinical use of α-Klotho in several pathophysiological conditions including chronic kidney disease, cancer, diabetes and HIV infection where age-related vascular alterations are implicated. High sodium intake has been shown to have a positive relationship with death [17]. Paczula et al. [18] discuss the role of endogenous cardiotonic steroids in mediating salt-induced hypertension and organ damage. Compelling lines of evidence are provided in the link between high-salt diet and organ damage and thus it can be inferred that control of dietary salt is of critical importance in the prevention and/or risk reduction. Irisin has been characterized as a myokine that has been linked to insulin resistance, obesity and other non-communicable adult diseases. However, information regarding the role of irisin in childhood and early adulthood is inconsistent. In this regard, Elizondo-Montemayor [19] discusses the potential role of irisin in cardiovascular and metabolic health and disease in the pediatric population.

Taking all contributions into account it is clear that nutrition plays an important role in cardiovascular health and disease. In general, nutrients exhibit a diverse range of properties including anti-oxidant effects, anti-inflammatory actions, modification of signal transduction mechanisms, as well as metabolic, molecular and membrane actions. Although the cardioprotective effects may not be due to a single nutrient, but a balanced and varied diet of food items that can provide different benefits may prove to be key to cardiovascular health. We are grateful to all contributors, who are highly regarded and well-recognized in their respective field of interest. They have helped to generate a valuable issue of the journal that is very much special. It is hoped that experts within the field and those that have a general interest in nutrition and human health will find the information presented in this Special Issue, educational and insightful to prompt further investigation and advancement in understanding into the essential role of nutrition in cardiovascular health and disease treatment and/or prevention.

**Acknowledgments:** Infrastructural support for this work was provided by the St. Boniface Hospital Research Foundation, Winnipeg, Canada.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Nutrition and Cardiovascular Health**

#### **Rosa Casas 1,2, Sara Castro-Barquero 1, Ramon Estruch 1,2 and Emilio Sacanella 1,2,\***

Received: 19 November 2018; Accepted: 6 December 2018; Published: 11 December 2018

**Abstract:** Cardiovascular disease (CVD) is the leading cause of death in Western countries, representing almost 30% of all deaths worldwide. Evidence shows the effectiveness of healthy dietary patterns and lifestyles for the prevention of CVD. Furthermore, the rising incidence of CVD over the last 25 years has become a public health priority, especially the prevention of CVD (or cardiovascular events) through lifestyle interventions. Current scientific evidence shows that Western dietary patterns compared to healthier dietary patterns, such as the 'Mediterranean diet' (MeDiet), leads to an excessive production of proinflammatory cytokines associated with a reduced synthesis of anti-inflammatory cytokines. In fact, dietary intervention allows better combination of multiple foods and nutrients. Therefore, a healthy dietary pattern shows a greater magnitude of beneficial effects than the potential effects of a single nutrient supplementation. This review aims to identify potential targets (food patterns, single foods, or individual nutrients) for preventing CVD and quantifies the magnitude of the beneficial effects observed. On the other hand, we analyze the possible mechanisms implicated in this cardioprotective effect.

**Keywords:** Mediterranean diet; cardiovascular disease; inflammation; nutrients; polyphenols; MUFA; PUFA; bioactive compounds; phytosterols; dietary pattern

#### **1. Introduction**

Data obtained in 2013 showed that the leading global cause of death in Western countries is cardiovascular disease (CVD), accounting for 17.3 million of all deaths worldwide per year (or 31.5% of all global deaths), despite steadily decreasing during the past 10 years [1,2]. One in three deaths in the United States and one in four deaths in Europe are caused by CVD [3]. So, in 2035, 45.1% (>130 million adults) of the US population are projected to have clinical expression of CVD [1,4]. CVD describes a range of disorders that affect the heart and blood vessels, such as hypertension, stroke, atherosclerosis, peripheral artery disease, and vein diseases [4]. The probability of developing CVD is associated with unhealthy dietary patterns (i.e., excessive intake of sodium and processed foods; added sugars; unhealthy fats; low intake of fruit and vegetables, whole grains, fiber, legumes, fish, and nuts), together with a lack of exercise, overweight and obesity, stress, alcohol consumption, or a smoking habit (Figure 1) [5–7]. Additionally, CVD often coincides with multiple co-morbidities, such as obesity, diabetes, hypertension, or dyslipidemia, which represent four of the 10 greatest risk factors for all-cause mortality worldwide [8]. Furthermore, the rising incidence of CVD over the last 25 years has become a public health priority, especially the prevention of CVD (or cardiovascular events) through lifestyle interventions [9]. On the one hand, a large body of scientific evidence has reported that nutrition might be the most preventive factor of CVD death [10], and could even reverse heart disease [8]. On the other hand, diet seems to play an important role in the management of

other risk factors, such as excess weight, hypertension, diabetes, or dyslipidemia [8]. In this sense, the identification and classification of nutrients, foods, or dietary patterns that can enhance CVD prevention is a priority.

**Figure 1.** Unhealthy dietary patterns, together with a lack of exercise, overweight and obesity, aging, gender, genetics, or a smoking habit, among others, might lead to the development of cardiovascular disease (CVD).

Atherosclerosis is an inflammatory disease that contributes to major incidence and mortality of CVD. Oxidative stress and systemic inflammation are modifiable by nutrition [10–13], with an excess energy intake and physical inactivity as contributors of pro-inflammatory cytokines' secretion [14]. Inflammatory processes involve the sub-endothelial area of the arterial wall, accumulating lipids and lipid-laden macrophages among other cell types [15,16]. Current scientific evidence shows that chronic inflammation plays a key role in the pathogenesis of coronary artery disease (CAD), including the initiation and progression of atheroma plaque and rupture, and post-angioplasty and restenosis [17]. The main mediators of CAD development are C-reactive protein (CRP), interleukin (IL)-1, IL-6, IL-8, IL-1β, IL-18, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α, among others. Moreover, those mediators are considered potential inflammation biomarkers and their expression may correlate with CAD severity [17–19].

As such, current evidence indicates that Western dietary patterns compared to healthier dietary patterns, such as the "Mediterranean diet" (MeDiet), leading to an excessive production of proinflammatory cytokines associated with a reduction of the synthesis of anti-inflammatory cytokines [20–23]. Therefore, the intake of fruits, vegetables, whole grains, nuts, seeds, and legumes is associated with lower inflammation [24–28], whereas red meat intake has been correlated with a higher inflammatory level [24,29–31]. Consequently, increased adherence to healthier dietary patterns, characterized by higher intake of fruits, vegetables, legumes, nuts, and whole grains, may mitigate low grade inflammation, preventing CVD [32–35].

In addition, microbiota has been linked to intestinal health, the immune, system and bioactivation and metabolism of nutrients, such as vitamins B and K and bioactive compounds. Recent clinical studies suggest a correlation between elevated plasma trimethylamine N-oxide (TMAO), which is produced by gut bacteria metabolism of dietary components, such as L-carnitine, betaine, and choline, and a higher risk of diabetes, hypertension, and atherosclerosis [36–38]. Therefore, it has been well

studied that diet affects the composition and activity of gut microbiota and situations of gut microbiota dysbiosis may be involved in the development of CVD.

This review aims to identify potential targets (food patterns, single foods, or individual nutrients) for CVD prevention, quantify the magnitude of the beneficial effects observed, and analyze the mechanisms implicated in these cardioprotective effects. Besides, studies were limited to humans with no time restriction. Relevant studies, systematic reviews, and meta-analyses were searched to obtain the reference lists. The Medical Subject Headings search terms included: Inflammation, oxidative stress, inflammatory markers, IL, CRP, TNF-α, IL-6, dietary pattern, Mediterranean diet, Dietary Approach to Stop Hypertension (DASH diet), atherosclerosis, fruits and vegetables, olive oil, nuts, wine, fiber, micronutrients, vitamins, minerals, omega-3 fatty acids, lycopene, phytosterols, and polyphenols.

#### **2. Atherosclerosis**

Early stages of atherosclerosis are involved the internalization of lipids in the intima, mainly low-density lipoproteins (LDL), which is translated to endothelial dysfunction [39]. The disruption of the endothelial function promotes the inflammatory response, thrombus formation, and multiple pathological consequences, such as calcifications, stenosis, rupture, or hemorrhage [15,40].

The inflammatory response is enhanced by the infiltration of low-density lipoproteins (LDL) particles in the extracellular matrix (EM) while circulating monocytes are attached to the endothelium and transformed into macrophages infiltrating into the sub-endothelial area. The retention of LDL in EM is mediated by proteoglycans, which facilitate retention in the intima [41]. The LDL particles attached in the intima are susceptible to oxidative modifications by reactive oxygen species (ROS) and enzymatic modification released from inflammatory cells. Macrophages are converted into foam cells after oxidized LDL (oxLDL) particles are absorbed by them. Additionally, endothelial dysfunction enhances platelets' adhesion, which secretes chemotactic substances and growth factors, promoting plaque progression [42]. Vascular smooth muscle cells (VSMC) are also involved in plaque progression. Foam cells' growth factors and cytokines' secretion enhance VSMC migration to the intima where they contribute to the formation of the fibrous cap [43]. If the progression of lipid accumulation persists, foam cells and macrophages' apoptosis is induced jointly with pro-thrombotic molecules' secretion [44,45]. Atherosclerotic plaque progression and plaque disruption, promoted by pro-thrombotic agents, initiate platelet activation and aggregation, which leads to the coagulation cascade and, consequently, thrombus formation [46]. The clinical manifestations of advanced atherosclerosis are coronary heart disease, ischemic stroke, peripheral artery disease, heart failure, or sudden death [47].

#### **3. Oxidative Stress and Inflammation**

Oxidative stress has been related to the pathogenesis of atherosclerosis [48]. ROS and reactive nitrogen species (RNS) are mainly produced through mitochondrial activity and other pathways, such as nitric oxide (NO) synthase, and oxidase enzymes, such as Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox), xanthine oxidase (XO), lipoxygenase, myeloperoxidase, uncoupled endothelial nitric oxide synthase (eNOS), and the mitochondrial respiratory chain via a one-electron reduction of molecular oxygen. Note the role of Nox in oxidative stress, as upregulated and overactive Nox enzymes contribute to oxidative stress and CVD. Several signaling pathways regulate inactivation and degradation of ROS and RNS, including catalase, glutathione peroxidase, and superoxide dismutase among others. An excess of ROS and RNS leads to oxidative stress, promoting cell proliferation, migration, autophagy, necrosis, DNA damage, endoplasmic reticulum stress, endothelial dysfunction, and higher levels of oxLDL [49,50]. Moreover, ROS activate the inflammatory response that directly affects plaque progression and endothelial function, increasing the levels of inflammatory cytokines, such as interleukins (IL-6, IL-8), TNF-α, and MCP-1, and adhesion molecules, such as intercellular adhesion molecule 1 (sICAM-1) and vascular cell adhesion molecule (sVCAM-1) [51]. Simultaneously, transcription factors activation, mainly nuclear factor kappa B

(NF-κβ) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and signal transduction cascades result in a high production of inflammatory cytokines and inducible nitric oxide synthase [52]. NO has important anti-inflammatory, antihypertensive, and antithrombotic actions due to its strong vasodilator activity and anti-platelet aggregation. Additionally, anti-inflammatory effects are enhanced by the ability of NO to inhibit NF-κβ expression and the subsequent adhesion molecules [53]. Oxidative stress contributes to endothelial eNOS dysfunction [54,55]. Dysfunctional eNOS generates superoxide anions instead of NO, which is translated to a higher ROS production and contributes to atherogenesis [56]. In the case of inducible NOS (iNOS), which is expressed in cells after cytokines or bacterial lipopolysaccharide stimulation, an excessive and sustained production of NO has been linked with inflammatory diseases and septic shock [57]. Therefore, a decrease of NO production by eNOS leads to endothelial dysfunction while an excessive NO production by iNOS may induce pro-inflammatory and pro-atherogenic factors.

The causes and risk factor of atherosclerosis and oxidative stress are not well defined. However, certain health conditions and habits may contribute to atherosclerosis development, such as high total cholesterol and low-high-density lipoprotein cholesterol (HDL-c) levels, hypertension, type 2 diabetes mellitus (T2DM), obesity, and physical inactivity. Additionally, healthy dietary patterns and lifestyle modifications are potential strategies for atherosclerosis and oxidative stress prevention.

#### **4. Dietary Patterns**

Several studies correlate healthy dietary patterns with lower plasmatic concentrations of pro-inflammatory markers [58], whilst a Western-type diet (meat-based dietary pattern) is associated with higher levels of low-grade inflammation [31]. For that reason, CVD guidelines recommend a healthy diet. Dietary intervention allows a better combination of multiple foods and nutrients. Therefore, healthy dietary patterns support a greater magnitude of beneficial effects than the potential effects of a single nutrient supplementation, because of the synergistic health effects among them. The current body of evidence shows that healthy dietary patterns share similarities, such as a high intake of fiber, antioxidants, vitamins, minerals, polyphenols, monounsaturated, and polyunsaturated fatty acids (MUFA and PUFA, respectively); low intake of salt, refined sugar, saturated, and trans fats; and carbohydrates of low glycemic load [59]. This translates to a high intake of fruits, vegetables, legumes, fish and seafood, nuts, seeds, whole grains, vegetable oils (mainly, extra virgin olive oil [EVOO]), and dairy foods together with a low intake of pastries, soft drinks, and red and processed meat [59,60].

