Rocío López-Sepúlveda

Quercetin inhibits vascular superoxide production induced by endothelin-1: Role of NADPH oxidase, uncoupled eNOS and PKC

Chronic administration of the most abundant dietary flavonoid quercetin exerts antihypertensive effects and improves endothelial function. We have investigated the effects of quercetin and its methylated metabolite isorhamnetin (1-10microM) on endothelial dysfunction and superoxide (O(2*)(-)) production induced by endothelin-1 (ET-1, 10nM). ET-1 increased the contractile response induced by phenylephrine and reduced the relaxant responses to acetylcholine in phenylephrine contracted intact aorta, and these effects were prevented by co-incubation with quercetin, isorhamnetin or chelerythrine (protein kinase C (PKC) inhibitor). This endothelial dysfunction was also improved by superoxide dismutase (SOD), apocynin (NADPH oxidase inhibitor) and sepiapterin (tetrahydrobiopterin synthesis substrate). Furthermore, ET-1 increased intracellular O(2*)(-) production in all layers of the vessel, protein expression of NADPH oxidase subunit p47(phox) without affecting p22(phox) expression and lucigenin-enhanced chemiluminescence signal stimulated by calcium ionophore A23187. All these changes were prevented by both quercetin and isorhamnetin. Moreover, apocynin, endothelium denudation and N(G)-nitro-l-arginine methylester (l-NAME, nitric oxide synthase inhibitor) suppressed the ET-1-induced increase in A23187-stimulated O(2*)(-) generation. Moreover, quercetin but not isorhamnetin, inhibited the increased PKC activity induced by ET-1. Taken together these results indicate that ET-1-induced NADPH oxidase up-regulation and eNOS uncoupling via PKC leading to endothelial dysfunction and these effects were prevented by quercetin and isorhamnetin.

Wine Polyphenols Improve Endothelial Function in Large Vessels of Female Spontaneously Hypertensive Rats

Red wine polyphenols (RWPs) have been reported to prevent hypertension and endothelial dysfunction. Several individual RWPs exert estrogenic effects. We analyzed the possible in vivo protective effects on blood pressure and endothelial function of RWPs in female spontaneously hypertensive rats (SHR) and its relationship with ovarian function. RWPs (40 mg/kg by gavage) were orally administered for 5 weeks. Ovariectomized rats showed both increased isoprostaglandin F2&agr; excretion and aortic superoxide production and reduced relaxant response to acetylcholine and contraction to the endothelial nitric oxide synthase (eNOS) inhibitor l-NAME measured in the aorta but similar blood pressure, as compared with sham-operated rats. Moreover, in ovariectomized rats aortic eNOS expression was unchanged, whereas caveolin-1, angiotensin II receptor (AT)-1, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox and p47phox expression was increased compared with sham-operated rats. In both ovariectomized and sham-operated SHR, RWPs reduced systolic blood pressure, urinary isoprostaglandin F2&agr; excretion, and aortic O2− production, improving the endothelium-dependent relaxant response to acetylcholine in SHR. These changes were associated with unchanged aortic eNOS expression, whereas caveolin-1 was increased and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox and p47phox expression was reduced. RWPs had no effect on the AT-1 overexpression found in ovariectomized animals. All these results suggest that a chronic treatment with RWPs reduces hypertension and vascular dysfunction through reduction in vascular oxidative stress in female SHR in a manner independent of the ovarian function.

Epicatechin lowers blood pressure, restores endothelial function, and decreases oxidative stress and endothelin-1 and NADPH oxidase activity in DOCA-salt hypertension.

