Antioxidant effects of resveratrol in the cardiovascular system

Abstract

The antioxidant effects of resveratrol (3,5,4'‐trihydroxy‐trans‐stilbene) contribute substantially to the health benefits of this compound. Resveratrol has been shown to be a scavenger of a number of free radicals. However, the direct scavenging activities of resveratrol are relatively poor. The antioxidant properties of resveratrol in vivo are more likely to be attributable to its effect as a gene regulator. Resveratrol inhibits NADPH oxidase‐mediated production of ROS by down‐regulating the expression and activity of the oxidase. This polyphenolic compound reduces mitochondrial superoxide generation by stimulating mitochondria biogenesis. Resveratrol prevents superoxide production from uncoupled endothelial nitric oxide synthase by up‐regulating the tetrahydrobiopterin‐synthesizing enzyme GTP cyclohydrolase I. In addition, resveratrol increases the expression of various antioxidant enzymes. Some of the gene‐regulating effects of resveratrol are mediated by the histone/protein deacetylase sirtuin 1 or by the nuclear factor‐E2‐related factor‐2. In this review article, we have also summarized the cardiovascular effects of resveratrol observed in clinical trials.

Linked Articles

This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc

Abbreviations

ApoE

apolipoprotein E

BH₄

tetrahydrobiopterin

eNOS

endothelial nitric oxide synthase

GCH1

GTP cyclohydrolase I

GPx1

glutathione peroxidase 1

HO1

haem oxygenase 1

KO

knockout

NOX

NADPH oxidase catalytic subunit

NQO

NAD(P)H:quinoneoxidoreductase

Nrf2

nuclear factor‐E2‐related factor‐2

SIRT1

histone/protein deacetylase sirtuin 1

SOD1

copper/zinc SOD

SOD2

mitochondrial manganese SOD

SOD3

extracellular SOD

Tables of Links

TARGETS
Nuclear hormone receptors a	Enzymes b
Oestrogen receptors (ER)	Dihydrofolate reductase
ERα	Endothelial NOS (eNOS)
	Haem oxygenase 1 (HO1)
	PDE1‐PDE5
	Sirtuin 1 (SIRT 1)
	Xanthine oxidase (XO)

LIGANDS
Angiotensin II	Nitric oxide (NO)
Bilirubin	Paraquat
Biliverdin	Resveratrol
Cysteine	Tetrahydrobiopterin (BH4)
Glutathione (GSH)	Uric acid
Hydrogen peroxide (H2O2)	Vitamin C
L‐NAME

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (^a,bAlexander et al., 2015a, 2015b).

Introduction

Resveratrol (3,5,4'‐trihydroxy‐trans‐stilbene) is a polyphenol phytoalexin present in a variety of plant species and in red wine. Preclinical studies have demonstrated that resveratrol has protective effects in a number of disease models, including cardiovascular disease, diabetes, cancer and neurodegenerative diseases (Baur and Sinclair, 2006; Juhasz et al., 2010b). Some beneficial effects have also been observed in clinical trials, although many discrepancies and conflicting information exist (Novelle et al., 2015). The mechanisms of action of resveratrol are complex. Among these, antioxidant properties contribute substantially to the health benefits of resveratrol. In the present article we have reviewed the molecular mechanisms of resveratrol's antioxidant effects in the cardiovascular system.

Role of oxidative stress in cardiovascular disease

ROS, including free oxygen radicals, oxygen ions and peroxides, may have both physiological and pathological roles that are concentration‐dependent. At moderate concentrations, ROS are important regulators of vascular homeostasis by acting as signalling molecules (Li et al., 2014). In contrast, a high ROS concentration, due to excessive ROS production or malfunctioning antioxidant defence systems, causes oxidative stress. All cardiovascular risk factors lead to oxidative stress, which represents an important pathomechanism for cardiovascular disease. Therefore, pharmacological prevention of oxidative stress is of therapeutic interest (Li et al., 2013).

ROS‐producing systems

Among the ROS‐producing enzyme systems in the vascular wall, NADPH oxidase, xanthine oxidase (XO), enzymes of the mitochondrial respiratory chain, and a dysfunctional endothelial NOS (eNOS) are of major importance (Li et al., 2013; 2014).

NADPH oxidases

NADPH oxidases are multi‐subunit enzyme complexes consisting of two membrane‐bound subunits (NOX and p22phox) and several cytosolic regulatory subunits (Bedard and Krause, 2007; Drummond et al., 2011; Wingler et al., 2011). In the vascular wall, vascular smooth muscle cells express NOX4 and NOX1, whereas endothelial cells express predominantly NOX4 and NOX2 (Li et al., 2013; Li et al., 2014). NADPH oxidases are major sources of ROS in the vasculature, producing superoxide (O₂ ^●¯) as well as hydrogen peroxide (by NOX4). Importantly, NADPH oxidase can trigger ROS production from other sources including uncoupled eNOS, XO (Landmesser et al., 2007) and mitochondria (Kroller‐Schon et al., 2014; Schulz et al., 2014). An up‐regulation of NADPH oxidase subunits has been observed in human atherosclerosis as well as in animal models of cardiovascular disease (Li et al., 2014).

