Nutritional improvement of the endothelial control of vascular tone by polyphenols: role of NO and EDHF
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- Schini-Kerth, V.B., Auger, C., Kim, J. et al. Pflugers Arch - Eur J Physiol (2010) 459: 853. doi:10.1007/s00424-010-0806-4
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Numerous studies indicate that regular intake of polyphenol-rich beverages (red wine and tea) and foods (chocolate, fruit, and vegetables) is associated with a protective effect on the cardiovascular system in humans and animals. Beyond the well-known antioxidant properties of polyphenols, several other mechanisms have been shown to contribute to their beneficial cardiovascular effects. Indeed, both experimental and clinical studies indicate that polyphenols improve the ability of endothelial cells to control vascular tone. Experiments with isolated arteries have shown that polyphenols cause nitric oxide (NO)-mediated endothelium-dependent relaxations and increase the endothelial formation of NO. The polyphenol-induced NO formation is due to the redox-sensitive activation of the phosphatidylinositol3-kinase/Akt pathway leading to endothelial NO synthase (eNOS) activation subsequent to its phosphorylation on Ser 1177. Besides the phosphatidylinositol3-kinase/Akt pathway, polyphenols have also been shown to activate eNOS by increasing the intracellular free calcium concentration and by activating estrogen receptors in endothelial cells. In addition to causing a rapid and sustained activation of eNOS by phosphorylation, polyphenols can increase the expression level of eNOS in endothelial cells leading to an increased formation of NO. Moreover, the polyphenol-induced endothelium-dependent relaxation also involves endothelium-derived hyperpolarizing factor, besides NO, in several types of arteries. Altogether, polyphenols have the capacity to improve the endothelial control of vascular tone not only in several experimental models of cardiovascular diseases such as hypertension but also in healthy and diseased humans. Thus, these experimental and clinical studies highlight the potential of polyphenol-rich sources to provide vascular protection in health and disease.
Diet, one of the most important lifestyle risk factors, can strongly influence the incidence of cardiovascular diseases [10, 96]. Indeed, accumulating data from numerous epidemiological studies indicate that regular intake of polyphenol-rich beverages and foods such as red wine, tea, and chocolate, and fruit and vegetables is associated with an improved cardiovascular prognosis [29, 48, 75, 95].
Studies aiming to characterize the beneficial effects of polyphenols on the cardiovascular systems have started several decades ago [14, 32]. The beneficial effects of polyphenols on the cardiovascular system have been attributed to their ability to reduce vascular oxidative stress, in particular, not only through their direct superoxide anion scavenging properties and interaction with other reactive oxygen species such as hydroxy and peroxy radicals [50, 81, 88, 102] but also through their stimulatory effect on endogenous antioxidant enzymes  and their inhibitory effect on xanthine oxidase and NAD(P)H oxidase, two major enzymes generating large amounts of reactive oxygen species [81, 82]. In addition to the antioxidant effects of polyphenols, both experimental and clinical studies indicate that polyphenols might also protect the cardiovascular system by improving the endothelial function. As it has been first demonstrated by Furchgott and Zawadzki , the endothelium plays a key role in the control of vascular tone by releasing several vasorelaxing factors, which have been identified later on as nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) [19, 38, 51, 83, 101]. Therefore, the aim of this review is to focus on the polyphenol-induced endothelial formation of NO and stimulation of EDHF-mediated responses, two major vasoprotective factors. In addition, this review will summarize both the experimental and the clinical evidences indicating that polyphenols might improve the endothelial function in health and disease.
Polyphenols cause NO-mediated endothelium-dependent relaxations
It was initially shown that various grape products including wines, grape juices, and grape skin extracts are able to induce concentration-dependent relaxations in rat aortic rings with endothelium but only minor relaxations in rings without endothelium . The fact that the polyphenol-induced relaxation is associated with an increase in the cyclic GMP content in intact aortic rings and that both the relaxation and the formation of cyclic GMP are prevented by NO synthase inhibitors indicates that grape-derived products increase the endothelial NO synthase activity leading to the formation of NO, which subsequently relaxes the vascular smooth muscle via the cGMP-mediated pathway. In addition, the endothelium-dependent relaxation appears to be strongly correlated with the concentration of polyphenols in red wines .
