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Resveratrol, a polyphenol phytoalexin, possesses diverse biochemical and physiological
actions, including estrogenic, antiplatelet, and anti-inflammatory properties. Several recent studies determined the cardioprotective abilities of resveratrol. Both in experiments (acute) and in chronic models, resveratrol attenuates myocardial ischemic reperfusion injury, atherosclerosis, and reduces ventricular arrhythmias. It appears that resveratrol-mediated cardioprotection is achieved through the preconditioning effect (the best yet devised method of cardioprotection), rather than direct protection. Thus, resveratrol likely fulfills the definition of a pharmacological preconditioning compound and gives hope to the therapeutic promise of alternative medicine.
Dipak K. Das and Nilanjana Maulik
Cardiovascular Research Center, University of Connecticut School of Medicine,
Farmington, CT 06030-1110
Resveratrol: A Preconditioning Agent Evolution Of Discovery To (10). Resveratrol can scavenge hydroxyl radicals with a reaction rate Cardioprotection constant of 9.45 x 108 M-1sec-1, which is slower than the potent scavenging demonstrated by ascorbic acid (13). Depending on the Almost 4500 years ago, Ayurveda, the ancient medicinal book of biological system, resveratrol can also scavenge the superoxide anion Hindus described "darakchasava" (fermented juice of red grapes) as 2 ) (13–15).
cardiotonic (1). Consequently, The Bible described grape juice or red Although resveratrol behaves as a poor in vitro scavenger of wine as a "gift of god," which was presumably used to purify body ROS, it functions as a potent antioxidant in vivo. The in vivo anti- and soul. In 1940, resveratrol was first identified as the medicinal oxidant property of resveratrol probably arises from its ability to component of grapes, and it was extracted from the dried roots of increase nitric oxide (NO) synthesis, which in turn functions as an Polygonum cuspidatum (popularly known as Ko-jo-kon in Japan) and in vivo antioxidant, scavenging superoxide radicals. In the ischemic used to treat hyperlipidemic diseases (2). In the modern era, resvera- reperfused heart, brain, or kidney, resveratrol induces NO synthesis trol has been rediscovered as an antiproliferative agent for cancer (3, and lowers oxidative stress (11, 16). Having an unpaired electron, 4). The cardioprotective ability of resveratrol stemmed from epidemi- NO behaves as a potent antioxidant in vivo, rapidly reacting near ological studies indicating that mild-to-moderate alcohol consump- the diffusion-limited rate (6.7 x 109 M-1s1) with O – 2 that is presum- tion is associated with reduced incidence of morbidity and mortality ably formed in the ischemic reperfused myocardium. The affinity of from coronary heart disease (5). The first experimental evidence 2 is far greater than the affinity of superoxide dismutase of cardioprotective ability of resveratrol was apparent from studies 2 . In fact, NO may compete with SOD for O2 , thereby showing that resveratrol could directly protect isolated hearts from 2 and sparing SOD for other scavenging duties.
ischemia reperfusion injury (6). Subsequently, resveratrol was found The effects of resveratrol have been tested in specific cells to protect most of the vital organs including kidney, heart, and brain lines and differentiated cell types. For example, resveratrol inhibits from ischemic reperfusion injury (7–9). The protective mechanism 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced free radical of resveratrol includes its role as intracellular antioxidant, anti- formation in cultured HL-60 cells (17). In DU 145 cells––a prostate inflammatory agent, its ability to induce nitric oxide synthase (NOS) cancer cell line––administration of resveratrol inhibited proliferation expression, and its ability to induce angiogenesis (10). A substantial that had been accompanied by a reduction in NO production and body of evidence strongly supports the notion that resveratrol medi- inhibition of inducible nitric oxide synthase (iNOS) (18). Resveratrol ated cardioprotection is achieved by preconditioning; the best yet also inhibited the formation of O – 2 and H2O2 produced by mac- devised method of myocardial protection (11, 12). rophages stimulated by lipopolysaccharide (LPS) or TPA (15). In a related study, resveratrol inhibited reactive oxygen intermediates and lipid peroxidation induced by tumor necrosis factor (TNF) in a wide Sources Of Resveratrol variety of cells (19). Resveratrol also scavenges peroxyl and hydroxyl radicals in the postischemic reperfused myocardium, thereby lower- The richest source of resveratrol is the roots of Polygonum cuspida- ing malonaldehyde formation, a presumptive marker of lipid peroxi- tum (Ko-jo-kon), mainly cultivated in China and Japan. The skins dation (6, 20).
