Donate Help Contact The AHA Sign In Home
American Heart Association
Hypertension
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Hypertension. 2003;42:1198-1205
Published online before print November 17, 2003, doi: 10.1161/01.HYP.0000103162.76220.51
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
42/6/1198    most recent
01.HYP.0000103162.76220.51v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, J.-C.
Right arrow Articles by Cheng, T.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, J.-C.
Right arrow Articles by Cheng, T.-H.
Related Collections
Right arrow Cardiovascular Pharmacology

(Hypertension. 2003;42:1198.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Inhibition of Cyclic Strain-Induced Endothelin-1 Gene Expression by Resveratrol

Ju-Chi Liu; Jin-Jer Chen; Paul Chan; Ching-Feng Cheng; Tzu-Hurng Cheng

From the Department of Medicine, Taipei Medical University, Wan Fang Hospital (J.-C.L., P.C., T.-H.C.), Taipei; the Institute of Biomedical Sciences, Academia Sinica (J.-J.C, C.-F.C., T.-H.C.), Taipei; the Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine (J.-J.C.), Taipei; and the Department of Pharmacology, National Defense Medical Center (T.-H.C.), Taipei, Taiwan, Republic of China.

Correspondence to Tzu-Hurng Cheng, PhD, Division of Cardiology, Department of Medicine, Taipei Medical University-Wan Fang Hospital, No. 111, Hsing Lung Road, Section 3, Wen-Shan District, Taipei 117, Taiwan. E-mail thcheng{at}gate.sinica.edu.tw


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Resveratrol is a phytoestrogen naturally found in grapes and is among the major constituents of wine thought to have a cardioprotective effect. Endothelin-1 (ET-1) is a potent vasopressor synthesized by endothelial cells both in culture and in vivo. The aims of this study were to test the hypothesis that resveratrol may alter strain-induced ET-1 gene expression and to identify the putative underlying signaling pathways in endothelial cells. We show that resveratrol indeed potently inhibits strain-induced ET-1 secretion, ET-1 mRNA level, and ET-1 promoter activity. Resveratrol also inhibits strain-increased NADPH oxidase activity, reactive oxygen species formation, and extracellular signal–regulated kinases1/2 (ERK1/2) phosphorylation. Furthermore, pretreating cells with resveratrol or antioxidant N-acetyl-cysteine decreases strain-increased or hydrogen peroxide–increased ET-1 secretion, ET-1 promoter activity, and ET-1 mRNA and ERK1/2 phosphorylation. Using both the electrophoretic mobility shift assay and a reporter gene assay, resveratrol and N-acetyl-cysteine also attenuated the strain-stimulated activator protein-1 binding activity and activator protein-1 reporter activity. In summary, we demonstrate for the first time that resveratrol inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK1/2 pathway through attenuation of reactive oxygen species formation. Thus, this study provides important new insights in the molecular pathways that may contribute to the proposed beneficial effects of resveratrol in the cardiovascular system.


Key Words: endothelin • gene expression • atherosclerosis • endothelium • oxygen • kinase


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Many epidemiological studies show a correlation between a low incidence of coronary heart disease and atherosclerosis and a moderate consumption of red wine.1,2 The vasoprotective effect of red wine, also known as the "French paradox," is currently best exemplified by trans-resveratrol (trans-3,5,4'-hydroxystilbene).3,4 Resveratrol has many biological activities, including protection from or reduction of the incidence of coronary heart disease. Resveratrol also has been found to protect the heart from ischemia-reperfusion injury.5 Antioxidant properties of resveratrol appear to be partly responsible for this activity.5–9 Moreover, resveratrol was shown to relax aortic rings in rats through an endothelium-mediated enhancement of the nitric oxide (NO)-cGMP cascade.10 Subsequent pharmacological studies indicate a direct relaxant effect of resveratrol on vascular smooth muscle that may exert beneficial effects in cardiovascular disease, though the mechanism of these effects is no known.11 Recently, Orallo et al12 demonstrated that the characteristic endothelium-dependent vasorelaxant effect of resveratrol in the rat aorta appeared to be caused by the inhibition of vascular NADH/NADPH oxidase and the subsequent decrease in the generation of basal cellular superoxide anions and, therefore, of NO biotransformation. However, little is known about the cellular/molecular mechanisms whereby resveratrol could protect against coronary heart disease. Indeed, whether resveratrol could inhibit the production of endogenous vasoconstrictors and thereby regulate vasomotion remains an intriguing possibility.

