Journal of Nutrition EB Program 2010 Early Registration

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanchez, M.
Right arrow Articles by Duarte, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanchez, M.
Right arrow Articles by Duarte, J.
© 2007 American Society for Nutrition J. Nutr. 137:910-915, April 2007

Biochemical, Molecular, and Genetic Mechanisms

Quercetin and Isorhamnetin Prevent Endothelial Dysfunction, Superoxide Production, and Overexpression of p47phox Induced by Angiotensin II in Rat Aorta1

Manuel Sanchez2, Federica Lodi3, Rocio Vera2, Inmaculada C. Villar2, Angel Cogolludo3, Rosario Jimenez2, Laura Moreno3, Miguel Romero2, Juan Tamargo3, Francisco Perez-Vizcaino3,* and Juan Duarte2

2 Department of Pharmacology, School of Pharmacy, Universidad de Granada, 18071 Granada, Spain and 3 Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain

* To whom correspondence should be addressed. E-mail: fperez{at}med.ucm.es.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The dietary flavonoid quercetin reduces blood pressure and improves endothelial function in several rat models of hypertension. We analyzed the effects of quercetin and its methylated metabolite isorhamnetin on the aortic endothelial dysfunction induced by incubation with angiotensin II (AngII) in vitro for 6 h. AngII diminished the relaxant responses to acetylcholine in phenylephrine-contracted aorta. Coincubation with quercetin or isorhamnetin, or addition of superoxide (O2–) dismutase or apocynin to the assay medium, prevented these inhibitory effects. At 6 h, AngII induced a marked increase in O2– production as measured by dihydroethidium fluorescence, which was prevented by quercetin and isorhamnetin. AngII also increased the expression of p47phox, a regulatory subunit of the membrane NADPH oxidase. Immunohistochemical analysis revealed that overexpression of p47phox occurred mainly in the medial layer. p47phox overexpression was also prevented by quercetin and isorhamnetin. Taken together, these results show for the first time, to our knowledge, that quercetin and isorhamnetin prevent AngII-induced endothelial dysfunction by inhibiting the overexpression of p47phox and the subsequent increased O2– production, resulting in increased nitric oxide bioavailability.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The flavonol quercetin is one of the most abundant polyphenolic compounds found in the human diet. Large epidemiological studies have shown an inverse association between dietary flavonol intake and mortality from coronary heart disease (1,2). Quercetin relaxes vascular smooth muscle (3) and its chronic daily treatment reduces blood pressure and endothelial dysfunction in spontaneously hypertensive rats (SHR)4 (4,5), in rats made hypertensive by chronic inhibition of nitric oxide (NO) synthase (6), or in renovascular hypertensive rats (7). In all these experimental models of hypertension, characterized by an activation of the renin-angiotensin-system, the protective effects exerted by quercetin were associated with a reduction of systemic and vascular oxidative status. However, the mechanisms involved are unclear.

Angiotensin II (AngII) increases arterial pressure, impairs endothelial function, induces vascular smooth muscle hypertrophy, and induces the expression of multiple vasoactive and inflammatory substances, playing a key role in the pathophysiology of cardiovascular diseases, including hypertension, atherosclerosis, and heart failure (8). A significant body of evidence supports a role for the intracellular production of reactive oxygen species (ROS), such as superoxide anion (O2–) and hydrogen peroxide, in the signal transduction of AngII (9–12). The major source of intracellular ROS in vascular cells is NADPH oxidase, a multisubunit enzymatic complex that comprises 2 membrane-bound subunits, Nox (Nox-1, Nox-2, also referred to as gp91phox, Nox-4 or Nox-5) and p22phox, which are regulated by cytoplasmic subunits such as p47phox, p67phox, and a low-molecular weight G protein (rac 2 or rac 1) (12). The translocation of cytosolic p47phox to the membrane is essential in the assembly process of this complex and plays a major role in NADPH oxidase activity in cardiovascular cells (13,14). AngII activates NADPH oxidases in vascular smooth muscle by inducing phosphorylation of p47phox and by de novo protein synthesis of p47phox and other NADPH oxidase subunits (8,9,15).

