Cardiovascular effects and molecular targets of resveratrol

Nitric Oxide 26 (2012) 102–110

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Nitric Oxide journal homepage: www.elsevier.com/locate/yniox

Review

Cardiovascular effects and molecular targets of resveratrol Huige Li ⇑, Ning Xia, Ulrich Förstermann Department of Pharmacology, University Medical Center, Johannes Gutenberg University, Mainz, Germany

a r t i c l e

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a b s t r a c t Resveratrol (3,5,40 -trihydroxy-trans-stilbene) is a polyphenol phytoalexin present in a variety of plant species and has been implicated to explain the health benefits of red wine. A wide range of health beneficial effects have been demonstrated for resveratrol in animal studies. In this review, we summarize the cardiovascular effects of resveratrol with emphasis on the molecular targets of the compound. In this regard, resveratrol stimulates endothelial production of nitric oxide, reduces oxidative stress, inhibits vascular inflammation and prevents platelet aggregation. In animal models of cardiovascular disease, resveratrol protects the heart from ischemia–reperfusion injury, reduces blood pressure and cardiac hypertrophy in hypertensive animals, and slows the progression of atherosclerosis. A number of direct and indirect target molecules mediating the aforementioned cardiovascular effects of resveratrol have been identified. These include, among others, the estrogen receptor a, the adenosine receptors, the cyclooxygenase 1, the histone/ protein deacetylase sirtuin 1, the AMP-activated protein kinase, the Akt kinase, the nuclear factor-E2related factor-2, and NF-jB. Molecular mechanisms involved in the signal cascades are discussed. Ó 2012 Elsevier Inc. All rights reserved.

Article history: Received 16 October 2011 Revised 11 December 2011 Available online 4 January 2012 Keywords: Resveratrol Cardiovascular disease Nitric oxide Oxidative stress Vascular inflammation

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . Endothelial dysfunction . . . . . . . . . . . . . Oxidative stress . . . . . . . . . . . . . . . . . . . . Vascular inflammation . . . . . . . . . . . . . . Platelet aggregation . . . . . . . . . . . . . . . . Ischemic heart disease . . . . . . . . . . . . . . Hypertension and cardiac hypertrophy . Smooth muscle cell hypertrophy . . . . . . Atherosclerosis . . . . . . . . . . . . . . . . . . . . Diabetes and metabolic syndrome . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest. . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: 4-HNE, 4-hydroxy-2-nonenal; ADMA, asymmetric dimethylarginine; AMPK, AMP-activated protein kinase; ApoE, apolipoprotein E; ARE, antioxidant response element; BH4, tetrahydrobiopterin; Cav-1, caveolin-1; COX-1, cyclooxygenase 1; CREB, cAMP response element-binding protein; eNOS, endothelial NO synthase; eNOS-P, eNOS phosphorylation at serine 1177; eNOS-Ac, eNOS acetylation; ER, estrogen receptor; GCH1, GTP cyclohydrolase I; GCLC, glutamate cysteine ligase catalytic subunit; Grx, glutaredoxin; GPx1, glutathione peroxidase 1; HO-1, heme oxygenase-1; ICAM-1, intercellular adhesion molecule 1; iNOS, inducible NO synthase; I/R, ischemia–reperfusion; Keap-1, Kelch-like erythroid cell-derived protein 1; LAD, left anterior descending coronary artery; MI, myocardial infarction; Nampt, Nicotinamide phosphoribosyltransferase; NO, nitric oxide; NOX, NADPH oxidase; NQO, NAD(P)H:quinone oxidoreductase; Nrf-1, nuclear respiratory factor-1; Nrf2, nuclear factor-E2related factor-2; oxLDL, oxidized low-density lipoprotein; PBEF, pre-B cell colony-enhancing factor; PGC-1a, proliferator-activated receptor-coactivator-1a; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PON, paraoxonase; ref-1, redoxfactor-1; ROS, reactive oxygen species; SIRT1, histone/protein deacetylase sirtuin 1; SERCA2a, sarcoplasmic calcium ATPase; SMC, smooth muscle cells; SOD1, copper/zinc superoxide dismutase; SOD2, mitochondrial manganese SOD; SOD3, extracellular SOD; STZ, streptozotocin; T2DM, type 2 diabetes mellitus; TBARS, thiobarbituric acid-reactive substances; Tfam, mitochondrial transcription factor A; TNF, tumor necrosis factor; Trx-1, thioredoxin-1; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor. ⇑ Corresponding author. Address: Department of Pharmacology, University Medical Center, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55131 Mainz, Germany. Fax: +49 6131 17 9329. E-mail address: [email protected] (H. Li). 1089-8603/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2011.12.006