Thereafter, NO-mediated endothelium-dependent relaxations induced by polyphenols from grape-derived products, including wines and grape seeds, have been observed in various types of blood vessels including the rat and rabbit aorta, the perfused rat mesenteric artery, and in the porcine and human coronary arteries [4, 24, 40, 43, 78, 94]. Electron paramagnetic resonance spectroscopy and NO-selective microsensor provided direct evidence that grape-derived polyphenols cause an increase in the endothelial formation of NO in intact segments of rat aorta and porcine coronary artery and in cultured endothelial cells [77, 98, 99]. In addition, polyphenols from various sources such as cocoa, tea, hawthorn, maritime pine bark, honey, and propolis have also been shown to induce endothelium-dependent NO-mediated relaxations in arteries [6, 21, 33, 39, 42, 57, 60, 67, 99].
Endothelium-dependent relaxations have also been observed in response to several authentic polyphenolic compounds including curcumin , apigenin , resveratrol [65, 87], and soy isoflavones .
Polyphenols increase endothelial NO synthase activity
Role of intracellular calcium
Role of the PI3-kinase/Akt pathway
Besides the calcium signal, the PI3-kinase/Akt pathway is another important signal pathway leading to the activation of endothelial NO synthase such as in response to the shear stress induced by blood flow, estrogens, and vascular endothelial growth factor [18, 30, 72]. The PI3-kinase/Akt causes a rapid activation of endothelial NO synthase through its phosphorylation at Ser1177 .
It has been shown that red wine polyphenols (RWPs) induce the activation of the PI3-kinase/Akt pathway in endothelial cells, which, in turn, causes phosphorylation of eNOS at Ser1177 (an activator site) and dephosphorylation of eNOS at Thr495 (an inhibitor site), leading to an increased formation of NO  (Fig. 2). The PI3-kinase/Akt signaling pathway is triggered by the polyphenol-induced intracellular formation of reactive oxygen species predominantly superoxide anions in endothelial cells [5, 68, 77]. A similar mechanism of eNOS activation has been observed in response to a grape seed extract , grape skin extract , purple grape juice , strawberry powder rich in polyphenols , and epigallocatechin-3-gallate , a major flavonoid from the flavan-3-ol subclass found in green tea. In addition, polyphenols cause within minutes the phosphorylation of eNOS, and this effect persists for several hours indicating a sustained activation of eNOS. In contrast, activation of G protein-coupled receptors by bradykinin or thrombin caused only a transient formation of NO, which reached a peak value within 1 min and thereafter vanished rapidly [27, 91].
Additional investigations identified Src kinase as a redox-sensitive mediator, which acts upstream of the PI3-kinase/Akt pathway leading to eNOS activation in response to grape-derived polyphenols [5, 6, 68] (Fig. 2). Moreover, Fyn, a member of the Src family, mediated the epigallocatechin gallate-induced PI3-kinase/Akt-mediated activation of eNOS .
The polyphenolic-rich fraction of black tea acutely enhanced NO formation subsequently to the phosphorylation eNOS at Ser1177 and the dephosphorylation of eNOS at Thr495 in porcine aortic endothelial cells . This stimulatory effect is calcium-dependent involving both intracellular and extracellular calcium and involves the p38 MAPK upstream of the PI3-kinase/Akt pathway . A calcium-dependent activation of eNOS has been shown in response to the tannin 1-α-O-galloylpunicalagin which is associated with the PI3-kinase/Akt pathway . Therefore, changes in [Ca2+]i in endothelial cells are likely to contribute to the redox-sensitive activation of eNOS in response to polyphenols via the PI3-kinase/Akt-dependent pathway.
Role of estrogen receptors
It has been shown that low concentrations (in the nanomolar range) of resveratrol, a polyphenolic phytoestrogen found in grapes and wine, as well as black tea polyphenols are able to activate estrogen receptors resulting in activation of p38 MAPK and eNOS in endothelial cells [7, 61] (Fig. 2). In contrast, the estrogen receptor antagonist ICI 182,780 did not alter relaxations to RWPs in intact aortic rings from both male and female rats . In addition, the RWP-induced phosphorylation of Akt and eNOS in porcine coronary artery endothelial cells was not affected by ICI 182,780 . Similar results were observed in the isolated porcine coronary arteries with the Crataegus (Hawthorn species) special extract WS 1442 . Therefore, these data indicate that an estrogen receptor-dependent pathway mediates activation of eNOS in response to some polyphenols including resveratrol and black tea polyphenols, but not to red wine polyphenols and Crataegus polyphenols.
