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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:419-426.)
© 1999 American Heart Association, Inc.

Original Contributions

Resveratrol, a Polyphenolic Compound Found in Wine, Inhibits Tissue Factor Expression in Vascular Cells

A Possible Mechanism for the Cardiovascular Benefits Associated With Moderate Consumption of Wine

Usha R. Pendurthi; J. Todd Williams; L. Vijaya Mohan Rao

From the Departments of Molecular Biology and Biochemistry, The University of Texas Health Center at Tyler.

Correspondence to Usha R. Pendurthi, PhD, Department of Molecular Biology, The University of Texas Health Center at Tyler, 11937 US Highway 271, Tyler, TX 75708. E-mail usha{at}uthct.edu

	Abstract

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Abstract—A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease mortality. The phytochemical resveratrol found in wine, derived from grapes, has been thought to be responsible for cardiovascular benefits associated with wine consumption because it was shown to have antioxidant and antiplatelet activities. In the present investigation, we examined the effect of resveratrol on induction of tissue factor (TF) expression in vascular cells that were exposed to pathophysiological stimuli. The data presented herein show that resveratrol, in a dose-dependent manner, inhibited the expression of TF in endothelial cells stimulated with a variety of agonists, including interleukin-1ß (IL-1ß), tumor necrosis factor- (TNF) and lipopolysaccharide (LPS). A similar inhibition of TF induction was also seen in LPS stimulated monocytes that were pretreated with resveratrol before their stimulation with LPS. In addition, resveratrol was shown to inhibit the LPS-induced expression of TNF mRNA in endothelial cells and of TNF and IL-1ß mRNA in monocytes. Nuclear run-on analysis in endothelial cells showed that resveratrol inhibited TF expression at the level of transcription. However, resveratrol did not significantly alter the binding of the transcription factors c-Fos/c-Jun and c-Rel/p65, the transcription factors required for the induction of TF promoter in both endothelial cells and monocytes. Similarly, resveratrol had no significant effect on the binding of NF-B in endothelial cells stimulated with IL-1ß, TNF, and LPS. Overall, our data show that resveratrol could effectively suppress the aberrant expression of TF and cytokines in vascular cells, but it requires further investigation to understand how resveratrol exerts its inhibitory effect.

Key Words: tissue factor • resveratrol • endothelial cell • monocytes • cardioprotective effect

	Introduction

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Several epidemiological studies suggest that coronary heart disease mortality is lowered by moderate consumption of alcohol, particularly red wine.^{1 2 3 4 5} Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol present in wine, has been thought to be responsible for cardiovascular benefits associated with moderate wine consumption.^{6 7 8} Resveratrol, a phytoalexin, is produced in grapes and a variety of other plants in response to fungal infection or other types of stress.^{9 10 11 12} Because of its high concentration in grape skin, most of the red wines contain significant amounts of resveratrol.¹³ Cardiovascular benefits of moderate wine consumption have been thought to stem, at least partly, from antioxidant⁶ and antiplatelet activities of resveratrol.^{8 14} Resveratrol has been shown to protect human LDL against copper-catalyzed oxidation⁶ and to inhibit human platelet aggregation and thromboxane B₂ synthesis.⁸ Recent studies in which humans consumed resveratrol-enriched grape juice showed that trans-resveratrol can be absorbed from grape juice in biologically active quantities and in amounts that are likely to cause reduction in the risk of atherosclerosis.¹⁵

Thrombosis plays a critical role in the development, progression, and clinical sequelae of atherosclerosis. Tissue factor (TF), a cell-surface glycoprotein, is the primary initiator of the coagulation cascade in both hemostasis and pathogenesis.^{16 17 18} TF is constitutively expressed in several extravascular cells, such as fibroblasts and pericytes, but not in cells within the vasculature, such as endothelial cells and monocytes.^{19 20} Little or no TF is found in the intima or media of normal arteries,^{19 20 21} whereas abundant TF is found in atherosclerotic plaques.²¹ TF antigen in human atherosclerotic plaques is localized within macrophages, smooth muscle cells, and endothelial cells and in the acellular lipid-rich core.^{21 22} Accumulation of TF in atherosclerotic plaques is thought to play a major critical role in determining plaque thrombogenicity.¹⁷

Because thrombosis is the integral part of the atherosclerosis and coronary artery disease, it is possible that the decreased risk of coronary heart disease associated with consumption of red wine, in part, could be caused by its ability to suppress the expression of TF in the arterial wall. In the present study, we have examined the effect of resveratrol on induction of TF expression in both endothelial cells and monocytes. The data show that resveratrol inhibits the induction of TF in these vascular cells exposed to various pathophysiological stimuli.