Among the earliest indications of vascular dysfunction in atherosclerosis is an impaired regulation of vasomotion, representing disturbed homeostasis of vascular cells.13 Key regulators of vasomotor function are the vasodilator NO and the vasoconstrictor endothelin-1 (ET-1). Among the endogenous mediators of cardiovascular disorders, ET-1, a 21–amino acid peptide, is a primary antecedent in coronary heart disease.14–17 Such effects are mediated by extremely potent vasopressors and mitogenic responses for ET-1 in the vasculature.18,19 Results from preclinical studies in humans as well as animals studies showed that plasma ET-1 levels are consistently elevated in many spasm-related cardiovascular diseases19,20 and that blockers for ET receptors can substantially alleviate complications of such diseases.20,21

ET-1 was originally isolated from a culture of porcine endothelial cells.21 Endothelial cells are constantly under the influence of mechanical forces, including cyclic strain, as a consequence of vessel contraction and relaxation. Recently, numerous studies have shown that oxidative stress, represented by reactive oxygen species (ROS), is capable of significantly altering vascular function.22–24 Thus, oxidative stress appears to be causally linked to the pathogenesis of atherosclerosis.22–24 Previous studies demonstrated that intracellular ROS levels are elevated in endothelial cells after cyclic strain treatment.25,26 Our previous study demonstrated that intracellular ROS mediate cyclic strain-induced ET-1 expression through the Ras/Raf/extracellular signal–regulated kinases1/2 (ERK1/2) signaling pathway.27 However, no studies exist that address the interference of resveratrol on ET-1 gene induction in vascular endothelial cells.

The present study was undertaken to identify potential signaling components underlying the protective actions of resveratrol in the vascular system. Because of the pathophysiological influence of endothelin on the cardiovascular system, the aims of this study were to investigate the effect of resveratrol on the strain-induced ET-1 gene expression and to identify signaling protein kinase cascades that may be responsible for the putative effect of resveratrol.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Imubind ET-1 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biochemica (ENDOTHELIN, Biomedica). ET-1 cDNA was obtained from a human endothelial cell cDNA library, as described previously.28 A full length of the ET-1 promoter region (4.4 kb) was fused to the chloramphenicol acetyltransferase (CAT) reporter gene.27 PBLCAT2 (containing CAT reporter gene with its promoter) and PBLCAT3 (containing CAT gene only) were constructed as described previously.29 2',7'-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Serva Co. Hydrogen peroxide (H2O2) was purchased from Acros Organics. Resveratrol, N-acetyl-cysteine (NAC), and all other chemicals of reagent grade were obtained from Sigma.

Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords, as described previously.27 All procedures involving human samples were conducted according to the Guidelines for Animal and Human Experimentation of Taipei Medical University. The transformed human cell line, ECV304 (ATCC CRL-1998), was purchased from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) before they were subcultured.

In Vitro Cyclic Strain on Cultured Endothelial Cells
Endothelial cells cultured on the flexible membrane base were subjected to cyclic strain produced by a computer-controlled application of sinusoidal negative pressure, as described previously.27

Measurement of ET-1 Concentration
ET-1 concentrations in culture media were assayed with Imubind-ET-1 ELISA kits (Biomedica), as described previously.27

Northern Blot Analysis
Preparation of total RNA and Northern blot analyses of ET-1 and 18S RNA were performed as described previously.27

Transfections
ECV304 were transiently transfected with different expression vectors by the calcium phosphate method, as described previously.27

CAT and ß-Galactosidase Assays
The CAT and ß-galactosidase assays were performed as described previously.27

NADPH Oxidase Activity Assay
NADPH oxidase was measured in HUVECs, as described previously.30

Detection of Intracellular ROS
Measurement of intracellular ROS formation in HUVECs was recorded by monitoring changes in dichlorofluorescein (DCF) fluorescence, as described previously.27 Chemiluminescence assay of superoxide production was measured as described previously.25

Western Blot Analysis
Rabbit polyclonal antiphosphospecific extracellular signal–regulated kinases1/2 (ERK1/2) antibody was purchased from New England Biolabs. Anti-ERK antibody was purchased from Santa Cruz Biotechnology. Western blot analysis was performed as described previously.27

Electrophoretic Mobility Shift Assay
The electrophoretic mobility shift assay was performed as described previously.31

Luciferase Assay
ECV304 plated on 3-cm-diameter culture dishes were transfected with the luciferase reporter construct having consensus AP-1 (AP-1-Luc) binding sites (Stratagene). After incubation for 24 hours in 2% serum DMEM, ECV304 were cultured under different treatments, as indicated for 48 hours. ECV304 were assayed for luciferase activity with a luciferase reporter assay kit (Stratagene). The firefly luciferase activities at AP-1 transcriptional activity were normalized for transfection efficiency to its respective ß-galactosidase activity and expressed as relative activity to control.

Statistical Analysis
Data are presented as mean±SEM. Statistical analysis was performed with the use of the Student t test and ANOVA, followed by a Dunnett multiple comparison test, with Prism version 3.00 for Windows (GraphPad Software). Probability values <0.05 were considered to be statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effect of Resveratrol on Strain-Induced ET-1 Gene Expression in Endothelial Cells
HUVECs cultured on flexible membrane bases were subjected to deformation to produce an average strain of 20%. ET-1 released into the culture media was measured. HUVECs under cyclic strain for 24 hours increased their ET-1 secretion into the culture medium (Figure 1A) 2-fold compared with unstrained endothelial cells. We then examined the effect of resveratrol on strain-increased ET-1 secretion. As shown in Figure 1B, treatment with cyclic strain for 24 hours increased ET-1 secretion. Resveratrol (1 to 100 µmol/L) significantly inhibited strain-increased ET-1 secretion (Figure 1B). These data indicate that resveratrol inhibits strain-increased ET-1 secretion in endothelial cells.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Resveratrol inhibits strain-induced ET-1 secretion. A, Increased ET-1 secretion from HUVECs subjected to cyclic strain. Culture media containing ET-1 from cells treated or not treated with cyclic strain (60 Hz; 20%) for various times were collected and analyzed by enzyme immunoassay. B, Resveratrol inhibits strain-induced ET-1 release. HUVECs were pretreated with resveratrol (0.1 to 100 µmol/L) 30 minutes before strain treatment. After strain (20%) for 24 hours, culture media were collected and concentrations were analyzed by enzyme immunoassay. Results are presented as mean±SEM (n=6). *P<0.05 vs unstrained control cells, #P<0.05 vs strained cells (ANOVA).

