ENOS Promoter Activation by Red Wine Polyphenols: an Interaction Study

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 F(2alpha) 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 p22(phox) and p47(phox) expression was increased compared with sham-operated rats. In both ovariectomized and sham-operated SHR, RWPs reduced systolic blood pressure, urinary isoprostaglandin F(2alpha) excretion, and aortic O(2)(-) 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 p22(phox) and p47(phox) 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.

Resveratrol (Resv), a red wine polyphenol, is known to exhibit vascular protective effects and reduce vascular smooth muscle cell mitogenesis. Vascular smooth muscle cell proliferation is a critical factor in the pathogenesis of restenosis, the renarrowing of vessels that often occurs after angioplasty and/or stent implantation. Although Resv has been shown to be an estrogen receptor (ER) modulator, the role of the ER in Resv-mediated protection against restenosis has not yet been elucidated in vivo. Therefore, with the use of a mouse carotid artery injury model, our objective was to determine the role of ER in modulating Resv-mediated effects on neointimal hyperplasia. Female wild-type and ER-α(-/-) mice were administered a high-fat diet ± Resv for 2 wk. A carotid artery endothelial denudation procedure was conducted, and the mice were administered a high-fat diet ± Resv for an additional 2 wk. Resv-treated wild-type mice exhibited a dramatic decrease in restenosis, with an increased arterial nitric oxide (NO) synthase (NOS) activity and NO production. However, in the ER-α(-/-) mice, Resv failed to afford protection and failed to increase NO production, apparently because of a decreased availability of the NOS cofactor tetrahydrobiopterin. To verify the role of NO in Resv-mediated effects, mice were coadministered Resv plus a nonselective NOS inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME). Cotreatment with l-NAME significantly attenuated the antirestenotic properties of Resv. These data thus suggest that Resv inhibits vascular proliferative responses after injury, predominately through an ER-α-dependent increase in NO production.

Genetic factors and the influence of superoxide are known to play roles in the etiology of glaucoma. We evaluated the association between primary open angle glaucoma (POAG) and two polymorphisms in the epithelial nitric oxide synthase (eNOS) gene, and one polymorphism in the myeloperoxidase (MPO) gene. We enrolled 66 patients with POAG and 100 healthy volunteers in this study. The polymorphisms in the eNOS and the polymorphism MPO -463 G-to-A in the MPO gene were resolved by polymorphism polymerase chain reaction (PCR). There were no significant differences in the distribution of the eNOS intron -4 (P=0.481), eNOS promotor -786 (P=0.555), and MPO -463 (P=0.292) gene polymorphisms between the POAG patients and the volunteers (P-values=0.481, 0.555, and 0.292, respectively). None of the alleles from either gene differed between the groups (P-values=0.483, 0.554, and 0.183, respectively). Superoxide is closely related to glaucoma, and eNOS and MPO are two important enzymes in the free radical pathway. However, polymorphisms of the eNOS intron-4, eNOS promotor -786, and MPO -463 gene polymorphisms did not reveal significant differences between POAG patients and controls in our study. The use of these agents and other superoxide-related genes for clinical applications requires further investigation.

In an ovine model of persistent pulmonary hypertension of the newborn (PPHN), endothelin-1 (ET-1) expression is increased, while endothelial nitric oxide synthase (eNOS) expression is decreased. However, the molecular mechanisms by which ET-1 attenuates eNOS expression in endothelial cells are not completely understood. Thus, the goal of this study was to determine if the overexpression of ET-1 decreases eNOS expression in pulmonary arterial endothelial cells isolated from fetal lambs. To increase the ET-1 expression, cells were transfected with a plasmid coding for Prepro-ET-1, a precursor of ET-1. After overexpression of Prepro-ET-1, ET-1 levels in the culture medium were significantly increased (control = 805.3 ± 69.8; Prepro-ET-1 overexpression = 1351 ± 127.9). eNOS promoter activity, protein levels, and NO generation were all significantly decreased by the overexpression of Prepro-ET-1. The decrease in transcription correlated with increased activity of protein kinase Cδ (PKCδ) and STAT3. Further, DNA binding activity of STAT3 was also increased by Prepro-ET-1 overexpression. The increase in STAT3 activity and decrease in eNOS promoter activity were inhibited by the overexpression of dominant negative mutants of PKCδ or STAT3. Further, a 2 bp mutation in the STAT3 binding site in the eNOS promoter inhibited STAT3 binding and led to enhanced promoter activity in the presence of Prepro-ET-1 overexpression. In conclusion, ET-1 secretion is increased by Prepro-ET-1 overexpression. This results in activation of PKCδ, which phosphorylates STAT3, increasing its binding to the eNOS promoter. This in turn decreases eNOS promoter activity, protein levels, and NO production. Thus, ET-1 can reduce eNOS expression and NO generation in fetal pulmonary artery endothelial cells through PKCδ-mediated activation of STAT3.

Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (NO) and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small-molecular-weight compounds with related structures, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild-type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by nitric-oxide inhibitor N(omega)-nitro-l-arginine methyl ester, indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin and reversed eNOS uncoupling. The combination of an up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.

Angiotensin ii increases VEGF expression and peroxynitrite-dependent MMP-2 activation in the rat aorta: Preventive effect of red wine polyphenols

Statins have many anti-atherogenic effects apart from reducing the serum level of low density lipoprotein cholesterol (LDL-C). For instance, statins can enhance the expression of endothelial nitric oxide synthase (eNOS), at least partly by upregulating its transcription. Although it has been reported that -786 T/C polymorphism of the promoter region has an important influence on statininduced transcription of the human eNOS gene, much remains unclear about statin-induced eNOS transcription. We tried to identify other statin-responsive promoter regions. A human endothelial cell line (EA.hy926 cells) was treated with pitavastatin, atorvastatin, or fluvastatin, after which eNOS mRNA levels were assessed by quantitative real-time RT-PCR. EA.hy926 cells were also transiently transfected with luciferase reporter genes driven by various lengths of the human eNOS promoter and were treated with statins before luciferase activity was measured. Statin treatment increased eNOS mRNA levels in EA.hy926 cells. In addition, cells transfected with the reporter gene driven by the eNOS promoter fragment starting from position -740 exhibited a pitavastatin-induced increase of luciferase activity, which was not observed in cells transfected with the reporter gene driven by the fragment starting from -727. Similar results were also obtained with atorvastatin and fluvastatin. Statins enhanced eNOS expression in EA.hy926 cells, at least partly by inducing its transcription. Although a statin-responsive sequence that could function even in a heterologous promoter was not precisely identified, the region of the human eNOS promoter around position -730 seems to be critical for statin-induced transcriptional activation.

Animal and clinical studies have suggested that polyphenols in fruits, red wine, and tea may delay the development of atherosclerosis through their antioxidant and anti-inflammatory properties. We investigated whether individual dietary polyphenols representing different polyphenolic classes, namely quercetin (flavonol), (-)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan), or chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the apolipoprotein E (ApoE)(-/-) gene-knockout mouse. Quercetin and theaflavin (64-mg/kg body mass daily) significantly attenuated atherosclerotic lesion size in the aortic sinus and thoracic aorta (P<0.05 versus ApoE(-/-) control mice). Quercetin significantly reduced aortic F(2)-isoprostane, vascular superoxide, vascular leukotriene B(4), and plasma-sP-selectin concentrations; and augmented vascular endothelial NO synthase activity, heme oxygenase-1 protein, and urinary nitrate excretion (P<0.05 versus control ApoE(-/-) mice). Theaflavin showed similar, although less extensive, significant effects. Although (-)-epicatechin significantly reduced F(2)-isoprostane, superoxide, and endothelin-1 production (P<0.05 versus control ApoE(-/-) mice), it had no significant effect on lesion size. Sesamin and chlorogenic acid treatments exerted no significant effects. Quercetin, but not (-)-epicatechin, significantly increased the expression of heme oxygenase-1 protein in lesions versus ApoE(-/-) controls. Specific dietary polyphenols, in particular quercetin and theaflavin, may attenuate atherosclerosis in ApoE(-/-) gene-knockout mice by alleviating inflammation, improving NO bioavailability, and inducing heme oxygenase-1. These data suggest that the cardiovascular protection associated with diets rich in fruits, vegetables, and some beverages may in part be the result of flavonoids, such as quercetin.

Red wine represents a source of polyphenols which exhibit a number of biological effects on various systems. In this respect, there is evidence that red wine polyphenols constitute one of the ingredients of the Mediterranean diet which is associated to a reduced risk of coronary heart disease according to current literature. Here, we have evaluated in vitro the molecular mechanisms elicited by polyphenols from red wine (Negroamaro) on human healthy mononuclear cells. In particular, we have investigated the involvement of polyphenols in the activation of p38 and ERK1/2 molecules belonging to the MAPK kinase family and on the expression of I kappaB alpha and p65/NF kappaB. Results will demonstrate that in cells both the expression of p38 and ERK1/2 augments in the presence of red wine polyphenols, but their expression drops in the presence of polyphenols plus lipopolysaccharides (LPS). This indicates that in Gram-negative infections polyphenols may attenuate triggering of inflammatory mediators as a response to LPS stimulation. Finally, the regulatory role of polyphenols on I kappaB alpha and p65/NF kappaB expression is discussed, pointing out that red wine might favor anti-atherogenic mechanisms in the course of cardiovascular disease.