Mediterranean and DASH dietary interventions are well studied for CV outcomes. Both dietary patterns may reduce the incidence CVD through the down-regulation of low-grade inflammation and better control of body weight, which also improve other risk factors, and are correlated with lower numbers of clinical events [59,60]. Thus, this will be the focus of this research.

#### *4.1. Mediterranean Diet*

Until now, the main benefits of the Mediterranean diet (MeDiet) against CVD (Figure 2) have been associated with a better control of risk factors to improve blood pressure (BP), lipid profile, glucose metabolism, arrhythmic risk, or gut microbiome [59]. Also, some authors have suggested that MeDiet may exert an anti-inflammatory effect (Table 1) in the vascular wall as a possible mechanism to explain the link between MeDiet and low CVD prevalence [61]. Interestingly, MeDiet seems to modulate the expression of pro-atherogenic genes as cyclooxygenase-2 (COX-2), MCP-1, and low-density lipoprotein receptor-related protein (LRP1) [62], reducing plasmatic levels of plaque stability and rupture related molecules as MMP-9, IL-10, IL-13, or IL-18 [63,64].

**Figure 2.** Main protection mechanisms of the Mediterranean diet against cardiovascular disease.

APOA2: Apolipoprotein A2; CETP: Cholesteryl ester transfer protein plasma; COX-2: Cyclooxygenase-2; CRP: C-reactive protein; IL: Interleukin; LRP1: Low-density lipoprotein receptor-related protein; MCP-1: Monocyte chemoattractant protein; MDA: Malondialdehyde; MMP-9: Metallopeptidase-9; oxLDL: Oxidized LDL; PAI-1: Plasminogen activator inhibitor 1; sICAM-1: Soluble intercellular adhesion molecule 1; sVCAM-1: Soluble vascular cell adhesion molecule; TNF-α: Tumor necrosis factor; TNFR: Tumor necrosis factor receptor; TCF7L2: Transcription factor 7-like 2; VEGF: Vascular endothelial growth factor.

The observational study, ATTICA, evaluated the link between MeDiet and the incidence of metabolic syndrome (MetS) in 1514 men and 1528 women (>18 y) without clinical evidence of CVD or any other chronic disease [65] during 10 years. Authors found that an increase of 10% in the MeDiet adherence score was associated with a 15% lower odds for CVD incidence. Nevertheless, the inflammatory factors studied (adiposity, CRP, IL-6), whose components are associated with a higher likelihood of CVD, showed a higher incidence (29%) in those subjects away from the MD [65]. Also, the Multi-Ethnic Study of Atherosclerosis (MESA) investigated if a dietary quality score based in a MeDiet pattern was related with regional adiposity [66]. Authors studied 5079 individuals free of CVD (61 ± 10 years) and found that a high quality dietary pattern was associated with less regional adiposity and a lower body mass index (BMI), CRP, and insulin resistance. Thus, Lahoz et al. [67] conducted a cross-sectional analysis of 1411 subjects of the Screening PRE-diabetes and type 2 DIAbetes (SPREDIA-2) study (mean age 61 years, 43.0% males) to assess whether the 14-point Mediterranean Diet Adherence Screener (MEDAS) was associated with serum CRP levels. After adjusting for confounders, the authors showed an inverse correlation between the adherence to MeDiet and CRP levels (*p* = 0.041). Also, a substudy of the MOLI-SANI cohort (6879 women and 6892 men) found that men with a higher adherence to a healthy high-antioxidant diet (HAC), vitamins, and phytochemicals enriched diet, inside a MeDiet pattern, were more protected against hypertension and inflammation than those with

a healthy low-antioxidant diet [68]. Authors found HAC was associated with a significant decrease in CRP levels (β = 0.03, *p* = 0.03). Finally, Sureda et al. [69] conducted a study of two cross-sectional nutritional surveys with men and women (219 males and 379 females) aged among 12–65 years old, who lived on the Balearic Islands. Results showed that the male adult population with a higher adherence to the MeDiet showed lower concentrations of pro-inflammatory biomarkers, such as TNF-α and hs-CRP. Also, in this population, lower levels of leptin or plasminogen activator inhibitor 1 (PAI-1) were observed, while adiponectine levels were increased. Moreover, females (young and old), with a higher adherence to the MeDiet, showed lower hs-CRP levels. Lower leptin levels were showed only in the young female group, while PAI-1 reduction was only observed in female adults.

On the other hand, the PREDIMED (Prevención con Dieta Mediterránea) study, the largest interventional study about MeDiet, which included 7447 subjects (55 to 80 years of age, 57% women) at high CV risk, without CVD at baseline, showed a lower prevalence of CV events in participants assigned to a MeDiet supplemented with extra-virgin olive oil (EVOO) or nuts than those assigned to a low-fat diet after five years intervention [70]. Focusing on the PREDIMED study, the MeDiet has reported an anti-inflammatory effect on the expression of adhesion molecules in leukocytes, but also improvements in the circulating levels of soluble adhesion molecules (sVCAM-1, sICAM-1, E- and P-selectin, cytokines (IL-1, IL-6, IL-8, IL-12p70, CRP, TNF-α, tumor necrosis factor receptor (TNFR)-60 and 80, etc.), chemokines (MCP-1, Regulated on Activation, Normal T Cell Expressed and Secreted [RANTES], macrophage inflammatory proteins [MIP-1β], etc.) and molecules related with vulnerability atheroma plaque (IL-10, IL-13, IL-18, or Matrix metallopeptidase-9 [MMP-9]) after three months, one, three, and five years intervention in those participants that followed a MeDiet supplemented with EVOO or nuts [63,64,71–74]. On the one hand, the results obtained support that MeDiet exerts an important immunomodulatory effect, reducing proinflammatory biomarkers, especially those related to atheroma stability plaque. On the other hand, these anti-inflammatory effects seem to appear in the short and medium-term (three months, one year) and later is maintained in the long-term (three and five years). In a Swedish randomized crossover study [75], the adherence to a Medierranean-style diet and its correlation with inflammatory biomarkers (CRP, IL-6), vasoregulation, vascular endothelial growth factor (VEGF), and serum phospholipid fatty acid composition was investigated. The 22 subjects free of CVD (10 women) received a Mediterranean-type diet or Swedish diet for four weeks. No changes were observed for CRP or IL-6 although the MeDiet group showed significant reductions in leukocytes and platelets levels (by 10% and 15%, respectively) and in VEGF levels (15%). Esposito and et al. [76] assessed the effects of a Mediterranean dietary pattern on endothelial function and vascular inflammatory markers in patients with the MetS during two years (90 patients/intervention group). Compared with controls, the MeDiet group showed lower serum concentrations of high-sensitivity ((hs)-CRP, IL-6, IL-7, and IL-18 (*p* ≤ 0.04; all)) and improved the endothelial function score (*p* < 0.001), defined as a measure of BP and platelet aggregation response to l-arginine. These results are in agreement with Azzini et al. [77], who also reported improvements in the CV risk profile and modulation of inflammatory levels (IL-10 and TNF-α), and a reduction in oxidative stress (malondialdehyde [MDA]).

The influence of polyphenols' content of a MeDiet pattern was also studied in the PREDIMED study [78]. Total polyphenol excretion (TPE) in urine was analyzed in 1139 participants homogenously and randomly in one of the three groups. Authors observed that MeDiet (supplemented with EVOO or nuts) significantly increased their TPE after one year of dietary intervention, decreasing inflammatory biomarkers compared with baseline (sVCAM-1, sICAM-1, IL-6 MCP-1, TNF-α).

Epigenetic studies have also reported similar results. Thus, Arpón et al. [79,80] conducted a substudy on 36 participants of the PREDIMED cohort after five years of intervention, where the main results were that a MeDiet supplemented with EVOO or nuts may influence the methylation status of peripheral white blood cell (PWBCs) genes. These changes were mainly observed in genes related to intermediate metabolism, diabetes, inflammation, and signal transduction. However, interactions among MeDiet and COX-2, IL-6, apolipoprotein A2 (APOA2), cholesteryl ester transfer

protein plasma (CETP), and transcription factor 7-like 2 (TCF7L2) gene polymorphisms have been demonstrated [81–84].

#### *4.2. DASH Diet*

A large body of evidence supports that adherence to a DASH dietary pattern is linked to improvements in BP [85], body weight [86], glucose-insulin homeostasis [87], blood lipids and lipoproteins [88], inflammation grade [89,90], endothelial function [91,92], the gut microbiome [93,94], CVD risk [95,96], and total mortality [97,98]. The DASH diet is characterized by a high intake of fruits and vegetables, legumes, low fat dairy, whole grain products, nuts, fish, and poultry; a reduced intake of saturated fat, red meat and processed meats, and sweet beverages; and a low intake of sodium and refined grains [89,99].

Focusing on inflammatory markers and oxidative stress, several studies have shown the protective effect of the DASH diet on CVD (Table 1). A recent systematic review and meta-analysis of randomized trials [89], which included six randomized control trials (RCT) with 451 participants who were followed for 3–24 weeks, studied the effect of the DASH diet on inflammatory biomarkers. Results showed that the DASH diet significantly reduced high hs-CRP concentrations (mean difference (MD) = −1.01, 95% confidence interval (CI): −1.64, −0.38; I-squared (I2) = 67.7%) compared to other diets. When the follow up of participants was longer, the reduction of hs-CRP serum levels was greater. However, when the DASH diet effect was compared to other healthy diets, no significant changes were observed. Besides, a meta-analysis conducted by Neale et al. [100] about 17 RCTs observed that following a healthy diet (MeDiet, Nordic diet, Tibetan diet, and DASH diet) was associated with a significant reduction of CRP levels (−0.75, 95% confidence interval (CI): −1.16, −0.35; *p* = 0.003). No changes were found for the other biomarkers (TNF-α, total adiponectin, high-molecular-weight adiponectin, adiponectin:leptin ratio, resistin, or retinol binding protein 4). Eichelmann et al. [101] studied the link between plant-based diets (Nordic diet, MeDiet, vegetarian diet, plant-based diet, Paleolithic diet, and DASH) and obesity-related pro-inflammatory markers (CRP, IL-6, TNF-α, sICAM-1, leptin, adiponectin, and resistin) on 29 interventional trials with a total of 2689 participants. Results showed improvements in obesity-related inflammatory profiles after following plant-based diets: CRP (−0.55 mg/L), IL-6 (−0.25 ng/L), and sICAM-1 (−25.07 ng/mL). No significant changes were observed for TNF-α, resistin, adiponectin, and leptin.

In a cross-sectional analysis of 1493 men and women (aged 50–69 years), potential associations between dietary quality (through the DASH dietary quality score), adiposity, and biomarkers of glucose metabolism, lipid profile, and inflammation were assessed [102]. Results showed that a higher adherence to the DASH dietary pattern was associated with improvements in adiposity measures (BMI, *p* < 0.05, waist circumference, *p* < 0.001), and lower concentrations of TNF-α, IL-6, CRP, WBC, and PAI-1 (*p* < 0.05; all), such as pro-inflammatory, pro-thrombotic, and pro-atherogenic markers. Improvements in lipoprotein profile parameters (LDL-c, HDL-c, and lower large very low density lipoprotein [VLDL] particles, *p* < 0.001 all) and glucose homeostasis biomarkers (HOMA, insulin and glucose, *p* < 0.05 all) were also shown.

With respect to interventional studies, the DASH diet has reported improvements on insulin resistance, inflammation, and oxidative stress in women with gestational diabetes [103]. The RCT was performed with 32 pregnant women diagnosed with gestational diabetes (GD) at 24- to 28-week gestation. All of them were randomly assigned to a DASH diet group or a control group (16 participants/ group) and were followed up for four weeks. The DASH diet compared with the control showed significant reductions in serum insulin levels (−2.62 μIU/mL, *p* = 0.03), fasting plasma glucose (FPG) (−7.62 mg/dL, *p* = 0.02), and the homeostasis model of assessment-insulin resistance (HOMA-IR) score (−0.8, *p* = 0.03). Furthermore, Kawamura et al. [104] also reported significant reductions in the BMI, BP, FPG, and fasting insulin level (*p* ≤ 0.003; all) after analyzing 58 Japanese participants with untreated high-normal BP or stage 1 hypertension (30 men and 28 women; mean age 54.1 ± 8.1 years), who followed a modified DASH diet (salt 8.0 g/day) during two months. Finally, the DASH diet also

increased the plasma total antioxidant capacity (45.2 mmol/L, *p* < 0.0001) and total glutathione levels (108.1 μmol/L, *p* < 0.0001). Neither group showed changes in serum hs-CRP levels. Saneei et al. [105] conducted a cross-over study, which examined the effects of the DASH diet on markers of systemic inflammation in 60 post-pubescent girls with MetS (aged 11–18 years and weight mean was 69 kg). Participants were randomized into two groups: The DASH diet or usual dietary advice (control group) and were followed for six weeks. Results did not show significant changes on TNF-α, IL-2, IL-6, and adiponectin levels, whereas hs-CRP levels were significantly lower (−0.09 mg/L, *p* = 0.002) in those participants with higher adherence to a DASH diet compared to the control group.

