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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1412-1420

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1412-1420.)
© 1999 American Heart Association, Inc.

Vascular Biology

Gallates Inhibit Cytokine-Induced Nuclear Translocation of NF-{kappa}B and Expression of Leukocyte Adhesion Molecules in Vascular Endothelial Cells

Takatoshi Murase; Noriaki Kume; Tadashi Hase; Yusuke Shibuya; Yoshinori Nishizawa; Ichiro Tokimitsu; Toru Kita

From the Biological Science Laboratories, Kao Corp, Ichikaimachi, Tochigi (T.M., T.H., Y.S., Y.N., I.T.), and the Department of Geriatric Medicine, Kyoto University, Kyoto (T.M., N.K., T.K.), Japan.

Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail nkume{at}kuhp.kyoto-u.ac.jp


*    Abstract
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 *Abstract
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Abstract—Gallates (gallic acid esters) belong to the class of phenolic compounds, which are abundant in red wine. In this study, we show that gallates can inhibit cytokine-induced activation of nuclear factor {kappa}B (NF-{kappa}B) and thereby reduce expression of endothelial-leukocyte adhesion molecules in cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of HUVECs with ethyl gallate (3 to 10 µmol/L) significantly suppressed interleukin-1{alpha} (IL-1{alpha})– or tumor necrosis factor-{alpha} (TNF-{alpha})– induced mRNA and cell-surface expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin, which was associated with reduced adhesion of leukocytes to HUVECs. Gel shift assays with the NF-{kappa}B consensus sequence showed the decreased densities of the shifted bands in gallate-treated HUVECs. Furthermore, gallate pretreatment inhibited cytokine-induced transcription of a fusion gene, which consisted of 4 repeats of the NF-{kappa}B consensus sequence and the luciferase reporter gene. Immunoblot analysis of nuclear extracts and whole-cell lysates demonstrated the decreased amounts of NF-{kappa}B p65 in nuclei but equal amounts of inhibitor-{kappa}B{alpha} (I-{kappa}B{alpha}) in whole-cell lysates of ethyl gallate–treated HUVECs. Incubation of the nuclear extracts from cytokine-activated HUVECs with ethyl gallate did not affect the NF-{kappa}B shifted bands induced by cytokines in gel shift assays. Taken together, these data demonstrate that ethyl gallate can inhibit cytokine-induced nuclear translocation of NF-{kappa}B p65 by way of a mechanism independent of I-{kappa}B{alpha} degradation and thereby suppress expression of VCAM-1, ICAM-1, and E-selectin, which was associated with reduced adhesion of leukocytes. These results in vitro demonstrate that gallates can exhibit anti-inflammatory properties by blocking activation of NF-{kappa}B and suggest that these natural compounds, abundant in red wine, may play important roles in the prevention of atherosclerosis and inflammatory responses in vivo.


Key Words: cell adhesion molecules • transcription factors • atherosclerosis • antioxidant • red wine


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Localized accumulation of monocytes/macrophages and T lymphocytes in the arterial intima appears to play a key role in early atherogenesis, as well as in plaque rupture in advanced atherosclerotic lesions. Molecular mechanisms involved in the recruitment of monocytes/macrophages and T lymphocytes into atherosclerotic lesions may depend on multiple and complex processes; however, adhesion of monocytes and T lymphocytes to the vascular endothelium is among the earliest and essential processes during atherogenesis, as well as in inflammatory responses. This process appears to be mediated by endothelial-leukocyte adhesion molecules (ELAMs) expressed on the surface of the vascular endothelium covering atherosclerotic and inflammatory lesions.1 2 3 4 5 These ELAMs include intercellular adhesion molecule 1 (ICAM-1),6 7 E-selectin,8 9 and vascular cell adhesion molecule-1 (VCAM-1),10 11 whose expression can be transcriptionally induced by inflammatory cytokines such as interleukin-1{alpha} (IL-1{alpha}) and tumor necrosis factor-{alpha} (TNF-{alpha}).