H. Li et al. / Nitric Oxide 26 (2012) 102–110

Introduction Resveratrol (3,5,40 -trihydroxy-trans-stilbene) is a polyphenol phytoalexin present in a variety of plant species, including white hellebore (Veratrum grandiflorum O. Loes), Polygonum cuspidatum, grapes, peanuts and mulberries [1,2]. It is the major red wine polyphenol and has been postulated to explain the lower incidence of myocardial infarction in France than in other comparable countries, the so-called ‘‘French paradox’’ [3–5]. Indeed, resveratrol has been shown to prevent or slow the progression of a wide variety of diseases, including cancer, cardiovascular disease, ischemic injuries and Alzheimer’s disease [1]. The compound has also been shown to mimic part of the caloric restriction effects extending the lifespan of various organisms [6]. Resveratrol exhibits versatile biological effects in animal studies. This is likely to be attributable to the fact that resveratrol is a molecule with many targets [7,8]. In this review, we summarize the cardiovascular effects of resveratrol and highlight the direct and indirect target molecules mediating these effects. Endothelial dysfunction Endothelial dysfunction (characterized as an impairment of endothelium-dependent relaxation) is an early event in atherogenesis and occurs even before structural changes in the vasculature. All major risk factors for atherosclerosis such as hyperlipidemia, diabetes, hypertension and smoking are associated with endothelial dysfunction [9]. Oral treatment with resveratrol results in the enhancement of agonist-stimulated, endothelium-dependent relaxations. This has been shown in animal models of disease, such as hypertensive rats [10], diabetic rats [11] and mice [12], and hypercholesterolemic rabbits [13]. In a double-blind, randomized cross-over study involving 19 overweight/obese men or post-menopausal women with untreated borderline hypertension, acute resveratrol consumption (30, 90 or 270 mg) increases plasma resveratrol concentrations (181, 532 and 1232 ng/ml, respectively) and flowmediated dilatation (FMD, from 4.1% to 6.6%, 6.6% and 7.7%, respectively) of the brachial artery [14]. The improvement of endothelial function by resveratrol is largely attributable to nitric oxide (NO) derived from endothelial NO synthase (eNOS). Resveratrol enhances endothelial NO bioactivity through multiple mechanisms [15–17]: (i) resveratrol enhances eNOS expression in endothelial cells by stimulating eNOS gene transcription and increasing eNOS mRNA stability [18]. Recent studies indicate that the upregulation of eNOS expression is, at least in part, mediated by the histone/protein deacetylase sirtuin 1 (SIRT1) (Table 1). An endothelium-specific overexpression of SIRT1 leads to an enhanced eNOS expression in mice [19]. In human coronary arterial endothelial cells, resveratrol-induced eNOS expression can be prevented by siRNA-mediated knockdown of SIRT1 [20]. (ii) Resveratrol enhances eNOS activity by stimulating eNOS phosphorylation at serine 1177 residue. This effect has been shown to be mediated by the estrogen receptor ERa and a signaling pathway involving the a-subunit of G-protein (Ga), caveolin-1 (Cav-1), the tyrosine kinase Src and the MAPK ERK1/2 [21,22]. In ER-a/ mice, resveratrol fails to increase NO production or to inhibit neointima formation [23]. (iii) Resveratrol enhances eNOS activity by inducing SIRT1-mediated deacetylation of lysines 496 and 506 in the calmodulin-binding domain of eNOS [24]. Cigarette smoke extract- or H2O2-induced reduction in SIRT1 expression/ activity and increase in eNOS acetylation can be reversed by resveratrol pretreatment [25]. (iv) Resveratrol enhances eNOS activity by decreasing the intracellular levels of the endogenous eNOS inhibitor asymmetric dimethylarginine (ADMA). This has been shown for resveratrol [26] and its derivative BTM-0512 [27,28].