#### **5. Foods**

#### *5.1. Fruits and Vegetables*

The European Society of Cardiology (ESC) and American Heart Association Nutrition Committee strongly endorse the daily consumption of multiple servings of both fruits and vegetables in order to reduce CVD risk [106,107]. These recommendations are based upon epidemiological studies and meta-analysis, mainly [106–113]. A recent meta-analysis [108] with 83 studies (71 clinical trials and 12 observational studies) showed that a higher intake of fruit or vegetable was significantly inversely associated with CRP and TNF-α levels (*p* < 0.05; both) and directly associated with an increased proliferation of γδ-T cell populations (*p* < 0.05). Also, Corley et al. [111] studied, in 792 participants aged 70 years from the Lothian Birth Cohort 1936, the association between biomarkers of systemic inflammation (such as CRP and fibrinogen) and specific single foods (fruits and vegetables). The dietary intake was measured using a 168-item Food Frequency Questionnaire (FFQ). Authors described that a higher fresh fruit intake was associated with lower CRP levels (≤3 mg/L) (β = 0.100, 95% CI 0.82, 0.99). No significant association was found between vegetables and CRP. Similar results (*p* < 0.05) were found between fibrinogen levels and fruit intake (β = 0.083) or combined fruits and vegetables intake (β = 0.084).

Also, in the cross-sectional study conducted by Holt et al. [112], in 285 healthy adolescent boys and girls aged 13 to 17 years, it was found that serum CRP levels were inversely associated with fruit intake (r = −0.19; *p* = 0.004), while IL-6 was inversely associated with fruit and vegetable intake and TNF-α only with vegetable consumption (*p* < 0.05; both). The HELENA Cross-Sectional Study [113], which aimed to demonstrate that a healthy diet might reduce adiposity and systemic inflammation, found that fruits and nuts were negatively linked with IL-4 (all subjects, *p* < 0.05; both) and TNF-α (only girls, *p* = 0.036). Contrastingly, vegetables showed only significant inverse correlations with sE-selectin (all subjects, *p* ≤ 0.0012; both). This study was carried out in 464 adolescents (13–17 years) of the European HELENA cohort. In a cross-sectional analysis [114], in 1005 Chinese women aged 40 to 70 years, the association between vegetable intake and inflammatory and oxidative stress markers was studied. Results showed that a higher intake of cruciferous vegetables was associated with lower concentrations of TNF-α (*p* trend = 0.001), IL-1β (*p* trend = 0.004), and IL-6 (*p* trend = 0.02). Additionally, the mean difference of concentrations among the highest and the lowest quintiles of cruciferous vegetables intake were 12.66% for TNF-α, 18.18% for IL-1β, and 24.68% for IL-6. Any association was observed between the consumption of cruciferous vegetable and oxidative stress markers (F2-isoprostanes and 2,3-dinor-5,6-dihydro-15-F2t-IsoP).

Finally, in a sub-study from the PREDIMED study, Urpí-Sardà et al. [77] found that the participants who increased more than 62.7 g/day of their consumption of vegetables after one year decreased their plasma concentration of TNFR60 from 1.7 μg/L to 1.5 μg/L (*p* < 0.05), as shown in Table 2.

CCR2: C-C chemokine receptor type 2; CD15: Sialil-Lewis X; CRP: C-reactive protein; IL: Interleukin; LFA: Lymphocyte function-associated antigen 1; MAC-1: T-lymphocytes and macrophage-1 receptor; MCP-1: Monocyte chemoattractant protein; MDA: Malondialdehyde; MDC: Macrophage-derived chemokine; MMP-9: Metallopeptidase-9; NT-proBNP: Pro-brain natriuretic peptide; oxLDL: Oxidized LDL; PAI-1: Plasminogen activator inhibitor 1; PF4: Platelet factor 4; sICAM-1: Soluble intercellular adhesion molecule 1; SOD: Superoxide dismutase; sVCAM-1: Soluble vascular cell adhesion molecule; TNF: Tumor necrosis factor; TNFR: Tumor necrosis factor receptor; IFN-γ: Interferon gamma.

#### *5.2. Olive Oil*

Several studies and meta-analyses have demonstrated the anti-inflammatory effects of olive oil (OO) rich diets [115–117]. Bioactive components of EVOO, the main key food of the MeDiet, have demonstrated improvements in inflammatory status, oxidative stress, and endothelial dysfunction [115]. A recent meta-analysis conducted by Schwingshackl et al. [118] in 30 RCT (3106 participants and daily consumption of 1 mg and 50 mg OO) found a significant decrease in CRP (−0.64 mg/L, *p* < 0.0001, n = 15 trials) and IL-6 (−0.29 mg/L, *p* < 0.04, n = 7 trials) compared to controls. Also, the flow-mediated dilatation (FMD) value was significantly increased in subjects with highest OO intake (0.6%, *p* < 0.002).

Several studies from the PREDIMED study and others related to the MeDiet have reported that a MeDiet supplemented with EVOO leads to decreased levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) [119], the progression of intima media thickness (IMT) in those with elevated baseline IMT [120,121], improved systolic and diastolic BP in both hypertensive and non-hypertensive patients [122–124], and decreased expression and concentration of circulating inflammatory biomarkers related to atherosclerosis [63,64,71–74]. Urpí-Sardà et al. [72] reported that those participants with a higher adherence to a MeDiet + EVOO and whose intake of VOO was higher than 24 g/day showed lower plasma concentrations of TNFR60 after one year of intervention. Moreover, Camargo et al. [125] observed that, after isolation of peripheral blood mononuclear cells (PBMCs), the MeDiet exerted an inhibitory effect on the expression of genes related to plaque progression, such as MMP-9, NF-κβ or MCP-1, by increasing IκB kinase (IκBα) expression after the intake of a MeDiet + EVOO compared with two other diets (*p* < 0.05; all). IκBα stabilizes the NF-κβ molecule in the cytoplasm, maintaining it in an "unactivated" state. Widmer et al. [126] observed that daily consumption of 30 mL of OO or 30 mL of OO supplemented with epigallocatechin 3-gallate (EGCG) in 82 subjects (≥18-y) with early atherosclerosis showed improvements of endothelial dysfunction, independently of EGCG supplementation, after four months of intervention. However, the OO + EGCG group showed significant reductions in inflammatory parameters, such as sICAM-1 (*p* ≤ 0.001), white blood cells (*p* < 0.05), monocytes, and lymphocytes (*p* < 0.05; both). Additionally, Oliveras-López et al. [127] showed an increase in plasma antioxidant capacity, antioxidant enzymes-catalase, and glutathione peroxidase, as well as an improvement in the superoxide dismutase (SOD) expression after analyzing 45 healthy adults (age: 21–45 years old, mean BMI: 21.4 ± 0.5 kg/m2) who ingested 50 mL of EVOO for 30 days. Along with the other studies, the intake of EVOO seems to have positive effects on endothelial function. Finally, in the VOLOS (Virgin Olive Oil Study), participants with mild dyslipemia were randomized in two groups of intervention (daily 40 mL of EVOO with 166 mg/L of hydroxytyrosol vs. refined OO with 2 mg/L of hydroxytyrosol) during seven weeks. Results showed significant reduction of thromboxane B2 (TXB2) levels of 20% in the EVOO groups [128], as shown in Table 2.

#### *5.3. Nuts*

Nuts, specifically peanuts and walnuts, have been demonstrated to reduce the CVD morbidity and mortality in numerous large prospective cohort studies [129,130]. Mente et al. [131] predicted that nut intake might offer a preventative risk reduction on heart disease (RR = 0.67 [95% CI: 0.57–0.77]). Also, nut intake is associated with weight loss improvements [132], lower LDL-c levels [71,133,134], hypertension risk [133,135], and T2DM, improving hyperglycemia and insulin resistance [136], and inflammatory and oxidants mediators [134,137–139].

In a recent extensive meta-analysis, 23 RCTs were evaluated [139] to investigate the effects of nut intake over some inflammatory biomarkers (CRP, sICAM-1, sVCAM-1, IL-6, E-selectin, TNF-α). Authors found significant reductions of sICAM-1 (−0.17 ng/mL, *p* = 0.01) after nut intake. No changes were observed among the others inflammatory markers. Similar results were found in the meta-analysis conducted by Neale et al. [138]. No significant differences were shown after analyzing a wide number of inflammatory biomarkers, such as CRP, adiponectin, IL-6, sICAM-1, sVCAM-1, and FMD, in a total of 32 RCT studies. A significant improvement in FMD after nut intake was observed.

On the one hand, Yu et al. [137] conducted a cross-sectional study to investigate if nut intake was correlated to inflammatory biomarkers levels (CRP, IL-6, and tumor necrosis factor receptor 2 (TNFR2)) from 5013 participants in the Nurses' Health Study (NHS) and Health Professionals Follow-Up Study (HPFS) who were non-diabetic. Results showed that a higher nut intake showed lower inflammatory biomarkers levels (CRP: RR = 0.80 [95% CI: 0.69, 0.90, *p*-trend = 0.0003]; and IL-6: RR = 0.86 [95% CI: 0.77, 0.97, *p*-trend = 0.006]).

However, a randomized, parallel-group study on 50 patients with MetS and supplemented with 30 g/day of raw nuts (15 g walnuts, 7.5 g almonds, and 7.5 g hazelnuts) showed significant reductions of plasma concentrations of IL-6 (−1.1 ng/L; *p* = 0.035) compared with a control diet [140]. These results are according with the data showed by Hernández-Alonso et al. [141], who reported, in a randomized cross-over study in 54 participants, significant reductions of fibrinogen, oxLDL, and platelet factor 4 levels in the pistachio-supplemented group compared with control group (*p* < 0.05; all) after four months of intervention. Additionally, the pistachio-supplemented group showed lower IL-6 (−9%) and resistin gene expression (−6%) (*p* < 0.05; both). Similar results (−10.3% for IL-6 and CRP, −15.7% for TNF-α) were showed in a randomized, crossover study for 12-weeks carried out by Liu et al. [142] in 20 Chinese subjects with T2DM with mild hyperlipidemia (nine men and 11 women, mean age of 58 years, and BMI of 26 kg/m2). See Table 2.

#### *5.4. Wine and Other Fermented Alcoholic Beverages*

Nowadays, there is enough evidence from both epidemiologic studies and RCTs to conclude that regular moderate consumption of fermented alcoholic beverages, mainly red wine and beer, has cardioprotective effects and can exert a positive effect on CV risk factors [143–145].

#### 5.4.1. Wine

Wine or wine-derived phenolic compounds that exert effects through mechanisms on atherosclerosis are clearly identified. On the one hand, wine and their phenolic compounds decrease oxidation of LDL-c and oxidation stress, and increase in NO, improving endothelial function. Also, ethanol increases HDL-c levels and inhibits platelet aggregation, promotes fibrinolysis, and reduces systemic inflammation [144,146].

Janssen et al. [147] investigated the relationship of wine consumption and CV risk markers (CRP, fibrinogen, factor VII, and PAI-1) in a multi-ethnic sample of 2900 healthy women of middle-age, who were followed up for seven years. Authors concluded that moderate wine consumption may protect against CVD, after observing lower concentrations of CRP (*p* < 0.001), fibrinogen (*p* < 0.001), factor VII (*p* < 0.01), and PAI-1 (*p* < 0.05) compared to abstainers or women that drink little wine.

On the other side, Estruch et al. [148] reported that both red wine and gin have anti-inflammatory properties in the atherosclerotic process through the reduction of fibrinogen levels (−9%) and IL-1α (−21%), as well as lower plasma hs-CRP (−21%), sVCAM-1 (−17%), and sICAM-1 (−9%) levels. Moreover, monocyte expression was significantly reduced by 27–32% (LFA-1, MAC-1, VLA-4). In another randomized, crossover consumption trial in 67 males at high risk of CVD, Chiva-Blanch et al. [149] investigated the effects of ethanol and phenolic compounds of 30 g alcohol/day of red wine, and the equivalent amount of dealcoholized red wine and gin (30 g alcohol/d) for four weeks on the expression of inflammatory biomarkers related to atherosclerosis. Thirty g/day of alcohol of red wine showed an increase in plasma concentrations of IL-10 and decreased macrophage-derived chemokine (MDC). On the other hand, sICAM-1, E-selectin, and IL-6 were reduced by phenolic compounds of red wine. Phenolic compounds also inhibited the expression of LFA-1 in T-lymphocytes and MAC-1, and CCR2 expressions in monocytes. Concentrations of CD40 antigen, CD40 ligand IL-16, MCP-1, and sVCAM-1 were downregulated in both groups: Ethanol and phenolic compounds of red wine. A current study conducted by Roth et al. [150] found that aged white wine presents a greater ability to repair and maintain endothelial integrity than gin. In this randomized, controlled, crossover study, 38 high-risk male volunteers (55–80 years), who received 30 g ethanol/day as aged white wine or gin for three weeks, were evaluated. After intervention, T-lymphocytes' expression of CD31 and CD40 and monocytes expression of CCR2 and CD36 were lower after consumption of aged white wine. Additionally, for aged white wine, a significant reduction was observed in plasma concentrations of IL-8 and IL-18, sICAM-1 and sVCAM-1. Both beverages showed significant reductions in LFA-1, MAC-1, VLA4, CD40, and CD31 expression, as well as lower concentrations of interferon gamma (IFN-γ). Finally, Estruch et al. [151], in a new study, where wine and gin were compared again, found that wine intake significantly decreased plasma SOD activity [8.1 U/gHb (95% CI: 138, 25; *p* = 0.009)] and MDA levels [11.9 nmol/L (95% CI: 21.4, 2.5; *p* = 0.020)] compared to the gin group, as shown in Table 2.