To determine whether resveratrol inhibits the strain-induced ET-1 gene expression, HUVECs were pretreated with resveratrol (1 to 100 µmol/L) for 30 minutes followed by 6 hours of cyclic strain. HUVECs under cyclic strain for 6 hours showed a 3.7-fold increase in ET-1 mRNA level compared with unstrained controls (Figure 2A). Resveratrol (1 to 100 µmol/L) pretreatment significantly reduced the strain-induced ET-1 expression. To further investigate whether the effect of resveratrol on strain-induced ET-1 gene expression is a transcriptional event, a plasmid containing an ET-1 upstream sequence in a CAT reporter construct (-4.4 kCAT) was cotransfected with pSV-ß'-galactosidase into ECV304. The cell extract was subsequently used to determine CAT activity. As shown in Figure 2B, increased ET-1 promoter activity was observed in endothelial cells under cyclic strain for 24 hours. In endothelial cells pretreated with resveratrol (1 to 100 µmol/L), the strain-induced ET-1 promoter activity was significantly reduced (P<0.05) (Figure 2B). To verify that resveratrol-mediated effects on vascular endothelial cells are not a consequence of cytotoxicity, we determined lactate dehydrogenase (LDH) activity as a parameter of necrosis in supernatants of resveratrol (1 to 100 µmol/L) and strain-treated HUVECs. Consistent with light microscopic observations, LDH activity in supernatants of treated and untreated cells remained unchanged (data not shown).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Resveratrol inhibits strain-induced ET-1 gene expression in endothelial cells. A, Inhibition of strain-induced ET-1 mRNA by resveratrol. HUVECs were either untreated controls (C, lane 1), or treated with different concentrations of resveratrol (1 to 100 µmol/L) in the absence (lanes 2 to 4) or presence of strain treatment (lanes 6 to 8). Total RNA was extracted and Northern hybridization was performed with 32P-labeled ET-1 as probe. 18S RNA was used to normalize the RNA applied in each lane. Data are presented as percentage changes of experimental groups compared with untreated controls. B, Resveratrol inhibits strain-induced ET-1 promoter activity. ECV304 cells were pretreated with resveratrol (1 to 100 µmol/L) for 30 minutes and then treated with cyclic strain for 24 hours. C (control), no drugs; CAT2 and CAT3 are shown as positive and negative control. CAT activities after normalizing to that of {alpha}-galactosidase activities are shown as relative activity compared with control groups. Results are shown as mean±SEM (n=6). *P<0.05 vs control, #P<0.05 vs strain.

Resveratrol Inhibits Strain-Increased NADPH Oxidase Activity and ROS Formation
Previous studies showed that cyclic strain increases NADPH oxidase activity30,32 and ROS formation in endothelial cells,25,26,30 which is involved in ET-1 induction.27 We next examined whether resveratrol prevents the strain-increased NADPH oxidase activity and ROS formation. HUVECs were treated with resveratrol (0.1 to 100 µmol/L) in the absence or presence of strain treatment. The addition of resveratrol (1 to 100 µmol/L) to cultured HUVECs significantly inhibited strain-induced NADPH oxidase activity and ROS formation as measured after strain treatment for 1 hour (Figure 3, A through C). The pretreatment of resveratrol (100 µmol/L) or NAC (10 mmol/L) to cultured HUVECs also significantly inhibited strain- or H2O2-induced ROS formation (Figure 3D). These findings clearly demonstrate that resveratrol inhibits strain-increased NADPH oxidase activity and intracellular ROS levels in endothelial cells.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Effects of resveratrol on strain-increased NADPH oxidase activity and ROS formation. A, Effect of resveratrol (0.1 to 100 µmol/L) on strain-increased NADPH oxidase activity. HUVECs after cyclic strain were lysed and immediately followed with NADPH oxidase activity assay. B, Effect of resveratrol (0.1 to 100 µmol/L) on strain-induced superoxide formation. HUVECs after cyclic strain were lysed and immediately followed with superoxide assay by lucigenin method. C, Effect of resveratrol (0.1 to 100 µmol/L) on strain-induced ROS generation. Strain-increased intracellular ROS levels were revealed by fluorescent intensities of DCF. D, HUVECs from either control (C; column 1) or treated with cyclic strain or H2O2 (25 µmol/L) in the presence of 100 µmol/L resveratrol, 10 mmol/L NAC for 1 hour. Fluorescence intensities of cells are shown as relative intensity of experimental groups compared with untreated control cells. Results are shown as mean±SEM (n=6). *P<0.05 vs control, #P<0.05 vs strain-treated (or H2O2-treated) cells (ANOVA).