Flavanol-rich diets have been reported to exert beneficial effects in preventing cardiovascular diseases, such as hypertension. We studied the effects of chronic treatment with epicatechin on blood pressure, endothelial function, and oxidative status in deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Rats were treated for 5 weeks with (-)-epicatechin at 2 or 10 mg kg(-1)day(-1). The high dose of epicatechin prevented both the increase in systolic blood pressure and the proteinuria induced by DOCA-salt. Plasma endothelin-1 and malondialdehyde levels and urinary iso-prostaglandin F(2α) excretion were increased in animals of the DOCA-salt group and reduced by the epicatechin 10 mg kg(-1) treatment. Aortic superoxide levels were enhanced in the DOCA-salt group and abolished by both doses of epicatechin. However, only epicatechin at 10 mg kg(-1) reduced the rise in aortic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and p47(phox) and p22(phox) gene overexpression found in DOCA-salt animals. Epicatechin increased the transcription of nuclear factor-E2-related factor-2 (Nrf2) and Nrf2 target genes in aortas from control rats. Epicatechin also improved the impaired endothelium-dependent relaxation response to acetylcholine and increased the phosphorylation of both Akt and eNOS in aortic rings. In conclusion, epicatechin prevents hypertension, proteinuria, and vascular dysfunction. Epicatechin also induced a reduction in ET-1 release, systemic and vascular oxidative stress, and inhibition of NADPH oxidase activity.

SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: Implications for vascular aging

Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD(+)-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22(phox) and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation.

Antihypertensive Effects of Peroxisome Proliferator-Activated Receptor-β Activation in Spontaneously Hypertensive Rats

Activation of nuclear hormone receptor peroxisome proliferator-activated receptor &bgr;/&dgr; (PPAR&bgr;) has been shown to improve insulin resistance and plasma high-density lipoprotein levels, but nothing is known about its effects in genetic hypertension. We studied whether the PPAR&bgr; agonist GW0742 might exert antihypertensive effects in spontaneously hypertensive rats (SHRs). The rats were divided into 4 groups, Wistar Kyoto rat-control, Wistar Kyoto rat-treated (GW0742, 5 mg · kg−1 · day−1 by oral gavage), SHR-control, and SHR-treated, and followed for 5 weeks. GW0742 induced a progressive reduction in systolic arterial blood pressure and heart rate in SHRs and reduced the mesenteric arterial remodeling, the increased aortic vasoconstriction to angiotensin II, and the endothelial dysfunction characteristic of SHRs. These effects were accompanied by a significant increase in endothelial NO synthase activity attributed to upregulated endothelial NO synthase and downregulated caveolin 1 protein expression. Moreover, GW0742 inhibited vascular superoxide production, downregulated p22phox and p47phox proteins, decreased both basal and angiotensin II–stimulated NADPH oxidase activity, inhibited extracellular-regulated kinase 1/2 activation, and reduced the expression of the proinflammatory and proatherogenic genes, interleukin 1&bgr;, interleukin 6, or intercellular adhesion molecule 1. None of these effects were observed in Wistar Kyoto rats. PPAR&bgr; activation, both in vitro and in vivo, increased the expression of the regulators of G protein–coupled signaling proteins RGS4 and RGS5, which negatively modulated the vascular actions of angiotensin II. PPAR&bgr; activation exerted antihypertensive effects, restored the vascular structure and function, and reduced the oxidative, proinflammatory, and proatherogenic status of SHRs. We propose PPAR&bgr; as a new therapeutic target in hypertension.

Endothelium-Dependent Vasodilator Effects of Peroxisome Proliferator-Activated Receptor β Agonists via the Phosphatidyl-Inositol-3 Kinase-Akt Pathway

Peroxisome proliferator-activated receptor β/δ (PPAR-β) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily that regulates the transcription of many target genes. More recently, acute, nongenomic effects of PPAR-β agonists have also been described. In the present study, we hypothesized that PPAR-β agonists might exert acute nongenomic effects on vascular tone. Here, we report that the structurally unrelated PPAR-β ligands [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid (L-165041) and 4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl] methyl]thio]-2-methylphenoxy]acetic acid (GW0742) induced vascular relaxation in phenylephrine-precontracted endothelium-intact rat aortic rings, which was significantly inhibited by endothelial denudation or nitric-oxide synthase (NOS) inhibition with NG-nitro-l-arginine methylester. These relaxant effects reached steady state within 15 min. The relaxation induced by L-165041 and GW0742 in aortic rings precontracted with the thromboxane A2 analog 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U-46619) was unaffected either by removal of extracellular calcium or by incubation with calcium-free solution containing the intracellular calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester. However, the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY-294002) inhibited the endothelium-dependent relaxant responses induced by both PPAR-β agonists. Blockade of PPAR-β with 3-[[[2-methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester (GSK0660) also partially inhibited these relaxant responses, although PPAR-γ blockade with 2-chloro-5-nitro-N-phenylbenzamide (GW9662) had no effect. In human umbilical vein endothelial cells, L-165041 and GW0742 increased nitric oxide (NO) production and Akt and endothelial NOS (eNOS) phosphorylation, which were sensitive to PI3K inhibition and PPAR-β blockade. In conclusion, the PPAR-β agonists acutely caused vasodilatation, which was partially dependent on endothelial-derived NO. The eNOS activation is calcium-independent and seems to be related to activation of the PI3K-Akt-eNOS pathway.