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(v) Resveratrol decreases Cav-1 levels and Cav-1/eNOS interaction [22,29]. (vi) Resveratrol prevents eNOS uncoupling by increasing the intracellular levels of eNOS cofactor tetrahydrobiopterin (BH4) [30]. Resveratrol enhances BH4 biosynthesis by upregulating GTP cyclohydrolase 1 (GCH1) and prevents BH4 oxidation by reducing oxidative stress. (vii) Resveratrol improves NO bioactivity by inhibiting superoxide-mediated NO inactivation (Fig. 1). Resveratrol has been identified as a SIRT1 activator in an in vitro assay with a fluorogenic acetylated peptide derived from a native SIRT1 substrate, p53 [31]. However, other in vitro studies demonstrate that once the fluorophore is removed from the peptide substrate, the induction of SIRT1 deacetylase activity by resveratrol is no longer detectable [32–34]. Resveratrol does not enhance SIRT1mediated deacetylation of native full-length protein substrates, including p53 and acetyl-CoA synthetase1 in in vitro assays [35], or PGC-1a in either in vitro or cell-based assays [34]. These observations lead to the conclusion that resveratrol is not a direct SIRT1 activator. On the other hand, many knockout and knockdown approaches clearly demonstrate that some (not all) effects of resveratrol in cell culture and animal experiments are SIRT1-dependent [36]. A recent study provides evidence that activation of SIRT1 by small molecule compounds is strongly dependent on structural features of the peptide substrate. SIRT1-catalyzed deacetylation is accelerated by such compounds only when the peptide bears specific ring systems which the authors have termed activation cofactors [37]. The fluorogenic moiety on SIRT1 substrate in in vitro assays may resemble the in vivo activation cofactors to facilitate resveratrol-mediated enhancement of SIRT1 deacetylase activity [37]. They speculate that in vivo, the activation cofactor might not reside on protein substrates but rather may be presented to SIRT1 during interaction with accessory proteins with which SIRT1 interacts [37]. Thus, an enzymatic activation of SIRT1 by resveratrol in vivo cannot be ruled out. In addition, the resveratrol effects may be also mediated by an upregulation of SIRT1 expression. Resveratrol enhances the expression of SIRT1 in endothelial cells [20], rat heart subjected to ischemia/reperfusion (I/R) injury [38] and in the heart of streptozotocin (STZ)-induced diabetic mice [39]. Another explanation is that the activation of SIRT1 by resveratrol in vivo is an indirect effect and is mediated by AMPK as an upstream target [40,41]. AMPK activation improves NAD+ availability for SIRT1 by switching from carbohydrate to lipid as the main energy source [42] and by enhancing the expression of the NAD+-producing enzyme, nicotinamide phosphoribosyltransferase (Nampt), also called pre-B cell colony-enhancing factor (PBEF) or visfatin [38,42]. The enhanced production of endothelial NO by resveratrol protects the vasculature through its antihypertensive (vasodilation), anti-thrombotic (inhibition of platelet aggregation) and antiatherosclerotic (prevention of leukocyte adhesion to vascular endothelium, reduction of LDL oxidation, and inhibition of vascular smooth muscle cell proliferation) properties [43,44]. Oxidative stress As a polyphenolic compound, resveratrol has been shown to be a scavenger of hydroxyl, superoxide, metal-induced radicals [45,46], and H2O2 [47]. However, the direct antioxidant effects of resveratrol are rather poor; resveratrol is less potent than other well established antioxidants such as ascorbate and cysteine [48]. Thus, the protective effects of resveratrol against oxidative injury are likely to be attributed to the upregulation of the endogenous cellular antioxidant systems rather than its direct ROS scavenging activity. Indeed, resveratrol induces antioxidant enzymes in cardiovascular tissues [49,50]. Superoxide dismutases (SOD) catalyze the dismutation of superoxide into hydrogen peroxide, which is further inactivated by glutathione peroxidases (GPx) and catalase

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Table 1 Effects of resveratrol in cultured cardiovascular cells. (See below-mentioned references for further information.)