#### 5.4.2. Beer

Among fermented beverages, beer has a moderate polyphenol content that confers grater cardioprotective effects than spirits and distilled beverages [145]. So, De Gaetano et al. [152] described in a consensus document the effects of moderate beer consumption on health and disease, where they concluded that epidemiological studies showed that low-moderate doses of beer intake protect against CV risk and its effect is comparable to that reported for moderate red wine consumption.

In 1999, Wannamethee et al. [153] studied 7735 British men during 17 years and found that regular beer intake was associated with lower total mortality (HR = 0.84 (CI: 0.71–10.01)).

Finally, Chiva-Blanch et al. [154] performed a randomized, crossover controlled clinical trial with 33 subjects to evaluate the effects of three beverages types: A non-phenolic alcoholic beverage, such as gin; beer, which is a moderate phenolic alcoholic beverage; and a non-alcoholic beer, with the same amount of phenolic compounds. Beer and gin showed improvements in the HDL cholesterol levels (around 5%) and adiponectin (around 7%) compared to the non-alcoholic beer intervention. Related to circulating inflammatory biomarkers, IL-1α levels increased (around 24%) and IL-5 levels decreased around 14% after beer and gin intervention. However, non-alcoholic beer showed significant improvements in homocysteine concentration (decreased by around 6%) and improved folic acid levels around 9%. Related to inflammatory biomarkers, non-alcoholic beer intervention showed significant decreases of IL-6r, IL-15, RANTES, and TNF-β levels, as shown in Table 2.

#### **6. Nutrients**

It is important to focus on the possible benefits of the intake of specific nutrients to avoid possible deficiencies of these nutrients, which can lead to the development of atherosclerotic disease. We have only included information about fiber, some vitamins, and minerals, but no other nutrients—such as carbohydrates, fats, or proteins—which have also been demonstrated to have a certain effect on the risk of developing atherosclerosis.

#### *6.1. Fiber*

A wide number of studies and scientific publications have reported the health benefits of dietary fiber intake decreasing cholesterol concentrations and BP, while a deficiency of fiber intake is associated with CVD development [155].

On the one hand, several meta-analyses have displayed that a higher dietary fiber intake is linked with a lower relative risk of total all-cause mortality among 16–23% [156–158]. On the other hand, several clinical trials have studied the link between diet and inflammation, and more specifically, the impact of dietary fiber. Although, to date, the implicated mechanisms are still unknown, the proposed mechanisms are that dietary fiber decreases the glucose absorption, and down-regulates the expression of oxidative stress related cytokines or the inflammatory response mediated by gut microbiota exposed to fiber [159].

In an observational study of 1958 postmenopausal women (age 50–79 years), dietary fiber consumption was associated with higher levels of inflammatory markers (CRP and IL-6) [160]. Also, in the Women's Health Initiative Observational Study (13,745 US men and women), a higher fiber intake (24.7 g/day) was associated with lower plasma concentrations of IL-6 and TNFR2 compared with the lowest fiber intake group (7.7 g/day) [161]. Similar results were expressed by Qi et al. [162], who observed that concentrations of CRP and TNFR2 were among 8% to 18% lower in the highest quintile of cereal fiber intake compared to the lowest quintile. Similar results were obtained by Estruch et al. [163] for CRP levels and other inflammatory cytokines (IL-6, sICAM-1, sVCAM-1), whose decrease was inversely related with dietary fiber intake, but not significantly. Additionally, cross-sectional data (1088 participants without T2DM at baseline and aged 40 y–60 y) from the Insulin Resistance Atherosclerosis Study [164] showed that whole grain products' intake was inversely related to PAI-1 (β = −0.102; SEM = 0.038; *p* = 0.0077) and CRP plasma concentrations (β = −0.102; SEM = 0.048; *p* = 0.0340).

In interventional studies, North et al. [165] studied, in 554 subjects (192 men, 362 women), the associations between dietary fiber and CRP levels, showing significantly lower CRP concentrations (−25–54%) with higher fiber intakes (≥3.3 g/MJ). An interventional cross-over study [166] with a total of 60 participants (50% patients with newly diagnosed T2DM, 50% nondiabetic subjects) received three isoenergetic meals separated by one week intervals: A high-fiber (16.8 g) meal; a high-fat meal; and a low-fiber (4.5 g) meal. Results showed that a high fiber intake showed lower plasma IL-18 concentrations and greater stimulation of adiponectin plasmatic levels. Finally, significant reductions of TNF-α (−3.7 pg/mL; *p* < 0.001), were observed after whole grain product intake in a randomized parallel arm feeding trial in 49 subjects who were overweight or obese, and low fruits, vegetables, and whole grain products' intake [167]. See Table 3.

**Table 3.** Potential inflammatory effects of different nutrients on CVD.

CRP: C-reactive protein; IL: Interleukin; PAI-1: Plasminogen activator inhibitor 1; sICAM-1: Soluble intercellular adhesion molecule 1; sVCAM-1: Soluble vascular cell adhesion molecule; tHcy: Total homocysteine; TNF-α: Tumor necrosis factor; TNFR: Tumor necrosis factor receptor.

#### *6.2. Micronutrients*

Nowadays, there are great experimental, epidemiological, and clinical evidence showing how micronutrients' ingestion may protect against CVD [168–170]. Micronutrients exert their protective effect through three possible ways: 1. Reducing endothelial cells damage; 2. improving the production of NO; and 3. inhibiting oxidation of LDL-c [168–170]. Both in adolescence and in adulthood, pro-inflammatory biomarkers have been associated with dietary antioxidants, such as Zn, Se, and vitamin C and E, whose deficiency leads to a higher CVD risk [171–175]. Also, a meta-analysis [176] suggested that Mg supplementation might significantly reduce serum CRP levels (−1.33 mg/L, 95% CI: −2.63, −0.02) after analyzing eight RCTs. Similar results were showed in another meta-analysis [177], where after stratifying by the baseline plasma CRP values of ≤3 and >3 mg/L, found significant reductions of CRP levels (1.12 mg/L, 95% CI: −2.05, −0.18, *p* = 0.019) for the last subgroup. Finally, a recent meta-analysis [178] of seven studies (all RCTs) showed that vitamin D-supplemented groups had lower levels of TNF-α (*p* = 0.04) compared with control groups. No differences between vitamin D and control groups were observed for CRP, IL-10, or IL-6. Relative to vitamin E, a recent meta-analysis [179] suggested that supplementation with vitamin E might reduce serum CRP levels (−0.62 mg/L, 95% CI = −0.92, −0.31; *p* < 0.001) after analyzing 12 trials with 246 participants in the intervention arms and 249 participants in control arms.

De Oliveira Otto et al. [180] investigated the association between dietary micronutrients (heme iron, nonheme iron, zinc (Zn), magnesium (Mg), β-carotene, vitamin C, and vitamin E) with inflammatory markers (CRP, IL-6, total homocysteine (tHcy), fibrinogen, coronary artery calcium, and common and internal carotid artery-IMT) and subclinical atherosclerosis in 5181 participants free of diabetes and CVD from the Multi-Ethnic Study of Atherosclerosis (aged 45 y–84 y). Authors found that Mg and nonheme iron were inversely associated with tHcy concentrations, whilst vitamin C was positively associated with tHcy concentrations. Besides, CRP levels were positively associated with Zn and heme iron, whereas Mg concentrations showed an inverse association with CCA-IMT. Finally, no significant association was observed between dietary intake of carotene or vitamin E and inflammatory markers. Wang et al. [181] also reported that serum vitamin D levels were negatively associated with IL-6 (r = −0.244, *p* = 0.002) and hs-CRP (r = −0.231, *p* = 0.004) levels after studying 152 acute stroke patients. Furthermore, for vitamin D, several observational studies have reported that reduced vitamin D levels are linked with endothelial dysfunction and higher arterial stiffness [182,183], and this deficiency might be related to foam cell formation and decreased expression of adhesion molecules in endothelial cells [184].

Recently, Tabesh et al. [185] examined the effects of co-supplementation of vitamin D and calcium on inflammatory biomarkers and adipokines in 118 diabetic participants with insufficient vitamin D levels. The placebo-controlled clinical trial after eight weeks with four intervention groups ((1) vitamin D + calcium placebo; (2) calcium + vitamin D placebo; (3) vitamin D + calcium; or (4) vitamin D placebo + calcium placebo) showed that supplementation with calcium and vitamin D decreased leptin (−9 ± 18 ng/mL), IL-6 (−4 ± 1 pg/mL, *p* < 0.001), and TNF-α (−3.4 ± 1.3, *p* < 0.05) concentrations compared with the placebo. Also, Shargorodsky et al. [186] studied the effect of vitamin C (500 mg), vitamin E (200 IU), coenzyme Q10 (60 mg), and selenium (120 μg) on inflammatory markers in the long-term (six months) in participants at high CVD risk. No significant changes were observed for homocysteine, endothelin, aldosterone, and renin in participants who received antioxidant supplementation, while there was a significant decrease in HbA1C and a significant increase in HDL-c. Large and small artery elasticity was also significantly increased after antioxidant supplementation intake. In addition, an RCT conducted by Ellulu et al. [187] in 64 obese patients, who were hypertensive and/or diabetic, reported the potential anti-inflammatory effect of 500 mg of vitamin C, twice daily. Vitamin C might decline hs-CRP (*p* = 0.01), IL-6 (*p* = 0.001), and fasting blood glucose (*p* < 0.01) after eight weeks of treatment. Christen et al. [188] also conducted a randomized, double-blind, placebo-controlled sub-study from the Women's Antioxidant and Folic Acid Cardiovascular Study. They tested a daily combination of folic acid (2.5 mg), vitamin B6 (50 mg), vitamin B12 (1 mg), or

placebo on 300 participants (half for each group). After 7.3 years, the supplemented group showed significant reductions in homocysteine concentrations (−18%), whereas no changes were observed in CRP, IL-6, sICAM-1, and fibrinogen levels, as shown in Table 3.

#### **7. Bioactive Compounds**

Multiple bioactive compounds (omega-3 fatty acids, lycopene, or polyphenols) present in the diet have been associated with beneficial effects on atherosclerosis development. All of them act to reduce levels of LDL-c, improving inflammatory and oxidative stress biomarkers. Next, we analyze those cited above.

#### *7.1. Omega-3 Fatty Acids*

PUFAs, as Omega-3 fatty acid (Ω-3 PUFA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have been reported as potential anti-atherogenic agents for the atherosclerotic process [189]. Mechanisms, through which they might reduce CV risk, include improvements in the lipid and lipoprotein profile, oxidation, thrombosis, endothelial function, BP, plaque stability, CV mortality, platelet aggregation, modulating concentration or expression of pro-inflammatory markers (adhesion molecules, cytokines, etc.), and immune cells [190–192].

In a meta-analysis conducted by Wang et al. [193] of 16 randomized placebo-controlled trials in 901 participants, it was reported that Ω-3 PUFA intake (0.45–4.5 g/day, for 56 days) increased FMD by 2.30% (95% CI: 0.89, 3.72%, *p* = 0.001) compared with the placebo group. Additionally, a meta-analysis of 38 RCTs [194] reported reductions by 20–30% in serum triglycerides levels in healthy participants after daily consumption of ≥4 g of EPA and DHA through either supplementation or consumption of enriched foods.

In an observational study [195], 102 Japanese individuals with acute coronary syndrome were analyzed and were stratified in three groups: ≤50, 51–74, and ≥75 years. It was found that low serum DHA concentrations leads to CVD, with DHA useful as a predictive marker of endothelial dysfunction. Similar results were reported by Kelley et al. [196].

On the one hand, Cawood et al. [197] showed that patients in the Ω-3 PUFA group (1.8 g EPA + DHA/day), followed up for 21 days, had a lower number of foam cells (*p* = 0.0390) and T-lymphocytes (*p* = 0.0097), less inflammation (*p* = 0.0108), and improved stability of atheroma plaque (*p* = 0.0209) after analyzing data obtained from a randomized placebo-controlled trial. On the other hand, patients who received Ω-3 PUFAs showed lower expression of mRNA for MMP-7 (*p* = 0.0055), -9 (*p* = 0.0048), -12 (*p* = 0.0044), and for IL-6 (*p* = 0.0395) and sICAM-1 (*p* = 0.0142). Similar results were reported by Thies et al. [198], where after administering dietary fish oil supplements (1.4 g EPA + DHA/day) in patients with advanced atherosclerotic plaque, less inflammation, inhibition of macrophages and lymphocytes infiltration and an increase in plaque stability was observed. Besides, several RCTs have pointed out that Ω-3 PUFAs might modulate the expression of cell adhesion molecules (sICAM-1, sVCAM-1, or sP-selectin) as well as CRP, IL-1β, IL-6, serum amyloid A (SAA), TNF-receptor concentrations, TNF-α, or PAI-1 levels among others [195,199], as shown in Table 4.