of grapes contain about 50–100 mg/resveratrol, and believed to be Resveratrol can maintain the concentrations of intracellular responsible for the cardioprotective properties of red wine, which antioxidants found in biological systems. For example, resveratrol contains about 0.2–7 mg/l of wine. In addition to grapes, a large maintained glutathione (GSH) amounts in oxidation-stressed periph- variety of fruits including mulberry, bilberry, lingonberry, sparkle- eral blood mononuclear cells isolated from healthy humans (21). In berry, deerberry, partridgeberry, cranberry, blueberry, and jackfruit, another study, resveratrol increased GSH amounts in human lym- peanut, and a wide variety of flowers and leaves including gnetum, phocytes that were activated by hydrogen peroxide (22). Similarly, white hellebore, corn lily, butterfly orchid tree, eucalyptus, spruce, resveratrol restored glutathione reductase in cells subjected to TPA- poaceae, scots pine, and rheum also contain resveratrol. Resveratrol mediated oxidative stress (23). In human lymphocytes, resveratrol is synthesized in response to environmental stressors that include increased the amounts of several antioxidant enzymes, including water deprivation, UV irradiation, and, especially, fungal infection. glutathione peroxidase, glutathione-S-transferase and glutathione Thus, the production of resveratrol in plants can be considered part reductase (24). of the defense mechanism.
A Natural Antioxidant Resveratrol has been recognized as a phytoestrogen based on its Resveratrol is capable of scavenging some intracellular reactive structural similarities to diethylstilbesterol (DES). Resveratrol can oxygen species (ROS). Although it possesses antioxidant proper- bind to the estrogen receptor (ER), thereby activating transcription ties, reseveratrol does not function as a strong antioxidant in vitro of estrogen-responsive reporter genes transfected into cells (25–31). Volume 6, Issue 1

Resveratrol functions as a superagonist when combined with estra- chronic models of cardiac injury, resveratrol reduced myocardial diol (E2) to induce the expression of estrogen-regulated genes (25); ischemic reperfusion injury (38, 39). In addition to hearts, resvera- however, several other studies showed conflicting results. Using trol also protects kidney and brain cells from ischemia-reperfusion the same cell line, Ashby et al. showed that resveratrol possessed injury. Similar to its effect on the heart, resveratrol's stimulation of antiestrogen activity because it suppressed progesterone receptor NO production during ischemia-reperfusion is believed crucial for expression induced by E2 (32). Another recent study showed that protection of the kidney from ischemic reperfusion injury (40). The both isomers of resveratrol possessed superestrogenic activity only at maintenance of constitutive NO release is a critical factor in the moderate concentration (>10 µM), whereas at lower concentrations recovery of function after an ischemic injury. Release of constitu- (<1 µM), antiestrogenic effects prevailed (33).
tive NO is significantly reduced after ischemia-reperfusion, thus the Most of the in vivo studies have failed to confirm the estrogenic maintenance of NO release by any means (e.g., induction of NO potential of resveratrol. At physiologic concentrations, resveratrol production with L-arginine) can restore myocardial function follow- does not appear to induce any changes in uterine weight uterine ing ischemia (41) (Figure 1).
epithelial cell height, or serum cholesterol (34). Only at very high Although resveratrol protects brain, kidney, and heart cells, it concentrations does resveratrol interfere with the serum cholesterol– preferentially kills cancer cells. For example, intraperitoneal adminis- lowering activity of E2. Elsewhere, resveratrol given orally or subcu- tration of resveratrol increased apoptosis and reduced tumor growth taneously did not affect uterus weight at any concentration (0.03 to (42). In oral squamous carcinoma cells, resveratrol inhibited growth; 120 mg/kg/day) (32). In a related study, resveratrol reduced uterine both alone and in combination with quercetin, an antioxidant found weight and decreased the expression of ER-α mRNA and protein in the skin of apples and red onions (43), and inhibited the growth and progesterone receptor (PR) mRNA (35). of highly metastatic B16-BL6 melanoma cells (44). In a rat colon In contrast, resveratrol possesses estrogenic properties in carcinogenesis model, resveratrol induced the expression of the stroke-prone spontaneously hypertensive rats (36). When ovariecto- proapoptotic protein Bax in aberrant cryptic foci of the colon (45). mized rats were fed resveratrol at the concentration of 5 mg/kg/day, In fact, resveratrol affects three major stages of carcinogenesis (i.e., treatment attenuated an increase in systolic blood pressure. In con- initiation, promotion, and progression) and inhibits the formation of cert, resveratrol enhanced endothelin-dependent vascular relaxation preneoplastic lesions in a mouse mammary organ culture model (4).
in response to acetylcholine and, in a manner similar to estradiol, Resveratrol inhibits the growth of cytotoxin-associated gene– prevented ovariectomy-induced decreases in femoral bone strength. carrying (CagA+) strains of Helicobacter pylori in vitro (46) as well as Resveratrol also acts as an estrogen receptor (ER) agonist in breast fifteen other strains of H. pylori (47), suggesting that its presumed cancer cells stably transfected with ERα (37). Although more data bacteriostatic activity might be part of its overall salutary effects. accumulate on the estrogenic behavior of resveratrol, the controversy Other beneficial effects of resveratrol include its ability to increase continues to persist.