Resveratrol Inhibits Strain-Activated ERK1/2 Phosphorylation in Endothelial Cells
To gain insight into the mechanism of action of resveratrol, we thus examined whether resveratrol affects intracellular protein kinase signaling pathways. Given that the ERK1/2 signaling pathway is involved in strain-induced ET-1 expression,27 we further investigated whether resveratrol inhibits the ERK1/2 pathway in strain-treated endothelial cells. We examined the phosphorylation of ERK1/2 in HUVECs exposed to resveratrol (1 to 100 µmol/L) in the absence or presence of strain treatment. As shown in Figure 4A, HUVEC exposure to strain treatment for 30 minutes rapidly activated phosphorylation of ERK1/2. However, HUVECs pretreated with resveratrol (1 to 100 µmol/L) showed significantly decreased strain-induced ERK1/2 phosphorylation. Moreover, HUVECs treated with H2O2 (25 µmol/L) showed increased ERK1/2 phosphorylation (Figure 4B). HUVECs pretreated with resveratrol (100 µmol/L) or NAC (10 mmol/L) showed significantly decreased strain- or H2O2-induced ERK1/2 phosphorylation. These findings imply that resveratrol inhibits the strain-activated ERK1/2 signaling pathway through attenuation of ROS formation in endothelial cells.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 4. Inhibitory effects of resveratrol on strain-increased ERK1/2 phosphorylation. A, Effect of resveratrol (1 to 100 µmol/L) on strain-activated ERK1/2 phosphorylation. B, Effect of resveratrol on strain- or H2O2-induced phosphorylation of ERK1/2. HUVECs were preincubated with either resveratrol (100 µmol/L) or NAC (10 mmol/L) for 30 minutes and stimulated with cyclic strain or H2O2 (25 µmol/L) for 30 minutes. Phosphorylation of ERK1/2 was detected by Western blotting, with anti-phospho-ERK1/2 used as antibody. Both resveratrol and NAC inhibited strain-induced activation of ERK1/2. Phosphorylation of ERK1/2 was detected and densitometric analyses were performed. White or black columns denote degree of ERK1 or ERK2 phosphorylation, respectively. Data are represented as fold increase relative to control groups. Results are shown as mean±SEM (n=6). *P<0.05 vs control, #P<0.05 vs strain (or H2O2) alone (ANOVA).

Resveratrol Inhibits Strain-Increased AP-1 Transcriptional Activity in Endothelial Cells
We next evaluated the effect of resveratrol on AP-1 activation, which is involved in ET-1 gene induction.27,31 With the use of the electrophoretic mobility shift assay, AP-1 binding to the consensus AP-1 binding sequence was assayed in HUVECs with strain treatment for 6 hours (Figure 5A). Pretreating HUVECs with resveratrol or NAC attenuated the strain-stimulated AP-1 binding activity. The effects of resveratrol on strain-induced AP-1 functional activity were also assessed in a reporter gene assay. Cyclic strain had no effect on luciferase activity of the background vector containing no AP-1 binding site (Figure 5B). On the contrary, cyclic strain significantly increased AP-1–luciferase activities in a time-dependent manner (Figure 5B). These results indicate that cyclic strain increases AP-1 transcriptional activity. We next examined the effects of resveratrol and NAC on strain-increased AP-1 transcriptional activity. Either resveratrol (10 µmol/L) or NAC (10 mmol/L) significantly attenuated strain- or H2O2-induced AP-1 reporter activation (Figure 5C). These results indicate that resveratrol inhibits strain-increased AP-1 transcriptional activation.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 5. Resveratrol attenuates strain-stimulated AP-1 binding activity in endothelial cells. A, Resveratrol or NAC attenuated strain-stimulated AP-1 binding activity in HUVECs. This binding activity was measured by using electrophoretic mobility shift assay. Cells were preincubated with resveratrol (1, 100 µmol/L) or NAC (10 mmol/L) for 30 minutes after incubation with strain treatment for 6 hours. C-Fos Ab denotes the anti–c-Fos antibody; 100xCold denotes 100-fold molar excess of unlabeled oligonucleotide relative to radiolabeled probe; this was added to the binding assay for competition with the unlabeled oligonucleotide. The experiment was repeated 2 times, with reproducible results. B, ECV304, transfected with empty vector or AP-1-Luc, was treated with cyclic strain under different time periods as indicated. Luciferase activity was expressed as relative activity to untreated control (C). C, ECV304, transfected with AP-1-Luc, was incubated for 24 hours with either no drug, 100 µmol/L resveratrol, or 10 mmol/L NAC in the absence or presence of strain treatment or H2O2 (25 µmol/L). Values are mean ± SEM of data for 6 experiments performed in triplicate. *P<0.05 vs untreated, #P<0.05 vs strain (or H2O2) alone (ANOVA).