Xanthine oxidase

XO‐catalysed chemical reactions lead to the production of O₂ ^●¯ and hydrogen peroxide. Endothelial cells express XO. In addition, XO can be released from the liver, and circulating XO can adhere to endothelial cells (White et al., 1996). Atherosclerosis in human and experimental animals is associated with increased activity of both endothelial XO and plasma XO (Patetsios et al., 2001; Guzik et al., 2006), suggesting a contribution of XO‐derived ROS to cardiovascular disease.

Mitochondria

Mitochondria produce substantial amounts of O₂ ^●¯ at electron transport chain complexes I and III. Complex I releases O₂ ^●¯ into the mitochondrial matrix and is considered the main producer of O₂ ^●¯. The matrix‐localized mitochondrial manganese SOD (SOD2) converts O₂ ^●¯ to H₂O₂, which in turn is reduced to water by glutathione peroxidase (GPx) or catalase (Lubos et al., 2011). The levels of mitochondrial ROS are of central importance to atherogenesis, heart function and other cardiovascular diseases. Loss of SOD2 causes perinatal lethality because of cardiac myopathy or congestive heart failure (Li et al., 1995; Nojiri et al., 2006). Moreover, mitochondrial ROS also promote the activity of other ROS sources (e.g. NADPH oxidases, eNOS uncoupling and XO) (Kroller‐Schon et al., 2014; Schulz et al., 2014).

Dysfunctional, uncoupled endothelial NOS

Under physiological conditions, eNOS produces NO, which represents a key element in the vasoprotective function of the endothelium (Li and Forstermann, 2000; Li et al., 2002). Under pathological conditions, however, eNOS may become dysfunctional (Forstermann and Munzel, 2006; Li and Forstermann, 2013; Li and Forstermann, 2014). Oxidative stress evidently contributes to endothelial dysfunction, primarily because of rapid oxidative inactivation of NO by an excess of O₂ ^●¯. In a second step, the persistant oxidative stress induces eNOS uncoupling (i.e. uncoupling of O₂ reduction from NO synthesis), thereby converting the eNOS enzyme to an O₂ ^●¯ producer.

A number of mechanisms are implicated in eNOS uncoupling (Forstermann and Munzel, 2006; Li and Forstermann, 2009; Forstermann and Li, 2011). Among these, tetrahydrobiopterin (BH₄) deficiency is likely to represent a major cause of eNOS uncoupling. BH₄ is biosynthesized from GTP with GTP cyclohydrolase I (GCH1) acting as the rate‐limiting enzyme (Schmidt and Alp, 2007). Under conditions associated with oxidative stress, peroxynitrite (and O₂ ^●¯ less effectively) oxidizes BH₄ to BH₂, leading to a deficiency of BH₄ (Laursen et al., 2001). BH₂ can be reduced back to BH₄ by the enzyme dihydrofolate reductase. Thus, a deficit in BH₄ can be caused by enhanced BH₄ oxidation, by reduced BH₄ de novo synthesis (i.e. due to the down‐regulation of GCH1) or by reduced BH₄ recycling from BH₂ (i.e. due to the down‐regulation of dihydrofolate reductase) (Chalupsky and Cai, 2005).

Uncoupling of eNOS is a crucial mechanism contributing to atherogenesis. It not only reduces NO production but also potentiates the pre‐existing oxidative stress. The overproduction of ROS (e.g. O₂ ^●¯ and subsequently peroxynitrite) by uncoupled eNOS in turn enhances the oxidation of BH₄, creating a vicious circle (Forstermann and Munzel, 2006; Li and Forstermann, 2013; Li and Forstermann, 2014).

Antioxidant systems

The vascular wall contains a variety of enzymes, which can act as antioxidant defence systems and reduce the ROS burden.

SOD

SOD enzymes catalyse the dismutation of O₂ ^●¯ into hydrogen peroxide, thereby providing a key antioxidant effect. There are three mammalian isoforms of SOD. The copper/zinc SOD (SOD1) is a soluble enzyme located in the cytoplasm and in the mitochondrial intermembrane space. SOD2 is found in the mitochondrial matrix. In contrast, the extracellular SOD (SOD3) is expressed in extracellular matrix, on the cell surface and in extracellular fluids (Li et al., 2014).

Catalase

Catalase is an important cellular antioxidant enzyme and catalyses the decomposition of hydrogen peroxide to oxygen and water. The overexpression of catalase reduces atherosclerosis in apolipoprotein E‐knockout (ApoE‐KO) mice (Yang et al., 2004).

Glutathione peroxidases

GSH peroxidase proteins convert hydrogen peroxide to water and lipid peroxides to their respective alcohols. GSH peroxidase 1 (GPx1) is the most abundant selenoperoxidase and is a key antioxidant enzyme in many cell types (Lubos et al., 2011).

NAD(P)H:quinone oxidoreductase 1

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyses two‐electron reduction of a broad range of substrates, including quinones. Quinonoid compounds generate aggressive ROS via redox cycling mechanisms and arylating nucleophiles. NQO1 reduces quinones to hydroquinones without the formation of semiquinones and ROS that are deleterious to cells. Therefore, the removal of quinones from a biological system by NQO1 is considered an important detoxification reaction (Ross and Siegel, 2004).

Haem oxygenase 1

Haem oxygenase 1 (HO1) catalyses the degradation of the pro‐oxidant haem into carbon monoxide, iron and biliverdin, which is subsequently converted to bilirubin. The antioxidant effects of HO1 also include the activation of transcriptional machinery that induces a range of antioxidant genes. The catabolism of haem provides protection to cells via numerous routes, including the induction of ferritin to store redox‐active iron, the antioxidant actions of biliverdin and bilirubin and the anti‐inflammatory and anti‐apoptotic effects of carbon monoxide (Dunn et al., 2014).