	Methods

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Reagents
Resveratrol, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS; Escherichia coli serotype O111:B4), human recombinant tumor necrosis factor- (TNF) and interleukin-1ß (IL-1ß), endothelial cell growth supplement (ECGS), fibronectin, and Histopaque were obtained from Sigma Chemical Co. A stock concentration (100 mmol/L) of resveratrol was made in ethanol. RPMI and F-12 culture media were obtained from GIBCO-BRL, Life Technologies. Fetal bovine serum, trypsin-versene mixture, penicillin-streptomycin, and L-glutamine were obtained from Bio-Whittaker. TRI reagent was from Molecular Research Center Inc. [³²P]ATP (3000 Ci/mmol) and [³²P]dCTP (3000 Ci/mmol) were from Dupont NEN. cDNA probes for TNF, IL-1ß, and others were obtained from American Type Culture Collection. Most of the molecular biology–grade chemicals were obtained from either Boehringer Mannheim or United States Biochemicals.

Coagulant Proteins
Recombinant VIIa was a gift from Novo-Nordisk. Human plasma factor X²³ and factor Xa²⁴ were purified as described earlier or purchased from Enzyme Research Laboratories Inc.

Human Umbilical Vein Endothelial Cells
Primary cultures of human umbilical vein endothelial cells (HUVEC) were obtained from Cell Systems and cultured at 37°C under 5% CO₂ in T-75 flasks in F-12 medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% L-glutamine, 40 µg/mL ECGS, and 15 U/mL heparin. The cells were subcultured by first detaching the cells with trypsin solution and replating them in 24-well culture dishes or in T-75 flasks that were coated with human fibronectin (0.65 µg/cm²). The monolayers were used within 24 hours after they reached confluency. Passages between 3 and 6 were used in the present experiments.

Monocytes
Mononuclear cells were isolated from blood as described earlier.²⁵ Briefly, 20 to 50 mL of blood was drawn from healthy volunteers into a plastic syringe with a 19-gauge needle and immediately added to a plastic tube containing heparin (a final concentration of 10 U/mL). Mononuclear cells were isolated by applying blood, which was diluted with an equal volume of sterile saline, on top of Histopaque 1077 (1.5 mL of Histopaque for 1 mL of blood) followed by density gradient centrifugation at 500g for 15 minutes at room temperature. The mononuclear cell band was collected and washed with sterile 0.15 mol/L NaCl by centrifugation at 1400g for 20 minutes. The cell pellet was suspended in RPMI medium in 1/5 volume of starting blood.

Cell Survival and Proliferation Assay
Cell survival and proliferation were determined using a tetrazolium-based colorimetric assay.²⁶ The assay is dependent on the reduction of tetrazolium salt 3-(4,5-dimethlythiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which results in formation of a blue formazan product, by various dehydrogenase enzymes of viable cells. Briefly, MTT solution (10 µL per 100 µL medium, 5 mg MTT/mL in PBS) was added to HUVEC that were cultured in a 96-well culture dish and pretreated for 2 hours with a control vehicle (ethanol) or varying concentrations of resveratrol (50 to 200 µmol/L). After 4 hours of further incubation at 37°C, acid isopropanol (100 µL of 0.04 N HCl in isopropanol) was added to the wells and mixed thoroughly, and the plates were read on a microplate reader (Molecular Devices Corp) using a test wavelength of 563 nm and a reference wavelength of 650 nm. The plates were normally read within 15 minutes of adding acid isopropanol.

Induction of TF
Confluent endothelial cell monolayers were washed twice with F-12 media. The monolayers were incubated with a control vehicle (ethanol, 0.1% vol/vol) or resveratrol for 2 hours and then stimulated with PMA (10 ng/mL), LPS (1 µg/mL), TNF (20 ng/mL), or IL-1ß (20 ng/mL). All incubations were carried out at 37°C and 5% CO₂ in an incubator. At the specific intervals, the medium was removed, and the monolayers were washed twice with F-12 medium or buffer A (10 mmol/L HEPES, 0.15 mol/L NaCl, 4 mmol/L KCl, 11 mmol/L glucose, pH 7.5) and processed further either to isolate nuclei and RNA (T-75 flasks) or to determine TF activity (12- or 24-well plates). To determine TF activity, cell lysates were prepared by solubilizing the monolayers in 15 mmol/L n-octyl ß-D-glucopyranoside. The cell lysates were frozen and thawed twice and sonicated for 1 minute (2x 30 s) before they were used in the assay.