Resveratrol Inhibits Strain-Induced ET-1 Expression, Which Is Compatible With Antioxidant Action
To further determine whether resveratrol affects the strain-induced ET-1 gene expression through attenuation of ROS formation, the effects of resveratrol on ET-1 gene induction were examined under cyclic strain or H2O2 stimulation. As demonstrated in Figure 6, either resveratrol or NAC alone had no effect on the basal ET-1 mRNA level (Figures 6A and 6B). However, endothelial cells treated with strain or H2O2 (25 µmol/L) significantly increased ET-1 mRNA and promoter activity (Figures 6A and 6B). In the presence of resveratrol (or NAC), strain- or H2O2-increased ET-1 mRNA and promoter activity were also significantly inhibited. Furthermore, either resveratrol or NAC alone had no effect on the basal ET-1 secretion. However, HUVECs treated with either strain or H2O2 (25 µmol/L) for 24 hours significantly increased ET-1 secretion (Figure 6C). In the presence of resveratrol (or NAC), both strain- and H2O2-increased ET-1 secretion were significantly inhibited. These data imply that resveratrol affects the strain-induced ET-1 gene expression, which is compatible with antioxidant action in endothelial cells.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Resveratrol modulates strain-induced ET-1 gene expression, which is compatible with antioxidant action. A, Resveratrol modulates strain-increased (or H2O2-increased) ET-1 mRNA. B, Resveratrol modulates strain-increased (or H2O2-increased) ET-1 promoter activity. Some cells were pretreated with resveratrol or NAC for 30 minutes. Endothelial cells were then treated with cyclic strain or H2O2 (25 µmol/L) for 24 hours. CAT2 (lane 10) and CAT3 (lane 11) are shown as positive and negative controls of CAT assay system. C, Resveratrol modulates strain-increased (or H2O2-increased) ET-1 secretion. Results are shown as mean±SEM (n=6). *P<0.05 vs control, #P<0.05 vs strain (or H2O2) alone (ANOVA).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major new finding of this work is that resveratrol inhibits strain-induced ET-1 gene expression in endothelial cells. It is supported by the observations that resveratrol inhibits strain-induced ET-1 protein secretion, mRNA level, and promoter activity in part through attenuation of ROS formation in endothelial cells. Previous studies, including ours, have indicated that hemodynamic forces, including shear flow33 and pressure-induced strain,27,34 can stimulate ET-1 gene expression. Recent studies provide evidence that ROS may act as second messengers in cells exposed to various stimuli.27,31 Previous studies from our collaborate laboratory and others have shown that cyclic-strain treatment of endothelial cells can induce intracellular ROS generation.25,26,32 This ROS generation is sustained at an elevated level as long as the mechanical forces are maintained and returns to basal levels with the removal of the forces. Matsushita et al32 further demonstrated a pivotal role for NADPH oxidase in cyclic strain–induced endothelial ROS production. Elevated ROS levels are involved in the release of ET-1,27 and this gene induction can be attenuated by antioxidant pretreatment of cells.

Studies have has shown that red wines strongly inhibit the synthesis of ET-1 and implicate that components specific to red wine may help to prevent coronary heart disease.35,36 Resveratrol is a major component of the polyphenols from grapes and red wine. A number of studies have demonstrated the antioxidant effects of resveratrol.37,38 This amphipathic molecule is capable of scavenging lipid hydroperoxyl free radicals as well as hydroxyl and superoxide anion radicals.5,39 Resveratrol was found to protect the kidney, heart, and brains from ischemia-reperfusion injury through its antioxidant ability.5,39,40 Moreover, resveratrol is a lipophilic substance and has been shown to accumulate in tissues such as the heart, liver, and kidney.41 Therefore, Bertelli et al41 concluded that an average drinker of wine can, particularly in the long term, absorb a sufficient quantity of resveratrol to explain the beneficial effect of red wine on health. Indeed, the results of our present study demonstrated that resveratrol reduces the strain-increased NADPH oxidase activity and ROS formation in endothelial cells, suggesting a reduction in the generation of intracellular ROS caused by strain treatment. In particular, it has been demonstrated that activation of ERK1/2 is redox-sensitive27,42,43 and that suppression of ROS inhibits strain-induced ET-1 gene expression.27 In porcine coronary arteries, short-term treatment with resveratrol substantially inhibited mitogen-activated protein kinase activity and reduction in the phosphorylation of ERK1/2.44 Recently, Haider et al45 demonstrated that resveratrol inhibits angiotensin II–induced vascular smooth muscle cell hypertrophy, possibly by interfering mainly with the ERK1/2 signaling pathway. One possible explanation for the inhibitory effect of resveratrol on strain-induced ET-1 gene expression may be its ability to attenuate ROS formation. In our previous study, deletion mapping revealed constructs containing 143 bp of the ET-1 5'-flanking sequence, allowing strain-induced transcription, and the presence of responsiveness elements for ROS- or strain-induced ET-1 expression located within 143 bp upstream of the transcription initiation site.27 Recently, we also found that the activation of AP-1 is redox-sensitive and might play a key role in ET-1 gene induction.31 Our present results indicate that resveratrol inhibits strain-induced AP-1 transcriptional activity. The inhibitory effect of resveratrol on strain-induced AP-1 transcriptional activation suggested that the attenuation of strain-induced ROS by resveratrol leads to inhibition of AP-1. Alternatively, resveratrol may inhibit strain-induced ET-1 gene expression by virtue of its increasing NO production.46 Resveratrol has been suggested to increase endothelial NO synthase gene expression in endothelial cells.46 Indeed, NO, acting through soluble guanylyl cyclase and cGMP formation, is a negative regulator of ET-1 gene induction.47–49 Thus, further experiments will be necessary to identify the mechanisms of the involvement of NO by which resveratrol exerts its inhibitory effects on strain-induced ET-1 gene expression.