Role of caveolin-1
A recent study has indicated that green tea polyphenols downregulate caveolin-1 protein expression and mRNA levels in both time- and concentration-dependent manners via activation of ERK1/2 and inhibition of p38 MAPK signaling pathways in bovine aortic endothelial cells  (Fig. 2). As caveolin-1 is a major negative regulator of eNOS activity, such an effect might contribute to increase eNOS activation by polyphenols.
Polyphenols increase endothelial NO synthase expression
In addition to causing a rapid and sustained activation of eNOS, polyphenols have been shown to increase the expression level of eNOS in cultured endothelial. Indeed, exposure of endothelial cells to red wine increased eNOS expression at both the mRNA and protein levels [105, 107]. The upregulation of eNOS is attributed to polyphenols contained in red wine because ethanol alone has no such effect . Moreover, eNOS upregulation is also observed in response to a red wine extract without alcohol . However, it must be underlined that large concentrations of red wine are required to increase eNOS expression (1% v/v in culture medium for 10 days, 3% v/v for 24 h, and 10% v/v for 12 h) . The analysis of the active polyphenols in red wine indicated the involvement of several polyphenolic compounds, in particular, trans-resveratrol and also to a lesser extent cinnamic and hydroxycinnamic acids, cyanidin, and several phenolic acids .
Resveratrol upregulated eNOS expression at concentrations ranging from 10 to 100 µM [49, 105]. The stimulatory effect is mainly mediated by an increase in the activity of the eNOS promoter (transcriptional effect) and a stabilization of eNOS mRNA (post-transcriptional effect) . In addition, lower concentrations of resveratrol such as 0.1 µM have also been shown to increase the expression of eNOS mRNA in cultured human umbilical vein endothelial cells . Moreover, it has been reported that in cultured human coronary arterial endothelial cells, resveratrol upregulates the expression of SIRT1, a NAD+-dependent protein deacetylase, and induces its enzymatic activation leading to an upregulation of eNOS mRNA expression and mitochondrial biogenesis; all these events might contribute to improve the endothelial function [25, 71].
The evaluation of the effectiveness of 33 different polyphenols (procyanidins, monomeric flavan-3-ols, flavonols, a flavone, a flavanone, a chalcone, a stilbene, and phenolic acids) to enhance eNOS mRNA expression at 100 µM in a hybrid human endothelial cell line EA.hy926 revealed a significant stimulatory effect only in response to four polyphenols including resveratrol, quercetin, epicatechin-gallate, and epigallocatechin-gallate. No clear relationship between the structure of the active polyphenols and the stimulatory activity was observed .
Polyphenols induce EDHF-mediated responses in arteries
The component of arterial endothelium-dependent relaxations, which is resistant to inhibitors of NO synthase and cyclooxygenases, has been attributed to EDHF. It is now commonly admitted that the importance of the EDHF phenomenon increases as the vessel size decreases . Several mechanisms have been proposed to explain the nature of the EDHF phenomenon (see related chapters in the present issue), but one hallmark of EDHF-mediated responses is that these relaxations are associated to an endothelium-dependent hyperpolarization of the vascular smooth muscle cells.
Although resveratrol has been shown to activate IKCa channels (24 pS) in MS1 cell line, a pancreatic islet endothelial cell line, by increasing their open probability , other mechanisms have been involved in the polyphenol-induced EDHF-mediated relaxation. Indeed, the RWP-induced EDHF-mediated relaxation and hyperpolarization are strictly dependent on an endothelial redox-sensitive mechanism involving the intracellular formation of superoxide anions in porcine coronary arteries  (Fig. 3). EDHF-mediated relaxations are reduced by antioxidants, such as N-acetylcysteine, and membrane permeant analogs of superoxide dismutase (SOD), such as Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and polyethylene glycol-SOD (PEG-SOD). In addition, exposure of cultured porcine coronary artery endothelial cells to RWPs induced the MnTMPyP-sensitive formation of superoxide anions in the presence of L-NA and indomethacin . Further investigations indicated that the RWP-induced EDHF-mediated relaxation of porcine coronary arteries involves the redox-sensitive activation of PI3-kinase leading to Akt phosphorylation in endothelial cells  (Fig. 3). The possibility that the PI3-kinase/Akt pathway modulates myo-endothelial gap junctions and/or potassium channel activity remains to be investigated.