(13-fold) the activity of the anti-aging gene SIRT1 [the mammalian homolog of the S. cerevisiae gene silent mating type information regulation 2 (Sir2)] (48). Thus, resveratrol-mediated activation of Health Benefits Of Resveratrol life-extending genes in human cells may open a new horizon of res-veratrol research (Figure 2).
As mentioned earlier, several recent studies determined the cardio-protective abilities of resveratrol. Both in acute experiments and Cardioprotection With Resveratrol Adenosine A /A receptors The role of NO in the cardioprotective abilities of grape products first became apparent when certain wines, grape juices, grape skins, or their components were found to relax precontracted smooth muscle of intact rat aortic rings but had no effect on aortas in which the endothelium had been removed (49). The extracts of wine or grape increased guanosine 3',5'–monophosphate (cGMP) amounts in intact vascular tissue, and both relaxation and the increase in cGMP were reversed by NG-monomethyl-L-arginine or by NG-nitro- L-arginine––competitive inhibitors of the synthesis of the endothe- lium-derived relaxing factor, NO––suggesting that vasorelaxation Figure 1. Molecular Targets of Resveratrol. A number of signaling pathways
induced by grape products is mediated by the NO-cGMP pathway. related to inflammation and gene expression are modulated by resveratrol.
A direct role for NO in vasorelaxation was identified when increased

Resveratrol: A Preconditioning Agent ished such beneficial effects of Antitumor activity, Chemoprevention resveratrol. The results support κB activation, Proliferation, Causes S-phase arrest, Induces apoptosis an anti-inflammatory action of myeloid leukomia cells of resveratrol through a NO- Prevents prostate and Reduces platelet adhesion, dependent mechanism. Pancreatic, gastric Monocyte adhesion The anti-inflammatory role and thyroid cancer of resveratrol is also evident from a research where resvera-trol effectively suppressed the aberrant expression of tissue Prevents LDL oxidation factor (TF) and cytokines in Protects lung from DNA vascular cells (54). Resveratrol, Damage and apoptosis in a dose-dependent manner, inhibited the expression of Protects cerebral TF in endothelial cells stimu- Reversible inhibition of herpes lated with a variety of agonists, Inhibited growth of simplex virus types 1 & 2 replication including interleukin-1β (IL- 1β), tumor necrosis factor–α (TNF-α), and lipopolysac- Figure 2. Health benefits of Resveratrol. Reveratrol has been implicated as a therapeutic in a number of major diseases
charide (LPS). Nuclear run-on analyses in endothelial cells NOS activity was found in cultured pulmonary artery endothe- showed that resveratrol inhibited the transcription of the TF gene. lial cells treated with resveratrol (28), suggesting that resveratrol In another recent study, resveratrol attenuated the agonist-induced could afford cardioprotection by affecting the expression of NOS. increase of TF mRNA in endothelial and mononuclear cells, result- Astringinin, an analog of resveratrol, also exhibits NO-mediated ing from the inhibition of IκBα degradation, thus decreasing the beneficial effects during ischemia and reperfusion damage in rat DNA-binding occupancy by the transcription factor c-Rel/p65 heart (50). Pretreating rats with astringinin significantly dampened (55). Elsewhere, resveratrol inhibited platelet aggregation induced the incidence ventricular tachycardia (VT) and ventricular fibrillation by collagen, thrombin, or ADP (56). Additionally, trans-resveratrol (VF) and decreased the concentrations of lactate dehyodrogenase interfered with the release of inflammatory mediators by activated (LDH) found in the carotid artery. Consistent with these results, polymorphonuclear cells (PMN) and decreased the adhesion-depen- resveratrol protected perfused working rat hearts through increased dent thrombogenic functions of PMN (57). ADP-induced platelet iNOS expression (11). The cardioprotective ability of resveratrol was aggregation in hypercholesterolemic rabbits can be blocked by abolished with an iNOS inhibitor, aminoguanidine. Resveratrol also treatment with resveratrol, suggesting a role for resveratrol in inter- failed to provide cardioprotection in iNOS knockout mice (51). In rupting the generation of eicosanoids involved in pathological pro- a recent study, however, resveratrol reduced myocardial ischemia cesses. Indeed, Pinto et al. showed that resveratrol could inhibit the reperfusion injury through both iNOS-dependent and iNOS-inde- dioxygenase activity of lipooxygenase and that oxidized resveratrol pendent mechanisms by increasing the expression of iNOS, endo- was as efficient as the reduced form in inhibiting lipoxygenase (58). thelial NOS (eNOS), and neuronal NOS (nNOS) (52).