In this study, we investigated the effects of quercetin and its methylated plasma metabolite isorhamnetin on the AngII-induced endothelial dysfunction in vitro and its relation with the production of O2– and the expression of p47phox.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
All of the procedures conform to the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85–23, revised 1996) and were approved by our Institutional Committee for the ethical care of animals. Male Wistar rats were obtained from Harlan Laboratories.

    Tissue culture. The descending thoracic aortae were dissected and cut into rings. Rings were incubated in Krebs solution (composition in mmol/L: NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2; KH2PO4, 1.2; and glucose, 11) containing an antibiotic-antimycotic mixture (penicillin, gentamycin, and anfotericin B) for 2, 4, 6, or 8 h in a cell culture incubator in the absence or presence of AngII (1 µmol/L) and in the presence of vehicle [dimethylsulfoxide (DMSO) 0.1%], quercetin (1 or 10 µmol/L), isorhamnetin (1 or 10 µmol/L), or losartan (10 µmol/L). Aortae were immediately used for contractile tension recording, frozen in liquid nitrogen, and stored at –80°C for Western blots or included in embedding medium and then frozen in liquid nitrogen and stored at –80°C for immunohistochemistry or O2– production analysis.

    Contractile tension recording. Aortic rings, previously incubated as mentioned above, were mounted in organ chambers, as previously described (3,4). The chamber was filled with Krebs solution at 37°C and gassed with 95% O2 and 5% CO2. Rings were stretched to 2 g of tension and equilibrated for 90–120 min. The contraction was recorded using data acquisition hardware and software (REGXPC computer program from Cibertec). After equilibration, arteries were stimulated with 1 µmol/L phenylephrine and a concentration-response curve was constructed by cumulative addition of acetylcholine (ACh). In some experiments, polyethyleneglycol O2– dismutase (PEG-SOD, 100 kU/L) or apocynin (100 µmol/L) were added to the organ chamber 60 min before the addition of phenylephrine. Endothelium-independent responses to sodium nitroprusside were also performed in the dark in rings precontracted with 1 µmol/L phenylephrine.

    In situ detection of vascular O2– production. Unfixed aortic rings were cryopreserved by incubation with PBS (0.1mol/L) containing 30% sucrose for 1–2 h, included in OCT, frozen, and 10-µm cross sections were obtained in a cryostat (Microm International Model HM500 OM) (16). Sections were incubated in a humidified chamber for 30 min in HEPES-buffered solution (in mmol/L: NaCl, 130; KCl, 5; MgCl2, 1.2; glucose, 10; and HEPES, 10, pH 7.3 with NaOH) at 37°C. Afterwards, the sections were further incubated for 30 min in HEPES solution containing dihydroethidium (DHE) (10 µmol/L) in the dark, counterstained with the nuclear stain 4',6-diamidino,2-phenylindol (DAPI), and mounted with a coverslip. Four sections of each preparation were examined on a fluorescence microscope (Leica DM IRB), photographed with a Leica DC300F color digital camera, and images were saved for off-line analysis. Ethidium and DAPI fluorescence were quantified using ImageJ (version 1.32j, NIH, http://rsb.info.nih/ij/). O2– production was estimated from the ratio of ethidium/DAPI fluorescence (16). Negative controls were obtained in the absence of DHE.

