Resveratrol in Cholesterol Metabolism and Atherosclerosis

Abstract

Resveratrol, a natural polyphenol produced by plants in response to environmental stress, has received great attention during the past few years due to its beneficial roles in longevity and glucose homeostasis. Resveratrol has been found to display antioxidant, anti-inflammatory, antifibrotic, and cardioprotective properties. Resveratrol reduces platelet aggregation, induces vasorelaxation, limits endothelial activation, and modulates lipid and lipoprotein metabolism. Although the mechanisms of action of resveratrol have not been completely defined, there is evidence that some of the effects of resveratrol may be mediated via activation of sirtuin 1 and AMP-activated protein kinase and through inhibition of the pleiotropic transcription factor nuclear factor κB. Pathways proposed to underlie resveratrol-mediated cardioprotection include reduction of oxidative stress and activation of endothelial nitric oxide synthase. Adenosinergic mechanisms may play a role in its atheroprotective activity. The ability of the nutraceutical resveratrol to positively influence the future treatment of cardiovascular disease is discussed.

Introduction

C ardiovascular disease is one of the chief causes of morbidity and mortality worldwide. Studies have shown that despite an increased prevalence of smoking and consumption of diets containing significant amounts of saturated fats, the incidence of cardiovascular disease is actually lower in the French population than in the American population.¹ This anomaly, otherwise known as the “French paradox,” is further supported by the Copenhagen Heart Study, which found an association between regular, moderate wine consumption and reduced coronary risk.² Thus, the relationship between wine consumption and coronary disease has generated increasing interest in recent years, inevitably raising the question: could this lower incidence of cardiovascular disease be attributed to the French consumption of red wine?

Resveratrol, a component of red wine, is thought to be the key ingredient responsible for offering many health benefits such as this lower incidence of cardiovascular disease among the French population.³ Although there remains scholarly dispute regarding the exact benefits of resveratrol, many studies have shown resveratrol to have cardioprotective (Table 1), anticarcinogenic, antioxidant, pain-modifying, and lipid-altering properties.^4
–6 Moreover, in addition to resveratrol's natural occurrence in grape-related foodstuffs and beverages, Vaccinium berries, peanuts, and soy, it is also an ingredient found in dietary supplements, nutraceuticals, and herbal medicines. Examples of this include Itadori tea, made from a plant known as Japanese knotweed, which is usually consumed in Japan and China as an herbal remedy for heart disease and stroke.⁷

Table 1.

Summary of the Cardioprotective Effects of Resveratrol

• Vasorelaxant activity

• Improves endothelial function

• Anti-inflammatory effects

• Anti-aggregatory action on platelets

• Antioxidant activity

• Scavenges free radicals

• Modulates lipoprotein metabolism

• Enhances reverse cholesterol transport

• Inhibits the expression of vascular adhesion molecules

This article will present an overview of the possible implications of resveratrol as a therapeutic agent for cardiovascular disease with an emphasis on resveratrol's effect on cholesterol metabolism.

Chemical Structure and Absorption

Resveratrol (trans-3,5,4′-trihydroxystilbene) is a type of natural phenol and a phytoalexin (antimicrobial substance synthesized de novo by plants to fight against fungal and bacterial infection). Resveratrol is a low-molecular-weight polyphenol stilbene with poor aqueous solubility. In nature, it exists as two geometric isomers (Fig. 1): cis and trans. The latter is more strongly associated with beneficial health effects.⁸ Resveratrol is synthesized from p-coumaroyl-coenzyme A and malonyl-coenzyme A, and its synthesis is enhanced by stress factors, mechanical injury, infection, or ultraviolet irradiation. It is also found in different polymer forms, such as vaticanol C, which exhibits potent cytotoxic effects compared with the other polymers.⁹

FIG. 1.

Chemical structure of the trans and cis isomers of resveratrol.