Nonenzymatic antioxidants

In addition to the antioxidant enzymes, nonenzymatic antioxidants are also important for the cell to control ROS levels. Nonenzyme low molecular weight antioxidant compounds include vitamins C and E, GSH, β‐carotene and uric acid.

Resveratrol as a ROS scavenger

As a polyphenolic compound, resveratrol has been shown in in vitro systems to directly scavenge a variety of oxidants, including hydroxyl radical (^●OH), O₂ ^●¯, H₂O₂ and peroxynitrite.

In a cell‐free system using the Fenton reaction as a source of ^●OH, resveratrol (at concentrations ≥300 μM) has been shown to act as a scavenger and not an inhibitor of the Fenton reaction (Leonard et al., 2003). The calculated resveratrol reaction rate of ^●OH (9.45 × 10⁸ M⁻¹·s⁻¹), however, is significantly less than that of well‐established antioxidants, including ascorbate (1.2 × 10¹⁰ M⁻¹·s⁻¹), GSH (1.5 × 10¹⁰ M⁻¹·s⁻¹) and cysteine (1.3 × 10¹⁰ M⁻¹·s⁻¹). The hydroxyl radical‐scavenging property of resveratrol is proposed to be due to its phenolic groups (Leonard et al., 2003).

Resveratrol (at concentrations ≥100 μM) has been shown to scavenge O₂ ^●¯ directly in a nonenzymatic, cell‐free system (the potassium O₂ ^●¯ system) (Jia et al., 2008). Interestingly, the O₂ ^●¯ scavenging activity of resveratrol is higher when the xanthine/XO system is used to generate O₂ ^●¯ (Table 1) (Hung et al., 2002; Jia et al., 2008). The difference in EC₅₀ values in these two systems can be explained by the fact that resveratrol not only has an O₂ ^●¯ scavenging activity but also suppresses O₂ ^●¯ generation by inhibiting XO activity (at concentrations ≥50 μM) (Jia et al., 2008).

Table 1.

Direct scavenging effects of resveratrol

	IC50 (μM)	Scavenging effect			Reference
		1 μM	10 μM	100 μM
O2 ●¯ (XXO)	245	n.d.	2.8%	n.d.	Hung et al. (2002)
O2 ●¯ (XXO)	252	n.d.	4%	23%	Jia et al. (2008)
O2 ●¯ (KO2)	458	n.d.	2%	18%	Jia et al. (2008)
H2O2	11	26%	48%	84%	Ungvari et al. (2007)
ONOO¯	63	10%	23%	57%	Holthoff et al. (2010)

Superoxide (O₂ ^●¯) is produced by either xanthine/xanthine oxidase (XXO) or by the potassium superoxide system (KO₂). ONOO¯, peroxynitrite; n.d., no data available.

In cell‐free assays, resveratrol effectively attenuates H₂O₂ levels (Table 1). Resveratrol (1 or 24 h incubation) also dose‐dependently decreases H₂O₂ concentration in cultured arteries treated with paraquat or UV light (Ungvari et al., 2007).

When incubated with authentic peroxynitrite in a cell‐free system, resveratrol directly scavenges peroxynitrite, blocking the nitration of bovine serum albumin 20‐fold more potently than N‐acetyl‐l‐cysteine (Holthoff et al., 2010). Resveratrol inhibits peroxynitrite‐induced LDL oxidation (Brito et al., 2002) and inhibits peroxynitrite‐induced cytotoxicity (Holthoff et al., 2010).

In general, however, the direct antioxidant effects of resveratrol are rather poor (Table 1). The effects of resveratrol against oxidative injury in vivo are more likely to be attributable to its effects as a gene regulator rather than its direct ROS scavenging activity (Li et al., 2012; Xia et al., 2014b).

Resveratrol as a gene regulator of the redox system

Many of resveratrol's protective effects in vivo are mediated by gene regulation. In whole‐genome microarray experiments using liver samples from mice fed a high‐calorie diet, the expression patterns of 782 out of 41 534 individual genes are changed significantly by resveratrol treatment. Remarkably, resveratrol prevents the effects of high caloric intake in 144 out of 153 significantly altered pathways (Baur et al., 2006). These results indicate that resveratrol is a powerful gene regulator (Xia et al., 2014b).

Direct molecular targets of resveratrol

Resveratrol has been shown to induce various biological effects in preclinical studies. This is probably because resveratrol is a molecule with many targets (Bollmann et al., 2014). Resveratrol is relatively hydrophobic because of its planar stilbene motif. Therefore, resveratrol has a relatively high affinity for hydrophobic pockets and binding sites in proteins. Moreover, the polar OH groups act as both hydrogen‐bond donors and acceptors, which can form multiple interactions with amino acid side chains as well as backbone amide groups (Britton et al., 2015). There have been around 20 proteins identified as having a specific affinity for resveratrol to date (Britton et al., 2015).