    Vascular p47phox expression. Aortae were frozen and homogenated in 200 µL of a buffer of the following composition (mmol/L): HEPES, 10 (pH 8); KCl, 10; EDTA, 1; EGTA, 1; dithiothreitol, 1; aprotinin, 0.006; leupeptin, 0.009; N{alpha}-p-tosyl-l-lysine chloromethyl ketone, 0.011; NaF, 5; Na2MoO4, 10; NaVO4 and phenylmethanesulfonyl fluoride, 0.5 and centrifuged. Western blots were performed with 30 µg of protein of the supernatant per lane (5,17). Sodium dodecyl sulfate-polyacrylamide (10%) electrophoresis was performed in a mini-gel system (Bio-Rad Laboratories). The proteins were transferred to nitrocellulose membranes overnight and incubated with rabbit anti- p47phox polyclonal antibodies (1:200 dilution, SantaCruz Biotechnology). The membranes were then washed 5 times for 10 min in Tris-buffered saline containing 0.1% Tween 20 and incubated with secondary peroxidase conjugated goat anti-rabbit antibody (1:2000, Santa Cruz Biotechnology). Antibody binding was detected by an enhanced chemiluminesce system (Amersham Pharmacia Biotech). Films were scanned and densitometric analysis was performed on the scanned images using Scion Image-Release Beta 4.02 software (http://www.scioncorp.com). Samples were reprobed for expression of {alpha}-actin. p47phox protein abundance/{alpha}-actin ratio was calculated and data are expressed as a percentage of the values in control aorta from the same gel.

    Immunohistochemistry. Sections (10 µm) of aorta were prepared as described above for DHE fluorescence, fixed with paraformaldehyde 4% for 1 h, and washed with PBS 5 times. Sections were blocked with 0.1 mol/L PBS + 0.3% Tween 20 + 5% bovine serum albumin for 1 h at 37°C in a humidified chamber, incubated with rabbit anti-p47phox polyclonal antibodies (1:50 dilution, SantaCruz Biotechnology), then washed 6 times for 5 min in 0.1 mol/L PBS + 0.3% Tween 20, incubated in the dark with secondary Cy3 conjugated goat anti-rabbit antibody (1:200, Jackson Immunoresearch Laboratories) and washed again 5 times. Then preparations were counterstained with DAPI, examined in a confocal microscope, and photographed. Negative controls were obtained in the absence of primary antibody.

    Drugs. All drugs and reagents were from Sigma, except DAPI from Calbiochem and isorhamnetin from Extrasynthese. Quercetin aglycone and isorhamnetin were initially dissolved in DMSO and all other drugs in distilled water.

    Statistical analysis. Results are expressed as means ± SEM and n reflects the number of animals. Significant differences between groups were calculated by ANOVA followed by a Newman Keuls test. P < 0.05 was considered significant. Concentration-response curves were fitted to a logistic equation and from these plots the maximal relaxant effect (Emax) and the negative logarithm of the concentration producing half maximal relaxation (pD2) were calculated.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Endothelial dysfunction. Incubation of the aortic rings for up to 8 h in the absence of AngII produced no significant changes in the contractile response to phenylephrine or in the relaxant response to ACh (Fig. 1; Table 1). Incubation of the aortic rings for 2, 4, 6, or 8 h with AngII produced no significant changes in the contractile response to phenylephrine but led to a progressive development of endothelial dysfunction, as indicated by the reduction in the Emax of ACh (Fig. 1; Table 1). The inhibition was maximal at 6 h and, therefore, this time point was chosen for further experiments. Coincubation with the AngII receptor type 1 (AT1) antagonist losartan (10 µmol/L) prevented AngII-induced endothelial dysfunction (Fig. 2). In AngII pretreated aorta, PEG-SOD (a membrane-permeable form of SOD, 100 kU/L added to the organ bath) and the NADPH oxidase inhibitor apocynin (100 µmol/L added to the organ bath) increased the relaxant response to ACh (Fig. 2).


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		Figure 1  Time course of AngII-induced endothelial dysfunction in rat aortic rings. Effects of incubation for 2, 4, 6, or 8 h with or without AngII (1 µmol/L) on endothelium-dependent relaxation to ACh in aortic rings precontracted with 1 µmol/L phenylephrine. Results are means ± SEM. Different from control rings at that concentration, * P < 0.05, ** P < 0.01. Calculated Emax and pD2 values and n in each set are shown in Table 1.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1 Contractile responses to phenylephrine (1 µmol/L) and pD2 and Emax values for ACh-induced relaxation in rat aortic rings incubated with AngII (1 µmol/L) for 0–8 h12