For induction of TF in monocytes, mononuclear cell fractions were incubated with a control vehicle or resveratrol for 2 hours and then stimulated with LPS (100 ng/mL) for 5 hours. The cells were continuously mixed (200 rpm) in a shaker incubator at 37°C during the incubation period. At the end of incubation, cells were sedimented by centrifugation and resuspended in RPMI medium. The cell suspension contained approximately 2.5x10⁶ cells/mL. The cell suspension was frozen at -20°C until tested to determine TF activity.

Measurement of TF Procoagulant Activity
TF activity was measured as the ability of cell lysates to support the activation of factor X with the addition of VIIa and CaCl₂. Measurement of TF activity was as follows: cell lysates (45 µL) were incubated with a reagent mixture (5 µL) containing factor VIIa (0.5 µg/mL), factor X (10 µg/mL), and CaCl₂ (5 mmol/L; all concentrations were final concentrations) in a 96-well plate. At the end of 15 minutes, 50 µL of Chromozym X (Boehringer Mannheim) containing 25 mmol/L EDTA was added to each well, and the initial rate of color development in mOD/min at 405 nm was measured with a microplate reader (Molecular Devices). This initial rate was converted to micrograms per milliliter of Xa from a standard curve prepared by adding 50 µL of Chromozym X to 50-µL serial dilutions of a 1-µg/mL sample of purified human factor Xa. Cell lysates from PMA treatments were diluted 100 times, whereas cell lysates from other treatments were diluted 10 times before they were used in the assay.

Analysis of TF mRNA
Total RNA was prepared from 2 to 3x10⁶ cells by the acid phenol method using TRI reagent according to the manufacturer's technical bulletin. Ten micrograms of total RNA was size fractionated by gel electrophoresis in 1% agarose/6% formaldehyde gels and transferred onto the nitrocellulose membrane by a capillary blot method. Northern blots were prehybridized at 42°C with a solution containing 50% formamide, 5xSSC, 50 mmol/L Tris HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mmol/L EDTA, 100 µg/mL denatured salmon sperm DNA, and 1% BSA and hybridized with ³²P-labeled TF cDNA probe and other cDNA probes (10⁶ cpm/mL) as described earlier.²⁷ The filters were exposed to either Dupont NEF or Fuji RX x-ray film.

Isolation of Nuclei and Run-on Transcription Assay
Nuclei from 4 to 6x10⁶ HUVEC were harvested as described earlier,²⁷ and run-on assays were performed with [-³²P]UTP-labeled RNA as described previously.²⁸

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from 4 to 6x10⁶ HUVEC as described in our earlier publication.²⁹ Protein concentration in nuclear extracts was determined using the Bio-Rad protein assay kit. Oligonucleotides for the proximal TF AP-1 site, 5'-CTGGGGTGAGTCATCCCTT-3'; a TF B-like site, 5'-GTCCCGGAGTTTCCTACCGGG-3'; and a prototypic NF-B site, 5'-CAGAGGGACTTTCCGAGA-3' (site-specific sequences underlined) were obtained from The Midland Certified Reagent Company. Double-stranded oligonucleotides were 5'-end labeled with [³²P]ATP. Nuclear extracts (10 µg) were preincubated for 20 minutes on ice in 20 µL of the binding buffer (10 mmol/L HEPES, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, and 4% glycerol) containing 250 µg/mL poly(dI:dC). After preincubation, 100 000 cpm of the ³²P-labeled oligonucleotide was added, and the mixture was incubated at room temperature for 30 minutes. The samples were electrophoresed in a 6% nondenaturing polyacrylamide gel. Electrophoresis was performed in 1x TBE buffer (89 mmol/L Tris HCl, 89 mmol/L boric acid, 2 mmol/L EDTA). After electrophoresis, the gel was dried and subjected to autoradiography. For quantification purposes, the dried gel was exposed to phosphor screens for 30 minutes to 2 hours, and the exposed screens were analyzed in a PhosphorImager (Molecular Dynamics) using "Image-quant" software. To calculate mean±SEM, the units (counts) obtained from different gels (experiments) were normalized with the counts present in the control lane of the same gel.