Nuclear factor {kappa}B (NF-{kappa}B) has been implicated in the transcriptional activation of numerous genes, including those relevant to atherogenesis and inflammatory responses.12 13 14 15 NF-{kappa}B activity is mediated by homodimeric or heterodimeric combinations of NF-{kappa}B family proteins, such as p50 (NF-{kappa}B1), p52 (NF-{kappa}B2), p65 (RelA), c-Rel, and RelB. NF-{kappa}B is found in an inactive form in the cytoplasm and appears associated with its inhibitor molecule (I-{kappa}B). On stimulation, I-{kappa}B is phosphorylated,16 and thereby NF-{kappa}B is released from I-{kappa}B, is translocated to the nucleus, and binds to the promoter DNA. In cultured human and bovine vascular endothelial cells, p50/p65 heterodimers of NF-{kappa}B appear to play a major role in cytokine-dependent transcription of E-selectin, ICAM-1, and VCAM-1 genes.17 18 19 20 21 22 Furthermore, a recent report has demonstrated that activation of NF-{kappa}B occurs in the vascular endothelium covering atherosclerotic lesions in humans.23

Oxidative modification of lipoproteins has been implicated in atherogenesis,24 and the efficacy of antioxidants has been suggested as a deterrent in the pathogenesis of atherosclerosis.25 Probucol, a cholesterol-lowering drug with antioxidant properties, has been shown to inhibit atherosclerotic progression in LDL receptor–deficient rabbits26 as well as oxidative modification of LDL.27 Antiatherogenic effects of vitamins with antioxidant properties have also been suggested.28 29 Because red wine contains large amounts of phenolic compounds that can act as antioxidants,30 31 32 33 34 consumption of red wine has also been suggested to be beneficial for the prevention of atherosclerosis. In fact, recent reports have demonstrated that intake of red wine can prevent oxidative modification of LDL in humans35 36 37 38 and prevent atherosclerotic progression in hypercholesterolemic mice.39

The phenolic compounds plentiful in red wine include galloyl compounds as well as flavonoids (catechins, anthocyanins, tannins, etc).30 31 32 33 34 In the present study, therefore, we have explored the effects of galloyl compounds on the expression of ELAMs and activation of NF-{kappa}B in cultured vascular endothelial cells.


*    Methods
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 up arrowIntroduction
 *Methods
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Reagents
Human umbilical vein endothelial cell (HUVEC) growth medium (E-300) was obtained from Kyokuto Pharm, and RPMI-1640 medium and the antibiotic-antimycotic mixture were obtained from GIBCO. Human TNF-{alpha}, IL-1{alpha}, and FCS were obtained from Boehringer Mannheim. Antibodies directed to the NF-{kappa}B subunit p65 and I-{kappa}B{alpha} were obtained from Santa Cruz Biotechnology. A double-stranded oligonucleotide having the NF-{kappa}B and activator protein 1 (AP-1) consensus sequence, the gel shift assay system, pGL2 promoter vector, pRL-TK vector, and the dual-luciferase reporter assay system were obtained from Promega. [32P]{gamma}-ATP was obtained from Amersham. Gallates were from Tokyo Kasei or Wako Pure Chemical. Anti–E-selectin and anti–ICAM-1 monoclonal antibodies were from R&D Systems. The anti–VCAM-1 monoclonal antibody was obtained from Pharmingen. The Micro-FastTrack mRNA isolation kit was obtained from Invitrogen.

Cells
Cultured HUVECs were obtained from Cell Systems (Kirkland, Wash) and were grown in E-300 medium in an atmosphere of 95% air, 5% CO2 at 37° in type I collagen–coated plastic flasks. E-300 medium consists of MCDB 107 and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 ng/mL endothelial growth factor, 10 ng/mL acidic fibroblast growth factor, 2% FCS, 10 mg/L heparin, and 10 mL/L antibiotic-antimycotic mixture. At confluence, the cells were subcultured at a 1:3 ratio and used at passage number 3 or 4. The human promyelomonocytic cell line HL-60 was obtained from the American Type Culture Collection (Manassas, Va), grown in suspension culture in RPMI-1640 containing 10% FCS, and routinely subcultured at a 1:10 ratio. Cultured bovine aortic endothelial cells (BAECs) were isolated by scraping the luminal surface of bovine aortas with a glass coverslip, and they were then cultured in DMEM containing 10% FCS in an atmosphere of 95% air and 5% CO2 at 37°C.