 

Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		Figure 2  AT1-receptor antagonism, O2– scavenging, and inhibition of NADPH oxidase inhibit AngII-induced endothelial dysfunction in rat aortic rings. Aortic rings were incubated for 6 h with or without AngII (1 µmol/L) in the absence or presence of losartan(A) (10 µmol/L) and then mounted in organ baths or incubated for 6 h with or without AngII (1 µmol/L) and then mounted in organ baths in the absence or presence of PEG-SOD (B) (100 kU/L) or the NADPH oxidase inhibitor apocynin (C) (100 µmol/L) for 60 min. Rings were stimulated with 1 µmol/L phenylephrine and endothelium-dependent relaxations were induced by ACh. Results are means ± SEM of 5–10 experiments. *Different from other treatment groups at that concentration, P < 0.05.

 
Incubation with quercetin or isorhamnetin (1 or 10 µmol/L) for 6 h had no significant effect on the contractile response to phenylephrine in either control or AngII-treated arteries (Table 2). However, both flavonoids prevented AngII-induced endothelial dysfunction (Fig. 3; Table 2); i.e. ACh-induced relaxation was significantly increased by 1 and 10 µmol/L quercetin and by 10 µmol/L isorhamnetin. No differences were observed in the endothelium-independent relaxant responses to the endothelium-independent vasodilator sodium nitroprusside in arteries from vehicle-, AngII-, AngII-quercetin-, and AngII-isorhamnetin-treated rings (pD2 = 7.78 ± 0.10, n = 5; 7.78 ± 0.13, n = 7; 7.98 ± 0.12, n = 9; 7.97 ± 0.10, n = 9, respectively; and Emax = 100 ± 1.0%; 99.0 ± 1.1%; 99.3 ± 0.3%; 99.9 ± 0.2%, respectively).


View this table:
[in this window]
[in a new window]

  		TABLE 2 Contractile responses to phenylephrine (1 µmol/L) and pD2 and Emax values for ACh-induced relaxation in rat aortic rings incubated with AngII (1 µmol/L) for 6 h in the presence of vehicle (DMSO), quercetin, or isorhamnetin12

 

Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Figure 3  Quercetin and isorhamnetin prevent AngII-induced endothelial dysfunction in rat aortic rings. Aortic rings were incubated with or without quercetin (Quer, 1 or 10 µmol/L) or isorhamnetin (Iso, 1 or 10 µmol/L) and with or without AngII (1 µmol/L) for 6 h and then mounted in organ baths, stimulated with 1 µmol/L phenylephrine, then endothelium-dependent relaxation was induced by ACh. Results are means ± SEM. *Different from other treatment groups at that concentration, P < 0.05. Calculated Emax and pD2 values and n in each set are shown in Table 2.

 
    In situ localization of O2– production. To characterize O2– production and localize it within the vascular wall, ethidium red fluorescence was analyzed in sections of aorta incubated with DHE that is converted into ethidium by O2–-induced oxidation. Positive red nuclei could be observed in adventitial, medial, and endothelial cells (Fig. 4A). Red fluorescence was quantified and the data were normalized by the blue fluorescence of the nuclear stain DAPI (Fig. 4B). At 6 h, Ang II induced an increase in O2– production that was most evident in the medial layer of the vessel. Quercetin or isorhamnetin (10 µmol/L) did not modify O2– production in control arteries but significantly prevented AngII-induced increase.


