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Keywords:

  • resveratrol;
  • cardioprotection;
  • quinone reductase 2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

Coronary heart disease (CHD) is a major and preventable cause of morbidity and death in the United States. Recently, significant research efforts have been directed at an epidemiological phenomenon known as the “French paradox.” This observation refers to the coexistence of high risk factors with unanticipated low incidence of CHD, and is postulated to be associated with low-to-moderate consumption of red wine. In vivo studies have shown that red wine intake is more CHD-preventative in comparison to other alcoholic drinks; enhanced cardioprotection may be attributed to grape-derived polyphenols, e.g., resveratrol, in red wine. This review summarizes results of in vitro and animal studies showing that resveratrol exerts multifaceted cardioprotective activities, as well as evidence demonstrating the presence of proteins specifically targeted by resveratrol, as exemplified by N-ribosyldihydronicotinamide:quinone oxidoreductase, NQO2. A mechanism encompassing nongenomic and genomic effects and a research roadmap is proposed as a framework for uncovering further insights on cardioprotection by resveratrol.


Introduction: red wine confers protection against CHD

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

Coronary heart disease (CHD), a primary cause of morbidity and death in developed countries, is regarded to be preventable through changes in lifestyle and diet. A documented decline in age-adjusted CHD-related mortality over the years has been observed in the United States, in concert with advances in strategies designed to control CHD, e.g., cessation in cigarette use, increase in exercise frequency, reduction in fat intake and increase in consumption of fruits and vegetables in the diet. Epidemiological studies of a phenomenon commonly referred to as the French paradox show an inverse relationship between low-to-moderate consumption of red wine and the risk for CHD (Fig. 1).1 The unanticipated low incidence of CHD co-existing with high risk factors has been associated with the cultural habit in France of drinking red wine in moderation with meals. In vivo studies have demonstrated that red wine is more effective in preventing CHD compared to other alcoholic beverages; enhanced protection may attribute to polyphenols in red wine, including resveratrol.2,3 Thus, a 30–45% reduction in CHD risk is found in population-based studies among low/moderate drinkers compared to those who drink excessively or individuals who abstain from use of alcohol-containing beverages. Animal and in vitro studies show that ethanol/red wine intake is beneficial to the cardiovascular system; rabbits fed ethanol or red wine and challenged with balloon angioplasty showed inhibition of neointimal hyperplasia in coronary arteries. Moreover, balloon-injured coronary arteries exposed to alcohol are accompanied by suppression of intimal hyperplasia. Exposure to ethanol or resveratrol in vitro also results in inhibition of vascular smooth muscle cells (VSMC) proliferation. In a recently published study, “Alcohol and the cardiovascular system: research challenges and opportunities,” CHD protection by moderate intake of red wine is identified as a research need of considerable urgency.35

image

Figure 1. Scheme depicting atherogenesis as the clinical manifestation of multiple cellular dysfunctions resulting from exposure to lifestyle and environmental events in the form of physical, chemical, and biological challenges. Cardioprotection by resveratrol may be attributed to its ability to act on the same multiple cellular targets adversely affected by extrinsic and intrinsic risk factors. These include the inhibition of LDL oxidation, suppression of platelet aggregation, and inhibition of smooth muscle and endothelial cell proliferation and function, by resveratrol.

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Studies supporting cardioprotection by resveratrol