Endothelial Cell–Leukocyte Adhesion Assay
Leukocyte adhesion assays were performed under static conditions as previously described,40 with minor modification. HL-60 cells were labeled with a fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), by incubation with 10 µmol/L BCECF-AM at 37°C for 1 hour in RPMI-1640 medium and were subsequently washed by centrifugation. Confluent HUVECs in 96-well plates were washed 3 times, and labeled HL-60 cells (2x105 cells per 200 µL) were added to each well of HUVECs. HL-60 cells were allowed to adhere to HUVECs by incubation at 37°C for 30 minutes, and unbound HL-60 cells were removed by low-speed centrifugation (250g, 5 minutes). HL-60 cells bound to HUVECs were lysed with 50 mmol/L Tris-HCl, pH 8, containing 0.1% SDS, and the fluorescence was measured in a Millipore cytofluorometer model 2350 at 485-nm excitation and 530-nm emission.

Fluorescence-Activated Cell Sorting (FACS)
Cell-surface expression of adhesion molecules was determined by indirect immunofluorescence followed by FACS (Becton Dickinson) analysis. Trypsinized cells were resuspended in E-300, incubated with primary monoclonal antibody for 30 minutes at 4°C, washed with PBS containing 0.1% BSA and 0.1% NaH, and then stained with FITC-conjugated goat anti-mouse IgG (Caltag Laboratories) for 30 minutes at 4°C. Cells were washed again, resuspended in PBS containing 0.1% BSA and 0.1% NaH, and applied to an FACScan analyzer (Becton Dickinson).

Northern Blot Analysis
HUVECs were washed with ice-cold PBS, and mRNA was isolated using a Micro-FastTrack mRNA isolation kit (Invitrogen). Equal amounts of mRNA were subjected to electrophoresis through 1% agarose/formamide gels and blotted onto Hybond-N+ membranes (Amersham). Blotted membranes were prehybridized for 6 hours at 42°C in a solution containing 50% (vol/vol) formamide, 5x SSPE, 0.5% SDS, 10% (vol/vol) Denhardt's solution, and denatured salmon sperm DNA and hybridized with a 32P-labeled DNA probe at 42°C overnight. Membranes were then washed twice in 2x SSPE/0.1% SDS for 15 minutes at 42°C and then autoradiographed and analyzed with a Fujix Bioimage Analyzer BAS2000 (Fuji Photo Film).

Nuclear Protein Extraction and Gel Shift Assay
Confluent HUVECs were either pretreated or left untreated with samples for 12 to 15 hours and then exposed to either TNF-{alpha} (1.25 ng/mL) or IL-1{alpha} (1.25 ng/mL) for 1 hour. Nuclear protein extracts were prepared by the method of Schreiber et al.41 42 In brief, after being washed with ice-cold PBS, cells were scraped off the plates with a cell scraper in 1 mL of ice-cold buffer A (10 mmol/L HEPES/NaOH, pH 7.9; 10 mmol/L KCl; 1.5 mmol/L MgCl2; 1 mmol/L DTT; 0.5 mmol/L PMSF; 2 µg/mL aprotinin; 2 µg/mL pepstatin; 1 mmol/L sodium orthovanadate; and 2 µg/mL leupeptin). After centrifugation at 300g for 10 minutes at 4°C, cells were resuspended in 80 µL of buffer B (buffer A containing 0.1% Triton X-100) by gentle pipetting. Cell lysates were allowed to stand on ice for 10 minutes and then centrifuged at 12 000g for 10 minutes at 4°C. Nuclear pellets were resuspended in 70 µL of ice-cold buffer C (20 mmol/L HEPES/NaOH, pH 7.9; 1.5 mmol/L MgCl2; 1 mmol/L DTT; 0.2 mmol/L EDTA; 420 mmol/L NaCl; 25% glycerol; 0.5 mmol/L PMSF; 2 µg/mL aprotinin; 2 µg/mL pepstatin; 1 mmol/L sodium orthovanadate; and 2 µg/mL leupeptin), incubated on ice for 30 minutes with intermittent mixing, and then centrifuged at 15 000g for 30 minutes at 4°C. The supernatant (nuclear extract; 2 µg protein) was incubated with a 22-bp oligonucleotide containing the NF-{kappa}B consensus sequence, 5'-AGTTGAGGGGACTTTCCCAGGC-3', which had been labeled with [32P]ATP with the use of T4 polynucleotide kinase for 20 minutes at room temperature in the presence of 50 µg poly(dI-dC) and 10 mmol/L Tris-HCl buffer, pH 7.5, containing 50 mmol/L NaCl, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 4% (wt/vol) glycerol, and 1 mmol/L MgCl2. In some experiments, excess unlabeled oligonucleotide containing either the NF-{kappa}B or the AP-1 consensus sequence was added to the nuclear extracts before incubation with the radiolabeled oligonucleotide. Nuclear extract–oligonucleotide mixtures were then subjected to electrophoresis through 5% (wt/vol) polyacrylamide gels, which were subsequently dried, autoradiographed, and analyzed with the Fujix Bioimage Analyzer BAS2000 (Fuji Photo Film).