	Results

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Suppression of TF Induction in Vascular Cells by Resveratrol
To test the effect of resveratrol on the induction of TF expression in endothelial cells, confluent monolayers of HUVEC were preincubated for 2 hours with resveratrol (100 µmol/L) and then stimulated for 6 hours with various agonists to induce TF expression. The results showed that resveratrol markedly suppressed LPS-, TNF-, IL-1ß-, and PMA-induced TF activity (Figure 1A). The inhibition varied from 60% to more than 90%. In a further experiment, HUVEC monolayers were preincubated for 2 hours with different concentrations of resveratrol (0 to 200 µmol/L) followed by PMA, IL-1ß, or TNF treatments to induce TF expression. The data showed that resveratrol inhibited the induction of TF expression in a dose-dependent manner. The suppression of TF expression was evident with as low as 5 µmol/L resveratrol, and a concentration of 20 µmol/L resveratrol inhibited the induction of TF activity by 50% (Figure 1).

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of resveratrol on the induction of TF activity in endothelial cells. Confluent monolayers of primary HUVEC were incubated with a fixed concentration, 100 µmol/L (A), or with various concentrations, 0 to 200 µmol/L (B), of resveratrol for 2 hours, and then the cells were stimulated for 6 hours with IL-1ß (20 ng/mL), TNF (20 ng/mL), LPS (1 µg/mL), or PMA (10 ng/mL). TF activity in cell lysates was measured as their ability to activate factor X in the presence of factor VIIa. For each stimulant, TF activity obtained in cells pretreated with a control vehicle (ethanol, 0.1% vol/vol) was taken as 100%. The data shown in A represent mean±SEM (n=4) and the data shown in B is from a representative experiment (n= 3).

Next, we investigated the treatment time required for resveratrol to be an effective inhibitor of TF induction. For these experiments, resveratrol (100 µmol/L) was added to HUVEC monolayers for varying times, either before or after the addition of LPS, and TF activity was analyzed 6 hours after the addition of LPS. The data showed that addition of resveratrol for 2 hours before the addition of LPS suppressed the TF activity by 75%, and the inhibition was about 95% in cells that were incubated with resveratrol for 6 hours before the addition of LPS. Resveratrol also markedly suppressed the induction of TF activity in endothelial cells when it was added at the time of LPS addition, and the suppression was minimal when it was added 2 hours after the addition of LPS (data not shown). Additional experiments were performed to determine how long the suppressive effect of resveratrol lasts after its removal from the endothelial cell surface. HUVEC were treated with resveratrol for 2 hours, washed with medium to remove resveratrol, and then stimulated with PMA at defined times after the wash. The data showed that a continuous presence of resveratrol was required to suppress the induction of TF because more than 60% of the PMA-induced TF activity was recovered when resveratrol was removed from the cell surface immediately before the addition of PMA. A full PMA-induced TF activity was obtained when the cells were stimulated 1 to 2 hours after the removal of resveratrol (data not shown). In this respect, resveratrol-mediated suppression of TF was similar to that of salicylate-mediated suppression of TF induction.³⁰

To test whether resveratrol can also inhibit the induction of TF activity in monocytes, mononuclear cell fraction was isolated by density gradient centrifugation from heparinized blood drawn from healthy human volunteers, and mononuclear cell fractions were preincubated with various concentrations of resveratrol (0 to 100 µmol/L) for 2 hours at 37°C before they were stimulated with LPS (100 ng/mL) for 5 hours to induce TF expression in monocytes. As shown in Figure 2, resveratrol inhibited the LPS-induced TF expression in monocytes in a dose-dependent manner. The half-maximal concentration of resveratrol required to inhibit the induction of TF activity in monocytes is approximately 10 µmol/L, similar to the concentration of resveratrol required to inhibit platelet aggregation and cyclooxygenase activity.^{11 31} One should note that resveratrol alone had no effect on the induction of TF, ie, the cells (both endothelial cells and monocytes) treated with resveratrol (up to 200 µmol/L), in the absence of the inducer, did not express TF activity. Further, the control vehicle, ethanol (0.1% vol/vol), had no effect on the agonist-induced TF expression in both endothelial cells and monocytes.

View larger version (15K): [in this window] [in a new window]   Figure 2. Inhibition of LPS-induced TF activity in monocytes by resveratrol. Mononuclear cells were isolated from heparinized human blood drawn from healthy volunteers by density gradient centrifugation. Mononuclear cells were incubated with various concentrations of resveratrol, 0 to 100 µmol/L, for 2 hours and then the cells were stimulated for 5 hours with LPS (100 ng/mL). TF activity in cell lysates was measured as their ability to activate factor X (10 µg/mL) in the presence of factor VIIa (0.5 µg/mL). The data represent mean±SEM (n=3).