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

Concerted effort by studies in our and other laboratories on the cardioprotective effects of resveratrol, and red wine with and without alcohol, have shown the following. First, resveratrol inhibits low-density lipoprotein (LDL) oxidation. Oxidation of LDL is considered a key primary event in the initiation of atherosclerosis. Because resveratrol has antioxidant properties, we tested whether it affected LDL oxidation. LDL isolated from normolipidemic adult males was oxidatively modified using Cu2+, with and without the addition of resveratrol. LDL oxidation was monitored by reactivity to thiobarbituric acid, agarose gel electrophoresis, and uptake into macrophages. Resveratrol significantly inhibited LDL oxidation.4–6 Secondly, resveratrol is shown to inhibit platelet aggregation—platelets are actively involved in the process of hemostasis, by which injury in the vascular endothelium is rapidly repaired in order not to compromise the fluidity of the blood. In normal endothelium injury, platelets adhere to the subendothelial matrix of a damaged vessel, spread over the surface and recruit additional platelets to form a thrombus. Improper regulation or over reactivity of this repair system can lead to pathological thrombosis. Studies show that platelet aggregation by a number of agonists is suppressed by resveratrol, both in vitro and in vivo.7–9 Administration of resveratrol (4 mg/kg/day) to rabbits fed a high cholesterol diet caused a 35% reduction in the average ADP-induced platelet aggregation rate (PAR), which was indistinguishable from animals fed a normal diet.7,10 Third, resveratrol suppresses proliferation of smooth muscle cells and pulmonary aortic endothelial cells. Migration and proliferation of smooth muscle cells in the intima of susceptible vessels is a requisite for atherogenesis. We found that exposure to resveratrol reduces proliferation of smooth muscle cells accompanied by G1[RIGHTWARDS ARROW]S block.11–13 Similarly, resveratrol also inhibits proliferation of cultured bovine pulmonary aortic endothelial cells (BPAEC) concomitant with induction of nitric oxide synthase in a dose-dependent manner.14–16 Fourth, in rabbits intragastrically fed resveratrol (4 mg/kg/day) for a duration of five weeks beginning one week prior to induced endothelial injury by denudation in the iliac artery, hyperplasia in the damaged endothelial vessel wall was inhibited, as evidenced by reduction of intimal proliferation index [scored as the ratio of intimal to (intimal + medial) area] from 0.41 ± 0.13 in control animals to 0.28 ± 0.07 in resveratrol-fed animals (P < 0.01). In addition, the relative content of smooth muscle cells, characterized by their “hills and valley” shape and positive reactivity for α-actin, in the intima of resveratrol-fed animals was also proportionately suppressed.17,18 Fifth, the mean area of atherosclerotic plaques was reduced from 56.4 ± 13.5 in rabbits fed a hypercholesterolemic diet to 33.6 ± 19.6 (arbitrary units) in animals fed resveratrol.19 Lastly, resveratrol pronouncedly inhibits proliferation of cultured human aortic smooth muscle cells,20,21 concomitant with dose-dependent increase in the expression of tumor suppressor gene p53, heat shock protein HSP27, quinone reductase 1 and 2, and altered subcellular distribution of nitric oxide synthase and apoptosis inducing factor.20

Taken together, these results provide support for the conclusion that resveratrol acts as a cardioprotective agent by a plethora of activities impinging on events key to the prevention of atherosclerosis and CHD, thereby reinforcing the notion that age-adjusted CHD deaths may be independently modulated by diet-based strategies including use of grape polyphenol resveratrol.

Discovering target proteins of resveratrol

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

How does resveratrol exert its multi-cell type CHD-protective activities? One might envisage possibilities that include transcriptional, posttranscriptional control of gene expression, differential cellular compartmentalization and trafficking of proteins, and co- or post-translational processing mechanisms, which can be studied by approaches that focus on DNA/gene aspects (by genomics), RNA transcription alterations (using microarrays), or protein level changes (by proteomics). Since proteins (as enzymes and structural/regulatory elements) play a direct role on maintaining and regulating the homeostatic and dynamic overture of biological systems, we have focused on the identification of protein targets that might underpin or contribute to cardioprotection by resveratrol using a ligand (resveratrol)-directed proteomic strategy. This strategy is based on the basic tenet that cardioprotective activities stemming from exposure to resveratrol relate to its ability to interact with distinct resveratrol target proteins, (RTPs).20,22–24 In this approach, resveratrol is appended to a solid matrix to facilitate retention, selection, and purification of distinct RTPs. Of note, affinity isolation of RTPs using resveratrol-directed proteomics from cells and tissues has several novel features: the discovery of RTPs with possible significant and integral links to observed cellular responses to resveratrol; the concentration and identification of scarce, low-abundance proteins not easily amenable to detection using crude extracts; and the facilitation of the generation of RTP profiles relevant to the biological effects of resveratrol.

To experimentally test and validate the presence of RTPs, resveratrol was immobilized on epoxy-activated agarose, generating a biospecific affinity platform for the rapid and specific capture of RTPs.20,22,24,25 Extracts prepared from various sources were fractionated on resveratrol affinity agarose columns by sequential elution with 0.35 M and 1 M NaCl, followed by 1 mM ATP and, finally, 2 mM resveratrol. The variously eluted fractions were concentrated and resolved by 10% SDS-PAGE, followed by silver staining. A specific protein migrating with a molecular weight of 22 kDa, denoted RTP-22 was found in the resveratrol-eluted fraction, based on our studies using extracts prepared from human prostate22,25 and melanoma cells,23 and from cultured human aortic smooth muscle cells.20 In all cell extracts tested, the retention of RTP-22 on the resveratrol affinity column was effectively and almost completely competed when extracts were first incubated with excess resveratrol prior to fractionation.20,22,23,25 The RTP-22 was subsequently identified as quinone reductase NQO2 by combining resveratrol-directed affinity chromatography purification strategy with MALDI-TOF mass spectrometry and cloning.26 Notably, purified recombinant NQO2 showed a high affinity for resveratrol, with a dissociation constant KD≤ 50 nM.26 Moreover, X-ray crystallographic analysis of NQO2 in complex with resveratrol shows that resveratrol binds to a hydrophobic interaction cavity located on the interface between dimeric NQO2.26 The boundary of this interaction cavity is defined by the isoalloxazine ring of cofactor FAD in NQO2, as well as by side chains of Y132′, F178′, F126′, M164′, and C121′ from the first protomer, and Y155 and F106 from the second protomer.26 It was found that the deep and narrow nature of the interaction cleft perfectly accommodates the physical dimension of resveratrol, in support of the notion that a stilbene-binding pocket exists within NQO2.26 In addition to hydrophobic and van der Waals interactions, all three hydroxyl groups of resveratrol form hydrogen bonds with key amino acid residues in the protein, thus providing not only enthalpy for the high affinity binding to NQO2, but also maintaining the planar conformation of resveratrol.26