Wine polyphenols are considered to have beneficial effects on CHD and atherosclerosis. The consumption of red wine is high in Italy and France, approximately four times greater than that in the UK. This disparity in red wine consumption is thought to be the reason for the 'French paradox', where France was shown to have a coronary mortality rate close to that of China or Japan despite saturated fat intakes and cholesterol levels similar to the UK and USA. In the present review, we discuss the effects of wine and some of its polyphenol constituents on early pathological indicators of CHD such as plasma lipids, the endothelium and vasculature, platelets and serum antioxidant activity. The review also examines whether the polyphenols or the alcohol in wine is responsible for the effects on markers of heart disease. The present review concludes that red wine polyphenols have little effect on plasma lipid concentrations but wine consumption appears to reduce the susceptibility of LDL to oxidation and increase serum antioxidant capacity. However, these effects do depend on the amount of wine and period of supplementation. Authors who have examined specific polyphenols suggest that some phenolics appear to have endothelium-dependent vaso-relaxing abilities and some a positive effect on NO concentrations. Red wine phenolics also have an inhibitory effect on platelet aggregation, and individual phenolics also have a similar effect in vitro, although it should be noted that there are often discrepancies as large as ten-fold between the concentrations of polyphenolics tested in vitro and their measured levels in vivo. Evidence suggests that alcohol has a positive synergistic effect with wine polyphenols on some atherosclerotic risk factors. Thus evidence that wine drinking is beneficial for cardiac health continues to accumulate but more research is required to understand fully and exactly the functions of red wine polyphenols.

Understanding transcription initiation of the endothelial nitric-oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present study therefore focused upon a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of constitutive expression of the human eNOS gene in vascular endothelium. Two tightly clustered cis-regulatory regions were identified in the proximal enhancer of the TATA-less eNOS promoter using deletion analysis and linker-scanning mutagenesis: positive regulatory domains I (-104/-95 relative to transcription initiation) and II (-144/-115). Analysis of trans-factor binding and functional expression studies revealed a surprising degree of cooperativity and complexity. The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional domain studies in Drosophila Schneider cells and endothelial cells revealed examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ. Therefore, multiprotein complexes are formed on the activator recognition sites within this 50-base pair region of the human eNOS promoter in vascular endothelium.

While sympathetic stimulation of the heart produces chronotropic, inotropic, and lusitropic effects, increased frequency alone causes a positive force-frequency relationship (FFR) and frequency-dependent acceleration of relaxation (FDAR).1 That is, contraction amplitude and relaxation rate are increased with increasing frequency in most species (including humans). The key mechanism involved in the positive FFR is increased sarcoplasmic reticulum (SR) Ca2+ load, due to increased Ca2+ influx and decreased Ca2+ efflux.1,2 Ca2+ influx increases due to more L-type Ca2+ current ( I Ca) per unit time, while Ca2+ efflux via Na+-Ca2+ exchange (NCX) decreases because the diastolic time is reduced and [Na+]i increases. Enhanced SR Ca2+-pump function causes FDAR and also augments SR Ca2+ loading. Various signaling pathways are involved (eg, CaMKII).3 In human heart failure, the FFR reverses (ie, from positive to negative) due to an inability of the SR to increase Ca2+ content.4 This negative FFR is a main contributor to the loss of contractile reserve in the failing heart. Of many pathways that can modify FFR, nitric oxide (NO) signaling is the topic addressed by Khan et al in this issue of Circulation Research .5 NO synthase (NOS) produces NO from l-arginine, and cardiac myocytes express all three NOS isoforms.6,7 NOS1 (nNOS) and NOS3 (eNOS) are constitutively expressed and produce low amounts of NO (regulated by [Ca2+-calmodulin]i levels). NOS2 (iNOS) …

Simultaneous LC Determination of Quercetin, Kaempferol and Isorhamnetin in Rabbits after Intragastric Administration of an Ethanol Extract from Pollen Typhae

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

1. Nitric oxide (NO) synthase inhibitors and Paf antagonists abrogate hypotension in septic shock. The latter may act by blocking intracellular transduction mechanisms in vascular smooth muscle cells and inflammatory cells. We examined the effect of Paf antagonists on expression of inducible NO synthase. 2. A murine macrophage cell line (J774.2) and rat vascular smooth muscle cells (VSMC) were stimulated with lipopolysaccharide (LPS), either alone or in combination with Paf or Paf antagonists, BN 50739 or E-6123. 3. NO synthase activity in J774.2 was measured by the conversion of [3H]L-arginine to [3H]L-citrulline. Nitrite accumulation was measured in the culture medium of J774.2 and VSM. 4. BN 50739 (10 mumol/L and E-6123 (1 mumol#L) both reduced the expression of calcium-independent NO synthase activity and nitrite accumulation, while Paf alone had no effect. 5. Inhibition of NO synthase induction by Paf antagonists might afford therapeutic benefits in the management of septic shock and possibly other cardiovascular disorders.