Similarly, trans-resveratrol inhibited, in a dose-dependent manner, the arachidonate-dependent synthesis of the inflammatory agents Thromboxane B2 (TxB2), hydroxyheptadecatrienoate (HHT), and 12-hydroxyeicosatetraenoate (12-HETE) (59). The cardioprotective ability of resveratrol also stems from its anti-inflammatory functions in the ischemic heart. Treatment with the polyphenol resveratrol significantly improved postischemic ventricu-lar function and reduced myocardial infarct size compared to non- Resveratrol protects the heart against ROS-mediated menadione (i.e., treated control group (53). The amount of proadhesive molecules, Vitamin K3) toxicity by inducing NAD(P)H:quinone oxidoreductase, including soluble intracellular adhesion molecule-1 (sICAM-1), also known as DT-diaphorase, a detoxifying enzyme for quinone- endothelial leukocyte adhesion molecule-1 (sE-Selectin), and vas- containing substances (60). The cardioprotective effect of resveratrol cular cell adhesion molecule-1 (sVCAM-1) were each significantly was also attributed to its ability to upregulate catalase activity in the decreased during reperfusion in the resveratrol-treated group. Nitro- myocardium. Resveratrol functions as in vivo antioxidant and can L-arginine methyl ester (L–NAME), an NO blocker, completely abol- scavenge peroxyl radicals in the heart (61, 62). A commercial prepa- Volume 6, Issue 1

ration of resveratrol made from P. Inhibits soluble adhesion cuspidatum root extract (Protykin®) molecule (ICAM, VCAM) formation also scavenges peroxyl radicals and protects the heart from ischemia Increases endogenous Anti-thrombin activity reperfusion injury (63). Prevents platelet aggregation Inhibits MAPK activation Resveratrol appears to induce an Inhibits LDL peroxidation Inhibits lipid peroxidation anti-apoptotic signal for the pro-tection of the heart. In porcine coronary arteries, short term treat- Inhibits Endothelin ment with resveratrol significantly Induces vasorelaxation inhibited mitogen-activated protein kinase (MAPK) activities, and Stimulates angiogenesis immunoblot analyses revealed Decreases ventricular Reduces ischemia/ Reperfusion injury consistent reduction in the phos-phorylation of extracellular signal-regulated kinases 1/2 Figure 3. Cardioprotection with resveratrol. The major contribution to health from resveratrol appears to be directed
(ERK1/2), Jun N-terminal toward limiting progression of heart disease and atherosclerosis. See text for details.
kinase (JNK-1), and p38 MAPK (64). The same study found that resveratrol attenuated basal and of adenosine-mediated regulation of coronary blood flow (i.e., the endothelin-1 (ET-1)-mediated protein tyrosine phosphorylation. adenosine hypothesis) remains controversial (74). This hypothesis Anti-apoptotic function of resveratrol is further supported by several is inconsistent with several findings that inhibition of KATP channel other studies, which have demonstrated a reduction of apoptotic blocks the effects of preconditioning and that a KATP channel opener cardiomyocytes in the ischemic reperfused heart that had been pre- can simulate ischemic preconditioning (73, 75). To reconcile the treated with resveratrol (51, 63) (Figure 3).
adenosine hypothesis, an argument has been made that adenosine could trigger a secondary mechanism such as activation of Gi pro-tein, which, in turn, could open the KATP channel. Pharmacological Preconditioning Another intriguing hypothesis has stemmed from the concept of stimulating an endogenous protective mechanism by myocar-dial adaptation to ischemic stress. Preconditioning induces the Preconditioning is the most powerful technique known to promote expression of endogenous antioxidant enzymes such as superoxide cardioprotection (64–69). The most generalized method of classical dismutase (SOD) and glutathione peroxidase (GSHPx-1) (76, 77) preconditioning is mediated by cyclic episodes of several short dura- and the heat shock proteins (HSP) HSP27, HSP32, and HSP70 (78, tions of reversible ischemia, each followed by another short duration 79). Additionally, preconditioning potentiates a signal transduction of reperfusion. In most laboratories including our own, precondi- cascade by inhibiting death signals and activating survival signals. tioning is achieved by four cycles of ischemia (five minutes each) fol- Thus, several proapoptotic and antiapoptotic genes and transcrip- lowed by ten minutes of reperfusion (66, 70). Such preconditioning tion factors, including JNK-1, c-Jun, NF-κB and AP-1, are likely to makes the heart resistant to subsequent lethal ischemic injury.
play a crucial role in preconditioning (80–82), as does NO (83, 84).