CRP: C-reactive protein; IL: Interleukin; LFA: Lymphocyte function-associated antigen 1; MCP-1: Monocyte chemoattractant protein; MMP: Metallopeptidase; PAI-1: Plasminogen activator inhibitor 1; SAA: Serum amyloid A; sICAM-1: Soluble intercellular adhesion molecule 1; sVCAM-1: Soluble vascular cell adhesion molecule; tHcy: Total homocysteine; TNF-α: Tumor necrosis factor; TNFR: Tumor necrosis factor receptor.

#### *7.2. Lycopene*

Lycopene is a lipophilic and an unsaturated carotenoid, present in red-colored fruits and vegetables, such as tomatoes, papaya, or watermelons among others. Epidemiological observational and interventional studies [200,201] suggest that lycopene might reduce atherosclerotic risk, particularly in early stages of atherosclerosis, preventing endothelial dysfunction (NO bioavailability and blood flow) and LDL oxidation. Others mechanisms through which lycopene might exert effects is improvement of the metabolic profile (by impairing cholesterol synthesis) and BP, through reductions in arterial stiffness, and modulation of the expression of pro-inflammatory markers and platelet aggregation [202]. Furthermore, dietary lycopene confers CV benefits and significant reduction in CV mortality and major CV events in postmenopausal women free of CVD or cancer [202]. Focusing on the risk of develops atherosclerosis, several studies have pointed out lycopene's antioxidant power as a possible mechanism to explain its health benefits [203].

Additionally, in a recent meta-analysis [204], dietary interventions supplemented with tomatoes significantly decreased LDL-c (−0.22 mmol/L; *p* = 0.006), IL-6 (−0.25; *p* = 0.03) and improved FMD by 2.53% (*p* = 0.01), while lycopene supplementation reduced SBP (−5.66 mmHg; *p* = 0.002). In another study with 40 participants with heart failure [205] (lycopene intervention, 29.4 mg/day of lycopene vs. control group), CRP levels decreased significantly in the intervention group, but only in women (*p* = 0.04).

Dietary data collected from the National Health and Nutrition Examination Survey (NANHES) 2003–2006 [206] showed significant inverse associations with tHcy and CRP for dietary lycopene intake (*p* < 0.05).

Therefore, Valderas-Martinez et al. [201] investigated the postprandial effects of a single dose of raw tomatoes (RT), tomato sauce (TS), and tomato sauce with refined olive oil (TSOO) on CVD. In this randomized, cross-over, controlled feeding trial in 40 subjects free of CVD, authors found that tomato intake significantly decreased some inflammatory biomarkers levels, such as LFA-1, IL-6, IL-18, MCP-1, and VCAM-1, and increased plasma IL-10 levels. In another interventional study [207], with 80 subjects, 40 early atherosclerosis cases, and 40 control subjects, the authors pointed out that serum carotenoids concentrations are linked with the risk of atherosclerosis development. They observed that serum lutein was negatively associated with IL-6 (*p* < 0.001) and directly associated with IFN-γ (*p* = 0.002). Moreover, zeaxanthin was inversely associated with VCAM-1 (*p* = 0.001) and apolipoprotein E (*p* = 0.022) levels, while lycopene was inversely associated with sVCAM-1 (*p* = 0.011) and LDL (*p* = 0.046). However, in a single-blind, randomized controlled intervention trial [208] with healthy volunteers (94 men and 131 women, aged 40 y–65 y), no changes were observed for inflammatory markers (oxLDL, sICAM-1, and IL-6), insulin resistance, and sensitivity markers after 12 weeks of dietary intervention, as shown in Table 4.

#### *7.3. Phytosterols*

A large body of scientific evidence has concluded that a daily dose of 2–3 g of plant sterols or phytosterols is associated with an LDL-c reduction of around 6–15% of the total concentration [209,210]. These reductions were also observed in a meta-analysis conducted by Demonty et al. [211], where after administering a daily dose of 2.15 g of phytosterols, LDL-c was reduced by 8.8%. In fact, plant sterols have been proposed as a complement of statins treatment in order to decrease the risk of CVD. However, available data is inconsistent, so more research is needed. In this other meta-analysis [212] of 20 RCTs with 1308 participants, the effect of phytosterols intake on pro-inflammatory markers was evaluated. Significant reductions of CRP levels (−0.10 mg/dL) were observed after plan sterols' intake.

In addition, clinical studies have evaluated the association between phytosterols' consumption and inflammatory markers, such as CRP and cytokines. Although the results showed by Ras et al. [213] are in aggreement with the data reported by Demonty, no changes in CRP levels were observed. In a new study conducted by Ras et al. [209], in 240 hypercholesterolaemic voluntaries who consumed a low-fat spread with added phytosterols (3 g/day) for 12 weeks, no changes in any of the markers evaluated (CRP, SAA, IL-6, IL-8, TNF-α, and sICAM-1) were observed. Devaraj et al. [214] described significant reductions of IL-6 and IL1β levels after the intake of an orange juice-based beverage fortified with sterols (2 g sterols/day). In addition, results of a double-blinded, randomized, crossover trial [215] with 58 hypercholesterolemic participants during 12 weeks of intervention with margarine supplemented with phytosterols (3 g/day) did not show changes in CRP, IL-6, or TNF-α levels, as shown in Table 4.

#### **8. Polyphenols**

Polyphenols are the most abundant dietary antioxidants present in most plant origin foods and beverages, which possess a wide range of health effects in the prevention of CVD [216]. The most relevant food sources are fruit and vegetables, red wine, black and green tea, coffee, EVOO, and chocolate, as well as nuts, seeds, herbs, and spices [217].

Numerous scientific reports accumulated in the last years suggest that polyphenols might exert their positive effects by delaying progression of atherosclerosis through several mechanisms: Regulation of signaling and transcription pathways, such as NF-κβ; antioxidant systems; prevention of leukocyte migration and later infiltration inside plaque; reduction of adhesion molecules levels; inhibition of the encoding of pro-inflammatory cytokines; reduction of BP because of the enhanced NO production; and improvements of lipid metabolism, coagulation activity, and endothelial function, among others [218,219].

Several epidemiological studies have reported a negative association between consumption of polyphenols or polyphenols-rich foods and CVD [220–222]. A meta-analysis [223], including 14 prospective cohort studies with 1,279,804 participants and 36,352 CVD cases, showed that moderate consumption of coffee (three to five cups/day) was associated with a lower CVD risk compared with non-consumers. Similar results were found in the meta-analysis conducted by Larsson et al. [224]. Additionally, in another meta-analysis (14 prospective studies, with 513,804 participants and a median of follow-up of 11.5 years), it was found that a daily intake of ≥3 cups of tea was associated with a lower risk of stroke (−13%) and ischemic stroke (−24%) [225].

Focusing on the action of polyphenols on endothelial function, a large number of studies that have tested red wine, grape juice, black tea, soy, or dark cocoa showed an increase in FMD [226]. Besides, a daily consumption of dark chocolate (50 g) was associated with an improvement of FMD by 3.99% in acute and 1.45% in chronic intake and reduced systolic (−5.88 mm Hg) and diastolic BP (−3.30 mm Hg) [227]. Additionally, and contrary to the expected, acute consumption of black tea was associated with an increase of SBP (5.69 mm Hg) and DBP (2.56 mm Hg) while FMD was increased by 3.40%. Contrary to black tea, green tea led to a significant reduction in LDL-c (−0.23 mmol/L) [227]. Furthermore, alcohol and red wine moderate consumption were associated with an increase of FMD after analyzing 801 individual foods with food frequency questionnaires (FFQs) from the 2000 Hoorn Study (women = 399; age 68.5 ± 7.2 years) [228].

So, several meta-analyses of RCTs [229,230] have reported significant reductions of LDL-c, SBP, fasting glucose, BMI, hemoglobin A1c, or TNF-α levels, and significant increments of HDL-c.

Additionally, hs-CRP, IL-1β, and sP-selectin levels were significantly decreased after anthocyanin extract interventions. A larger cross-sectional study (BELSTRESS) with observational data from 1031 healthy men (49 years on average) found that drinking tea might reduce the inflammatory processes underlying CVD, as tea intake leads to lower levels of CRP (*p* = 0.004), SAA (*p* = 0.001), and haptoglobin (*p* = 0.02) [231].

Anti-inflammatory and immune-modulating effects of polyphenols have also been suggested as a potential pathway for polyphenols' health benefits. Several RCTs showed the relationship between cocoa polyphenols and inflammatory biomarkers related to atherosclerosis disease progression. In this sense, Vazquez-Agell et al. [232] suggested that the acute consumption of 40 g of cocoa with water might inhibit the NF-κβ transcription and down-regulate adhesion molecules' production (sICAM-1 and sE-selectin). Moreover, Monagas et al. [233], in a cross-interventional trial, showed that cocoa

powder intake led to a reduced expression of adhesion molecules on monocyte surfaces (VLA-4, CD40, and CD36, *p* ≤ 0.028; all) and lower serum levels of soluble adhesion molecules (sP-selectin and sICAM-1; both *p* = 0.007) in 42 subjects (mean age 69.7 years) at high risk of CVD and after four weeks of intervention. Additionally, Basu et al. [234] found that green tea consumption, as a beverage or extract, did not alter inflammatory biomarkers (adiponectin, CRP, IL-6, IL-1β, sVCAM-1, sICAM-1, leptin, or leptin:adiponectin ratio) related to MetS after eight weeks of intervention. Only SAA was significantly decreased after green tea beverage and extracts' intake (*p* < 0.005). Also, Zhang et al. [235] reported a significant decrease of CXCL7 by 12.32%, CXCL5 by 9.95%, CXCL8 by 6.07%, CXCL12 by 8.11%, and CCL2 levels by 11.63% after 320 mg intake of purified anthocyanins during 24 weeks. Similar results were observed after administering 320 mg of purified anthocyanins in 150 hypercholesterolemic patients during 24 weeks [236]. In this case, significant reductions of β-thromboglobulin, sP-selectin, and RANTES (regulated on activation, normal T cell expressed and secreted) were observed. Anthocyanins have also been correlated with lower levels of oxidative stress biomarkers. In an interventional study with 42 overweight and smoker participants, significant reductions in oxLDL and 8-iso-prostaglandin F2α were observed after the supplementation with an extract of maqui berry (162 mg anthocyanins) for 40 days [237]. Isoflavones and stilbens (mainly resveratrol) have also shown anti-inflammatory and immunomodulatory effects. Isoflavones improved CRP concentrations of 117 healthy European postmenopausal women after 50 mg/day of isoflavones intake [238]. Other RCT on postmenopausal American women who received daily 25 g of soy protein supplementation, showed reductions of subclinical atherosclerosis progression by 16% as well as reductions of carotid thickness progression by a mean of 68% [239]. Relative to stilbens, Tomé-Carneiro et al. [240] found significant reductions of oxLDL, apolipoprotein B (ApoB), and LDL-c after analyzing the effect of the intake of a grape supplement containing 8 mg resveratrol for six months. Also, a significant reduction of inflammatory markers levels (IL-18, sICAM-1, and sVCAM-1; *p* ≤ 0.037; all) related with atheroma plaque development was observed in 44 healthy participants who ingested the resveratrol extract during four weeks [241]. Additionally, a randomized, double-blind, placebo-controlled clinical trial conducted by Seyyedebrahimi et al. [242] with 48 diabetics type 2 participants and supplemented with 800 mg/day of resveratrol for eight weeks showed a reduction in plasmatic levels of protein carbonyl content and ROS in PBMCs. Furthermore, after resveratrol supplement intake, an increase in the expression of Nrf2 and SOD was observed, as shown in Table 5.

**Table 5.** Potential inflammatory effects of polyphenols on CVD.

ApoB: Apolipoprotein B; CRP: C-reactive protein; CXCL: Chemokine (C-X-C motif) ligand; IL: Interleukin; NF-κβ: Nuclear factor kappa B; Nrf2: Nuclear factor (erythroid-derived 2)-like 2; oxLDL: Oxidized LDL; RANTES: Regulated on activation, normal T cell expressed and secreted; ROS: Reactive oxygen species; SAA: Serum amyloid A; sICAM-1: Soluble intercellular adhesion molecule 1; SOD: Superoxide dismutase; TNF-α: Tumor necrosis factor.