The resveratrol-induced suppression of cyclic strain–induced ET-1 expression, as observed presently in cultures, if it does occur in vivo, for example, as a result of consumption of this agent, may have several consequences; that is, it is known that ET-1 is a primary antecedent in coronary heart disease and atherosclerosis.14–17 If indeed resveratrol consumed as a constituent of red wine is responsible for lowering the incidence of coronary heart disease and atherosclerosis, this may be one of its possible mechanisms of action. The effects of resveratrol on endothelial cells observed in the present study, for example, downregulation of ET-1 expression, suppression of ROS formation, and inhibition of ERK1/2 phosphorylation, all are compatible with its putative cardioprotective and vasoprotective effects.

Perspectives
Within the past decade, it has been repeatedly suggested that oxidative stress is related to and a cause of endothelial dysfunction in atherosclerosis. The present study delivers important new insights to the molecular mechanisms of action of resveratrol in endothelial cells. Our results for the first time clearly show that resveratrol remarkably influences important cyclic strain–activated pathways in endothelial cells. Moreover, they suggest that resveratrol acts partially by interfering with the ERK1/2 pathway to reduce strain-induced ET-1 gene expression. It is plausible that the strain-activated signaling pathway consists of redox-sensitive steps and that resveratrol treatment could modulate the redox state of the cell. In summary, our data show that resveratrol inhibits strain-induced ET-1 gene expression in part through attenuation of ROS formation and the suppression of the ERK1/2 pathway in endothelial cells. These findings support the proposed beneficial effects of resveratrol in the cardiovascular system.


*    Acknowledgments
 
This work was supported in part by National Science Council Grants (NSC 92-2314-B-038-051-; NSC 92-2314-B-038-052-), Taiwan, Republic of China.