Red wine polyphenols prevent endothelial dysfunction induced by endothelin-1 in rat aorta: role of NADPH oxidase

RWPs (red wine polyphenols) exert antihypertensive effects and improve endothelial function by reducing the plasma levels of ET-1 (endothelin-1) and the subsequent vascular production of O(2)(•-) (superoxide anion). Our present study was designed to evaluate whether RWPs act directly in the vascular wall improving endothelial dysfunction and O(2)(•-) production induced by ET-1 and to analyse the compounds responsible for these protective effects. We incubated rat isolated aortic rings in the presence or absence of ET-1 (10 nM) and RWPs (10(-4) to 10(-2) g/l) or catechin (0.2 μM), epicatechin (10 μM) and resveratrol (0.1 μM). ET-1 reduced the relaxant responses to acetylcholine, increased intracellular O(2)(•-) production, NADPH oxidase activity and protein expression of NADPH oxidase subunit p47phox. All these changes were prevented by RWPs. The preventive effects of RWPs were unaffected by co-incubation with either ICI-182780, an ER (oestrogen receptor) antagonist, or GW9662, a PPARγ (peroxisome-proliferator-activated receptor γ) antagonist. RWPs inhibited the phosphorylation of the mitogen-activated protein kinase, ERK1/2 (extracellular signal-regulated kinase 1/2), a key regulator of p47phox expression in response to ET-1. When the isolated polyphenols were tested, at the concentrations found in 10(-2) g/l RWPs, only epicatechin prevented endothelial dysfunction and all biochemical changes induced by ET-1 in the vascular wall. Taken together, these results indicate that RWPs prevent ET-1-induced vascular O(2)(•-) production by reducing overexpression of p47phox and the subsequent increased NADPH oxidase activity, leading to improvement in endothelial function. The effects of RWPs appear to be independent of ER and PPARγ activation and are related to ERK1/2 inhibition.

Lack of beneficial metabolic effects of quercetin in adult spontaneously hypertensive rats

Insulin sensitivity is partly dependent on insulin-mediated nitric oxide (NO) release and antioxidants may decrease insulin resistance by amelioring NO bioavailability. The effects of chronic therapy with the antioxidant quercetin on blood pressure, vascular function and glucose tolerance in male spontaneously hypertensive rats (SHR), a model of genetically hypertension and insulin resistance, were analyzed. Rats were divided into four groups, WKY vehicle, WKY quercetin, SHR vehicle and SHR quercetin. Animals were daily administered by gavage for four weeks: vehicle, quercetin in vehicle (10mg/kg body weight). Blood pressure was followed by tail-cuff plethysmography. Chronic quercetin treatment reduced systolic blood pressure, and significantly reduced left ventricular (-10%) and renal (-6%) hypertrophy. However, oral glucose tolerance test, homeostatic model assessment of insulin resistance, total cholesterol and triglycerides were unaffected by quercetin in both strains of rats. It also improved the blunted aortic endothelium-dependent relaxation to acetylcholine, without affecting both endothelium-dependent relaxation to insulin and endothelium-independent relaxation to sodium nitroprusside in SHR. In WKY rats, quercetin in vitro and in vivo, impaired the relaxation to insulin. Quercetin reduced both plasma malondialdehyde levels and aortic superoxide production in SHR. Furthermore, quercetin inhibited insulin-stimulated protein kinase B (Akt)- and endothelial NO synthase (eNOS) phosphorylation. In conclusion, quercetin reduced blood pressure, left ventricular and renal hypertrophy and improved NO-dependent acetylcholine relaxation. However, and despite its antioxidant effects, quercetin was unable to improve insulin sensitivity possibly through its specific interference with the insulin signalling pathway.