Resveratrol is rapidly and efficiently absorbed following oral administration, although its bioavailability is low because of its first-pass metabolism in the liver.¹⁰ Peak concentration of resveratrol in plasma is reached after 30 min. Plasma elimination half-lives vary between 4.77 and 9.70 h.¹¹ Resveratrol is metabolized to glucuronide and sulfate conjugates, and these metabolites may contribute to the pharmacological activity of the parent agent.¹² Approximately 75% of this polyphenol is excreted via feces and urine. However, because of its lipophilic nature, resveratrol may be bound to the cellular fraction in blood or in lipophilic tissues, leading to an underestimation of its concentration.

Overview of Cholesterol Metabolism and Resveratrol

Macrophages play a pivotal role in the development of atherosclerosis. Lipid metabolism in macrophages is an important process in the context of hypercholesterolemia. Uptake of excessive amounts of native and modified lipoproteins leads to their conversion into foam cells, which accumulate to create fatty streaks, a central feature of the early phase of atherosclerotic lesion development. Networks of proteins associated with macrophage lipid metabolism have been found in recent years to be affected by resveratrol. Among these are the peroxisome proliferator-activated receptor (PPAR) γ, liver X receptors (LXRs), the ATP binding cassette (ABC) transporters A1 and G1, and, most recently, cholesterol 27-hydroxylase (Fig. 2).

FIG. 2.

Pathways of lipid metabolism regulated by resveratrol. Uptake by subendothelial macrophages of excessive amounts of modified lipoproteins, such as oxidized low-density lipoprotein (oxLDL), via the major scavenger receptors CD36, lectin-like oxLDL receptor (LOX-1), and scavenger receptor (SR)-A1 leads to foam cell formation. Macrophages overload with lipid, become foam cells, and accumulate to form fatty streaks, a hallmark of the early phase of atherosclerotic lesion development. Resveratrol activates silent information regulator 1 (SIRT1) with subsequent positive regulation of peroxisome proliferator-activated receptor (PPAR) action. SIRT1 binds directly to endothelial nitric oxide synthase (eNOS), thereby stimulating nitric oxide production and fatty acid oxidation. Activation of the PPARγ/liver X receptor (LXR) cascade and the AMP-activated protein kinase (AMPK) pathway by resveratrol up-regulates ATP binding cassette (ABC) transporters A1 and G1 expression and function. The LXR cascade is also turned on by resveratrol via stimulation of cholesterol 27-hydroxylase, which produces oxysterols that are LXR ligands. HDL, high-density lipoprotein; HODE, hydroxyoctadecadienoic acid.

PPARγ, a member of the nuclear receptor superfamily, has numerous anti-atherogenic effects in macrophages, endothelial cells, and smooth muscle cells.¹³ This receptor has been linked to macrophage maturation and uptake of modified (oxidized) low-density lipoprotein (LDL). PPARγ agonists also exert diverse anti-inflammatory actions important in atherosclerosis.¹⁴

LXRs are key transcriptional regulators of lipid metabolism and direct transcriptional targets of PPARγ.¹⁵ LXRs are commonly characterized as “cholesterol sensors.” Treatment of hypercholesterolemic LDL receptor-deficient mice with a synthetic LXR agonist significantly reduces development of atherosclerosis.¹⁶ LXRs form obligate heterodimers with retinoid X receptors, related members of the nuclear receptor superfamily activated by retinoic acid. Various oxysterols and intermediates of the cholesterol biosynthetic pathway act as LXR ligands.¹⁷ Oxysterols are derivatives of enzymatic or nonenzymatic cholesterol oxidation. They are bioactive lipids that act as regulators of lipid metabolism and inflammation. Oxysterols, including 27-hydroxycholesterol, 24(S),25-epoxycholesterol, 22(R)-hydroxycholesterol, and 24(S)-hydroxycholesterol, are natural ligands of LXRs. LXRs induce lipid transporters such as ABCA1 and ABCG1.¹⁸

The cytochrome P450 27-hydroxylase catalyzes the first step in the oxidation of the side chain of sterol intermediates in the bile acid synthesis pathway: 27-hydroxylation of cholesterol to form the oxysterol 27-hydroxycholesterol.¹⁹ 27-Hydroxycholesterol, a component of the major circulating lipoproteins, exits cells through lipid membranes orders of magnitude faster than cholesterol. 27-Hydroxycholesterol behaves like a lipid-lowering statin drug by inhibiting the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase. 27-Hydroxylase activity in human macrophages counteracts lipid overload by ridding cells of excess cholesterol.^20,21 27-Hydroxycholesterol is an oxysterol signaling molecule and LXR ligand. LXRs respond to increases in 27-hydroxycholesterol and other oxysterols with up-regulated transcription of gene products that control cholesterol catabolism and efflux, including ABCA1 (Fig. 2).