[51]. There are three mammalian SOD isoforms: copper/zinc SOD (SOD1), mitochondrial manganese SOD (SOD2), and extracellular SOD (SOD3). Induction of SOD enzymes by resveratrol has been shown in endothelial cells [30,52] for SOD1; in endothelial cells

[30,53] and cardiac myoblasts [54] for SOD2, and in endothelial cells for SOD3 [30]. Resveratrol also upregulates GPx1 and catalase in aortic segments [47] and cultured aortic smooth muscle cells [50]. We have recently reported that all the aforementioned anti-

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Fig. 1. Cardiovascular effects and molecular targets of resveratrol.

oxidant enzymes are induced by resveratrol in human EA.hy 926 endothelial cells and in the heart of apolipoprotein E (ApoE) knockout mice [30] (Fig. 1). In addition, resveratrol has been shown to enhance the expression levels of thioredoxin-1 (Trx-1) [55], Trx-2, glutaredoxin (Grx)1, Grx-2 [56], heme oxygenase-1 (HO-1) [57,58], NAD(P)H:quinone oxidoreductases (NQO1 and NQO2), and c-glutamylcysteine synthetase (glutamate cysteine ligase catalytic subunit, GCLC), the rate-limiting enzyme for glutathione (GSH) synthesis [58–60]. The molecular mechanisms underlying the induction of antioxidant enzymes by resveratrol are not completely understood. Recent studies have demonstrated that SIRT1 and the nuclear factor-E2-related factor-2 (Nrf2) play crucial roles in this process. In cultured human coronary arterial endothelial cells, resveratrolinduced SOD2 upregulation can be blocked by siRNA-mediated knockdown of SIRT1. An overexpression of SIRT1 leads to SOD2 upregulation [53]. Nrf2 is a transcription factor involved in the regulation of a number of ROS detoxifying enzymes. Under basal conditions, Nrf2 interacts with Kelch-like erythroid cell-derived protein 1 (Keap-1), a cytosolic repressor protein that limits Nrf2-mediated gene expression. Upon stimulation, Nrf2 is released from Keap-1 and translocates to the nucleus. It binds to antioxidant response element (ARE) and activates ARE-dependent transcription of phase II and antioxidant defense enzymes. In cultured endothelial cells,

resveratrol induces NQO1, HO-1 and GCLC in an Nrf2-dependent manner [58]. Relatively lower concentrations of resveratrol (0.1– 1 lM) are sufficient for this effect than that for activation of SIRT1 [58]. Resveratrol also reduces oxidative stress by inhibiting ROS production. NADPH oxidases (NOX) are the major ROS producing enzymes in the cardiovascular system [51]. The expression of NOX2 and NOX4 in the heart of hypercholesterolemic mouse [30], as well as NOX1, NOX2 and NOX4 in the aorta of trauma hemorrhagic rats [61] is reduced by resveratrol treatment. In addition, resveratrol also reduces the activity of the NADPH oxidase enzyme complex by targeting the regulatory subunits. This has been shown for NOX activation induced by angiotensin II or oxLDL in cultured endothelial cells [62]. In platelets, protein kinase C (PKC)-mediated phosphorylation of p47phox is prevented by resveratrol [63]. Vascular inflammation In coronary arterial endothelial cells, both TNF-a- and cigarette smoke extract (CSE)-induced ROS production, NF-jB activation, and upregulation of inflammatory markers (ICAM-1, iNOS, IL-6, and TNF-a) can be abrogated by resveratrol treatment [64,65]. NF-jB is likely to be the major molecular target for the antiinflammatory effects of resveratrol in the vasculature. Several mechanisms may be involved in the inhibition of NF-jB by