The mechanisms underlying cardiac preconditioning have been Recent studies from our laboratory determined that resveratrol studied extensively, and several regulatory pathways have been iden- may function as a pharmacological preconditioning agent as it fulfills tified in different systems. Three important factors: the adenosine several criteria for preconditioning, including activation of adenosine A1 receptor, multiple kinases [including protein kinase C (PKC), A1 and A3 receptors, PKC, MAPKs, and the KATP channel. Adenosine MAPKs, and tyrosine kinases], and the mitochondrial ATP-sensitive accumulates in tissues under metabolic stress. In myocardial cells, potassium (KATP) channel are known to play a crucial role in precon- the nucleoside interacts with various receptor subtypes (i.e., A1, ditioning-mediated cardioprotection. For example, cardioprotection A3, and perhaps A2A and A2B) that are coupled, via G proteins, to achieved by preconditioning can be abolished by A1 receptor antag- multiple effectors, including enzymes, channels, transporters and onists (71, 72), whereas A1 receptor agonists can limit myocardial cytoskeletal components. Studies using adenosine receptor agonists infarct size (73). Although there is general agreement regarding the and antagonists, as well as animals overexpressing the A1 recep- beneficial role of adenosine on ischemic tissue, Berne's hypothesis tor indicate that adenosine exerts anti-ischemic action. Adenosine Resveratrol: A Preconditioning Agent all of which can be elicited by preconditioning challenges.
Box 1. The Two Phases of Cardioprotection
The intracellular signaling mechanisms that mediate precondi- Acute or classical preconditioning can produce tioning require the activation of one or more MAPKs. Among these, marked reduction of myocardial infarct size and the stress-activated protein kinases (SAPKs, also termed JNKs, see cardiomyocytes apoptosis in all species tested so above) and p38 are known to be regulated by extracellular stresses, far; however, its clinical potential is limited because including environmental stress, oxidative stress, heat shock, and UV of its short duration of approxiamtely 2–3 hours. radiation (100). JNKs and p38 appear to be involved in distinct cel- A delayed protective effect reappears after 12–24 lular functions, because they possess different cellular targets and are hours, following early preconditioning and this car- located on different signaling pathways. Thus, JNKs activate c-Jun dioprotective effects last up to 72 hours.
whereas p38 stimulates MAPK-activated protein kinase 2 (MAPKAP kinase 2) (101). A recent study demonstrated that preconditioning triggered a tyrosine kinase-regulated signaling pathway that leads to released during preconditioning, by short periods of ischemia fol- the translocation and activation of p38 and MAPKAP kinase 2 (87). lowed by reperfusion, induces cardioprotection to subsequently Activation of KATP channels appears to be an adaptive mechanism sustained ischemia. This protective action is mediated by A1 and A3 that protects the myocardium against ischemic reperfusion injury (102). receptor subtypes and involves the activation and translocation of The activation of this ion channel is at least partially responsible for the PKC to sarcolemmal and to mitochondrial membranes, leading to increase in outward K+ currents, shortening of action potential duration PKC-dependent KATP channels. Other effectors possibly contribut- (APD), and increasing extracellular K+ concentrations during anoxic ing to cardioprotection by adenosine or preconditioning that seem and globally ischemic conditions (103). It appears that delayed precon- particularly involved in the delayed (i.e., second) window of cardio- ditioning, irrespective of preconditioning stimulus, is always mediated protection (Box 1) include MAPKs, heat shock proteins, and iNOS by KATP channels (104). The mitochondrial KATP channel opener diazox- and eNOS. Because of its anti-ischemic effects, adenosine has been ide significantly reduced the rate of cell death following simulated tested as a protective agent in clinical interventions such as percu- ischemia in adult ventricular cardiac myocytes (73). Intravenous injec- taneous transluminal coronary angioplasty (PTCA), coronary artery tion of diazoxide ten minutes before induced ischemia greatly reduced bypass grafting (CABG), and tissue preservation, and was found in infarct