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		Figure 4  Quercetin and isorhamnetin prevent AngII-induced O2– overproduction in rat aortic rings. Aortic rings were incubated with or without quercetin or isorhamnetin (10 µmol/L) and with or without AngII (1 µmol/L) for 6 h. (A) Left pictures show arteries incubated in the presence of DHE, which produces a red fluorescence when oxidized to ethidium by O2– and right pictures show blue fluorescence of the nuclear stain DAPI. Both types of images are merged with green elastin autofluorescence. Negative controls were obtained in the absence of DHE. (B) Values of red ethidium fluorescence normalized to blue DAPI fluorescence. Results are means ± SEM of 6–10 sections analyzed. Means without a common letter differ, P < 0.05.

 
    p47phox Protein expression and localization. In aortic segments, incubation with AngII for 6 h increased the expression of p47phox as measured by western blot (Fig. 5). Preliminary experiments showed that this increase was maximal at 6 or 8 h. Coincubation with quercetin or isorhamnetin had no effect by themselves but prevented AngII-induced increase. Immunohistochemical analysis revealed that overexpression of p47phox occurred mainly in the medial layer and was also prevented by quercetin and isorhamnetin (10 µmol/L) (Fig. 6).


Figure 5
View larger version (31K):
[in this window]
[in a new window]

  		Figure 5  Induction of p47phox by AngII in rat aortic rings is prevented by quercetin and isorhamnetin. Aortic rings were incubated with or without quercetin or isorhamnetin (10 µmol/L) and with or without AngII (1 µmol/L) for 6 h and protein expression was analyzed by western blot. Values are means ± SEM, n = 4, of densitometric values normalized to actin and expressed as a percentage of control. Means without a common letter differ, P < 0.05.

 

Figure 6
View larger version (78K):
[in this window]
[in a new window]

  		Figure 6  Immunohistochemical localization of p47phox in rat aortic rings Aortae were incubated for 6 h in the absence or presence of AngII (1 µmol/L), quercetin (10 µmol/L), or isorhamnetin (10 µmol/L). Pictures show p47phox protein (red fluorescence) merged with green elastin autofluorescence. Negative control in the absence of primary antibody is also shown. Pictures are representative of 10–12 sections from at least 3 different aortae.

 
    Role of PPAR{gamma}. Incubation with the PPAR{gamma} antagonist GW9662 (1 µmol/L) (18) did not modify ACh-induced relaxation in control arteries (pD2 = 6.88 ± 0.13 and Emax = 80 ± 3%, n = 8) and did not prevent AngII-induced endothelial dysfunction (pD2 = 6.78 ± 0.09 and Emax = 66.0 ± 6, n = 9). Moreover, quercetin and isorhamnetin (10 µmol/L) improvement of AngII-induced impaired ACh relaxation were unaffected by GW9662 (pD2 = 7.12 ± 0.18, n = 10; 7.43 ± 0.18, n = 10; and Emax = 92.6 ± 3.9%; 89.2 ± 3.2% in rings coincubated with quercetin in the presence or absence of GW9662, respectively; and pD2 = 7.04 ± 0.17, n = 10; 7.08 ± 0.16, n = 10; and Emax = 85.6 ± 4.5%; 86.5 ± 3.1% in rings coincubated with isorhamnetin in the presence or absence of GW9662, respectively).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
AngII is a well-known trigger for increased vascular oxidative stress and the subsequent O2– driven NO inactivation plays a major role in the genesis of clinical endothelial dysfunction and hypertension (9–12). In this study, we show that AngII-induced endothelial dysfunction in rat aortic rings in vitro can be prevented by the dietary flavonol quercetin and its methylated metabolite isorhamnetin. Moreover, the flavonols also prevented AngII-induced overexpression of p47phox in the vessel media and the increase in vascular O2– production.

Endothelial dysfunction is present in animals made hypertensive by infusion of AngII as well as in mice that chronically overexpress renin and angiotensinogen (9,19). The fact that AngII can also induce endothelial dysfunction in vitro in the mouse carotid artery (20) and rat aorta (this study) indicates that these changes are due to direct effects of AngII on the vessel wall, independent of circulating hormones, neurogenic mechanisms, or changes in arterial pressure. This alteration was suppressed by coincubation with the AT1 receptor antagonist losartan, demonstrating that endothelial dysfunction is mediated by activation of AT1 receptors. Chronic oral administration of quercetin reduces blood pressure and restores endothelial function in several animal models of hypertension, including SHR, NO-deficient, deoxycorticosterone acetate-salt, and Goldblatt hypertensive rats (4–7). However, it was unclear whether the effects on endothelial function were due to a direct effect on the vessel wall, secondary to the blood pressure-lowering effect, or driven by neurohumoral mechanisms of quercetin or its metabolites. Our results show that both quercetin and isorhamnetin are also effective in vitro, reducing endothelial dysfunction in aortic rings. Moreover, it should be noted that the effective concentration of quercetin was as low as 1 µmol/L, which is in the range achieved in plasma after a regular meal containing flavonoid (21), indicating that this effect appears to be physiologically relevant.