For the antioxidant effects of resveratrol in the cardiovascular system, the NAD⁺‐dependent histone/protein deacetylase sirtuin 1 (SIRT1) and the nuclear factor‐E2‐related factor‐2 (Nrf2) are particularly important (see below). Another important resveratrol target for its cardiovascular effects is the oestrogen receptor (ER). A subpopulation of ERα is associated with caveolae in the endothelial plasma membrane and coupled to the eNOS in endothelial cells via a G protein (Wyckoff et al., 2001). Resveratrol has been shown to rapidly activate eNOS by stimulating the membrane ER (Klinge et al., 2005; Klinge et al., 2008). This represents one of the many mechanisms by which resveratrol enhances endothelial NO production (Xia et al., 2014a). In addition to the direct vasoprotective effects of endothelial NO (Xia et al., 2014a), ER‐mediated NO production is likely to be involved in resveratrol‐induced up‐regulation of antioxidant proteins such as thioredoxin‐1 and also HO1 (Thirunavukkarasu et al., 2007; Yu et al., 2010).

SIRT1 as a resveratrol target

Among the known resveratrol targets, SIRT1 has received much attention. Resveratrol has been identified as a SIRT1 activator in an in vitro assay (Howitz et al., 2003). However, later studies indicate that resveratrol directly activates SIRT1 only on certain substrates (Hubbard et al., 2013). Indirectly, resveratrol may activate SIRT1 either through a signalling cascade involving PDE inhibition and subsequent elevation of cellular NAD⁺ (Park et al., 2012) or by enhancing the binding of SIRT1 to lamin A, a protein activator of SIRT1 (Liu et al., 2012). Resveratrol inhibits PDE1, PDE3 and PDE4 with IC₅₀ values of 6, 10 and 14 μM, respectively, without affecting the activity of PDE2 or PDE5 (Park et al., 2012). Finally, the SIRT1‐dependent effects of resveratrol in vivo may be also partially attributable to an up‐regulation of SIRT1 expression (Csiszar et al., 2009; Xia et al., 2013).

Like resveratrol, SIRT1 is also a molecule with many targets, which is the molecular basis by which SIRT1 regulates a broad range of biological processes. SIRT1 modulates gene expression by targeting molecules such as histones, non‐histone substrates (e.g. transcription factors and co‐regulators) and SIRT1‐interacting proteins (Zhang and Kraus, 2010). For instance, SIRT1 deacetylates the RelA/p65 subunit of NF‐κB, thereby suppressing inflammation (Yeung et al., 2004). By targeting p53, sterol regulatory element‐binding proteins, forkhead box O (FOXO) transcription factors and proliferator‐activated receptor–coactivator (PGC)‐1α, SIRT1 modifies the expression of a number of enzymes involved in cell cycle/apoptosis, stress defence, anti‐ageing processes, lipid metabolism and metabolic adaptation (Liang et al., 2009; Sinclair and Guarente, 2014). In addition, SIRT1 target molecules also include some cytosolic proteins that are not transcription factors or cofactors including eNOS (Mattagajasingh et al., 2007).

SIRT1‐dependent up‐regulation of antioxidant enzymes

Resveratrol regulates the expression and activity of a number of redox enzymes, thereby inhibiting ROS production and facilitating ROS detoxification (Figure 1).

Figure 1.

Antioxidant effects of resveratrol. Resveratrol inhibits NADPH oxidase‐mediated ROS production by down‐regulation of the catalytic subunits (NOX proteins) and by inhibiting membrane translocation of Rac1 and inhibiting phosphorylation of p47phox. Resveratrol directly activates SIRT1 on certain substrates. It can also activate SIRT1 indirectly by potentiating the activation effect of lamin A or via a pathway involving PDE inhibition that leads to elevation of cellular NAD⁺. Among the established SIRT1 targets, FOXO transcription factors contribute to the antioxidative effects of resveratrol by up‐regulating antioxidative enzymes (e.g. SOD2 and catalase, CAT) and eNOS. SIRT1 inhibits mitochondrial O₂ ^●¯ production by stimulating mitochondrial biogenesis, which is mediated by proliferator‐activated receptor–coactivator‐1α (PGC‐1α) deacetylation and by NO‐dependent mechanisms. The up‐regulation of GCH1 leads to enhancement of BH₄ biosynthesis and prevention of eNOS uncoupling. In addition, resveratrol up‐regulates a number of antioxidant enzymes by activating Nrf2.

The up‐regulation of SOD enzymes by resveratrol has been observed in cultured cells (Spanier et al., 2009; Ungvari et al., 2009; Xia et al., 2010) as well as in laboratory animals in vivo (Xia et al., 2010). In cultured human endothelial cells, resveratrol (10–100 μM) increases the mRNA and protein levels of all three SOD enzymes (Spanier et al., 2009; Ungvari et al., 2009; Xia et al., 2010). The up‐regulation of SOD1 and SOD2, but not that of SOD3, by resveratrol is likely to be mediated by SIRT1 (Ungvari et al., 2009; Xia et al., 2010). A recent study has shown that the SIRT1‐induced up‐regulation of SOD2 is partially mediated by FOXO1, a transcription factor regulated by SIRT1 (Hsu et al., 2010).

Resveratrol enhances the expression of GPx1 and catalase in cultured human endothelial cells (Ungvari et al., 2007; Xia et al., 2010) as well as in cardiac tissue of ApoE‐KO mice (Xia et al., 2010). SIRT1 is likely to be involved in the up‐regulation of GPx1 (Xia et al., 2010) and catalase (Alcendor et al., 2007) by resveratrol. The transcription factor FOXO3a, which is a target molecule of SIRT1, has been implicated in the SIRT1‐mediated up‐regulation of catalase (Hasegawa et al., 2008; Liang et al., 2009).