Polyphenols and endothelial function in experimental models of cardiovascular diseases
A polyphenol-rich cocoa powder (up to 300 mg/kg bodyweight) also reduced blood pressure similarly to 50 mg/kg of captopril, an angiotensin converting enzyme inhibitor, in the spontaneously hypertensive rat .
Several studies have also reported an antihypertensive effect in response to several purified polyphenols from fruits and vegetables. Indeed, quercetin, a flavonol found widely in fruits and vegetables, reduced blood pressure in several experimental models of hypertension including spontaneously hypertensive rats, NG-nitro l-arginine methyl ester-treated rats, two-kidney one clip Goldblatt rats, DOCA-salt, and Dahl salt-sensitive rats . Similarly, genistein, an isoflavone found mainly in vegetables, as well as hesperitin and glucosyl-hesperidin, two flavonones, decreased blood pressure and improved endothelium-dependent relaxations of aortic rings of spontaneously hypertensive rats [104, 112, 113]. Furthermore, catechin prevented endothelial dysfunction in the prediabetic stage of Otsuka Long-Evans Tokushima Fatty rats by reducing vascular NADPH oxidase activity and expression .
Altogether, these experimental studies indicate that ingestion of polyphenol-rich products has a beneficial effect in several experimental models of major cardiovascular diseases including hypertension, diabetes, and atherosclerosis. The beneficial effect is associated with the restoration of the vasoprotective effect of endothelial cells most likely by reducing the excessive NADPH oxidase-dependent vascular oxidative stress. In addition, the ability of polyphenols to increase the endothelial formation of NO and the EDHF-mediated responses might also contribute to the vasoprotective effect either directly or indirectly by preventing the oxidative stress-induced inactivation of NO.
Polyphenols and endothelial function in healthy and diseased humans
In healthy subjects, ingestion of 250 to 500 ml of red wine or de-alcoholized red wine increased, in some but not all studies, flow-mediated vasodilatation as assessed in the brachial artery by plethysmography [1, 31, 47]. Intake of 5–7 ml/kg of purple grape juice by healthy subjects for 7–14 days reduced ex vivo platelet aggregation and increased platelet-derived NO formation [44, 58]. In patients with coronary artery diseases, flow-mediated dilatation was improved after intake of 4 ml/kg of either white or red wine during a light meal  and also after intake of about 8 ml/kg of purple grape juice [22, 97]. The beneficial effect of purple grape juice on the endothelial function was associated with a reduced susceptibility of LDL particles to oxidation . In addition, intake of 5.5 ml/kg of purple grape juice daily by hypertensive Korean patients for 8 weeks reduced both systolic and diastolic blood pressure by, respectively, 7.2 and 6.2 mmHg . Besides grape-derived products, an improved endothelial function in patients with coronary artery diseases has been observed in response to intake of short- and long-term black tea consumption  and after dietary supplementation with epigallocatechin gallate, a major catechin in tea (300 mg) . In addition, consumption of a flavonoid-rich dark chocolates and cocoa beverages improved endothelial function in healthy subjects, hypercholesterolemic postmenopausal women, and in diabetic patients associated to an increased circulating level of NO species [13, 37, 92, 108]. Moreover, an improved endothelial function was observed after administration of (–)-epicatechin (1 mg/kg, a major flavanol of cocoa) to healthy humans . Chronic intake of flavanol-rich dark chocolate reduced blood pressure in patients with upper-range hypertension or stage 1 hypertension without concomitant risk factors [46, 100].
Altogether, these experimental and clinical studies highlight the potential of polyphenol-rich sources to ameliorate or restore vascular protection in health and disease by enhancing the two major endothelial vasoprotective mechanisms, i.e., the formation of NO and EDHF-mediated responses and also by reducing oxidative stress in the arterial wall which promotes pro-inflammatory and pro-thrombotic responses (Fig. 4).