Excess of O2– generation is critically involved in the breakdown of NO associated to endothelial dysfunction in aortic rings from AngII-infused rats (9,22). In our experiments, the presence of SOD in the organ chamber restored the relaxant response induced by ACh in aortic rings exposed to AngII. Similarly, overexpression of SOD prevented, while downregulation of SOD potentiated, AngII-induced endothelial dysfunction (20). We also found that AngII increased intracellular O2– production, measured by ethidium red fluorescence, especially in the smooth muscle cells of the medial layer. Furthermore, quercetin and isorhamnetin diminished the increased O2– production in the smooth muscle cells. In addition, the endothelium-independent vasodilation induced by the soluble guanylyl cyclase activator nitroprusside was similar in control and AngII-treated rings and unaffected by the flavonoids, indicating that endothelial dysfunction is due to changes in endothelium-derived NO bioactivity rather than downstream effects on vascular smooth muscle.

The maximal effect of AngII was observed after 6–8 h of incubation even when AngII was absent during the challenge with ACh. Similarly, quercetin and isorhamnetin were present during the exposure to AngII but absent during the endothelial function test. These slow and persistent changes induced by AngII are consistent with the involvement of changes in gene expression. Several studies have shown that NADPH oxidase is critically involved in AngII-induced endothelial dysfunction (9,15). In fact, the NADPH oxidase inhibitor apocynin, which impedes the assembly of the p47phox and p67phox subunits within the membrane NADPH oxidase complex, diminished endothelial dysfunction in AngII-infused mice (23), SHR (5), and AngII-exposed aortic rings in vitro (our results). These results suggest that O2– generated by NADPH oxidase is also involved in the alteration of aortic endothelial function induced by in vitro incubation with AngII. The p47phox subunit of NADPH oxidase plays a pivotal role of in the vascular oxidant stress and blood pressure response in AngII-dependent hypertension (24,25). Moreover, AngII-induced changes in p47phox expression and phosphorylation have been widely analyzed in smooth muscle and endothelial cells in culture (12,13). In this study, in aortae incubated for 6 h with AngII, we found higher protein levels of this NADPH oxidase component, measured by western blot, than in control aortae. Immunohistochemical analysis revealed that overexpression of p47phox occurred mainly in the medial layer. This increased p47phox protein expression is consistent with the increased O2– production found in aortae stimulated by AngII. Coincubation with either quercetin or isorhamnetin decreased the levels of this protein in AngII-treated aortae and the immunohistochemical staining of p47phox without a significant effect in control rings. These results suggest that quercetin and its methylated metabolite decreased O2– production stimulated by AngII by downregulating the expression of the p47phox subunit of vascular NADPH oxidase. In a recent study, we demonstrated that in SHR, the improvement of endothelial function by chronic oral administration of quercetin is associated with a reduction in the NADPH oxidase activity and p47phox expression that is abnormally high in these animals as compared with normotensive Wistar-Kyoto rats (5). These results suggest that decreased NADPH oxidase derived O2– and, thus, diminished NO inactivation may be an important mechanism contributing to the prevention of endothelial dysfunction by quercetin, independently of its blood pressure-lowering properties.