Nrf2‐dependent up‐regulation of antioxidant enzymes

Nrf2 is an indirect target of resveratrol (Ungvari et al., 2010). Under quiescent conditions, Nrf2 is localized in the cytoplasm through binding to Kelch‐like erythroid cap‘n’collar homologue (ECH) associated protein 1 (Keap 1). This interaction facilitates the ubiquitination and subsequent degradation of Nrf2. Treatment of cells with resveratrol leads to Nrf2 release from Keap 1 and Nrf2 translocation to the nucleus. The binding of Nrf2 to antioxidant response elements triggers antioxidant response element‐dependent transcription of phase II and antioxidant defence enzymes. In cultured endothelial cells, resveratrol up‐regulates gene expression of antioxidant defence enzymes NQO1 and HO1 in an Nrf2‐dependent manner (Ungvari et al., 2010), although the molecular mechanism by which resveratrol activates Nrf2 is still unclear.

Lower concentrations of resveratrol (0.1–1 μM) are able to produce this effect (Ungvari et al., 2010), whereas higher concentrations are needed to activate SIRT1 (high μM) (Howitz et al., 2003; Milne et al., 2007). The in vivo relevance of resveratrol‐induced Nrf2 activation has been demonstrated in mice fed a high‐fat diet, in which the endothelial protective effects of resveratrol are largely diminished by genetic Nrf2 depletion (Ungvari et al., 2010). Nrf2 may be also involved in GPx1 up‐regulation (Gounder et al., 2012; Howden, 2013), although direct evidence is still unavailable.

Effects of resveratrol on nonenzymatic antioxidants

Nrf2 activation by resveratrol also leads to an up‐regulation of γ‐glutamylcysteine synthetase, the rate‐limiting enzyme for GSH synthesis (Ungvari et al., 2010). Consistently, resveratrol increases endothelial GSH content (Ungvari et al., 2009).

Reduction of ROS production from vascular NADPH oxidases

The expression of NOX2 and NOX4 in the heart of hypercholesterolemic ApoE‐KO mouse is reduced by resveratrol (100 mg·kg⁻¹) (Xia et al., 2010). This effect is likely to be independent of SIRT1; the down‐regulation of NOX4 by resveratrol in endothelial cells (by 10–100 μM resveratrol) was not affected by SIRT1 inhibition or SIRT1 knockdown (Spanier et al., 2009; Xia et al., 2010). Trauma haemorrhage in rats leads to an up‐regulation of vascular NOX1, NOX2, NOX4, p22phox and p47phox. All these expressional changes can be normalized by resveratrol (30 mg·kg⁻¹ i.v.) treatment (Yu et al., 2010). The effect of resveratrol in trauma haemorrhagic rats is abolished by an ER antagonist or by a haem oxygenase enzyme inhibitor. Thus, it is possible that an ER‐dependent up‐regulation of HO1 is involved in the regulation of NADPH oxidase by resveratrol (Yu et al., 2010).

In addition to its effect on NOX expression, resveratrol also modulates the activity of the NADPH oxidase enzyme complex. The activity of NOX4 relies on its association with p22phox, whereas the activity of NOX1 and NOX2 in vascular cells requires not only p22phox but also p47phox (or NOXO1), p67phox (or NOXA1) and Rac proteins (Brandes and Kreuzer, 2005). Resveratrol (5 μM) reduces angiotensin II‐ and oxLDL‐induced NADPH oxidase activation in cultured endothelial cells by inhibiting the membrane translocation of Rac (Chow et al., 2007). In platelets, protein kinase C‐mediated phosphorylation and activation of p47phox are prevented by resveratrol (0.15–0.25 μM) (Shen et al., 2007).

Reduction of ROS production from mitochondria

Resveratrol stimulates mitochondrial biogenesis and thereby decreases mitochondrial ROS generation because mitochondrial proliferation reduces the flow of electrons per unit of mitochondria (Csiszar et al., 2009; Beauloye et al., 2011). Mitochondrial biogenesis is impaired in the aorta of type 2 diabetic db/db mice, and this impairment can be normalized by resveratrol treatment (20 mg·kg⁻¹) (Csiszar et al., 2009). Resveratrol (10 μM) increases mitochondrial mass and mitochondrial DNA content and up‐regulates the electron transport chain constituents and mitochondrial biogenesis factors in human cultured coronary arterial endothelial cells (Csiszar et al., 2009). SIRT1‐dependent NO production (Csiszar et al., 2009; Xia et al., 2013) and SIRT1‐mediated PGC‐1α deacetylation (Beauloye et al., 2011) are implicated in resveratrol‐stimulated mitochondrial biogenesis in endothelial cells.

Resveratrol decreases mitochondrial ROS levels not only by reducing ROS production but also by up‐regulating antioxidant defence systems and thus accelerating ROS detoxification. The expression of SOD2 is enhanced by resveratrol in a SIRT1‐dependent manner (Ungvari et al., 2009). An up‐regulation of SOD2 per se increases mitochondrial generation of H₂O₂, which can easily penetrate mitochondrial membranes and diffuse into the cytoplasm. Interestingly, resveratrol treatment results in lower cytoplasmic H₂O₂ levels (Ungvari et al., 2009), which may result from increased H₂O₂ detoxification by GPx1 in mitochondria and/or by enhanced H₂O₂ inactivation by GPx1 and catalase in the cytoplasm. Both antioxidant enzymes are up‐regulated by resveratrol (see above).