#### **9. Conclusions**

We have shown the intimate relationship between nutrition and CVD. Thus, the challenge is in promoting healthy dietary habits as well as an active lifestyle as early as possible in children and young adults. The evidence favors consumption of healthy dietary patterns, such as the Mediterranean diet or DASH diet, against other unhealthy dietary patterns, such as the Western diet, based on a high consumption of salt, added sugars, and saturated and trans-fats. Despite the fact that strong evidence shows the potential health benefits of a great amount of foods, nutrients, bioactive compounds, and dietary antioxidants, such as polyphenols, may exert on CV risk factors or directly on CVD development, it is necessary to conduct more interventional studies with a higher number of cases and

longer follow up. To date, a lot of results obtained have produced few conclusions and sometimes, even contradictions. Therefore, due to a lack of information about possible mechanisms implicated in the cardioprotective effect of diet, foods, nutrients, or bioactive compounds, this needs to be more investigated.

**Author Contributions:** Conceptualization, R.C. and E.S.; Methodology, R.C. and R.E.; Investigation, R.C. and S.C.-B.; Writing—Original Draft Preparation, R.C. and S.C.-B.; Writing—Review & Editing, R.C. and R.E.; Visualization, E.S.; Supervision, E.S.; project administration, E.S.; funding acquisition, R.E.

**Funding:** This research was funded by the Instituto de Salud Carlos III, Spain, grant number PIE14/00045; by the Instituto de Salud Carlos III, Spain, grant number PI044504; by Fundació la Marató de TV3, Spain, grant number PI044003; and the Sociedad Española de Medicina Interna (SEMI), spain, grant number DN40585.

**Acknowledgments:** This research was funded by the Instituto de Salud Carlos III, Spain. CIBER OBN is an initiative of the Instituto de Salud Carlos III, Spain.

**Conflicts of Interest:** R.E. reports serving on the board of and receiving lecture fees from the Research Foundation on Wine and Nutrition (FIVIN); serving on the boards of the Beer and Health Foundation and the European Foundation for Alcohol Research (ERAB); receiving lecture fees from Cerveceros de España and Sanofi-Aventis; and receiving grant support through his institution from Novartis. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. No other potential conflict of interest relevant to this article was reported.

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Marine Omega-3 (N-3) Fatty Acids for Cardiovascular Health: An Update for 2020**

**Jacqueline K. Innes 1 and Philip C. Calder 1,2,\***

Received: 25 January 2020; Accepted: 14 February 2020; Published: 18 February 2020

**Abstract:** The omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are found in seafood (especially fatty fish), supplements and concentrated pharmaceutical preparations. Long-term prospective cohort studies consistently demonstrate an association between higher intakes of fish, fatty fish and marine n-3 fatty acids (EPA + DHA) or higher levels of EPA and DHA in the body and lower risk of developing cardiovascular disease (CVD), especially coronary heart disease (CHD) and myocardial infarction (MI), and cardiovascular mortality in the general population. This cardioprotective effect of EPA and DHA is most likely due to the beneficial modulation of a number of known risk factors for CVD, such as blood lipids, blood pressure, heart rate and heart rate variability, platelet aggregation, endothelial function, and inflammation. Evidence for primary prevention of CVD through randomised controlled trials (RCTs) is relatively weak. In high-risk patients, especially in the secondary prevention setting (e.g., post-MI), a number of large RCTs support the use of EPA + DHA (or EPA alone) as confirmed through a recent meta-analysis. This review presents some of the key studies that have investigated EPA and DHA in the primary and secondary prevention of CVD, describes potential mechanisms for their cardioprotective effect, and evaluates the more recently published RCTs in the context of existing scientific literature.

**Keywords:** eicosapentaenoic acid; docosahexaenoic acid; omega-3 polyunsaturated fatty acids; cardiovascular disease; coronary heart disease

#### **1.  They are characterised by, and named according to, the presence of the closest double bond to the methyl end of the hydrocarbon (acyl) chain being on carbon number three, if the methyl carbon is counted as number one. The most functionally important n-3 fatty acids appear to be eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) [1]; however, roles for docosapentaenoic acid (22:5n-3) have also emerged now [2]. The best dietary source of EPA and DHA (and also docosapentaenoic acid) is seafood, especially fatty fish (also called 'oily fish'). The blubber and tissues of sea mammals, such as whales and seals, also contain EPA and DHA in significant amounts. Various supplements, including fish oils, cod liver oil, krill oil and some algal oils, contain EPA and DHA. Finally, concentrated pharmaceutical-grade preparations of EPA and DHA, or EPA alone, are available. Typical values for the EPA and DHA content of selected fish, n-3 fatty acid supplements and pharmaceutical preparations are shown in Table 1. EPA and DHA are often referred to as marine n-3 fatty acids because of their association with seafood.

 DHA, docosahexaenoic acid; EPA, eicosapentaenoic

The various n-3 fatty acids are related metabolically to one another, and the pathway of conversion of plant-derived n-3 fatty acids (e.g., α-linolenic acid (ALA; 18:3n-3)) to EPA and then to DHA is shown in Figure 1. Studies in humans have identified that there is a fairly low rate of conversion of ALA along this pathway, especially all the way to DHA [3,4]. It is now recognised that this conversion is influenced by several factors, including the stage of life course, age, sex, various hormones, genetics and diseases [4].

The intake of EPA and DHA from diet is strongly influenced by fish consumption because fish, in general, and fatty fish, in particular, are the richest dietary source of these fatty acids. The intake of fish and fatty fish is high in some countries, such as Japan, but it is low in many Western countries, including the USA and the United Kingdom. As a result, the intake of EPA + DHA among adults varies among different populations and is low in most Western countries; it is generally considered that in non-fatty fish eaters, the intake of EPA + DHA is <0.2 g/day [5,6]. This is lower than the recommended intake for the general population [7–9]. Nevertheless, despite these low intakes, it is evident that the recommendations (typically 0.2–0.5 g/day depending upon the authority making the recommendation) can be met by including fatty fish in the diet on a regular basis or, if that is not possible, by using supplements that contain EPA and DHA (Table 1).

**Figure 1.** Metabolic pathway of conversion of the plant essential n-3 fatty acid, α-linolenic acid (18:3n-3), to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3).

#### **2. Strong Evidence for a Protective E**ff**ect of EPA and DHA Towards Cardiovascular Disease Emerges from Ecological, Case Control and Cohort Studies**

The potential for EPA and DHA to have a role in reducing the risk of cardiovascular disease (CVD) was first identified by studies in the Greenland Inuit, where the low rate of mortality from myocardial infarction (MI) and ischaemic heart disease [10,11] was linked to the very high dietary intake of EPA and DHA [12]. These observations were replicated in other native Arctic populations [13] and the Japanese population [14]. Subsequently, substantial evidence accumulated from epidemiological and case-control studies in Western populations indicating that consumption of fish, fatty fish, or EPA and

DHA is associated with reduced risk of mortality from CVD, especially coronary heart disease (CHD) (reviewed in [15]). For example, in the Nurse's Health Study, there was an inverse dose-dependent association of risk for developing CHD, having a non-fatal MI or dying from CHD across quintiles of intake of EPA + DHA [16]. The risk for all three outcomes was about 50% in the group with the highest intake compared with the group with the lowest intake of EPA+DHA. The intake of EPA and DHA is highly correlated with their concentrations in blood lipids and red blood cells [17]. A number of studies have associated the concentrations of EPA + DHA (often expressed as a proportion of total fatty acids) in blood plasma or serum, plasma lipid fractions, whole blood, red blood cells and adipose tissue with lower cardiovascular morbidity and mortality (reviewed in [15]). For example, in the Physician's Health Study, there was an inverse dose-dependent association of risk for sudden death across quartiles of whole blood EPA + DHA, with an 80% lower risk in those with the highest whole blood EPA+DHA concentration compared to those with the lowest whole blood EPA + DHA concentration [18]. More recently, the largest prospective cohort study conducted to date included ~420,000 participants from the National Institutes of Health AARP Diet and Health Study with a 16-year follow-up and reported a significant inverse association between fish and EPA + DHA intake and various mortality outcomes [19]. Comparing the highest with lowest quintiles of fish intake, both men and women had 10% lower CVD mortality. EPA + DHA intake was associated with 15% and 18% lower CVD mortality in men and women, respectively, across extreme quintiles.

Cohort studies associating the intake of fish or marine n-3 fatty acids with cardiovascular or coronary outcomes have been subject to a number of meta-analyses. These include a 2012 aggregation of seven prospective cohort studies, including 176,441 participants, which investigated the association between dietary fish, EPA+DHA intake or plasma EPA + DHA concentrations and heart failure [20]. The investigators found a 15% risk reduction of heart failure associated with the highest versus lowest fish intake and a 14% lower risk of heart failure for those with the highest intake compared to those with the lowest dietary intake or plasma concentrations of EPA + DHA. A comprehensive meta-analysis, published in 2014, investigated the association between dietary intakes or blood levels of different classes of fatty acids (including n-3 fatty acids) and combined coronary disease outcomes [21]. The aggregation of data from 16 studies involving over 422,000 individuals showed a risk reduction of 13% for those in the top tertile of dietary EPA + DHA intake compared with those in the lower tertile of intake. Furthermore, the aggregation of data from 13 studies involving over 20,000 individuals showed risk reductions of 22%, 21% and 25% for those in the top tertile of circulating EPA, DHA and EPA + DHA, respectively, compared with those in the lower tertile. Alexander et al. [22] brought together data from prospective cohort studies examining the association of dietary EPA and DHA with risk of various coronary outcomes. The aggregation of data from 17 studies showed an 18% risk reduction for any CHD event for those with higher dietary intake of EPA + DHA compared to those with lower intake. There were also significant reductions of 23%, 19% and 47% in the risk for fatal coronary events, coronary death and sudden cardiac death, respectively.

The association between EPA or DHA concentration in a body compartment, such as plasma, serum, red blood cells or adipose tissue, and risk of future CHD in adults who were healthy at study entry was investigated by pooling data from 19 studies involving over 45,000 individuals [23]. EPA and DHA were each independently associated with a lower risk of fatal CHD, with a 10% lower risk for each one standard deviation increase in content [23]. The omega-3 index is the red blood cell content of EPA+DHA expressed as a proportion of total fatty acids [24]. Omega-3 index is a marker of both long-term dietary intake of these fatty acids and their tissue levels and is suggested to be a marker of CHD risk [24]. Harris et al. [25] used data from 10 cohort studies and identified a 15% reduction in risk of fatal CHD for each one standard deviation increase in omega-3 index.

#### **3. Mechanisms by which EPA and DHA Reduce the Risk of Cardiovascular Disease**

Prospective cohort studies have the advantage of a very long follow-up time to observe health outcomes in what starts as a generally healthy study population, something which is typically not possible in randomised control trials (RCTs). There are well-recognised limitations of such cohort studies, including the lack of ability to show causation. Despite this significant limitation, the considerable number of large prospective cohort studies conducted to date that have consistently shown an inverse association between dietary, blood or tissue EPA and DHA and incidence of mortality from CVD provide important evidence for the key role of marine n-3 fatty acids in the prevention of CVD. As such, there has been much interest in the mechanisms by which n-3 fatty acids, specifically EPA and DHA, achieve their cardioprotective action, with much attention being focused on the potential modulation of key cardiovascular risk factors. These risk factors include high blood pressure, high serum triglycerides, low high-density lipoprotein (HDL)-cholesterol, elevated post-prandial lipaemia, endothelial dysfunction, cardiac arrhythmia, heart rate and heart rate variability and a tendency towards thrombosis and inflammation. Large numbers of studies, including many RCTs, in humans have investigated the effect of the combination of EPA and DHA on these risk factors and many of these studies have been included in a number of meta-analyses performed in recent years (Table 2). These meta-analyses demonstrate that EPA and DHA lower triglycerides [26], lower the blood pressure (both systolic and diastolic) [26,27], reduce the heart rate and increase heart rate variability [26,28–30] and reduce platelet aggregation [31], whilst appearing to increase both low density lipoprotein (LDL)- and HDL-cholesterol [26]. Regarding vascular endothelial function, EPA and DHA have been demonstrated to improve flow-mediated dilatation [32,33] and arterial compliance [34]. Concerning the effect of EPA and DHA on inflammation, several meta-analyses have reported that they lower blood concentrations of the acute phase protein, C-reactive protein (CRP), and the pro-inflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-6 [26,35], although the effect may be dependent on the health status of the individual. Furthermore, EPA and DHA have been reported to decrease the plasma or serum concentrations of pro-inflammatory eicosanoids like thromboxane B2 and leukotriene B4 [36].