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resveratrol: (i) resveratrol may inhibit ROS-mediated NF-jB activation through its antioxidant activities (reducing H2O2 levels) [66]; (ii) IjB kinases (IKK) are upstream kinases known to activate NF-jB via phosphorylation-dependent degradation of IjBa. Resveratrol has been shown to inhibit NF-jB activation by targeting IKK, resulting in inhibition of IKK-mediated I-jB phosphorylation/degradation in skin tumor models [67]. (iii) The efficient transcriptional activation of NF-jB also depends on the phosphorylation of its active subunit p65/RelA [67]. Resveratrol has been shown to block p65 phosphorylation, nuclear translocation and its interaction with CBP/p300, rendering NF-jB transcriptionally inactive [67,68]; (iv) the inhibitory effect of resveratrol on NF-jB may be also mediated by SIRT1. SIRT1 has been shown to physically interact with the p65 subunit of NF-jB and to inhibit its activity by deacetylating p65 at lysine 310 [69]. Indeed, the contribution of SIRT1 has been demonstrated in a recent study. The anti-inflammatory effects of resveratrol in CSE-treated endothelial cells are abolished by knockdown of SIRT1, whereas the overexpression of SIRT1 mimicks the effects of resveratrol [65]. Platelet aggregation Platelet aggregation plays a crucial role in the atherothrombotic process and antiplatelet agents reduce the incidence of myocardial infarction and stroke. Resveratrol has been shown to inhibit the aggregation of platelets isolated from healthy subjects [70,71]. Notably, efficient inhibitory effects are also evident in platelets from aspirin-resistant patients [72]. In vivo treatment of hypercholesterolemic rabbits with resveratrol (4 mg/kg/day) inhibits platelet aggregation without any effect on serum lipid levels [71]. Diverse mechanisms are involved in the antiplatelet action of resveratrol. The compound enhances endothelial NO production (see above). This NO may diffuse into platelets and inhibits platelet aggregation via activation of guanylyl cyclase and production of cyclic guanosine monophosphate (cGMP). Interestingly, resveratrol also stimulates cGMP synthesis in isolated human platelets [63], indicating an additional mechanism independent of endothelial NO. This may be a result of enhanced platelet NO production, and/or improved NO bioactivity due to reduction of oxidative stress. Resveratrol prevents PKC-mediated phosphorylation of p47phox and reduces hydroxyl radical levels in human platelets [63]. In a cell-free system, resveratrol has been identified as potent inhibitor of cyclooxygenase 1 (COX-1), but not COX-2 [73]. The selective inhibition of COX-1 by resveratrol is irreversible and non-competitive, and requires the presence of the m-hydroquinone moiety (3,5-di-OH group) of the compound [73]. In addition to this direct COX-1 inhibition, resveratrol may also attenuate thromboxane synthesis by inhibition of a pathway involving p38 MAP kinase [63]. Recent studies demonstrate that apoptosis can also occur in anuclear platelets. Whereas resveratrol inhibits collagen-induced platelet activation at lower concentrations (0.15–0.25 lM), at higher concentrations (>5 lM), however, resveratrol induces platelet apoptosis [74]. Also this mechanism may contribute to the antiplatelet activities of resveratrol. Ischemic heart disease Resveratrol protect against ischemic heart disease through multiple mechanisms. It prevents myocardial infarction by inhibiting platelet aggregation; protects the myocardial tissue from ischemia–reperfusion injury through a preconditioning-like mechanism; and it also potentiates the regeneration of infarcted myocardium.

Preconditioning is a protective and adaptive phenomenon whereby brief episodes of ischemia and reperfusion (I/R) render the heart resistant to subsequent ischemic injury [4]. Pretreatment of rats with resveratrol results in a cardioprotection when the isolated heart is subjected to 30 min global ischemia followed by 2 h reperfusion [38,75], or permanent left anterior descending coronary artery (LAD) occlusion [55]. Resveratrol-provided cardioprotection is evidenced by superior postischemic ventricular recovery, reduced myocardial infarct size, and decreased number of apoptotic cardiomyocytes [55,75] (Table 2). The mechanisms underlying the preconditioning effect of resveratrol are complex. NO and the antioxidant enzyme HO-1 has been identified as crucial factors mediating the protective effects of resveratrol. Resveratrol induces eNOS expression in the ischemic heart [76] and NO can induce the expression of HO-1 [4]. Another mechanism is the survival signal pathway, which is mediated by the adenosine receptors A1 and A3, with subsequent activation of PI3K/Akt and cAMP response element-binding protein (CREB), resulting in phosphorylation and upregulation of Bcl-2, respectively [77] (Fig. 1). This anti-apoptotic molecule protects the cardiac tissue from cell death. The anti-apoptotic action of resveratrol in cardiomyocytes may also involve the SIRT1–FOXO1 pathway [38,78]. In addition, resveratrol-mediated survival of cardiac myoblasts through induction of autophagy also contributes, along other enhanced survival signals, to the recovery of cells from injury [79]. Furthermore, resveratrol has also been shown to potentiate the regeneration of infracted myocardium. A major problem in the effectiveness of stem cell therapy is the death of stem cells due to the oxidative environment [4]. In a rat LAD occlusion model with direct injection of adult cardiac stem cells on the border zone of the myocardium, a resveratrol pretreatment significantly reduces oxidative stress, enhances stem cell survival and proliferation, and improves cardiac function [80]. In addition, resveratrol may also enhance regeneration of the infarcted myocardium by stimulating neovascularization [55]. Interestingly, I/R of rat myocardium leads to differential expression of over 50 microRNAs. Pretreatment with resveratrol largely restores the altered microRNA expression in the ischemic heart [81]. Resveratrol-regulated miRNAs in I/R includes, for example, miR-21, miR-20b, miR-27a, and miR-9. These miRNAs have been previously shown to regulate the ERK-MAP kinase signaling pathway in cardiac fibroblasts (miR-21), to modulate VEGF and angiogenesis through HIF-1a (miR-20b), or to regulate FOXO1 (miR-27a) and SIRT1 (miR-9) [82].