Quercetin has been suggested to show agonistic effects on PPAR{gamma} (26). Because the PPAR{gamma} ligands reduce O2– generation stimulated by AngII in human coronary artery endothelial cells (27), we hypothesized that quercetin and isorhamnetin might also prevent endothelial dysfunction via activation of these receptors. However, the PPAR{gamma} antagonist GW9662 did not affect the effects induced by quercetin and isorhamnetin, suggesting that these protective effects are unrelated to PPAR{gamma} activation. Thus, 2 potential mechanisms might be involved in the effects of quercetin. First, ROS can activate its own production via increased expression of NADPH oxidase subunits (28). Thus, quercetin and isorhamnetin, via scavenging ROS, might inhibit this positive feedback mechanism. Additionally, several protein kinases (e.g. protein kinase C, mitogen-activated protein kinases, and Src) have been reported to be involved in AngII-induced activation of NADPH oxidase (29). Because quercetin and related flavanoids are broad protein kinase inhibitors (30), it might also be a possible role of protein kinase inhibition in preventing AngII-induced endothelial function.

Taken together, these results indicate that quercetin and isorhamnetin prevent AngII-induced endothelial dysfunction by inhibiting the overexpression of p47phox and the subsequent increased O2– production resulting in increased response to NO.


   FOOTNOTES
 
1 Supported by CICYT grants (AGL2004-06685 and SAF2005-03770) and by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (Red HERACLES RD06/0009). Back

4 Abbreviations used: ACh, acetylcholine; AngII, Angiotensin II; AT1, AngII receptor type 1; DAPI, 4',6-diamidino,2-phenylindol; DHE, dihydroethidium; DMSO, dimethylsulfoxide; Emax, maximal relaxant effect; NO, nitric oxide; O2–, superoxide; pD2, half maximal relaxation; PEG-SOD, polyethyleneglycol superoxide dismutase; ROS, reactive oxygen species; SHR, spontaneously hypertensive rats; SOD, superoxide dismutase. Back

Manuscript received 25 November 2006. Initial review completed 11 December 2006. Revision accepted 9 January 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet. 1993;342:1007–11.[Medline]

2. Huxley RR, Neil HA. The relation between dietary flavonol intake and coronary heart disease mortality: a meta-analysis of prospective cohort studies. Eur J Clin Nutr. 2003;57:904–8.[Medline]

3. Duarte J, Perez-Vizcaino F, Utrilla P, Jimenez J, Tamargo J, Zarzuelo A. Vasodilatory effects of flavonoids in rat aortic smooth muscle. Structure-activity relationships. Gen Pharmacol. 1993;24:857–62.[Medline]

4. Duarte J, Perez-Palencia R, Vargas F, Ocete MA, Perez-Vizcaino F, Zarzuelo A, Tamargo J. Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol. 2001;133:117–24.[Medline]

5. Sanchez M, Galisteo M, Vera R, Villar IC, Zarzuelo A, Tamargo J, Perez-Vizcaino F, Duarte J. Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats. J Hypertens. 2006;24:75–84.[Medline]

6. Duarte J, Jimenez R, O'Valle F, Galisteo M, Perez-Palencia R, Vargas F, Perez-Vizcaino F, Zarzuelo A, Tamargo J. Protective effects of the flavonoid quercetin in chronic nitric oxide deficient rats. J Hypertens. 2002;20:1843–54.[Medline]

7. Garcia-Saura MF, Galisteo M, Villar IC, Bermejo A, Zarzuelo A, Vargas F, Duarte J. Effects of chronic quercetin treatment in experimental renovascular hypertension. Mol Cell Biochem. 2005;270:147–55.[Medline]

8. Kim S, Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev. 2000;52:11–34.[Abstract/Free Full Text]

9. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation:contribution to alterations of vasomotor tone. J Clin Invest. 1996;97:1916–23.[Medline]

10. Berry C, Hamilton CA, Brosnan MJ, Magill FG, Berg GA, McMurray JJ, Dominiczak AF. Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation. 2000;101:2206–12.[Abstract/Free Full Text]

11. Didion SP, Faraci FM. Angiotensin II produces superoxide-mediated impairment of endothelial function in cerebral arterioles. Stroke. 2003;34:2038–42.[Abstract/Free Full Text]