Received June 30, 2003; first decision July 22, 2003; accepted October 15, 2003.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Mukamal KJ, Conigrave KM, Mittleman MA, Camargo CA Jr, Stampfer MJ, Willett WC, Rimm EB. Roles of drinking pattern and type of alcohol consumed in coronary heart disease in men. N Engl J Med. 2003; 348: 109–118.[Abstract/Free Full Text]
  2. German JB, Walzem RL. The health benefits of wine. Annu Rev Nutr. 2000; 20: 561–593.[CrossRef][Medline] [Order article via Infotrieve]
  3. Sato M, Maulik N, Das DK. Cardioprotection with alcohol: role of both alcohol and polyphenolic antioxidants. Ann N Y Acad Sci. 2002; 957: 122–135.[Abstract/Free Full Text]
  4. Sun AY, Simonyi A, Sun GY. The "French Paradox" and beyond: neuroprotective effects of polyphenols. Free Radic Biol Med. 2002; 32: 314–318.[CrossRef][Medline] [Order article via Infotrieve]
  5. Ray PS, Maulik G, Cordis GA, Bertelli AA, Bertelli A, Das DK. The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury. Free Radic Biol Med. 1999; 27: 160–169.[CrossRef][Medline] [Order article via Infotrieve]
  6. Olas B, Wachowicz B. Resveratrol and vitamin C as antioxidants in blood platelets. Thromb Res. 2002; 106: 143–148.[CrossRef][Medline] [Order article via Infotrieve]
  7. Hung LM, Su MJ, Chu WK, Chiao CW, Chan WF, Chen JK. The protective effect of resveratrols on ischaemia-reperfusion injuries of rat hearts is correlated with antioxidant efficacy. Br J Pharmacol. 2002; 135: 1627–1633.[CrossRef][Medline] [Order article via Infotrieve]
  8. Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, Forti L, Pagnoni UM, Albini A, Prosperi E, Vannini V. Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. J Biol Chem. 2001; 276: 22586–22594.[Abstract/Free Full Text]
  9. Jang JH, Surh YJ. Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheochromocytoma (PC12) cells. Mutat Res. 2001; 496: 181–190.[Medline] [Order article via Infotrieve]
  10. Fitzpatrick DF, Hirschfield SL, Coffey RG. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol. 1993; 265: H774–H778.[Medline] [Order article via Infotrieve]
  11. Chen CK, Pace-Asciak CR. Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. Gen Pharmacol. 1996; 27: 363–366.[Medline] [Order article via Infotrieve]
  12. Orallo F, Alvarez E, Camina M, Leiro JM, Gomez E, Fernandez P. The possible implication of trans-resveratrol in the cardioprotective effects of long-term moderate wine consumption. Mol Pharmacol. 2002; 61: 294–302.[Abstract/Free Full Text]
  13. Gielen S, Schuler G, Hambrecht R. Exercise training in coronary artery disease and coronary vasomotion. Circulation. 2001; 103: E1–E6.[Medline] [Order article via Infotrieve]
  14. Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens. 2001; 14: 83S–89S.[CrossRef][Medline] [Order article via Infotrieve]
  15. Wenzel RR, Czyborra P, Luscher T, Philipp T. Endothelin in cardiovascular control: the role of endothelin antagonists. Curr Hypertens Rep. 1999; 1: 79–87.[Medline] [Order article via Infotrieve]
  16. Mundhenke M, Schwartzkopff B, Kostering M, Deska U, Klein RM, Strauer BE. Endogenous plasma endothelin concentrations and coronary circulation in patients with mild dilated cardiomyopathy. Heart. 1999; 81: 278–284.[Abstract/Free Full Text]
  17. Schiffrin EL, Touyz RM. Vascular biology of endothelin. J Cardiovasc Pharmacol. 1998; 32 (suppl 3): S2–S13.[Medline] [Order article via Infotrieve]
  18. Tonnessen T, Giaid A, Saleh D, Naess PA, Yanagisawa M, Christensen G. Increased in vivo expression and production of endothelin-1 by porcine cardiomyocytes subjected to ischemia. Circ Res. 1995; 76: 767–772.[Abstract/Free Full Text]
  19. Hoffmann E, Assennato P, Donatelli M, Colletti I, Valenti TM. Plasma endothelin-1 levels in patients with angina pectoris and normal coronary angiograms. Am Heart J. 1998; 135: 684–688.[CrossRef][Medline] [Order article via Infotrieve]
  20. Pernow J, Wang QD. Endothelin in myocardial ischaemia and reperfusion. Cardiovasc Res. 1997; 33: 518–526.[Abstract/Free Full Text]
  21. Webb DJ, Monge JC, Rabelink TJ, Yanagisawa M. Endothelin: new discoveries and rapid progress in the clinic. Trends Pharmacol Sci. 1998; 19: 5–8.[CrossRef][Medline] [Order article via Infotrieve]
  22. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994; 344: 793–795.[CrossRef][Medline] [Order article via Infotrieve]
  23. Drexler H. Endothelial dysfunction: clinical implications. Prog Cardiovasc Dis. 1997; 39: 287–324.[CrossRef][Medline] [Order article via Infotrieve]
  24. Griendling KK, Alexander RW. Oxidative stress and cardiovascular disease. Circulation. 1997; 96: 3264–3265.[Medline] [Order article via Infotrieve]
  25. Wung BS, Cheng JJ, Hsieh HJ, Shyy YJ, Wang DL. Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1. Circ Res. 1997; 81: 1–7.[Abstract/Free Full Text]
  26. Cheng JJ, Wung BS, Chao YJ, Wang DL. Cyclic strain-induced reactive oxygen species involved in ICAM-1 gene induction in endothelial cells. Hypertension. 1998; 31: 125–130.[Abstract/Free Full Text]
  27. Cheng TH, Shih NL, Chen SY, Loh SH, Cheng PY, Tsai CS, Liu SH, Wang DL, Chen JJ. Reactive oxygen species mediate cyclic strain-induced endothelin-1 gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in endothelial cells. J Mol Cell Cardiol. 2001; 33: 1805–1814.[CrossRef][Medline] [Order article via Infotrieve]
  28. Wang DL, Tang CC, Wung BS, Chen HH, Hung MS, Wang JJ. Cyclical strain increases endothelin-1 secretion and gene expression in human endothelial cells. Biochem Biophys Res Commun. 1993; 195: 1050–1056.[CrossRef][Medline] [Order article via Infotrieve]
  29. Cheng TH, Shih NL, Chen SY, Wang DL, Chen JJ. Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes. Cardiovasc Res. 1999; 41: 654–662.[Abstract/Free Full Text]