Although the concentrations of resveratrol used in the above experiments are similar or lower than the concentrations of resveratrol used by other investigators with different cell types,^{8 11 32} we wanted to exclude the possibility that resveratrol used in the above experiments was toxic to vascular cells. Therefore, we evaluated the cytotoxic effect of resveratrol on both HUVEC and monocytes. Monolayers of HUVEC were incubated for 6 hours with various concentrations of resveratrol (0, 50, 100, and 200 µmol/L), the last 4 hours in the presence of MTT. We found no differences between the control and resveratrol (up to 100 µmol/L) -treated cells in their ability to cleave MTT (Absorbance_{563–650 nm}: control, 0.291±0.042; 50 µmol/L resveratrol-treated, 0.301±0.013; 100 µmol/L resveratrol-treated, 0.286±0.014; n=4). MTT cleavage was slightly inhibited, by approximately 20%, in cells treated with the 200 µmol/L concentration of resveratrol. Resveratrol also had no effect on mononuclear cell viability as determined in trypan blue exclusion staining. The cell viability varied between 90% and 99% in cells treated with resveratrol (0 to 100 µmol/L). Further, to rule out the possibility that the inhibition of induced expression of TF in resveratrol-treated cells represents a nonspecific effect of resveratrol on protein synthesis in general, we evaluated the effect of resveratrol on [³⁵S]methionine incorporation in endothelial cells. The results showed that a similar percentage of [³⁵S]methionine was incorporated into proteins in ethanol- (control vehicle) and resveratrol-treated cells, up to 200 µmol/L. These data establish that resveratrol has no effect on overall cellular metabolism and protein synthesis.

Resveratrol Suppresses Induction of TF mRNA
We next examined whether resveratrol inhibits the induction of TF mRNA in endothelial cells. HUVEC monolayers were preincubated with various concentrations of resveratrol (0, 5, 20, 100, and 200 µmol/L) for 2 hours and then treated with PMA for 2 hours to induce TF mRNA. Northern blot analysis revealed a dose-dependent inhibition of TF mRNA accumulation in resveratrol-treated cells. A concentration of 100 µmol/L resveratrol inhibited the PMA-induced TF mRNA accumulation by more than 95%. Endothelial cells that were treated with resveratrol (200 µmol/L) but not stimulated with PMA expressed no measurable TF mRNA (data not shown). Resveratrol also suppressed LPS-, TNF-, and IL-1ß-induced TF expression in HUVEC (Figure 3). Pretreatment of HUVEC with resveratrol also inhibited LPS-induced accumulation of TNF mRNA (Figure 4). Next, we investigated the effect of resveratrol on LPS-induced expression of TF, TNF, and IL-1ß mRNA in monocytes. As shown in Figure 5, pretreatment of mononuclear cells with resveratrol markedly inhibited the expression of TF, TNF, and IL-1ß mRNA in monocytes. (Because there was no significant induction of IL-1ß mRNA in stimulated HUVEC under our experimental conditions, we could not test the effect of resveratrol on the induction of IL-1ß mRNA in endothelial cells.)

View larger version (77K): [in this window] [in a new window]   Figure 3. Resveratrol suppresses the induction of TF mRNA in HUVEC. HUVEC monolayers were treated for 2 hours with 100 µmol/L concentration of resveratrol. Then, the monolayers were stimulated for 2 hours with PMA (10 ng/mL), LPS (1 µg/mL), TNF (20 ng/mL), or IL-1ß (20 ng/mL) to induce TF mRNA. Higher molecular weight bands observed on the Northern blot represent unprocessed or partially processed nuclear TF mRNA transcripts that could contain one or more introns.54 The part of the blot that contained PMA-induced samples was exposed to x-ray film for 4 hours whereas the remaining blot was exposed to film for 24 hours.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect of resveratrol on TNF mRNA expression in HUVEC. HUVEC monolayers were treated with resveratrol and stimulated with various agonists as described in Figure 3. Northern blot analysis was performed using radiolabeled human TNF cDNA probe. N denotes no inducer (unstimulated).

View larger version (37K): [in this window] [in a new window]   Figure 5. Effect of resveratrol on TF, TNF, and IL-1ß mRNA expression in LPS-stimulated monocytes. Total RNA was extracted from mononuclear cells exposed to LPS (100 ng/mL) for 2 hours with or without pretreatment (for 2 hours) with resveratrol (50 µmol/L). Levels of various mRNAs were determined by Northern blot analysis using radiolabeled human cDNA probes. The blot was first probed with TF cDNA and then reprobed with TNF cDNA followed by IL-1ß cDNA. R denotes resveratrol treatment.