12. Lyle AN, Griendling KK. Modulation of vascular smooth muscle signaling by reactive oxygen species. Physiology (Bethesda). 2006;21:269–80.[Medline]

13. Li JM, Shah AM. Mechanism of endothelial cell NADPH oxidase activation by angiotensin II. Role of the p47phox subunit. J Biol Chem. 2003;278:12094–100.[Abstract/Free Full Text]

14. Touyz RM, Yao G, Schiffrin EL. c-Src induces phosphorylation and translocation of p47phox: role in superoxide generation by angiotensin II in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2003;23:981–7.[Abstract/Free Full Text]

15. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res. 2002;90:1205–13.[Abstract/Free Full Text]

16. Lodi F, Cogolludo A, Duarte J, Moreno L, Coviello A, Peral De Bruno M, Vera R, Galisteo M, Jimenez R, et al. Increased NADPH oxidase activity mediates spontaneous aortic tone in genetically hypertensive rats. Eur J Pharmacol. 2006;544:97–103.[Medline]

17. Cogolludo A, Moreno L, Lodi F, Frazziano G, Cobeno L, Tamargo J, Perez-Vizcaino F. Serotonin inhibits voltage-gated K+ currents in pulmonary artery smooth muscle cells: role of 5-HT2A receptors, caveolin-1, and KV1.5 channel internalisation. Circ Res. 2006;98:931–8.[Abstract/Free Full Text]

18. Fu M, Zhang J, Zhu X, Myles DE, Willson TM, Liu X, Chen YE. Peroxisome proliferator-activated receptor gamma inhibits transforming growth factor beta-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3. J Biol Chem. 2001;276:45888–94.[Abstract/Free Full Text]

19. Didion SP, Sigmund CD, Faraci FM. Impaired endothelial function in transgenic mice expressing both human renin and human angiotensinogen. Stroke. 2000;31:760–4.[Abstract/Free Full Text]

20. Didion SP, Kinzenbaw DA, Faraci FM. Critical role for cuzn-superoxide dismutase in preventing angiotensin II-induced endothelial dysfunction. Hypertension. 2005;46:1147–53.[Abstract/Free Full Text]

21. Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr. 2005;81 Suppl 1:S230–42.

22. Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS. Effects of ANG II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expression. Am J Physiol Regul Integr Comp Physiol. 2003;285:R117–24.[Abstract/Free Full Text]

23. Virdis A, Neves MF, Amiri F, Touyz RM, Schiffrin EL. Role of NAD(P)H oxidase on vascular alterations in angiotensin II-infused mice. J Hypertens. 2004;22:535–42.[Medline]

24. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003;111:1201–9.[Medline]

25. Li JM, Wheatcroft S, Fan LM, Kearney MT, Shah AM. Opposing roles of p47phox in basal versus angiotensin II-stimulated alterations in vascular O2– production, vascular tone, and mitogen-activated protein kinase activation. Circulation. 2004;109:1307–13.[Abstract/Free Full Text]

26. Liang YC, Tsai SH, Tsai DC, Lin-Shiau SY, Lin LK. Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferator-activated receptor-gamma by flavonoids in mouse macrophages. FEBS Lett. 2001;496:12–8.[Medline]

27. Mehta JL, Hu B, Chen J, Li D. Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation. Arterioscler Thromb Vasc Biol. 2003;23:2203–8.[Abstract/Free Full Text]

28. Cai H. NADPH oxidase-dependent self-propagation of hydrogen peroxide and vascular disease. Circ Res. 2005;96:818–22.[Abstract/Free Full Text]

29. Lassegue B, Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003;285:R277–97.[Abstract/Free Full Text]

30. Middleton E Jr, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 2000;52:673–751.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
F. Jiang, N. Guo, and G. J. Dusting
Modulation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Expression and Function by 3',4'-Dihydroxyflavonol in Phagocytic and Vascular Cells
J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 261 - 269.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanchez, M.
Right arrow Articles by Duarte, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanchez, M.
Right arrow Articles by Duarte, J.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Copyright © 2007 by American Society for Nutrition