Reduction of ROS production from uncoupled eNOS

In the hypercholesterolaemic, atherosclerosis‐prone ApoE‐KO mice, uncoupled eNOS contributes significantly to the oxidative stress in cardiovascular tissues. ApoE‐KO mice show increased ROS production in the aorta (Alp et al., 2004; Wohlfart et al., 2008) and the heart (Xia et al., 2010). The NOS inhibitor L‐NAME decreases O₂ ^●¯ production in both organs (Alp et al., 2004; Wohlfart et al., 2008; Xia et al., 2010), indicating eNOS uncoupling in this pathological model. The major molecular mechanism of eNOS uncoupling in ApoE‐KO mice has been found to be a deficiency of BH₄, very likely due to increased oxidative degradation of the molecule (Alp et al., 2004).

Treatment of ApoE‐KO mice with resveratrol (30 or 100 mg·kg⁻¹) enhances the expression of the BH₄‐synthesizing enzyme GCH1, increases the biosynthesis of BH₄ and reverses eNOS uncoupling (Xia et al., 2010). Findings from cell culture studies demonstrate that the up‐regulation of GCH1 by resveratrol is a SIRT1‐dependent effect, because it can be reduced by the SIRT1 inhibitor sirtinol or by siRNA‐mediated SIRT1 knockdown (Xia et al., 2010). At the same time, resveratrol also prevents BH₄ oxidation by reducing ROS levels, through both SIRT1‐dependent (up‐regulation of SOD1, SOD2, GPx1 and catalase) and SIRT1‐independent (up‐regulation of SOD3 and down‐regulation of NOX4) mechanisms (Xia et al., 2010). Resveratrol‐induced up‐regulation of GCH1 and elevation of BH₄ levels have also been observed in superior thyroid arteries obtained from patients with hypertension and dyslipidaemia (Carrizzo et al., 2013).

Resveratrol doses and pharmacokinetics

The optimal resveratrol dose is not known. Because of the low bioavailability of resveratrol (Baur and Sinclair, 2006; Cottart et al., 2014), very high resveratrol doses (up to 3000 mg) have been used in some clinical trials (Table 2). A recent study indicates that such high doses may be unnecessary. Interestingly, the low dose (5 mg in humans or 0.07 mg·kg⁻¹ in mice) has been shown to have even superior cancer chemopreventive efficacy than the high dose (1000 mg in humans or 14 mg·kg⁻¹ in mice) (Cai et al., 2015). Under certain conditions, resveratrol may display a hormetic action, protecting cells at lower doses while being detrimental at higher doses (Juhasz et al., 2010a).

Table 2.