Whilst the majority of the scientific evidence base to-date has focused on the administration of EPA and DHA in combination (as occurs naturally in fish and most supplements), there has been much interest in the potential for EPA and DHA to have independent roles in cardiovascular risk reduction. A recent systematic review of the scientific literature concluded that EPA and DHA appear to have differential effects on a number of cardiometabolic outcomes [37]. For example, regarding modulation of blood lipids, whilst both EPA and DHA lowered blood triglycerides, there was evidence for a slightly larger triglyceride-lowering effect for DHA [38,39]. Whilst neither EPA nor DHA affected total cholesterol concentrations to a significant degree, there was an independent effect on other blood lipid parameters, with EPA lowering the HDL3-cholesterol subfraction and DHA increasing the more cardioprotective HDL2-cholesterol [40,41]. DHA also increased LDL-cholesterol more than EPA, an effect observed more in men than in women, and increased LDL particle size, an effect which was not observed with EPA [39–41]. From the more limited trial data, DHA appears to be more effective than EPA at lowering blood pressure and heart rate in normotensive individuals, whilst neither EPA nor DHA had any effect in hypertensive diabetic patients [40–43]. DHA also appeared to increase vasodilatory effects and reduce constrictor effects in the vasculature [44]. Both EPA and DHA were equally effective at increasing systemic arterial compliance [45]. In terms of platelet function, only EPA decreased platelet count and volume [46], whilst only DHA decreased collagen-stimulated platelet aggregation and platelet-derived thromboxane B2 [47]. Interestingly, neither EPA nor DHA had any effect on fibrinolytic function [47]. Furthermore, from the limited comparative studies available, DHA seemed to be more effective than EPA at lowering a wide range of pro-inflammatory biomarkers in subjects with subclinical inflammation [39,48]. Both EPA and DHA, however, were effective at reducing biomarkers of oxidative stress (F2 isoprostanes) [49–51]. Thus, whilst there are relatively few trials that have been conducted to date that directly compare EPA and DHA, the limited data suggest that EPA and DHA have different effects with regard to cardiovascular risk factors. More research, however, is necessary to be more certain about this.

*Int. J. Mol. Sci.* **2020**, *21*, 1362

**Table 2.** *Cont*.

IL-6, interleukin-6; weighted mean difference.

 LDL, low-density lipoprotein; MD, mean difference; RCT, randomized controlled trial; SMD, standard mean difference; TNF-α, tumour necrosis factor-α; WMD,

#### **4. RCTs of Primary Prevention of Cardiovascular Disease with Marine n-3 Fatty Acids**

When compared to the large number of observational studies investigating the association between marine n-3 fatty acid exposure and cardiovascular outcomes that have been conducted to date (Section 2), only a few RCTs of sufficient size or duration have investigated the cardioprotective effects of marine n-3 fatty acids in generally healthy populations. The open-label, Japan EPA Lipid Intervention Study (JELIS) directly investigated the use of 1.8 g/d EPA (as an ethyl ester) plus statin versus statin alone in 18,645 hypercholesterolaemic participants [52]. A number of the participants were on existing cardiovascular medication (in addition to statin) and the study included hypercholesterolemic, but otherwise, healthy subjects as well as those with pre-existing CHD, with all patients being followed up for ~5 years. The primary outcome was any major coronary event, including sudden cardiac death, fatal and non-fatal MI and other non-fatal events, including unstable angina pectoris, angioplasty, stenting, and coronary artery bypass grafting. The addition of EPA to statin had no effect over statin alone on the primary outcome in the primary prevention arm of the trial. Two large primary prevention RCTs were published in late 2018 [53,54]. The ASCEND (A Study of Cardiovascular Events iN Diabetes) trial randomised 15,480 people with diabetics and no evidence of CVD to receive either marine n-3 fatty acids (840 mg/d EPA + DHA) or olive oil placebo [53]. The primary outcome was the first serious vascular event and after a mean follow-up of 7.4 years, there was no difference in the primary outcome between the two groups. In exploratory analyses, there were significantly fewer deaths from vascular events in the marine n-3 fatty acid arm (rate ratio: 0.81; 95% CI: 0.67–0.99), as well as a trend towards reduced risk of death from CHD (rate ratio: 0.79; 95% CI: 0.61–1.02). The Vitamin D and Omega-3 Trial (VITAL) trial randomised 25,871 healthy participants aged over 50 years (men) and 55 years (women) to receive marine n-3 fatty acids (840 mg/d EPA+DHA) and/or vitamin D (2000 IU/d) or placebo [54]. After a median follow-up of 5.3 years, there was no difference in the primary outcome of major cardiovascular events (a composite of MI, stroke or death from cardiovascular causes) in those participants supplemented with marine n-3 fatty acids versus placebo. An analysis of the individual components of the composite showed a significant reduction in the n-3 fatty acid arm in MI (hazard ratio: 0.72; 95% CI: 0.59–0.90) and CHD (hazard ratio: 0.83; 95% CI: 0.71–0.97). Correspondingly, there was also a reduced risk of death from these two non-prespecified outcomes (for MI—hazard ratio: 0.50, 95% CI: 0.26–0.97; for CHD—hazard ratio: 0.76, 95% CI: 0.49–1.16). Thus, whilst RCT evidence in primary prevention is less clear than that from the prospective cohort studies, there is now some indication of benefit from marine n-3 fatty acids towards cardiovascular health, especially CHD, from recent large and long RCTs, such as ASCEND and VITAL.

#### **5. RCTs of Secondary Prevention of Cardiovascular Disease with Marine n-3 Fatty Acids**

A number of large, randomized, controlled, secondary prevention trials or trials in high-risk patients have been conducted to investigate the effect of EPA and DHA in patients with established CVD. These trials generate a changing picture with time.

#### *5.1. Secondary Prevention Trials and Meta-Analyses Published Prior to 2010*

Several large, secondary prevention trials of marine n-3 fatty acids were conducted prior to 2010. The Diet and Reinfarction Trial (DART) included 2,033 recent (mean: 41 days) MI survivors, who were given dietary advice concerning fat, fish and fibre intake and followed up for 2 years [55]. Those patients advised to eat at least two portions of fatty fish per week (or to take fish oil supplements) had a 29% reduction in total mortality as well as a reduced risk of death from ischaemic heart disease at 2 years compared to those patients given other advice. The landmark Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico (GISSI)–Prevenzione trial investigated the effect of supplementation with 840 mg/d EPA+DHA in 11,324 recent (≤3 months) MI survivors versus vitamin E supplementation, supplementation with both EPA + DHA and vitamin E, and placebo control [56]. After 3.5 years, patients who received EPA + DHA had a 20% reduction in total mortality, 30% reduction

in cardiovascular death and 45% decrease in sudden death compared to those that did not receive EPA + DHA. There was no benefit reported on non-fatal MI or stroke. The beneficial effect of EPA + DHA on total mortality and sudden cardiac death was observed after 3 months and 4 months of supplementation, respectively, and raised interest in the potential anti-arrhythmic action of EPA and DHA [57]. The GISSI investigators undertook a separate RCT in 6,975 patients with chronic heart failure (GISSI-HF) to investigate the effect of 840 mg/d EPA+DHA versus placebo over a period of ~4 years in this patient population [58]. The investigators reported a small (9%), but significant, reduction in all-cause mortality and a small (8%) reduction in combined all-cause mortality or admission to hospital for cardiovascular reasons in this high-risk population following supplementation with marine n-3 fatty acids. In line with the increased prescription of statins to prevent all-cause mortality and cardiovascular events at this time, JELIS directly investigated the use of 1.8 g/d EPA (as an ethyl ester) plus statin versus statin alone in 18,645 hypercholesterolaemic participants who were followed up for ~5 years [52]. As mentioned above, the addition of EPA to statin had no effect over statin alone in the primary prevention arm of JELIS, but in the secondary prevention arm, EPA caused a 19% decrease in non-fatal coronary events compared with the statin alone group [52]. Unlike GISSI, JELIS found no beneficial effect on cardiovascular mortality. A subsequent analysis found an inverse association between plasma EPA levels and risk of major coronary events; participants with the highest levels of plasma EPA (≥150 μg/mL) were 20% less likely to experience a major coronary event [59].

Meta-analyses of RCTs conducted prior to 2010 confirmed the reductions in mortality seen in individual trials of marine n-3 fatty acids ([60–63]; Table 3). For example, a 2002 meta-analysis of 11 RCTs in 15,806 patients with CHD found 20% lower risk of non-fatal MI, 30% lower risk of fatal MI, 30% lower risk for sudden death and 20% lower risk for all-cause mortality in patients who received marine n-3 fatty acids versus control [60]. Another meta-analysis from 2002 of 14 RCTs and 20,260 participants, both with and without CVD, found a 23% reduction in overall mortality and 32% reduction in cardiac mortality for those patients who were given marine n-3 fatty acids versus controls [61]. A further meta-analysis in 2009 focused on 8 RCTs and 20,997 patients with CHD found a 57% reduction in sudden death in patients with prior MI who were taking marine n-3 fatty acids compared with placebo [62]. In 2009, a meta-analysis of 11 RCTs representing 39,044 patients with all stages of CVD, including both low- and high-risk patients, found a 13% reduction in cardiovascular death and in sudden death and an 8% reduction in all-cause mortality in high-risk patients who were taking marine n-3 fatty acids compared to controls [63]. The investigators also found an 8% reduction in non-fatal cardiovascular events in moderate-risk patients.

#### *5.2. RCTs with Marine n-3 Fatty Acids in High-Risk Patients Published in the Period 2010–2013*

Three RCTs published in 2010 failed to replicate the findings of earlier trials [64–66]. The OMEGA study investigated the effect of supplementation with 840 mg/d EPA + DHA for 1 year in 3,851 early post-MI patients, with the primary endpoint of sudden cardiac death [64]. Marine n-3 fatty acids did not decrease the rate of sudden cardiac death, total mortality, major adverse cerebrovascular and cardiovascular events or revascularisation compared to control. The Supplémentation en Folates et Omega-3 (SU.FOL.OM3) trial investigated the effect of supplementation of B vitamins and/or marine n-3 fatty acids (600 mg/d EPA + DHA) in 2,501 patients with documented MI, unstable angina or ischaemic stroke for ~5 years [65]; the primary outcomes were cardiovascular death, stroke and non-fatal MI. Marine n-3 fatty acids did not have any effect on these outcomes. The Alpha Omega study included 4,837 post-MI patients who were given margarine fortified with low doses of EPA + DHA (400 mg/d), ALA (2 g/d), EPA + DHA + ALA or placebo, and followed up for 40 months [66]. There was no reduction in cardiovascular events in any group. On further analysis, those patients with diabetes in the EPA + DHA as well as ALA-fortified margarine groups, however, experienced a reduction in fatal CHD and arrhythmia-related events [66].

The Outcome Reduction with an Initial Glargine Intervention (ORIGIN) trial, published in 2012, assessed the effect of supplementation with 840 mg/d EPA + DHA in 12,536 dysglycaemic patients at high risk of cardiovascular events, together with insulin glargine or standard care for a median of 6 years [67]. No effect of marine n-3 fatty acids was reported for total mortality, death from cardiovascular causes or arrhythmia compared to placebo. The Risk and Prevention Study, published in 2013, investigated the effect of supplementation with 840 mg/d marine n-3 fatty acids in 12,513 patients with multiple cardiovascular risk factors or atherosclerotic vascular disease (but no MI) for a median of 5 years, and reported no effect on hospitalisation or death from cardiovascular causes compared to placebo [68]. In a prespecified subgroup analysis, compared with placebo, marine n-3 fatty acids resulted in an 18% lower incidence of the revised primary endpoint among women. Most of the secondary endpoints (e.g., fatal or non-fatal MI or stroke, fatal or non-fatal coronary event, and sudden death) did not differ between groups; however, admissions for heart failure were significantly lower in the marine n-3 fatty acid group.

Whilst these RCTs failed to show any benefit of marine n-3 fatty acids, they did have acknowledged limitations [69,70], which are worthy of mention. OMEGA was underpowered to detect any benefit on its primary outcome, sudden cardiac death, as power calculations were based on earlier RCTs in patients without more recent treatments and hence with higher background rates of sudden cardiac death compared to those seen in OMEGA. Furthermore, reported fish consumption was relatively high among the enrolled patients and increased during the trial, thereby increasing dietary EPA + DHA intake and introducing a confounding factor. The trial also had a relatively short follow-up period of just one year. Regarding SU.FOL.OM3, the length of time between the occurrence of primary cardiovascular event and the commencement of supplementation (median of 101 days) was longer than that of GISSI-Prevenzione (≤3 months) and the dose of EPA + DHA used was lower than that of GISSI-Prevenzione (840 mg/d EPA + DHA) and JELIS (1.8 g/d EPA). There were also fewer than expected background cardiac deaths, perhaps also due to more effective pharmacological management of cardiovascular risk factors. With respect to Alpha Omega, despite the long follow-up time, the dose of EPA+DHA used was modest and enrolment to the trial occurred on average at 4 years post-MI, despite the existence of data suggesting that early intervention with EPA + DHA is required to see a beneficial effect post-MI. ORIGIN included a large number of participants taking effective cardiovascular risk-reducing medications compared to earlier trials, and this may have made the cardioprotective effect of EPA + DHA harder to detect. The Risk and Prevention Study set out to examine the effect of marine n-3 fatty acids on the composite primary outcome of death, non-fatal MI and non-fatal stroke; however, the event rate at one year was lower than anticipated and the primary outcome was revised to hospitalisation or death from cardiovascular causes over the follow-up period.