A coordinated lipid transport pathway is likely to be regulated by these receptors. The regulatory region of LXRα contains both a PPAR response element and an LXR response element.²² Linking of the two receptor systems, PPARγ and LXRα, provides an attractive but not well-understood pathway to explain lipid and cholesterol uptake and efflux from macrophages.

Physiological or pharmacological manipulations that reduce macrophage lipid ingestion may be effective for preventing or reversing atherosclerosis. There is significant evidence, both from our lab and others, that supports the theory that the beneficial effects of resveratrol on atherosclerosis may involve increased induction and expression of reverse cholesterol transport proteins such as PPARγ, LXRα, 27-hydroxylase, and ABCA1, which promote cholesterol efflux, thereby impeding cholesterol accumulation. In addition, it has been demonstrated that resveratrol exerts statin-like effects by down-regulating 3-hydroxy-3-methylglutaryl-coenzyme A reductase in hyperlipidemia and increasing the ratio of apolipoprotein (apo) A-I to apoB, which is negatively related to cardiovascular risks.²³ Studies have also shown that resveratrol reduces the oxidation of LDL into oxidized LDL (the form of LDL that promotes endothelial dysfunction and atherogenesis).^24
–26 Resveratrol also inhibits foam cell formation and counters atherogenic changes provoked by the cytokine interferon γ (IFN-γ), signifying possible clinical utility of resveratrol as a cardioprotective agent (authors' unpublished data).

The PPAR, LXR, and ABCA Loop

PPARs are ligand-dependent transcription factors belonging to the nuclear receptor family that regulate an array of physiological processes involved in regulating adipose tissue metabolism.²⁷ The PPAR nuclear receptor subfamily is composed of three members: PPARα, PPARγ, and PPARβ.

The PPARγ receptor plays an important role in the regulation of adipocyte gene expression and differentiation, as well as glucose homeostasis.²⁸ Recent evidence indicates that PPARγ is expressed at high levels in macrophages, including the foam cells of atherosclerotic lesions.²⁹ A study by Chawla et al.³⁰ provided evidence that PPARγ promotes cholesterol efflux through a transcriptional cascade involving LXRα and ABCA1. PPARα also induces LXRα expression and thereby stimulates ABCA1-dependent cholesterol efflux to apoA-I.³¹

LXRα limits cholesterol accumulation through the expression of genes for the cholesterol efflux proteins ABCA1³² and the high-density lipoprotein (HDL)-associated lipoprotein apoE.³³ ApoE and apoE-containing HDL work in concert with ABCA1 and ABCG1, respectively, to promote cellular cholesterol efflux (Fig. 2). LXRα senses excess cholesterol by binding to oxysterol ligands,³⁴ a mechanism amplified in humans and mice by the ability of LXRα to autoregulate its own promoter.³⁵

Owing to this cascade, PPARγ and LXR ligands cooperate and promote ABCA1 expression and cholesterol efflux from macrophages. Chawla et al.³⁰ provided in vivo evidence that loss of PPARγ function in macrophages accelerates the process of atherogenesis in mice. Bone marrow transplanted from PPARγ null mice into hypercholesterolemic LDL receptor-deficient mice significantly increases atherosclerosis.