Proposed mechanisms of cardioprotection by resveratrol

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

Our current hypothesis regarding cardioprotection by grape polyphenol resveratrol is that it acts by both nongenomic and genomic mechanisms, directly and indirectly. The nongenomic cardioprotective activities of resveratrol may encompass its direct binding and interaction with cellular RTPs, as well as occur indirectly via its ability to function as effective modulator of enzyme activities (Fig. 2). In the direct mechanistic theme, for instance, resveratrol has been shown to bind to integrins on cell membrane,27,28 leading to integrin-coupled signaling cascades and subcellular protein distribution. A key RTP we have chosen to focus on is NQO2. Our working hypothesis is that binding of resveratrol to NQO2 induces a conformational change that directly alters its molecular fate in cellular trafficking and gene expression; conformationally altered NQO2.resveratrol complex may facilitate additional interaction with NQO2-binding proteins to effect subsequent changes in gene expression. In contrast, the indirect nongenomic mechanism of resveratrol action may be by its ability to exert potent inhibition of enzymes possibly linked to the development of atherogenesis.

image

Figure 2. Proposed genomic and nongenomic activities of resveratrol contributing to its cardioprotective activities, both directly and indirectly. Cardioprotection by resveratrol is hypothesized to occur by multi-active genomic mechanisms. For instance, resveratrol may modulate transcription by binding to estrogen response elements (EREs) as a phytoestrogen. In addition, resveratrol may target antioxidant-response-element (ARE) and modulate transcription by promoting the cytosol-to-nucleus translocation of transcription factor Nrf2. The nongenomic activity of resveratrol may involve its direct and indirect interaction with target protein NQO2; NQO2 may modulate the cardioprotective effects of resveratrol by acting as a direct and indirect cardio-active sensor and mediator of resveratrol.

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Cardioprotection by resveratrol may also involve genomic mechanisms (Fig. 2), postulated as involving multiple transcription regulatory features that include (1) its phytoestrogenic attributes, where it affects transcription via the classical mechanism of binding to estrogen response elements (EREs);29,30 (2) its ability to regulate antioxidant-response-element (ARE)-dependent transcription by promoting translocation of transcription factor Nrf2 from the cytosol-located Nrf2/Keap1 complex to the nucleus to facilitate transcription of antioxidant response element, ARE-responsive genes;31–34 and (3) ERE/ARE-independent, RTP-dependent or mediated transcriptional events, details of which remain to be elucidated in future studies.

Proposed future research maps

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

We propose herewith a limited research roadmap that might facilitate further discovery and elucidation of the mechanism of cardioprotection by resveratrol: (1) structure/functional, uptake and biotransformation experiments combined with in silico analysis, using resveratrol analogues in appropriate tissue culture and animal models systems; (2) structural studies of freshly isolated and preserved specimens from animals challenged with cardiovascular damaging pharmacological agents and diets, with and without dietary supplementation by resveratrol ± other red wine polyphenols; (3) use of laser capture microdissection (LCM) microsystems to isolate stable and unstable plaques from archived diseased human tissues; (4) resveratrol affinity chromatography directed proteomic analysis to identify qualitative and quantitative RTP profile differences between normal and diseased tissues, for further identification by mass spectrometry, interactome, and microgenomic analysis; and (5) small inhibitory RNA and pharmacological agent intervention experiments. It is hoped that these proposed strategies and approaches will provide a better understanding of the role NQO2 plays in cardioprotection as well as illuminating insights on the mechanism of cardioprotection by resveratrol in red wine.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References

We thank Dr. Ole Vang in the Department of Life Sciences and Chemistry, Roskilde University, Denmark, for the invitation to write this review. A portion of this review was presented by JMW as an invited speaker at the 1st International Conference of Resveratrol and Health, held in Elsinore, Denmark, September 13–15, 2010. Studies on cardioprotection by resveratrol in our laboratories over the years were supported in part by the Intramural Sponsored Research Program of New York Medical College, California Grape Trade Commission, Phillip Morris USA Inc., and Phillip Morris International, to JMW.

References

  1. Top of page
  2. Abstract
  3. Introduction: red wine confers protection against CHD
  4. Studies supporting cardioprotection by resveratrol
  5. Discovering target proteins of resveratrol
  6. Proposed mechanisms of cardioprotection by resveratrol
  7. Proposed future research maps
  8. Acknowledgments
  9. Conflicts of interest
  10. References
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