Resveratrol Inhibits Induction of TF Gene Transcription
To determine whether the lower number of TF mRNA transcripts in stimulated endothelial cells that were treated with resveratrol was caused by a decrease in transcriptional activation of the TF gene, nuclear run-on experiments were performed. As shown in Figure 6, there was minimal transcription of the TF gene in unstimulated endothelial cells, and PMA treatment markedly enhanced the transcriptional rate. The data also showed that treatment of endothelial cells with resveratrol before the addition of PMA markedly suppressed the PMA-induced transcriptional activation of the TF gene. Quantitative analysis of the hybridization signal using a PhosphorImager showed that resveratrol suppressed the transcriptional rate of the TF gene by more than 70%. Resveratrol also inhibited transcriptional activation of tissue plasminogen activator. In contrast, resveratrol had no effect on PMA-induced transcriptional activation of IL-8, plasminogen activator inhibitor (PAI)-2, and urokimase plasminogen activator (Figure 6). Transcription rate of the TF pathway inhibitor (TFPI) gene was unchanged with PMA treatment, and resveratrol had no effect on the transcription of the TFPI gene. These data illustrate that the resveratrol suppresses TF gene transcription by a specific mechanism and not by acting as a nonspecific inhibitor of transcription.

View larger version (79K): [in this window] [in a new window]   Figure 6. Effect of resveratrol on transcription of TF and other genes in PMA-stimulated endothelial cells. Nuclei were isolated from unstimulated cells (Con), cells stimulated with PMA (10 ng/mL) for 1 hour (PMA), and cells treated with resveratrol (100 µmol/L) for 2 hours before stimulation with PMA for 1 hour (Res+PMA).Three identical blots containing target DNAs were hybridized with equal amounts of labeled transcripts of nuclear RNA. Housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPD) and tubulin, were used as controls. Other DNAs used were UPA, urokinase-type plasminogen activator; IL-8, interleukin 8; Tsp, thrombospondin; tPA, tissue plasminogen activator; UPAR, urokinase-type plasminogen activator receptor; PAI-2, plasminogen activator inhibitor-2; and TFPI, tissue factor pathway inhibitor.

Resveratrol Does Not Affect DNA Binding of c-Fos/c-Jun and c-Rel/p65 Transcription Factors
Earlier studies showed that induction of TF gene transcription in endothelial cells and monocytes is regulated by the interaction of c-Fos/c-Jun and c-Rel/p65 bound to TF promoter,³³ and electrophoretic mobility shift assays (EMSA) have been used to demonstrate the binding of these transcription factors to specific sequences in the TF promoter.³⁴ To determine the mechanism by which resveratrol suppresses the induction of TF gene transcription in endothelial cells, we examined the binding of nuclear proteins to oligonucleotides containing sequences of the AP-1 site and the B-like site of the TF promoter. HUVEC monolayers were treated with a control vehicle or resveratrol (100 µmol/L) for 2 hours and then stimulated for 1 hour with TNF, IL-1ß, LPS, and PMA. Nuclear extracts were analyzed by EMSA using radiolabeled oligonucleotides containing AP-1 (proximal) and B-like sites in the TF promoter. In addition, we also examined the binding of nuclear extracts to a prototypic B site from the mouse immunoglobulin light-chain enhancer (Ig kappa).

As shown in Figure 7, nuclear extracts from both unstimulated and stimulated endothelial cells formed a DNA–protein complex with the AP-1 site, and resveratrol treatment had no significant effect on AP-1 binding activity of both unstimulated and stimulated endothelial cells. Quantitative analysis of the data from several experiments (n=3 to 6) showed no statistically significant differences in the levels of AP-1 binding in nuclear extracts from cells pretreated with resveratrol or a control vehicle (probability value varied between 0.48 and 0.89 in different treatments). EMSA performed to examine the binding of nuclear proteins to an oligonucleotide containing the TF B-like site showed a minimal DNA–protein complex in unstimulated cells, whereas a prominent DNA–protein complex was observed in TNF- and IL-1ß-stimulated cells (Figure 8). Treatment of cells with resveratrol (100 µmol/L) for 2 hours before the addition of stimuli had no effect or only a minimal effect on the formation of the inducible complex. Quantitative analysis of the data showed no statistically significant differences between the levels of c-Rel/p65 in nuclear extracts from cells treated with either TNF or IL-1ß in the presence and absence of resveratrol (IL-1ß treatment, P=0.89; TNF treatment, P=0.66; n=4). Similar results were also obtained using a B site that binds to p50/p65, the heterodimers that regulate TNF gene transcription (Figure 9). No differences were found in their binding activity to the B site between the nuclear extracts isolated from the cells treated with the control vehicle and the cells treated with resveratrol before they were stimulated with TNF, IL-1ß, or LPS. However, a moderate inhibition was observed in binding activity to the B site in cells that were pretreated with resveratrol and stimulated with PMA.