Furthermore, it was demonstrated that resveratrol offered protection to the brain against ischemic stroke in mice through a PPARα-dependent mechanism.³⁶ The protective effect of resveratrol against ischemic injury in the brain was lost in PPARα-null mice. PPARβ also regulates the PPARα co-activator-1α, a key regulator of cellular energy expenditure in peripheral tissues, which affects insulin sensitivity and is involved in oxidation in the mitochondria.^37,38

ABCA1

ABC proteins actively transport numerous substrates, most importantly lipids, across extra- and intracellular membranes against a concentration gradient.³⁹ Unidirectional (inward or outward) transport by these integral membrane proteins requires energy that is provided by ATP hydrolysis.⁴⁰ This process is essential to maintain cholesterol homeostasis in the body.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I, and is essential for the formation of HDL principally in the liver and, to a lesser extent, in the small intestine.⁴¹ Intracellular cholesterol levels tightly control the presence and expression of this transporter in macrophages.⁴² Plasma levels of HDL cholesterol are inversely proportional to the occurrence of coronary artery disease.⁴³ In humans, lack of ABCA1 caused by mutations in the ABCA1 gene on chromosome 9q31 results in Tangier's disease, a rare autosomal codominant disease associated with marked reductions in HDL plasma levels and increased risk of cardiovascular disease.⁴⁴ ABCA1 is expressed through a sequence loop of PPARγ and LXRα activation (Fig. 2). ABCA1 has been identified as a major transporter to facilitate cholesterol efflux, reduce cholesterol accumulation in macrophages, and thereby prevent toxicity associated with cholesterol overload.⁴⁵ ABCA1 with apoA-I controls the rate-limiting step in cholesterol and phospholipid transport. Lipid-poor apoA-I, the most abundant apo in HDL, interacts with ABCA1 on the cell surface. ABCA1 promotes cholesterol outflow and transfer to apoA-I.⁴⁶ In baby hamster kidney cells, ABCA1 overexpression redistributes cholesterol to cell surface domains where it can be removed by apos.⁴⁷ Overexpression of macrophage ABCA1 in LDL receptor-deficient mice reduces atherosclerotic lesion development.⁴⁸ ABCA1 expression is up-regulated by agonists of PPARγ and LXRα through a transcriptional cascade ultimately dependent on the activation of LXRα.^30,49

In a study conducted by Trasino et al.,⁵⁰ resveratrol induced expression of both ABCA1 and ABCG1 in a human prostate cancer cell line in an LXR-independent manner. In another study by Berrougui et al.,²⁶ incubating J774 mouse macrophages in the presence of resveratrol resulted in an increase in ABCA1 protein expression. When the J774 macrophages were preincubated with increasing concentrations of resveratrol (0–25 μM), apoA-I-mediated cholesterol efflux was enhanced in a resveratrol concentration-dependent manner, thereby suggesting that resveratrol promotes cholesterol efflux by up-regulating ABCA1. This proposal was complemented by the findings of Sevoy et al.,⁵¹ showing that resveratrol induces LXRα and elevates ABCA1 and ABCG1 mRNA levels. These studies strengthened the proposition that up-regulation of ABCA1 protein may be the key pathway involved in cholesterol efflux, thus reducing atherosclerosis following polyphenol consumption. The study also demonstrated reduced influx of cholesterol into macrophages in a dose-dependent manner, which further signified that resveratrol down-regulates lipoprotein lipase and scavenger receptor-AII, which promote lipid uptake by macrophages. Resveratrol has also been shown to attenuate foam cell formation.⁵²

Resveratrol and IFN-γ

IFN-γ, a pro-atherogenic cytokine expressed at high levels in atherosclerotic lesions,⁵³ is a potent activator of macrophages for inflammatory response and cellular immunity.⁵⁴ Atherosclerosis-promoting functions of this cytokine include activation of monocytes and macrophages with a consequent increase in apoptosis and expression of adhesion molecules in the endothelium and synthesis of other pro-inflammatory cytokines. It is believed that IFN-γ modulates the inflammatory response associated with atherosclerosis. Plasma levels of this cytokine are higher in patients with coronary diseases such as stable and unstable angina and myocarditis.⁵⁵