  30. Cheng JJ, Chao YJ, Wang DL. Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (PYK2) in endothelial cells. J Biol Chem. 2002; 277: 48152–157.[Abstract/Free Full Text]
  31. Cheng CM, Hong HJ, Liu JC, Shih NL, Juan SH, Loh SH, Chan P, Chen JJ, Cheng TH. Crucial role of extracellular signal-regulated kinase pathway in reactive oxygen species-mediated endothelin-1 gene expression induced by endothelin-1 in rat cardiac fibroblasts. Mol Pharmacol. 2003; 63: 1002–1011.[Abstract/Free Full Text]
  32. Matsushita H, Lee KK, Tsao PS. Cyclic strain induces reactive oxygen species production via an endothelial NAD(P)H oxidase. J Cell Biochem. 2001; 81: 99–106.[CrossRef]
  33. Ziegler T, Bouzourene K, Harrison VJ, Brunner HR, Hayoz D. Influence of oscillatory and unidirectional flow environments on the expression of endothelin and nitric oxide synthase in cultured endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 686–692.[Abstract/Free Full Text]
  34. Wang DL, Wung BS, Peng YC, Wang JJ. Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells. J Cell Physiol. 1995; 163: 400–406.[CrossRef][Medline] [Order article via Infotrieve]
  35. Corder R, Douthwaite JA, Lees DM, Khan NQ, Viseu Dos Santos AC, Wood EG, Carrier MJ. Endothelin-1 synthesis reduced by red wine. Nature. 2001; 414: 863–864.[CrossRef][Medline] [Order article via Infotrieve]
  36. Khan NQ, Lees DM, Douthwaite JA, Carrier MJ, Corder R. Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells. Clin Sci (Lond). 2002; 103 (suppl 48): 72S–75S.[Medline] [Order article via Infotrieve]
  37. Martinez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production. Biochem Pharmacol. 2000; 59: 865–870.[CrossRef][Medline] [Order article via Infotrieve]
  38. Olas B, Zbikowska HM, Wachowicz B, Krajewski T, Buczynski A, Magnuszewska A. Inhibitory effect of resveratrol on free radical generation in blood platelets. Acta Biochim Pol. 1999; 46: 961–966.[Medline] [Order article via Infotrieve]
  39. Das DK, Sato M, Ray PS, Maulik G, Engelman RM, Bertelli AA, Bertelli A. Cardioprotection of red wine: role of polyphenolic antioxidants. Drugs Exp Clin Res. 1999; 25: 115–120.[Medline] [Order article via Infotrieve]
  40. Bastianetto S, Zheng WH, Quirion R. Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons. Br J Pharmacol. 2000; 131: 711–720.[CrossRef][Medline] [Order article via Infotrieve]
  41. Bertelli AA, Giovannini L, Stradi R, Urien S, Tillement JP, Bertelli A. Evaluation of kinetic parameters of natural phytoalexin in resveratrol orally administered in wine to rats. Drugs Exp Clin Res. 1998; 24: 51–55.[Medline] [Order article via Infotrieve]
  42. Sano M, Fukuda K, Sato T, Kawaguchi H, Suematsu M, Matsuda S, Koyasu S, Matsui H, Yamauchi-Takihara K, Harada M, Saito Y, Ogawa S. ERK and p38 MAPK, but not NF-kappaB, are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts. Circ Res. 2001; 89: 661–669.[Abstract/Free Full Text]
  43. Tanaka K, Honda M, Takabatake T. Redox regulation of MAPK pathways and cardiac hypertrophy in adult rat cardiac myocyte. J Am Coll Cardiol. 2001; 37: 676–685.[Abstract/Free Full Text]
  44. El-Mowafy AM, White RE. Resveratrol inhibits MAPK activity and nuclear translocation in coronary artery smooth muscle: reversal of endothelin-1 stimulatory effects. FEBS Lett. 1999; 451: 63–67.[CrossRef][Medline] [Order article via Infotrieve]
  45. Haider UG, Sorescu D, Griendling KK, Vollmar AM, Dirsch VM. Resveratrol suppresses angiotensin II-induced Akt/protein kinase B and p70 S6 kinase phosphorylation and subsequent hypertrophy in rat aortic smooth muscle cells. Mol Pharmacol. 2002; 62: 772–777.[Abstract/Free Full Text]
  46. Wallerath T, Deckert G, Ternes T, Anderson H, Li H, Witte K, Forstermann U. Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase. Circulation. 2002; 106: 1652–1658.[Abstract/Free Full Text]
  47. Ohkita M, Takaoka M, Shiota Y, Nojiri R, Matsumura Y. Nitric oxide inhibits endothelin-1 production through the suppression of nuclear factor kappa B. Clin Sci (Lond). 2002; 103 (suppl 48): 68S–71S.[Medline] [Order article via Infotrieve]
  48. Smith AP, Demoncheaux EA, Higenbottam TW. Nitric oxide gas decreases endothelin-1 mRNA in cultured pulmonary artery endothelial cells. Nitric Oxide. 2002; 6: 153–159.[CrossRef][Medline] [Order article via Infotrieve]
  49. Mitsutomi N, Akashi C, Odagiri J, Matsumura Y. Effects of endogenous and exogenous nitric oxide on endothelin-1 production in cultured vascular endothelial cells. Eur J Pharmacol. 1999; 364: 65–73.[CrossRef][Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. Lekli, G. Szabo, B. Juhasz, S. Das, M. Das, E. Varga, L. Szendrei, R. Gesztelyi, J. Varadi, I. Bak, et al.
Protective mechanisms of resveratrol against ischemia-reperfusion-induced damage in hearts obtained from Zucker obese rats: the role of GLUT-4 and endothelin
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H859 - H866.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S.-H. Juan, J.-J. Chen, C.-H. Chen, H. Lin, C.-F. Cheng, J.-C. Liu, M.-H. Hsieh, Y.-L. Chen, H.-H. Chao, T.-H. Chen, et al.
17{beta}-Estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells
Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1254 - H1261.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
42/6/1198    most recent
01.HYP.0000103162.76220.51v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, J.-C.
Right arrow Articles by Cheng, T.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, J.-C.
Right arrow Articles by Cheng, T.-H.
Related Collections
Right arrow Cardiovascular Pharmacology

Hypertension Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2003 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.