Cardiovascular effects of resveratrol in humans

	Resveratrol treatment		Resveratrol effects						References
Study subjects	Daily dose	Duration	BP	Lipid	Glucose	Anthropometry	Inflammation	Other effects
Healthy subjects (n = 20)	400 mg	30 days			Glucose ↔		TNF‐α ↔	Plasma from resveratrol‐treated subjects down‐regulates, in vitro, endothelial VCAM and ICAM expression	Agarwal et al. (2013)
					Insulin ↓		IL‐6 ↔
					HOMA‐IR ↓		IFN‐γ ↓
Healthy firefighters (n = 30)	100 mg	90 days		TG ↔	Glucose ↔		IL‐6 ↓	ALT ↔; AST ↔; GGT ↔; thiol ↔; 8‐isoprostane ↔; 8‐OHdG ↔; erythrocyte GPx activity ↓	Macedo et al. (2015)
				TC ↔
				LDL ↔			TNF‐α ↓
				HDL ↔
Non‐obese, postmenopausal women (n = 15)	75 mg	84 days	SBP ↔	TG ↔	Glucose↔	BW ↔	CRP ↔		Yoshino et al. (2012)
				TC ↔	Insulin↔		IL‐6 ↔
			DBP ↔	LDL ↔	HOMA‐IR↔	BMI ↔	Leptin ↔
				HDL ↔			ADPN ↔
Healthy aged physically inactive men (n = 14)	250 mg	56 days	MAP ↔	TG ↔	Glucose ↔	BW ↔	SIRT1 ↔	Resveratrol blunts the positive effects (MAP, TG and LDL) of exercise training in aged men	Gliemann et al. (2013)
				TC ↔
				LDL ↔		BMI ↔
				HDL ↔
Healthy aged physically inactive men (n = 9)	250 mg	56 days	MAP ↔	TG ↔	Glucose ↔	BW ↔	CRP ↓	Resveratrol impairs exercise training‐induced effects in skeletal muscle	Olesen et al. (2014)
				TC ↔			TNF‐α ↔
				LDL ↔		BMI ↔	IL‐6 ↔
				HDL ↔
Healthy obese men (n = 11)	150 mg	30 days	SBP ↓	TG ↓	Glucose ↓	BW ↔	CRP ↔	Metabolic rate ↓; activation of AMPK and SIRT1	Timmers et al. (2011)
			DBP ↔		Insulin ↓		IL‐6 ↔
							TNF‐α ↓
					HOMA‐IR ↓		Leptin ↓
							ADPN ↔
Healthy obese men (n = 28)	75 mg	42 days	SBP ↔			BMI ↔		FMD ↑	Wong et al. (2013)
			DBP ↔
Healthy obese men (n = 12)	500 mg	28 days	SBP ↔	TG ↔	Glucose ↔	BMI ↔	hsCRP ↔	No effect on resting energy expenditure or lipid oxidation rates	Poulsen et al. (2013)
				TC ↔	Insulin ↔		IL‐6 ↔
			DBP ↔	LDL ↔	HOMA‐IR ↔		TNF‐α ↔
				HDL ↔	HbA1c ↔		MCP‐1 ↔
Healthy obese men (n = 10)	150 mg	30 days						GLP‐1 ↔	Knop et al. (2013)
								GIP ↔
								Glucagon ↓
Overweight older adults (n = 10–12)	300 or 1000 mg	90 days	SBP ↔		Glucose ↓	BW ↔			Anton et al. (2014)
			DBP ↔			BMI ↔
						WC ↔
Overweight/obese individuals with mild hypertriglyceridaemia (n = 8)	1–2 g	14 days		TG ↔	Glucose ↔			Intestinal and hepatic lipoprotein particle production ↓	Dash et al. (2013)
				TC ↔	Insulin ↔
				HDL ↔	HOMA‐IR ↔
Older adults with impaired glucose tolerance (n = 10)	1–2 g	28 days	SBP ↔	TG ↔	Glucose ↔	BW ↔	hsCRP ↔		Crandall et al. (2012)
				TC ↔	Insulin ↔
			DBP ↔	LDL ↔	HOMA‐IR ↓		ADPN ↔
				HDL ↔
Patients with metabolic syndrome (n = 34)	100 mg	90 days	SBP ↔	TG ↔	Glucose ↔	BW ↔	hsCRP ↔	FMD↑	Fujitaka et al. (2011)
				LDL ↔	Insulin ↔	BMI ↔
			DBP ↔	HDL ↔	HOMA‐IR ↔	WC ↔	IL‐6 ↔
					HbA1c ↔
Patients with metabolic syndrome (n = 12)	1.5 g	90 days			Insulin ↓	BW ↓			Mendez‐del Villar et al. (2014)
						BMI ↓
						WC ↓
Patients with type 2 diabetes mellitus (n = 28)	250 mg	90 days	SBP ↓	TG ↔	Glucose ↓	BW ↔			Bhatt et al. (2012)
				TC ↓
			DBP ↓	LDL ↓	HbA1c ↓	BMI ↔
				HDL ↔
Patients with type 2 diabetes (n = 33)	1 g	45 days	SBP ↓	TG ↔	Glucose ↓	BW ↔			Movahed et al. (2013)
				TC ↔	Insulin ↓
			DBP ↔	LDL ↔	HOMA‐IR ↓	BMI ↔
				HDL ↑	HbA1c ↓
Patients with type 2 diabetes (n = 5)	3 g	84 days		TG ↔	Glucose ↔	BW ↔	ADPN ↔	SIRT1 ↑; p‐AMPK/AMPK ↑ in skeletal muscle	Goh et al. (2014)
				TC ↔	Insulin ↔
				LDL ↑	HbA1c ↔	BMI ↔
				HDL ↔	HOMA‐IR ↔
Patients with type 2 diabetes and hypertension (n = 13)	8 mg	365 days	SBP ↔	TG ↔	Glucose ↓		hsCRP ↔	Expression changes of cytokines and microRNAs in PBMC	Tome‐Carneiro et al. (2013b)
			DBP ↔	TC ↔	HbA1c ↓		ADPN ↔
				LDL ↔			TNF‐α ↔
				HDL ↔			PAI‐1 ↔
							IL‐6 ↓
Patients undergoing primary CVD prevention (n = 25)	8 mg	365 days		TG ↔			hsCRP ↓		Tome‐Carneiro et al. (2012)
				TC ↔			TNF‐α ↓
				LDL ↔			ADPN ↔
				HDL ↔			IL‐6 ↔
							PAI‐1 ↓
Patients with stable CAD (n = 25)	8–16 mg	365 days					hsCRP ↓	Expression of pro‐inflammatory gene in PBMCs ↓	Tome‐Carneiro et al. (2013a)
							TNF‐α ↔
							ADPN ↓
							IL‐6 ↔
							PAI‐1 ↓
Patients with stable angina pectoris (n = 29)	20 mg	60 days		TG ↓			hsCRP ↓	NT‐proBNP ↓	Militaru et al. (2013)
				TC ↓
				LDL ↓
				HDL ↑
Post‐infarction patients with stable CAD (n = 20)	10 mg	90 days	SBP ↔	TG ↔	HbA1c ↔		CRP ↔	Left ventricular diastolic function ↑; FMD ↑; platelet activity ↓	Magyar et al. (2012)
				TC ↔
			DBP ↔	LDL ↓			TNF‐α ↔
				HDL ↔
Overweight or obese men with NAFLD (n = 10)	3 g	56 days	SBP ↔	TG ↔	Glucose ↔	BW ↔	CRP ↔	ALT ↑; AST ↔	Chachay et al. (2014)
				TC ↔	Insulin ↔	BMI ↔	TNF‐α ↔	Liver steatosis ↔
			DBP ↔	LDL ↔	HOMA‐IR ↔		IL‐6 ↓	F2‐isoprostanes ↔
				HDL ↔				Total antioxidant capacity ↔
Overweight patients with NAFLD (n = 25)	500 mg	84 days	SBP ↔	TG ↔	Glucose ↔	BW ↓	hsCRP ↓	ALT ↓; AST ↓; GGT ↓	Faghihzadeh et al. (2015), Faghihzadeh et al. (2014)
				TC ↔	Insulin ↔	BMI ↓	TNF‐α ↔
			DBP ↔	LDL ↔	HOMA‐IR ↔	WC ↓	IL‐6 ↓	Liver steatosis ↓
				HDL ↔
Patients with NAFLD (n = 30)	600 mg	90 days	SBP ↔	TG ↔	Glucose ↓	BW ↔	hsCRP ↓	ALT ↓; AST ↓; GGT ↔	Chen et al. (2015)
				TC ↓	Insulin ↔	BMI ↔	ADPN ↑
			DBP ↔	LDL ↓	HOMA‐IR ↓	WC ↔	IL‐6 ↓
				HDL ↔