The stimulation of IFN-γ leads to the activation of receptor-associated Janus kinase-1 and -2, which in turn leads to the activation of signal transducers and activation of transcription proteins, which play a pivotal role in regulating the immune system.^56,57 Knockout of signal transducer and activation of transcription 1 in bone marrow attenuates development of atherosclerosis in the atherosclerosis-susceptible apoE knockout mouse.⁵⁸ Overproduction and hyperresponsiveness to IFN-γ are implicated in pathologic conditions like autoimmunity and tissue damage secondary to excessive inflammation.^59,60

Resveratrol has also been reported to inhibit IFN-γ production in human peripheral monocytes and in splenic lymphocytes through inhibition of nuclear factor κB (NF-κB) activation. This highlights its potential application as a blocker of IFN-γ action and a protective agent against inflammatory and immune disorders.^61,62 IFN-γ exhibits atheroma-promoting properties in macrophages by disabling reverse cholesterol transport pathways through suppression of 27-hydroxylase, ABCA1, and ABCG1 expression. Our group recently showed that resveratrol can overcome down-regulation of all three of these key proteins in IFN-γ-treated THP-1 human monocytes; in this model system, resveratrol nullifies pro-atherogenic effects of IFN-γ and restores a critical defense mechanism against atherosclerosis.⁶³

Resveratrol and Silent Information Regulator 1 of the Sirtuin Family

The sirtuin family of NAD-dependent histone deacetylases is composed of seven mammalian proteins, silent information regulator (SIRT) 1–7. Resveratrol was discovered to be a strong activator of SIRT1, a class III protein deacetylase that regulates lipid metabolism by de-acetylation of modified lysine residues on histones and various transcriptional regulators.^64
–66 SIRT1 has several effects associated with protection from the development of atherosclerosis. SIRT1 deacetylates histones, transcription factors, and co-regulators and thereby controls activity of regulatory protein and, ultimately, gene expression. SIRT1 regulates the actions of the PPARs, the PPARγ co-activator PPARα co-activator-1α, and NF-κB.^67,68 SIRT1 deacetylates and therefore activates LXRα, enhancing ABCA1 expression.⁶⁹ Loss of SIRT1 reduces apoA-I-mediated cholesterol efflux in primary murine macrophages.

SIRT1 also affects the lectin-like oxidized LDL receptor-1 (LOX-1), a scavenger receptor that allows uptake of oxidized LDL into endothelial cells and macrophages. SIRT1 inhibits tumor necrosis factor α-induced expression of LOX-1 in cultured murine macrophages. Murine studies further suggest that down-regulation of LOX-1 in macrophages by SIRT1 occurs through suppression of NF-κB signaling.⁷⁰

SIRT1 is an important signaling molecule in endothelium. SIRT1 improves endothelial function. It binds directly to endothelial nitric oxide synthase (eNOS) and has been shown to target eNOS for deacetylation, thereby stimulating nitric oxide production and promoting vascular relaxation.^71,72 Endothelial-derived nitric oxide controls vascular tone and has atheroprotective effects. In apoE knockout mice, endothelium-specific SIRT1 overexpression reduced atherosclerotic lesion formation while up-regulating eNOS.⁷³ In human coronary arterial endothelial cells, a recent study showed that resveratrol induces SIRT1 and up-regulates eNOS. Knockdown of SIRT1 prevents eNOS up-regulation.⁷⁴

Although studies have shown that resveratrol functions as an agonist for SIRT1, these findings are not altogether conclusive. Moreover, these findings have been disputed by other studies, which have challenged the role of resveratrol as a direct activator of SIRT1.^75
–77 Thus, the exact role of resveratrol on influencing the promotion, expression, and increase in activity of SIRT1 remains unclear.

Gracia-Sancho et al.⁷⁸ found that resveratrol increases the expression of the transcription factor Krüppel-like factor 2 in human vascular endothelial cells via SIRT1. Endothelial vasoprotective gene targets of Krüppel-like factor 2 such as eNOS, thrombomodulin, and C-type natriuretic peptide are not induced by resveratrol if Krüppel-like factor 2 is silenced.