Unsurprisingly, several meta-analyses conducted over the last ten years reflect the inclusion of these null RCTs and report more mixed conclusions than earlier meta-analyses ([21,22,71–79]; Table 3). A meta-analysis from 2013 (representing 11 RCTs and 15,348 patients with CVD) reported a 32% reduction in cardiac death, 33% reduction in sudden death and 25% reduction in myocardial infarction, with no effect on all-cause mortality or stroke [75]. This meta-analysis included only those RCTs with EPA + DHA dosages of at least 1 g/d and with a duration of at least 1 year. Another meta-analysis published in 2014, which included a broader range of marine n-3 fatty acid dosages and durations and focused only on patients with ischaemic CHD, reported that whilst there were no effects on cardiovascular events, there was a 12% reduction in death from cardiac causes, 14% reduction in sudden cardiac death and 8% reduction in all-cause mortality in those taking EPA + DHA versus placebo [76]. Published in 2017, a meta-analysis focusing solely on cardiac death as the outcome examined 14 RCTs (involving 71,899 subjects in both mixed and secondary prevention settings) with marine n-3 fatty acid supplementation for a duration of at least 6 months [77]. The study found an 8% lower risk of cardiac death in the primary analysis and a 13%–29% lower risk of cardiac death in those participants with intakes of EPA + DHA of at least 1g/d and for certain subgroups, such as subjects with high plasma triglycerides or cholesterol, and where <40% subjects were taking statins.

**Table 3.**Meta-analyses published up to 2018 of RCTs investigating the effect of marine n-3 fatty acids on cardiovascular outcomes.

**Table 3.** *Cont*.

randomised controlled trial.

#### *5.3. An Important RCT and a New Meta-Analysis were Published in 2019*

The Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention Trial (REDUCE-IT), published in early 2019, included 8,179 participants (29% in a primary prevention cohort with diabetes plus another cardiovascular risk factor and 71% in a secondary prevention cohort) supplemented daily with 4 g of icosapent ethyl (a highly purified EPA ethyl ester) or mineral oil placebo and followed up for a median of 4.9 years [80]. All patients were being treated with statins and had triglyceride concentrations of 135–499 mg/dL (1.52–5.63 mmol/L). The primary outcome was a composite of cardiovascular death, non-fatal MI, non-fatal stroke, coronary revascularisation or unstable angina. The patients who received icosapent ethyl had a statistically significant reduction in the primary outcome compared to placebo (hazard ratio: 0.75; 95% CI: 0.68–0.83; *p* < 0.001), the pre-specified secondary outcome (composite of cardiovascular death, non-fatal MI or non-fatal stroke) (hazard ratio: 0.80; 95% CI: 0.66–0.98; *p* = 0.03) and a whole range of other pre-specified outcomes (Figure 2). This effect was greater in the secondary prevention cohort than in the primary prevention cohort [80]. Interestingly, as with JELIS, REDUCE-IT used EPA only, but at a much higher dose (4 g daily versus 1.8 g daily) and included participants with high triglyceride levels and on statin medication. Accordingly, the participants in the icosapent ethyl arm had a significant reduction in triglycerides (a decrease of 0.5 mmol/L; *p* < 0.001) and LDL-cholesterol (a decrease of 0.13 mmol/L; *p* < 0.001) relative to placebo at 1 year. Of note, the improvement in the primary outcome in the icosapent ethyl arm of the trial was not dependent on the baseline triglyceride level or the degree of subsequent triglyceride lowering, suggesting that the reduction in cardiovascular risk in this population may be via a different mechanism independent of (or in addition to) triglyceride lowering. REDUCE-IT is of key interest as it demonstrates that even in at-risk populations that are well managed with modern pharmacological treatments, a suitably high dosage of EPA (i.e., 4 g daily) can provide an additional benefit in reducing cardiovascular-related events and mortality.

**Figure 2.** Effect of icosapent ethyl compared with placebo on different endpoints in REDUCE-IT [80]. "From New England Journal of Medicine, Bhatt D.L., Steg P.G., Miller M., Brinton E.A., Jacobson T.A., Ketchum S.B., Doyle R.T. Jr., Juliano R.A., Jiao L., Granowitz C., Tardif J.C., Ballantyne C.M.; REDUCE-IT Investigators, Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia, Volume No. 380, Page 11–22, Copyright © (2020) Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society."

A recent meta-analysis, published in 2019, included data from 13 RCTs, including GISSI-Prevenzione, JELIS, GISSI-HF, SU.FOL.OM3, Alpha Omega, OMEGA, ORIGIN, VITAL, ASCEND and REDUCE-IT [81]. Trials had to have a sample size of at least 500 patients and a follow-up for at least one year to be included. The sample sizes ranged from 563 [82] to 25,871 [54], the mean duration of follow-up ranged from 1.0 [64] to 7.4 [53] years and the mean dose of EPA+DHA ranged from 0.37 [66] to 4.0 [80] g/d. The total sample size of the aggregated trials was 127,477, while the mean duration of follow-up was 5 years. The outcomes of interest included MI, CHD death, total CHD, total stroke, CVD death, total CVD and major vascular events. In the analysis excluding REDUCE-IT, marine n-3 fatty acid supplementation was associated with a significantly lower risk of MI, CHD death, CVD death and total CVD (Table 4). Inverse associations for all outcomes were strengthened after including REDUCE-IT (Table 4). Statistically significant linear dose–response relationships were found for several outcomes: for example, every 1 g/d EPA + DHA corresponded to 9% and 7% lower risk of MI and total CHD, respectively. The meta-analysis concluded that "marine [n-3 fatty acid] supplementation lowers risk for MI, CHD death, total CHD, CVD death, and total CVD, even after exclusion of REDUCE-IT. Risk reductions appeared to be linearly related to marine [n-3 fatty acid] dose."

**Table 4.** Summary of findings from the meta-analysis published by Hu et al. [81] in 2019.

Abbreviations: CHD, coronary heart disease; CVD, cardiovascular disease.

#### **6. Trusted Authority Views on Marine n-3 Fatty Acids and Cardiovascular Disease**

In 2010, the European Food Safety Authority (EFSA) concluded that EPA and DHA help to maintain normal cardiac function, normal blood pressure and normal blood concentrations of triglycerides in the general population [9]. In terms of the intake of EPA and DHA required to bring about these health claims, EFSA recommend an intake of 250 mg/day of EPA+DHA to maintain normal cardiac function, 3 g/d to maintain normal blood pressure and 2 g/d to maintain normal blood concentrations of triglycerides, as part of a healthy diet [9]. The American Heart Association (AHA) also supports the use of marine n-3 fatty acids. In 2018, the AHA published guidance in support of the dietary intake of fish in primary prevention [83]; specifically, the AHA advisory recommends 1 to 2 seafood meals per week to reduce the risk of congestive heart failure, CHD, ischemic stroke and sudden cardiac death. In recognition of the triglyceride-lowering effect of EPA and DHA, the AHA recently updated its earlier recommendation for the use of 2 to 4 g/d EPA + DHA for this indication: "we conclude that prescription n-3 fatty acids, whether EPA + DHA or EPA-only, at a dose of 4 g/d, are clinically useful for reducing triglycerides, after any underlying causes are addressed and diet and lifestyle strategies are implemented, either as monotherapy or as an adjunct to other triglyceride-lowering therapies" [84]. On the basis of the positive outcomes of REDUCE-IT, the European Society of Cardiology and the European Atherosclerosis Society issued an update to the "Clinical Practice Guidelines for the Management of Dyslipidaemias" specifically recommending that "in high-risk patients with [triglyceride] levels between 1.5 and 5.6 mmol/L (135–499 mg/dL) despite statin treatment, n-3 polyunsaturated fatty acids (icosapent ethyl 2 × 2 g/day) should be considered with a statin" [85]. Furthermore, the American Diabetes Association makes a recommendation "that icosapent ethyl be considered for patients with diabetes and atherosclerotic cardiovascular disease or other cardiac risk factors on a statin with controlled low-density cholesterol, but with elevated triglycerides (135–499 mg/dL) to reduce cardiovascular risk" [86]. Finally, the National Lipid Association's position is "that for patients

aged ≥45 years with clinical [atherosclerotic cardiovascular disease] ASCVD, or aged ≥50 years with diabetes mellitus requiring medication plus ≥1 additional risk factor, with fasting triglycerides 135 to 499 mg/dL on high-intensity or maximally tolerated statin therapy (±ezetimibe), treatment with icosapent ethyl is recommended for ASCVD risk reduction" [87]. In 2017, the AHA reinforced its earlier support for EPA+DHA in people with CVD [88] and extended [89] that "the recommendation for patients with prevalent CHD such as a recent MI remains essentially unchanged: Treatment with n-3 fatty acid supplements is reasonable for these patients. Even a potential modest reduction in CHD mortality (10%) in this clinical population would justify treatment with a relatively safe therapy. We now recommend treatment for patients with prevalent heart failure without preserved left ventricular function to reduce mortality and hospitalizations (9%) on the basis of a single, large RCT. Although we do not recommend treatment for patients with diabetes mellitus and prediabetes to prevent CHD, there was a lack of consensus on the recommendation for patients at high CVD risk. Because there are no reported RCTs related to the primary prevention of CHD, heart failure, and atrial fibrillation, we were not able to make recommendations for these indications." In contrast to these well thought out and carefully worded statements by the AHA, in 2014, the National Institute for Health and Clinical Excellence in the UK recommended not to use marine n-3 fatty acids for the primary prevention of CVD, for the secondary prevention of CVD or in people with diabetes [90].

#### **7. Summary, Discussion and Conclusions**

There is a large body of evidence gathered from long-term prospective cohort studies that consistently demonstrates an association between higher intakes of fish, fatty fish and marine n-3 fatty acids (EPA+DHA) or higher levels of EPA and DHA in the body and lower risk of developing CVD, especially CHD, having an MI and cardiovascular mortality in the general population. This cardioprotective effect of EPA and DHA is plausible considering the robust identification of mechanisms that show that EPA and DHA beneficially modulate a number of known risk factors for CVD, such as blood lipids, blood pressure, heart rate and heart rate variability, platelet aggregation, endothelial function and inflammation. Despite this, evidence for primary prevention of CVD through RCTs is relatively weak. In high-risk patients, especially in the secondary prevention setting (e.g., post-MI), a number of large RCTs support the use of EPA + DHA (or EPA alone) as confirmed through a very recent meta-analysis [81]. Surveying these trials serves to highlight a number of factors which may have contributed to the positive outcomes reported and why other trials have had less conclusive or even null findings. Such factors include a sufficiently high dose of EPA alone or of a combination of EPA + DHA; sufficient duration of supplementation; supplementation in post-MI patients to begin relatively soon post-MI; and the adequate powering of studies to detect an effect on the primary outcome which may have a low rate of occurrence. Given these considerations, it is unsurprising that studies of short duration or using low doses of EPA + DHA conducted in the setting of multiple pharmaceutical (and other) approaches to controlling risk have failed to demonstrate the effects of EPA + DHA. As discussed elsewhere [91,92], there are also other factors to consider when evaluating and interpreting the literature in this field. The first is that the marine n-3 fatty acid naive condition is unlikely to occur, such that any placebo-controlled trial of EPA and DHA is conducted against a certain background intake of those fatty acids in all participants, although that background intake may be very low [5,6]. Nevertheless, background intakes of EPA and DHA can be highly variable both within and between individuals, with significant changes occurring simply by eating a single meal of fatty fish. Finally, the bioavailability of EPA and DHA can vary [93] (a) among individuals due to physiological differences, (b) according to the intake of meals in relation to supplements and (c) perhaps according to the time of day, thus influencing how much of these bioactive fatty acids is actually available to exert their effects. Taken together, these factors highlight the challenges to conducting robust trials in humans with supplemental n-3 fatty acids, and they are likely to contribute to the variable outcomes from such trials, especially when low doses of EPA and DHA are used.

Recent AHA advisories support the use of marine n-3 fatty acids in the treatment of hypertriglyceridemia [84] and in a range of patients with CVD [89] and the use of fish for primary prevention of CVD [83]. Both EPA and DHA beneficially modify a range of risk factors, although DHA may be more effective [37]. Nevertheless, the highly successful REDUCE-IT used pure EPA, although at a high dose [80]. Differentiating the dose-dependent effects of EPA and DHA on cardiovascular risk factors and on clinical outcomes is going to be important. Furthermore, robust primary prevention trials are still needed. In the meantime, recent trials reviewed herein and the most recent meta-analysis support the use of marine n-3 fatty acids to reduce cardiovascular mortality.

**Author Contributions:** Conceptualization, P.C.C.; writing—original draft preparation, J.K.I.; writing—review and editing, J.K.I. and P.C.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** J.K.I. has no conflicts of interest to declare. P.C.C. has research funding from BASF AS; acts as an advisor/consultant to BASF AS, DSM, Cargill, Smartfish and Pfizer (now GSK) Consumer Healthcare; and has received reimbursement for travel and/or speaking from Fresenius Kabi, Pfizer (now GSK) Consumer Healthcare and Smartfish.

#### **Abbreviations**

#### **References**

fatty acids on top of modern guideline-adjusted therapy after myocardial infarction. *Circulation* **2010**, *122*, 2152–2159. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Fish, Fish Oils and Cardioprotection: Promise or Fish Tale?**

#### **Akshay Goel 1, Naga Venkata Pothineni 1, Mayank Singhal 2, Hakan Paydak 1, Tom Saldeen 1 and Jawahar L. Mehta 1,\***

Received: 8 November 2018; Accepted: 20 November 2018; Published: 22 November 2018