View larger version (53K): [in this window] [in a new window]   Figure 7. Effect of resveratrol on the binding of transcription factor AP-1 to the TF promoter. Nuclear extracts were isolated from HUVEC pretreated with resveratrol (100 µmol/L) or control vehicle for 2 hours before the addition of IL-1ß (20 ng/mL), LPS (1 µg/mL), or PMA (10 ng/mL) for 1 hour. Nuclear extracts were incubated with radiolabeled DNA probe containing proximal TF AP-1 site, and protein–DNA complexes were analyzed on 6% polyacrylamide gel electrophoresis. To demonstrate specificity of the binding, a 100-fold molar excess of cold competitor oligonucleotides containing either the AP-1 site (SC) or Sp1 site (NC) were added to the incubation mixture containing radiolabeled DNA probe and nuclear extracts isolated from the unstimulated cells.

View larger version (89K): [in this window] [in a new window]   Figure 8. Effect of resveratrol on c-Rel/p65 activation. Nuclear extracts were obtained as described in Figure 7 and incubated with radiolabeled DNA probe containing TF-B site. The c-Rel/p65 complex migrated as a doublet (the faster migrating band could be the result of a rapid proteolytic truncation of p65 in nuclear extracts that retains dimerization and DNA binding). To demonstrate specificity of the complex formation, a 100-fold molar excess of cold competitor oligonucleotides containing either TF-B site (SC) or an unrelated site (NC) was used.

View larger version (80K): [in this window] [in a new window]   Figure 9. Effect of resveratrol on NF-B activation. Nuclear extracts were isolated as described in Figure 7 and incubated with radiolabeled DNA probe containing prototypic NF-B consensus sequence. Protein–DNA complexes were analyzed on 6% polyacrylamide gel electrophoresis. To demonstrate specificity of the binding, a 100-fold molar excess of cold competitor oligonucleotides containing either the B site (SC) or an unrelated site (NC) was added to the incubation mixture containing radiolabeled DNA probe. The autoradiograph shows data from two separate experiments (lanes 1 to 10, and lanes 11 to 16).

	Discussion

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Several epidemiological studies have suggested that coronary heart disease mortality can be decreased by moderate consumption of alcohol, particularly red wine.^{1 2 3} However, it is unclear how the moderate consumption of red wine provides the protective effect against heart disease. Although the cardioprotective effects of most alcoholic beverages are probably because of ethanol-induced elevation of HDLs^{35 36} and lowering of platelet aggregation,^{37 38} factors other than ethanol in alcohol-containing beverages may also be responsible for the protective effect of alcoholic beverages.^{7 39 40} Resveratrol, a phenolic compound found primarily in the skin of grapes and relatively abundant in red wine, has been thought to be one such factor because it has been shown to have antioxidant⁶ and antiplatelet^{8 14} activities. However, in-depth knowledge on whether and how resveratrol provides the protective effect against heart disease is lacking.

Thrombotic disorders are the main cause of myocardial infarction and stroke.⁴¹ Activation of the TF-mediated coagulation pathway not only plays a major role in determining plaque thrombogenicity but could also have other effects on the vessel wall.¹⁷ For example, thrombin⁴² and factor Xa,⁴³ intermediary products generated in the TF-mediated pathway of coagulation, are shown to promote vascular smooth cell proliferation and thus may play a role in the development of intimal hyperplasia.¹⁷ Furthermore, recent studies show that TF may also function as a true receptor in generating intracellular signals^{44 45 46 47} and thus may participate in other biological effects, including cell adhesion and migration.⁴⁶ Macrophages, smooth muscle cells, and endothelial cells in atherosclerotic arterial wall were shown to express TF.^{21 22} Therefore, our present observations that show resveratrol suppresses the induction of TF expression in both endothelial cells and monocytes suggest an alternative or an additional mechanism by which the moderate consumption of red wine could contribute to the decreased risk of coronary artery disease observed in epidemiological studies. However, we should add a caveat to the above interpretation that HUVEC, although commonly used as a model for endothelial cell function, are not representative of the vascular tree affected by atherosclerosis. Given the heterogeneity of endothelial cell function and response to agonists, it would be important to extend the above data with resveratrol to other endothelial cell types before reaching a generalized conclusion.