Role in Angiogenesis

Angiogenesis, or neovascularization, is the formation of new capillaries from preexisting blood vessels. Angiogenesis has the potential to increase blood perfusion in ischemic tissue. On the other hand, neovascularization may destabilize advanced plaques and promote plaque rupture.

Studies have shown that resveratrol therapy inhibits endothelial dysfunction by regulating vascular endothelial growth factor, eNOS, caveolin-1, and heme oxygenase-1, thereby leading to neovascularization of the myocardium and protection against myocardial injury caused by ischemia-reperfusion.^79,80 However, another study stated that resveratrol inhibits or possibly prevents angiogenesis and therefore can decrease or prevent the formation of collateral vessels,^81,82 thereby causing a point of conflict on the use of resveratrol in cases of hypoxic myocardium. Inhibition or prevention of angiogenesis can be attributed to reduced endothelial proliferation and migration.

Overall, effects of resveratrol on angiogenesis are multifaceted as resveratrol is pro-angiogenic in the ischemic myocardium but anti-angiogenic in multiple cancers and tumors.⁸³ SIRT1 activation through natural polyphenols like resveratrol may be beneficial in diseases where endothelial dysfunction occurs such as diabetes, arteriosclerosis, and some pulmonary disorders.

AMP-Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and a key controller in the regulation of whole-body energy homeostasis.⁸⁴ AMPK is a serine/threonine protein kinase and a heterotrimeric enzyme consisting of an α-catalytic and β-and γ-regulatory subunits. It plays an integral role in lipid metabolism by switching on the oxidative process for fatty acids and by inhibiting the synthesis of lipids.⁸⁵ It also aids in endothelial relaxation and dilation. Resveratrol activates AMPK (Fig. 2), which in turn directly phosphorylates eNOS, thereby increasing nitric oxide production.⁸⁶ Resveratrol and structurally related phenols have been shown to directly increase the activation of AMPK and its related activators.⁸⁷ In fact, some investigators have suggested AMPK as a target for resveratrol (Fig. 3).^88,89 After resveratrol pharmacologically inhibits AMPK, the compound's ability to improve endothelium-dependent vasodilation in mouse aortic rings is essentially abolished,⁹⁰ thus providing compelling evidence of the importance of AMPK in resveratrol-mediated effects on vasculature.

FIG. 3.

Summary of resveratrol's effects on cell pathways and metabolic processes. Resveratrol regulates the gene expression of pro-oxidative and antioxidative enzymes in human endothelial cells. By decreasing the expression and activity of vascular NADPH oxidases and enhancing the expression of superoxide dismutase, resveratrol reduces oxidative stress. Resveratrol increases endothelial nitric oxide production by SIRT1-dependent eNOS up-regulation, SIRT1-dependent eNOS deacetylation, and extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated eNOS phosphorylation. Resveratrol activates AMPK, which enhances phosphorylation of eNOS, leading to vasorelaxation. Anti-inflammatory activity of resveratrol could be mediated by its interference with nuclear factor κB (NFκB). The ability of resveratrol to activate the PPARγ/LXRα pathway promotes cholesterol efflux and prevents foam cell formation. Resveratrol counteracts tumor necrosis factor α (TNFα)- and interferon γ (IFNγ)-mediated changes in cell metabolism. Overall, changes brought about by resveratrol prevent oxidative stress, defend against lipid overload, induce mitochondrial biogenesis, and improve endothelial function.

AMPK was recently shown to suppress macrophage foam cell formation by inducing ABCG1 expression in macrophages without regulating ABCA1 expression.⁹¹ Furthermore, treatment with resveratrol and other phenols dramatically decreases lipid accumulation in cultured hepatocytes and in animal livers by stimulating SIRT1 and AMPK activity.^92,93 Resveratrol also increases metabolic rate, insulin sensitivity, and glucose tolerance while reducing fat mass in control mice but not in AMPK-deficient mice.⁹⁰

