II.4.2.a. Endothelial cells and regulation of vascular homeostatis
Endothelial cells of healthy blood vessels form a monolayer at the luminal surface and mediate chemically the vascular homeostasis. These cells prevent the contact of circulating blood with the underlying prothrombotic arterial wall. The endothelium plays a critical in the control of vascular tone by synthesizing and releasing various substances into the bloodstream which are classified as vasodilators and vasoconstrictors, based on their mechanism of action. Vasodilators are generally referred to as endothelium-derived relaxing factor (EDRF), represented mainly by nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostacyclin (PGI2), and vasoconstrictors, known as
endothelium-derived contracting factors (EDCFs), represented by endothelin-1.
The gas NO is generated from L-arginine by the enzyme endothelial NO synthase (eNOS) and diffuses toward the underlying vascular smooth muscle, where it stimulated guanylate cyclase in the sarcoplasm and increase the production of cyclic guanosine monophosphate (cGMP) (Vanhoutte 2009). NO is the most potent of all endogenous compounds causing vascular muscle relaxation, but also displays paracrine action by preventing platelet aggregation, adhesion and exerting an anti-proliferative effect on smooth muscle cells. In addition, NO also limits the oxidation of low-density lipoproteins (LDLs) and can lower the activity of reactive oxygen species (Frankel et al. 1995; Teissedre et al. 1996). The physiological effect of NO is very short (less than 20 sec) because of the inactivation of haemoglobin (Stankevicius et al. 2003; Stoclet et al. 2004).
PGI2 can be synthesized through transcellular metabolism. Similarly to NO, PGI2
dilates blood vessels, causing smooth muscle relaxation. This is mainly attributed to the stimulation of adenylate cyclase and thus to an increased content of intracellular adenosine monophosphate (cAMP) and the activation of potassium channels in smooth muscle cells (Kawashima et al. 2004; Fetalvero et al. 2007; Vanhoutte 2009).
Mechanical damages to endothelium or a loss of functional integrity disturbs the fragile multifactorial equilibrium ensured by endothelial cells and thus, causing the
61
development of pathological changes such as hypertension, atherosclerotic lesions, thrombi and disturbed tissues perfusion (Goch et al. 2009). Endothelial dysfunction is often associated with pronounced oxidative stress which is due, at least in part, to an increased expression of NADPH oxidase, an enzyme generating superoxide anions on the arterial wall (Griendling et al. 2000; Griendling et al. 2003). Superoxide anions react with NO to reduce its bioavailability and, hence, vascular protective effects. Furthermore, the dysfunction can activate the endothelium-dependent contractile responses involving the contractile actions of endothelin-1 and vasoconstrictor factors acting on tromboxane receptors. The latter stimulates platelet aggregation and vessel constriction.
II.4.2.b. Vascular protection by (poly)phenols
Pioneer study by Fitzpatrick et al. (1993), using rat aorta with various grape products including wines, grape juices and grape skin extracts, have showed that (poly)phenols increase the formation of NO by endothelial NO synthase action. Using another animal model, in porcine coronary artery ring, the endothelium-dependent relaxation induced by red wine (poly)phenols is observed at concentration of 3 µg/mL (Ndiaye et al. 2004). Several other aortic ring experiments using physiological concentrations of (poly)phenols have also shown that (poly)phenols induce endothelium-dependent relaxation (Fitzpatrick et al. 1993; Karim et al. 2000; Chin-Dusting et al. 2001; Woodman et al. 2004). This regulation of vascular nitric oxide is thought to involve the ability of (poly)phenols to interact with kinase signaling pathways such as the PI3-kinase/Akt pathway and intracellular Ca+2 on eNOS phosphorylation and subsequent NO production (Lorenz et al. 2004; Stoclet et al. 2004). However, in vitro experiment results are of limited value and (poly)phenol in vivo effects might not be effectively evaluated.