ADPN, adiponectin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BW, body weight; CAD, coronary artery disease; CRP, C‐reactive protein; CVD, cardiovascular disease; DBP, diastolic blood pressure; FMD, flow‐mediated dilatation; GGT, γ‐glutamyl transferase; GIP, glucose‐dependent insulinotropic polypeptide; GLP‐1, glucagon‐like peptide‐1; GPx, GSH peroxidase; HOMA‐IR, homeostasis model assessment of insulin resistance; hsCRP, high‐sensitivity CRP; ICAM, intercellular adhesion molecule; MAP, mean arterial pressure; MCP‐1, monocyte chemoattractant protein‐1; n, number of subjects treated with resveratrol; NAFLD, non‐alcoholic fatty liver disease; NT‐proBNP, N‐terminal prohormone of brain natriuretic peptide; PAI‐1, plasminogen activator inhibitor type 1; PBMC, peripheral blood mononuclear cells; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride; VCAM, vascular cell adhesion molecule; WC, waist circumference.

About 1 h after oral ingestion of a single resveratrol dose by healthy volunteers, the maximum peak plasma concentrations (C_max) of 0.6 and 137 μM (for intakes of 5 mg and 1 g respectively) are reached (Cai et al., 2015). Circulating resveratrol is still detectable as late as 24 h after resveratrol administration (average resveratrol concentrations 0.08 and 14 μM in the 5 mg and 1 g dose groups respectively) (Cai et al., 2015).

The following mechanisms may contribute to the phenomenon that low resveratrol doses are effective, despite the rapid and extensive metabolism of resveratrol into sulfate and glucuronide conjugates. (i) Some resveratrol metabolites are biologically active, although not as effective as the parent molecule (Miksits et al., 2009; Lu et al., 2013). (ii) Resveratrol and its metabolites can accumulate in tissues, resulting in enhanced concentrations compared to those in serum (Bresciani et al., 2014; Cai et al., 2015). (iii) Some metabolites can be converted back to resveratrol in tissues (Miksits et al., 2009).

Cardiovascular effects of resveratrol in humans

Preclinical studies have demonstrated a variety of protective effects in animal models of cardiovascular disease, including hypertension (Mizutani et al., 2000; Dolinsky et al., 2009; Dolinsky et al., 2013), hypercholesterolaemia (Penumathsa et al., 2007; Juhasz et al., 2011), atherosclerosis (Wang et al., 2005; Do et al., 2008), ischaemic heart disease (Andreadou et al., 2015; Novelle et al., 2015), diabetes (Su et al., 2006; Um et al., 2010) and metabolic syndrome (Novelle et al., 2015; Pechanova et al., 2015). These cardiovascular effects of resveratrol in laboratory animals have been reviewed in our previous article (Li et al., 2012) and in a recent publication (Zordoky et al., 2015). Therefore, here we have summarized only the cardiovascular effects of resveratrol observed in clinical trials.

As shown in Table 2, the results of these studies are not always consistent, sometimes even contradictory. Moreover, the antioxidant effect of resveratrol does not always lead to a beneficial effect on cardiovascular health. For instance, exercise training induces a number of beneficial cardiovascular effects in healthy aged men, probably mediated partly through ROS‐dependent mechanisms. A concomitant oral resveratrol supplementation, however, blunts part of these positive effects of exercise training (Gliemann et al., 2013; Olesen et al., 2014).

Overall, the major limitation of the clinical studies currently available is the small sample size (Table 2). Large clinical trials are clearly warranted to establish the clinical significance of resveratrol in humans.

Conclusion

The antioxidant effects of resveratrol are implicated in the health benefits of the compound. The direct ROS‐scavenging effects of resveratrol are relatively poor. Resveratrol's antioxidant effects in vivo are more likely to be attributable to its regulation of redox genes leading to reduced ROS production from NADPH oxidases, uncoupled eNOS and the mitochondria. At the same time, an up‐regulation of antioxidant enzymes by resveratrol accelerates the detoxification of ROS.

Author contributions

N.X. and H.L. wrote the initial draft of the manuscript. All authors critically reviewed and revised the manuscript and agreed to its publication.

Conflict of interest

The authors declare no conflicts of interest.

Xia, N. , Daiber, A. , Förstermann, U. , and Li, H. (2017) Antioxidant effects of resveratrol in the cardiovascular system. British Journal of Pharmacology, 174: 1633–1646. doi: 10.1111/bph.13492.