size in the rabbit heart (105); however, the protective effects of most cases to enhance the post-ischemic recovery of function. The diazoxide could be blocked by 5-hydroxydecanoate (5-HD), a selective mechanisms underlying the role of adenosine and of mitochondrial blocker of the mitochondrial KATP channel. function in preconditioning are not completely clear, and uncertain-ties remain concerning the role played by newly identified potential effectors such as free radicals, the sarcoplasmic reticulum, etc. In Making A Case For Reseveratrol As A addition, more studies are needed to clarify the signaling mecha- Pharmacological Preconditioning Agent nisms by which A3 receptor activation or overexpression may pro-mote apoptosis and cellular injury. It should be clear from the above discussion that unlike pharma- Several investigators have proposed a unifying hypothesis that cological therapeutic interventions, preconditioning protects the activation of PKC represents a link between cell surface receptor heart by increasing its endogenous defense mechanisms (106). activation and a putative end-effector sarcolemmal or mitochondrial Unfortunately, ischemic preconditioning–mediated cardioprotection KATP channels (85, 86). Possible involvement of protein tyrosine has a limited duration––early preconditioning lasts for several hours, kinases in preconditioning was proposed for the first time by Maulik and delayed preconditioning lasts for several days (107). There is a et al. (87). Now, it is increasingly clear that protein tyrosine kinases definite need to identify a pharmacological preconditioning agent play a crucial role in mediating preconditioning in some animal to render the preconditioning stimulus everlasting. Recently, mono- species (88). Protein tyrosine kinases may act in parallel to (89, 90), phosphoryl lipid A (MLA) was found to induce dose-dependent downstream of (91, 92) or upstream of (93) PKC in eliciting pre- cardioprotection against myocardial infarction (108, 109). Such car- conditioning; however, the identity of these protein tyrosine kinases dioprotection was achieved through the ability of MLA to increase remains unclear. Among a large number of tyrosine kinases, Src endogenous NO formation, which participates in mediating ischemic family tyrosine kinases have received much attention (94). Src has preconditioning. Similarly, resveratrol protects the ischemic myocar- been implicated in the mechanism of cell survival and death, which dium through NO (11). In this study, preconditioning of the hearts is regulated by complex signal transduction processes (95). Rapid with resveratrol provided cardioprotection, as evidenced by improved activation of Src family tyrosine kinases after ischemia has also been postischemic ventricular functional recovery (i.e., developed pressure documented in the isolated guinea pig heart (96). Members of the and aortic flow), reduced myocardial infarct size, and cardiomyocyte Src family can be activated by many effectors, including stimulation apoptosis. Additionally, resveratrol diminished the amount of ROS of G protein–coupled receptors (97), increased intracellular Ca2+ activity, which was demonstrated through reduced malonaldehyde (98), oxidative stress (99), and enhanced nitric oxide synthesis (84), formation. These results suggest resveratrol may act to pharmacologi- Volume 6, Issue 1

cally precondition the heart in a Modules NF-κB/AP-1 A report by Imamura et al. showed that iNOS knockout Activates adenosine Triggers survival signal through mouse could not be precondi- PI3K/Akt signaling tioned with resveratrol, further indicating that this polyphe- Modulates Bcl-2/Bax/Bad nol provides cardioprotection through NO and specifically through the induction of iNOS Inhibits LDL peroxidation (51). In this study, control experi-ments were performed with wild type mouse hearts and with Potentiates redox signaling Potentiates MAPK signaling iNOS-null mouse hearts that were not treated with resveratrol. Resveratrol treated wild-type Induces iNOS/eNOS mouse hearts displayed signifi- Decreases ventricular Reduces ischemia/ Reperfusion injury cant improvement in post-isch-emic ventricular functional Figure 4. Preconditioning with resveratrol. Mechanisms of reveratrol preconditioning involve a number of well-defined
recovery compared to those of molecular signaling events. The therapeutic implications of preconditioning are discussed in the text.