Chronic ( − )-epicatechin improves vascular oxidative and inflammatory status but not hypertension in chronic nitric oxide-deficient rats

The present study analysed the effects of the flavanol (-)-epicatechin in rats after chronic inhibition of NO synthesis with NG-nitro-L-arginine methyl ester (L-NAME), at doses equivalent to those achieved in the studies involving human subjects. Wistar rats were randomly divided into four groups: (1) control-vehicle, (2) L-NAME, (3) L-NAME-epicatechin 2 (L-NAME-Epi 2) and (4) L-NAME-epicatechin 10 (L-NAME-Epi 10). Rats were daily given by oral administration for 4 weeks: vehicle, (-)-epicatechin 2 or 10 mg/kg. Animals in the L-NAME groups daily received L-NAME 75 mg/100 ml in drinking-water. The evolution in systolic blood pressure and heart rate, and morphological and plasma variables, proteinuria, vascular superoxide, reactivity and protein expression at the end of the experiment were analysed. Chronic (-)-epicatechin treatment did not modify the development of hypertension and only weakly affected the endothelial dysfunction induced by L-NAME but prevented the cardiac hypertrophy, the renal parenchyma and vascular lesions and proteinuria, and blunted the prostanoid-mediated enhanced endothelium-dependent vasoconstrictor responses and the cyclo-oxygenase-2 and endothelial NO synthase (eNOS) up-regulation. Furthermore, (-)-epicatechin also increased Akt and eNOS phosphorylation and prevented the L-NAME-induced increase in systemic (plasma malonyldialdehyde and urinary 8-iso-PGF2α) and vascular (dihydroethidium staining, NADPH oxidase activity and p22phox up-regulation) oxidative stress, proinflammatory status (intercellular adhesion molecule-1, IL-1β and TNFα up-regulation) and extracellular-signal-regulated kinase 1/2 phosphorylation. The present study shows for the first time that chronic oral administration of (-)-epicatechin does not improve hypertension but reduced pro-atherogenic pathways such as oxidative stress and proinflammatory status of the vascular wall induced by blockade of NO production.

Vascular superoxide production by endothelin-1 requires Src non-receptor protein tyrosine kinase and MAPK activation.

ET-1 induces vascular O(2)(*-) production via activation of NADPH oxidase. We have investigated whether c-Src and MAPKs activation are involved in ET-1-induced vascular oxidative response. At 2 h, ET-1 induced an increase in NADPH oxidase-driven O(2)(*-) production in rat isolated aortic rings, which was completely suppressed in PP2 (c-Src inhibitor)-pretreated rings, whereas PP3 (inactive analogue of PP2) was without effect. ET-1 increased the levels of phospho-c-Src, the active form of c-Src, and the phosphorylation of cortactin, a Src-specific substrate. Both c-Src and cortactin phosphorylation induced by ET-1 were prevented by PP2. The increased expression of p47(phox), the main cytosolic subunit of NADPH oxidase, induced by ET-1 was also prevented by PP2. The increased vascular O(2)(*-) production and p47(phox) up-regulation induced by ET-1 was only inhibited in aortic rings coincubated with the ERK1/2 inhibitor, PD98059; being without effects both the p38 MAPK inhibitor, SB203580, and JNK inhibitor, SP600125. Aortic rings incubation with ET-1 increased the phosphorylation of ERK1/2. This effect was suppressed by coincubation with PP2 showing that this event is down-stream of c-Src activation. In conclusion, ET-1 induces NADPH oxidase-driven O(2)(*-) generation through increase of p47(phox) protein expression. The signalling pathway for this effect involves c-Src activation and ERK1/2 phosphorylation.