Hypertension and cardiac hypertrophy Antihypertensive effects of resveratrol have been demonstrated in several animal models. In rat with partial nephrectomy-induced cardiac hypertrophy, oral treatment with resveratrol (50 mg/kg/ day) results in a significant reduction in systolic blood pressure (20 mm Hg) and a reduction in cardiac hypertrophy [83]. These protective effects are associated with a decrease in serum endothelin-1 (and a moderate, not significant reduction in angiotensin II), and an increase in serum NO levels [83]. In ovariectomized, stroke-prone spontaneously hypertensive rats, a diet supplemented with resveratrol (5 mg/kg/day) improves endotheliumdependent vascular relaxation and lowers the systolic blood pressure by 15% after 3 weeks of administration [10]. In fructose-fed rats, an experimental model of insulin resistance syndrome, chronic treatment with resveratrol (10 mg/kg/day) prevents the increase in systolic blood pressure and cardiac hypertrophy, restores mesenteric and cardiac eNOS activities, and decreases the elevated TBARS levels [84] (Table 2).

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Table 2 Effects of resveratrol in animal models of cardiovascular disease. (See below-mentioned references for further information.)

When a lower dose (2.5 mg/kg/day) is used, resveratrol has only minor effects on blood pressure but still significantly prevents the development of concentric cardiac hypertrophy [85,86]. This indicates that the beneficial effects of resveratrol on cardiac hypertrophy are not only mediated by a reduction of blood pressure, but also involve factors other than changes in hemodynamic load. These may include the activation of the anti-hypertrophic AMPKsignaling pathway and inhibition of the prohypertrophic Akt signaling pathway [87]. AMPK (and its upstream kinase LKB1) not only antagonizes the hypertrophic response, it also delays the transition from cardiac hypertrophy to heart failure. Importantly, AMPK additionally inhibits cardiac remodeling by preventing angiotensin II-induced myocardial fibrosis [88]. An important regulator of the LKB1/AMPK pathway is oxidative stress. In patients with hypertension or heart failure as well as in hypertensive rats, oxidative stress and lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE) are elevated [85]. 4-HNE forms covalent adducts with LKB1. The resulting inhibition of the LKB1/AMPK signaling pathway leads subsequently to induction of mTOR/p70S6 kinase-mediated protein synthesis and cardiac myocyte cell growth. Treatment of cardiomyocytes with resveratrol prevents 4-HNE modification of the LKB1/AMPK signaling axis, blunts the prohypertrophic p70S6 kinase response, and reduces left ventricular hypertrophy [85].

Smooth muscle cell hypertrophy Hypertrophy and hyperplasia of vascular smooth muscle cells are hallmarks of atherosclerosis, restenosis, and hypertension. Akt signaling is essentially involved in angiotensin II- and epidermal growth factor (EGF)-induced hypertrophy [89,90] and migration [91] of vascular smooth muscle cells (SMC). Resveratrol effectively blocks both angiotensin II- and EGF-induced SMC hypertrophy through inhibition of Akt [89,90]. Although NOX1 (but not NOX4) is involved in angiotensin II-induced Akt phosphorylation, neither NOX1 nor NOX4 is required for EGF-induced Akt activation [92]. Interestingly, the mono-methylation derivative of resveratrol at the 40 -OH group, which is largely redox inactive, inhibits Akt phosphorylation to a similar extent as native resveratrol, whereas the trimethylation resveratrol analog has no effect on Akt activity [92]. These data indicate that the direct antioxidant capacity of resveratrol is not required for the inhibition of Akt phosphorylation, and the 3- and 5-OH groups of resveratrol seem to be critically involved in mediating Akt inhibition [92]. The inhibition of Akt by resveratrol may involve the adapter molecule Gab1, and the protein tyrosine phosphatase Shp2. Resveratrol activates Shp2. In fibroblasts with Shp2 deficiency, resveratrol does not inhibit EGF-stimulated Akt activation, indicating that Shp2 is necessary for the inhibitory effect of resveratrol on the PI3K/Akt pathway [89] (Table 1).