Other Cardioprotective Properties

Endothelial dysfunction is a marker for development and progression of atherosclerosis. Atherosclerosis is initiated by functional alterations in the vascular endothelial monolayer that influence proliferation, platelet adhesion, and free radical production.⁹⁴ Moreover, there is reasonable consensus that oxidative stress plays a central role in the development of atherosclerosis and vascular inflammation in aging.⁹⁵ In atherosclerotic animal models and in human patients with atherosclerosis, endothelial dysfunction has been shown to be associated with an increase in reactive oxygen species.⁹⁶ Despite this, recent large randomized clinical trials showed no significant benefit when antioxidants targeted to the cell membranes (such as vitamin E) were given to patients with a high-risk coronary artery disease profile.^97,98 The reasons for the failure of antioxidants to reduce mortality or myocardial infarction may be attributed to inadequate dosage or inactivation due to oral route administration. Targeting antioxidants to the site of oxidant formation in the mitochondria or inhibiting oxidant formation at its source is a possible approach for consideration. Although generation of reactive oxygen species is important in the molecular mechanisms associated with endothelial damage, more data are needed before it can be determined which approach may be clinically useful in reducing oxidative stress in humans.⁹⁹ Oral antioxidants cannot be recommended as a cardioprotective therapy at this time.

Apart from altering gene expression and modulating different factors in cholesterol metabolism, resveratrol is being considered as a cardioprotective agent through its antioxidant, vasodilator, and platelet-altering properties (Fig. 3). Further trials in humans are needed to establish whether resveratrol has definitive benefit in humans and, if so, which of resveratrol's multiple mechanisms of action may be involved.

Antioxidant Activity

When LDL is modified, particularly by oxidative reactions, cholesterol uptake by macrophage scavenger receptors is triggered, leading to foam cell formation and progression to atherosclerosis.¹⁰⁰ There is evidence to suggest that oxidative stress not only can encourage influx, but also can impair efflux. In THP-1 macrophages, stress conditions brought about using iron-ascorbate to induce lipid peroxidation decreased cholesterol efflux without any change in influx. Efflux changes were accompanied by diminished levels of ABCA1, LXR, and PPAR.¹⁰¹ This iron-ascorbate model measures the impact of oxidative stress, without participation of oxidized LDL. There is also significant evidence indicating a role for oxidized LDL in both early and advanced inflammatory stages of atherosclerosis.¹⁰² Atherosclerosis is slowed when LDL oxidation is stopped, and there is a strong relationship between the ability of the LDL to resist oxidation and the severity of atherosclerosis.^103,104

In macrophages, resveratrol restores cholesterol efflux to HDL3 in a dose-dependent manner.²⁶ This same effect is observed with two other antioxidants, vitamin E and butylhydroxytoluene.^101,105 Resveratrol significantly decreases iron-ascorbate oxidant system-induced lipid peroxide levels in heart mitochondria.¹⁰⁶

Berrougui et al.²⁶ studied the kinetics of resveratrol's free radical scavenging activity by allowing it to react with 1,1-diphenyl-2-picrylhydrazyl, a stable free radical. The study found that both resveratrol and vitamin E (α-tocopherol), at similar concentrations, exhibited equivalent 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity. This study also showed that resveratrol protected HDL3 against copper-induced oxidation in a concentration-dependent manner by decreasing conjugated diene formation.

Resveratrol has three phenolic hydroxyl groups and is very lipophilic. This lipophilic property enables it to associate with the lipid moiety of lipoproteins and prevent the oxidation of their unsaturated fatty acids.⁸ Resveratrol increases the expression of antioxidant enzymes like superoxide dismutase, catalase, and glutathione peroxidase, thereby reducing the formation of free radicals and preventing endothelial injury (Fig. 3).¹⁰⁷ Treatment of apoE knockout mice with resveratrol for 7 days results in the up-regulation of superoxide dismutase, glutathione peroxidase 1, and catalase in heart tissue.¹⁰⁸ Some researchers believe that the expression of these enzymes is the actual mechanism by which resveratrol prevents oxidative injury rather than the direct scavenging activity of reactive oxygen species. Through this inhibitory effect on the oxidation of LDL, resveratrol blocks the internalization of oxidized lipoproteins. Based on its ability to prevent oxidation of lipids, resveratrol may be considered as a possible therapeutic agent in atherosclerotic patients.¹⁰⁹