In humans, 30 min after the consumption of red wine, circulating NO concentration increased to 30-40 nM, respectively. In addition, a reduction of the blood pressure (11 mm Hg) and an increase of heart rate was observed (Matsuo et al. 2001). A study using olive oil, showed a reduction of blood pressure in hypertensive patients, possibly through enhanced NO levels stimulated by (poly)phenols. (Ferrara et al. 2000). Short-term and long-term effects of tea consumption were also investigated (Duffy et al. 2001). This study used water as a control beverage and results showed that both short-term and long-term consumption improved endothelial function. However, this study did not demonstrated any effect on plasma antioxidant capacity, on plasma concentration of F2-isoprostanes, a marker of
62
These findings are consistent with several other well-conducted studies that failed to demonstrate a reduction in markers of oxidative stress after tea consumption (O'Reilly et al. 2001).
Several other studies demonstrated that flavonoid-containing beverages have beneficial effects on endothelial function. Stein et al. (1999) observed that consumption of grape juice for 14 days was associated with improved brachial artery flow-mediated dilation among 15 adults with angiographically proven coronary artery disease. A second study from the same group also indicated beneficial effects of purple grape juice on endothelial function (Chou et al. 2001). The effect of cocoa on flow-mediated dilation was also investigated. Among patients with at least one cardiovascular disease risk factor, impaired endothelial function was observed. Two hours after the patients consumed cocoa containing 176 mg/dL flavan-3-ols, the investigators observed a significant increase in flow-mediated dilation. They also observed increased in nitrosylated and nitrosated species in plasma, which suggested an increase in nitric oxide production (Heiss et al. 2003).
Endothelium- and NO-dependent relaxation has been reported for several isolated flavonoids, especially the anthocyanin delphinidin (Andriambeloson et al. 1998) and the flavone chrysin (Duarte et al. 2001). These effects are related to a pro-oxidant effect because it can be inhibited by superoxide dismutase and catalase and a subsequent increased in endothelial cystolic Ca2+ levels (Andriambeloson et al. 1998). Using ERα deficient mice, a recent study evidenced an activation of NO pathway leading to the induction of endothelial vasodilatation in aorta endothelial cells by the anthocyanin delphinidin (Chalopin et al. 2010). The authors also demonstrate the implication of the alpha isoform estrogen receptor (ERα) in the transduction of the vascular benefits of (poly)phenols. Actually, silencing the effects of ERα completely prevented the effects of delphinidin to activate NO pathway (Chalopin et al. 2010). Some group have also described that the effects of quercetin were partially endothelium-dependent and related to the release of endothelium-derived relaxing factors (Ajay et al. 2003; Khoo et al. 2010). A pro-oxidant mechanism involving the release of H2O2
has been proposed (Khoo et al. 2010).
A complex array of information has been reviewed by Stoclet et al. (2004), proposing a global view of vascular protection by dietary (poly)phenols. Briefly, plant (poly)phenols firstly act on endothelial cells by enhancing the production of vasodilating factors (i.e., NO, EDHF and prostacyclin) and inhibit the synthesis of vasoconstrictor endothelin-1. This
63
mechanism involves an increase of Ca2+ level and redox-sensitive activation of the phosphoinositide 3 (PI3)-kinase/Akt pathway (leading to rapid and sustained activation of nitric oxide synthase and formation of EDHF) and enhance expression of nitric oxide synthase. Secondly, (poly)phenols can operate in smooth muscle cells by inhibiting the expression of two major pro-angiogenic factors: vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2). The mechanism requires both redox-sensitive inhibition of the p38 mitogen-activated protein kinase (p38 MAPK) pathway activation (leading to inhibition of platelet-derived growth factor (PDGF)-induced VEGF gene expression) and redox-insensitive mechanisms (leading to inhibition of thrombin-induced MMP-2 formation). A more recent review by Schini-Kerth et al. (2011), describes the in vitro and in vivo vascular protection by natural product-derived (poly)phenols during the last 15 years. Grape-derived products (i.e. juices, extracts, wines and marc extracts), berries, tea and plants are able to improve the endothelial function in both in vitro and in vivo mostly by stimulating the endothelial formation of NO. The in vitro experiments are of limited value and cannot be generalized to in vivo effects.