non-treated wild-type hearts. Both resveratrol-treated and non-treated iNOS-knockout mouse hearts exhibited relatively poor yl-8-cyclopenthylxanthine) and an adenosine A3 receptor blocker recovery in ventricular function as compared to that in wild-type resveratrol-treated hearts. Myocardial infarct size and the number of 1,4-(+/–)-dihydropyridine-3,5-dicarboxylate], but not adenosine apoptotic cardiomyocytes were lower in the resveratrol-treated wild- A2A receptor blocker [CSC; 8-(3-chlorostyryl)caffeine], abrogated the type mouse hearts as compared to results from the other groups cardioprotective abilities of resveratrol, suggesting a role of adenos- of hearts. Cardioprotective effects of resveratrol were abolished ine A1 and A3 receptors in resveratrol preconditioning. Resveratrol when the wild-type mouse hearts were simultaneously perfused induced the expression of Bcl-2 and caused its phosphorylation with aminoguanidine, an iNOS inhibitor. Resveratrol induced the along with phosphorylation of cyclic AMP response element binding expression of iNOS (for several hours) in the wild-type mouse hearts protein (CREB), Akt, and Bad. CPT blocked the phosphorylation but not in the iNOS-knockout hearts, after only thirty minutes of of Akt and Bad without affecting the phosphorylation of CREB, reperfusion. Additionally, resveratrol-treated wild-type mouse hearts whereas MRS 1191 blocked phosphorylation of all these proteins. were subjected to lower amount of oxidative stress, as evidenced A phosphatidylinositol 3'-kinase (PI3K) inhibitor LY 294002 par- by reduced amount of malonaldehyde content in these hearts com- tially blocked the cardioprotective abilities of resveratrol (12). These pared to findings in iNOS-knockout and untreated hearts. These results indicate that resveratrol preconditions the heart through the results demonstrated that resveratrol was unable to precondition activation of adenosine A1 and A3 receptors, the former transmitting iNOS-knockout mouse hearts, but it could successfully precondition a survival signal through PI3K-Akt-Bcl-2 signaling pathway, while the the wild-type mouse hearts, indicating an essential role of iNOS in latter protects the heart through a CREB-dependent Bcl-2 pathway resveratrol preconditioning of the heart. The above results strongly in addition to the Akt-Bcl-2 pathway.
support the notion that resveratrol mediated cardio-protection is achieved by preconditioning (Figure 4). In normal tissue, resveratrol decreases the expression of iNOS; Angiogenic Properties Of Resveratrol however, as seen from the above studies, in ischemic heart, resve-ratrol induces iNOS, an observation that is consistent with isch- Therapeutic angiogenesis describes the method of improving blood emic preconditioning (101, 108). Recently, resveratrol was found flow to ischemic tissue by the induction of neovascularization by to protect the ischemic heart through the increased expression of angiogenic agents administered as recombinant protein or by gene adenosine A1 and A3 receptors (12), a property shared by ischemic therapy. Indeed, therapeutic angiogenesis has recently emerged as a preconditioning. The results of this study demonstrated signifi- promising investigational strategy for the treatment of patients with cant cardioprotection with resveratrol treatment, as evidenced by ischemic limb and heart disease. In the past ten years, alternative improved ventricular recovery, reduced infarct size, and cardiomyo- revascularization/angiogenesis strategies have progressed from bench cyte apoptosis. Specifically, an A1 receptor blocker (CPT; 1,3-dimeth- to bedside, focusing on the capillary sprouting and growth of new Resveratrol: A Preconditioning Agent vessels to replace the old. However, most of the strategies involve and physiological function including regulation of vascular tone and the delivery of growth factors and, unfortunately, very little suc- vascular remodeling (123). One potential therapeutic target for NO is cess with these strategies has been demonstrated so far for various angiogenesis (124). Incubation of human vascular smooth muscle cells reasons. The approach of using resveratrol to induce angiogenesis with NO donors enhances the synthesis of VEGF and inhibition of and increased expression of growth factors and their receptors is an NOS abrogates VEGF production (125). Not surprisingly, eNOS inhib- exciting and potentially very important strategy for myocardial pro- itors block VEGF-induced endothelial cell migration, proliferation, tection. We have shown in the rat myocardial infarction model that, and tube formation in vitro and VEGF-induced angiogenesis in vivo. three weeks after infarction, resveratrol treatment leads to increased Dimmelers et al. found that in the absence of eNOS inhibition, VEGF expression of vascular endothelial growth factor (VEGF) and its stimulates PI3K- and Akt-dependent phosphorylation of eNOS, result- tyrosine kinase receptor Flk-1 (110). Pretreatment with resveratrol ing in an activation of eNOS and increased NO production (126). also increases the amounts of iNOS and eNOS expressed as well as Although both tyrosine kinase receptors Flt-1 and Flk-1 are necessary increases the expression of anti-apoptotic and pro-angiogenic factors for VEGF signaling, there are differences between the