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Atherosclerosis In the hypercholesterolemic rabbit model, resveratrol treatment reduces the size, density, and mean area of atherosclerotic plaques [93]. Interestingly, red wine and dealcoholized red wine have similar effects as resveratrol, indicating that wine polyphenolics, rather than alcohol present in red wine, are mainly responsible for the cardiovascular protective properties of red wine [93]. In ApoE/ mice, resveratrol [94] and a resveratrol-containing mixture [95] have been shown to reduce atherosclerotic lesions. Whereas no effects on plasma lipid levels are observed in the hypercholesterolemic rabbits (resveratrol daily dose 3 mg/kg bodyweight, via drinking water for 12 weeks), resveratrol reduces total and LDL cholesterol and increases HDL cholesterol in the ApoE/ mice (0.02% or 0.06% (w/w) resveratrol supplemented in chow for 20 weeks) fed a normal diet [94]. In animal models of intimal hyperplasia, resveratrol treatment inhibits neointima formation [96,97] (Table 2). The following mechanisms may contribute to the anti-atherosclerotic properties of resveratrol: (i) stimulation of NO production [23]; (ii) inhibition of LDL oxidation [98]; (iii) reduction of vascular inflammation and prevention of leukocyte adhesion; (iv) inhibition of smooth muscle cell proliferation [60,96,99]. A contribution of SIRT1 in the inhibitory effects of resveratrol on atherogenesis is likely. Endothelium-specific overexpression of SIRT1 decreases atherosclerosis in ApoE/ mice [19]. Diabetes and metabolic syndrome Resveratrol has been shown to decrease blood glucose and to protect pancreatic beta-cells from oxidative damage in animal studies [100]. Moreover, resveratrol reduces diabetic vascular complications (e.g. restores vascular responsiveness of cerebral arterioles [101]) and decreases diabetic cardiomyopathy [39]. The anti-hyperglycemic effects of resveratrol have been demonstrated in STZ-induced (type 1) as well as in STZ-nicotinamide-induced (type 2) diabetic rats [102]. The reduction in blood glucose is likely to result mainly from the stimulatory action of resveratrol on glucose uptake by peripheral tissues. Interestingly, in experiments on isolated cells, resveratrol is able to stimulate glucose uptake in the absence of insulin [102]. Moreover, resveratrol has been shown to potentiate the glucose-stimulated insulin secretion in beta-cells through SIRT1-dependent mechanisms [103]. Insulin resistance is the most critical factor for the development of type 2 diabetes. Resveratrol enhances insulin sensitivity in vitro in SIRT1-dependently and attenuates high fat diet-induced insulin resistance in vivo at a dose of 2.5 mg/kg/day [104]. A recent study demonstrates that AMPK is essential for resveratrol to increase insulin sensitivity [105]. Resveratrol is likely to stimulate SIRT1 activity indirectly through an increase in NAD-to-NADH ratio in an AMPK-dependent manner [105]. In a randomized double-blind crossover study, treatment of 11 healthy, obese men with 150 mg/day resveratrol for 30 days induces modest metabolic changes mimicking the effects of calorie restriction [106]. Resveratrol reduces sleeping and resting metabolic rate, and lowers systolic blood pressure by 5 mmHg. In muscle, resveratrol activates AMPK, increases SIRT1 and PGC-1a protein levels. Furthermore, resveratrol decreases intrahepatic lipid content, circulating glucose, triglycerides, and inflammation markers. The improved HOMA index suggests favorable effects of resveratrol on insulin sensitivity [106]. Conclusion An expanding body of preclinical evidence suggests that resveratrol has the potential to provide health benefits. Even though the

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