Platelet Function

Platelets play a key role in the pathogenesis of acute thrombotic events in coronary artery disease when silent chronic disease transitions to acutely symptomatic events such as onset of acute myocardial infarction after atherosclerotic plaque rupture.¹¹⁰ Damaged endothelium promotes platelet activation and aggregation. Studies have shown that resveratrol inhibits platelet aggregation.^111,112 This notable contribution to the in vivo effects of resveratrol on platelets is likely mediated by direct and irreversible inhibition of cyclooxygenase-1 activity, resulting in decreased production of thromboxane A2, a potent inducer of clotting and vasoconstriction.¹¹³ Inhibition of cycloxygenase-1 is the same mechanism proposed to account for the cardioprotective effects of low-dose aspirin.¹¹⁴ A significant point in favor of considering resveratrol in therapy is that resveratrol remains effective even in platelets from aspirin-resistant individuals.¹¹⁵ Resveratrol has also been shown to inhibit transcription of the cyclooxygenase-1 and -2 genes that are required to produce prostaglandins, which in turn play an important part in platelet aggregation. Resveratrol has been shown to induce platelet apoptosis via both intrinsic and extrinsic pathways. It stimulates caspase-9, caspase-3, and caspase-8 activation as well as cytochrome c release in washed human platelets, which promote platelet destruction and prevent pathological clotting.¹¹⁶

Vasodilation

Impaired endothelium-dependent vasodilation is a hallmark of endothelial dysfunction and is associated with increased risk of developing cardiovascular diseases. Resveratrol has been shown to improve flow-mediated vasodilation in patients with existing coronary heart disease.¹¹⁷ The vasodilator properties of resveratrol are mediated by increased bioavailability of nitric oxide through the elevation of eNOS expression and activity.¹¹⁸ In addition, resveratrol reduces the expression of molecules that promote vasoconstriction such as endothelin-I and the angiotensin-II type 1 receptor.^119,120

In a recent randomized double-blind clinical trial, 40 patients post-myocardial infarction were given either 10 mg of resveratrol or placebo daily for 3 months. Flow-mediated vasodilation was measured before and after the treatment, and a significant improvement in endothelial function was observed in the resveratrol-treated group (P<.05).¹²¹

Perspective

Resveratrol is produced by several plants and is found abundantly in the skin of red grapes and as a component of red wine. Resveratrol is considered to be a nutritional supplement and is not regulated as a drug in the United States. At present, it is not known for sure whether administration of phenols would affect progression of cardiovascular disease in elderly patients. In various experimental settings, including studies in aged laboratory rodents, resveratrol was shown to attenuate free radical production and aid reverse cholesterol transport. Resveratrol has potent anti-atherosclerotic effects in vitro and in vivo that indicate potential clinical utility in preventing the onset and/or progression of atherosclerotic cardiovascular disease. Despite a good deal of preclinical evidence, data on cardiovascular effects in humans are quite limited.¹²² Purported effects have not been proven in clinical trials thus far.

Because resveratrol has a strong safety profile, is well tolerated, and can be used in combination with multiple other therapies without contraindication, it is of interest as an approach to management of cardiovascular disease. Further studies on resveratrol absorption, bioavailability, effects on oxidative stress and lipid-altering capabilities, and their role in cardiovascular pathology are warranted. The role of resveratrol as a therapeutic agent in cardiovascular disease has not yet been fully explored. Long-term clinical studies in humans are in progress or in the process of being planned and may yield clearer answers in the next 3–5 years.¹²³

Footnotes

Acknowledgments

This work was supported by the Elizabeth Daniell Fund, by an Innovative Research Grant from the Arthritis Foundation National Center, and by a Winthrop-University Hospital Pilot and Feasibility Grant.

Author Disclosure Statement

The authors confirm that there are no conflicts of interest.

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