The half-maximal concentration of resveratrol required to inhibit the induction of TF activity in monocytes and endothelial cells (10 to 20 µmol/L) is similar to the reported concentrations of resveratrol required to inhibit platelet aggregation and eicosanoid synthesis.^{8 14} Goldberg and colleagues⁸ found that resveratrol inhibited thromboxane B₂ synthesis and thrombin-induced platelet aggregation with IC₅₀s of 7 µmol/L and 160 µmol/L, respectively. Bertelli et al³¹ reported that resveratrol at the concentration of 10 µmol/L decreased collagen-induced platelet aggregation by 44%. Recent studies of Jang et al¹¹ showed that resveratrol inhibited cyclooxygenase activity with an IC₅₀ of 15 µmol/L. It is not clear whether resveratrol could be present in this range in vivo during the consumption of red wine. We are not aware of any data on serum levels of resveratrol after wine consumption or its absorption and metabolism. In general, the concentration of trans-resveratrol in red wines is in the range 10 to 20 µmol/L, although as high as 100 µmol/L has been recorded.^{13 48} Furthermore, in addition to free trans-resveratrol, most red wines also contain high concentrations of cis-resveratrol and resveratrol glycosides that could contribute to the biologically available dose.⁴⁹ Therefore, it is possible that moderate consumption of red wine could provide resveratrol in sufficient doses to influence the expression of TF expression in vascular cells. In this context, it may be important to point out that recent studies of Goldberg and colleagues¹⁵ with healthy human subjects who consumed red wine and grape juice enriched with resveratrol showed that trans-resveratrol can be absorbed in biologically active quantities and in amounts that are likely to reduce the synthesis of thromboxane B₂ synthesis and probably thrombin-induced platelet aggregation.

Resveratrol-mediated inhibition of TF activity in endothelial cells is caused by inhibition of transcriptional activation of the TF gene. Suppression of TF activity is associated with the lower accumulation of TF mRNA. Nuclear run-on assays demonstrated that the rate of TF gene transcription is reduced by 70% in resveratrol-treated endothelial cells. Induction of TF gene transcription in both endothelial cells and monocytes is mediated by the functional interaction between c-Fos/c-Jun and c-Rel/p65 heterodimers.³³ c-Fos/c-Jun heterodimers bind to two AP-1 sites, and c-Rel/p65 heterodimers bind to a B-like site in the TF promoter. Although c-Fos/c-Jun heterodimers bind to AP-1 sites in both unstimulated and stimulated endothelial cells, the binding is an absolute requirement in stimulated endothelial cells for c-Rel/p65 heterodimers to facilitate activation of TF gene transcription. Therefore, either blocking of c-Fos/c-Jun binding or activation of c-Rel/p65 could downregulate TF transcription. However, our present data show that resveratrol had no significant effect on the DNA binding activity of c-Fos/c-Jun or c-Rel/p65 heterodimers. Therefore, it is unlikely that the resveratrol-mediated suppression of TF gene expression in endothelial cells is caused by reduced DNA binding of c-Fos/c-Jun or c-Rel/p65 in endothelial cells. Nonetheless, it is possible that the resveratrol-mediated suppression of TF gene expression involves the repression of AP-1- or NF-B-mediated transcription, because resveratrol also suppressed the expression of TNF and IL-1ß, both of which are regulated by AP-1- and NF-B-dependent transcription.^{50 51 52 53} It is possible that resveratrol may modulate NF-B activity by altering phosphorylation of p65 or cofactors that are required for transactivation or inducing synthesis or activation of inhibitors of NF-B-mediated transcription. Further work is required to elucidate how resveratrol suppresses the induction of TF expression in vascular cells.

	Acknowledgments

 
This study was supported in parts by grant HL-58869 from the National Heart, Lung, and Blood Institute and a grant from the American Heart Association, Texas Affiliate. We thank Jason Voigt for his excellent technical assistance. Sameer Shirodkar, a summer volunteer from Robert E. Lee High School, Tyler, Tex, contributed to data obtained with mononuclear cells.

Received July 2, 1998; accepted September 15, 1998.

	References

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