intracellular NF-κB and Sp-1. We also demonstrated increased capillary density functions of the two receptors (126). Flk-1, rather than Flt-1, is pre- and improved left ventricular function (again, three weeks after dominantly involved in eNOS phosphorylation, based on mutation infarction) by pharmacological preconditioning with resveratrol. studies. Stimulation of Flt-1 (another VEGF receptor) is linked to cell In one of our previous studies, we documented hypoxia- migration, whereas Flk-1 activation is associated with both cell migra- reoxygenation–mediated myocardial angiogenesis through an NF- tion and proliferation through the MAPK cascade (127). Interestingly, κB-dependent mechanism in a rat model of chronic myocardial induction of the expression of VEGF and Flt-1 occurs within a very infarction (111). In this study, the administration of pyrrolidine short time, but induction of the Flk-1 does not occur until days dithiocarbamate (PDTC), an NF-κB inhibitor, blocked angiogenesis, later (128). One of our very recent studies showed that resveratrol thus demonstrating the essential and important role of NF-κB in could induce, in chronological order, the expression of iNOS and myocardial angiogenesis. Again, notable induction of eNOS and VEGF, followed by Flk-1, and lastly eNOS. Immunohistochemistry iNOS by resveratrol in the myocardium provides evidence support- detected increased expression of these proteins in the hearts from ing the hypothesis that resveratrol regulates endothelial cell growth resveratrol-fed rats subjected to thirty minutes of ischemia and two as also evident by increased perfused capillaries (111). The eNOS hours of reperfusion as compared to those in non-fed hearts (129). isoform is constitutively expressed and its expression can be stimu- Several other reports exist in the literature to show that resveratrol can lated by pharmacological interventions whereas, iNOS is a cyto- induce eNOS and iNOS expression. For example, resveratrol induced kine-inducible isoenzyme (112). The eNOS isoform participates in an expression of eNOS in the human umbilical vein endothelial cells circulatory function during heart failure; the iNOS isoform may have (HUVEC) (130). In addition, to its long-term effects on eNOS expres- an important role in the hemodynamics early after myocardial infarc- sion, resveratrol also enhanced the rapid production of bioactive NO, tion (113). iNOS modulates arterial hemodynamics in large, conduit which suggests a role for iNOS. Our results support these previous arteries whereas eNOS is supposed to regulate peripheral resistance observations; we have observed iNOS expression within twenty-four of the vessel (114). hours of resveratrol treatment, but eNOS expression was not apparent Numerous experimental results have demonstrated that very until three days later. Additionally, Hsieh et al. found that resveratrol low concentrations of NO produced from eNOS or pharmacological induced the expression of iNOS in cultured bovine pulmonary artery concentrations of exogenous NO produced by NO donors reduce endothelial cells (131). apoptotic cell death (115, 116). In our study, besides eNOS induc-tion, iNOS was significantly induced in all resveratrol-treated groups. Recent studies have demonstrated that eNOS/NO play an important Future Perspectives role in many VEGF-induced actions. VEGF induces the produc-tion of nitric oxide (NO) from rabbit, pig, bovine, and human It is becoming increasingly clear that resveratrol has two faces. On vascular endothelial cells (117). The inhibition of NO production one hand, it protects cells by potentiating a survival signal. On the by eNOS inhibitors significantly reduced VEGF-induced mitogenic other hand, it selectively kills cancer cells. It behaves as an antioxi- and angiogenic effects (118). eNOS/NO have been implicated as dant, yet it can induce redox signaling. It is an antiproliferative agent important mediators for VEGF-induced hemodynamic changes and for cancer; it induces apoptosis in tumor cells and sensitizes cancer microvascular permeability. Recent studies indicate that a variety cells by inhibiting cell survival signal transduction and antiapoptotic of stimuli, including hypoxia, shear stress, inflammatory cytokines, pathways. In contrast, the same compound triggers a survival signal high glucose, and injury can modulate eNOS expression and activity in the ischemic tissue by inducing antiapoptotic genes and blocks (119, 120). In vitro experiments have shown that activation of Flk-1 apoptosis in ischemic heart. At low doses, resveratrol stimulates induces proliferation and migration of endothelial cells as well as angiogenesis, but at higher doses, it blocks angiogenic response. expression of eNOS and iNOS (121, 122). Resveratrol has been recognized as a phytoestrogen based on its NO is a pleiotropic molecule that affects diverse biochemical structural similarities to diethylstilbesterol (DES), yet many in vivo Volume 6, Issue 1 provides evidence that resveratrol was used centuries ago in
studies failed to confirm estrogenic potential of resveratrol. At low concentrations, resveratrol scavenges ROS, but at higher concentra- Vastano, B.C., Chen, Y., Zhu, N., Ho, C.T., Zhou, Z., and Rosen, R.T. tions, it behaves like a prooxidant.
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