Western Blot Analysis
To measure the amounts of NF-{kappa}B p65 in nuclei, nuclear protein extracts prepared as described above were subjected to SDS-polyacrylamide (8%) gel electrophoresis, followed by electroblotting onto an Immobilon polyvinylidene transfer membrane (Millipore). To examine amounts of I-{kappa}B{alpha}, cells were lysed in a buffer containing 62.5 mmol/L Tris-HCl, 2% SDS, 10% glycerol, 0.5 mmol/L PMSF, 2 µg/mL aprotinin, 2 µg/mL pepstatin, 1 mmol/L sodium orthovanadate, and 2 µg/mL leupeptin. The whole-cell lysates were subjected to SDS-polyacrylamide (8%) gel electrophoresis, followed by electroblotting onto an Immobilon polyvinylidene difluoride transfer membrane. Membranes were probed with a rabbit polyclonal antibody directed to NF-{kappa}B p65 or a rabbit antibody directed to I-{kappa}B{alpha}, incubated with horseradish peroxidase–labeled anti-rabbit Ig for 1 hour, and then washed with PBS containing 0.1% Tween 20. Bands were visualized by ECL Western blotting detection reagents (Amersham).

Plasmid Constructs
A pGL2 luciferase vector containing 4 repeats of the consensus NF-{kappa}B binding sequence adjacent to the SV40 promoter, which was designated (NF-{kappa}B)x4-Luc, was constructed. In brief, 2 synthetic oligonucleotides, 5'-CGGGGAATTTCCGGGGAATTTCCGGGGAATTTCCGGGGAATTTCCGGGGA-3' and 5'-CGCGTCCCCGGAAATTCCCCGGAAATTCCCCGGAAATTCCCCGGAAATTCCCCGAGCT-3', were annealed in 10 mmol/L Tris-HCl (pH 7.5)/100 mmol/L NaCl by heating at 95°C for 3 minutes followed by a gradual cooling to room temperature. These double-stranded (NF-{kappa}B)x4 oligonucleotides were subcloned into the SacI/MluI site of the pGL2 promoter vector (Promega).

DNA Transfection and Luciferase Assay
BAECs cultured in 12-well plates were transfected with 1 µg (NF-{kappa}B)x4-Luc and 0.5 µg pRL-TK vector (Promega), which contains the herpes simplex virus thymidine kinase promoter and Renilla luciferase, by the lipofection method with the SuperFect reagent (Qiagen). Three hours after transfection, cells were washed with PBS and incubated in DMEM with 1% FCS for 4 hours. After pretreatment with or without 10 µmol/L ethyl gallate for 12 hours, cells were treated for an additional 10 hours with or without TNF-{alpha} (10 ng/mL) in the presence or absence of ethyl gallate. Cells were then lysed, and firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega).


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Gallates Inhibit HL-60 Adhesion to Cytokine-Activated Endothelial Cells
To explore the effects of gallates on endothelial cell–leukocyte interactions, we examined adhesion of HL-60, a promyelomonocytic cell line, to cytokine-activated HUVECs under static conditions. Unstimulated confluent HUVECs exhibited minimal binding to HL-60; however, HL-60 adhesion was substantially increased when the HUVECs were treated with inflammatory cytokines, such as IL-1{alpha} and TNF-{alpha}. Concurrent incubation of confluent HUVECs with ethyl gallate (3,4,5-trihydroxybenzoic acid ethyl ester) inhibited HL-60 adhesion to HUVECs treated with IL-1{alpha} (Figure 1ADown) or TNF-{alpha} (Figure 1BDown) in a dose-dependent manner. Half-maximal inhibition was achieved at 3 µmol/L ethyl gallate, and the maximal effect was observed at 10 µmol/L. Other gallic acid alkyl esters (methyl gallate, propyl gallate, octyl gallate, etc) and galloyl compounds (hamameritannin; tannic acid; 1,2,3,4,6-pentagalloylglucose, etc) similarly inhibited HL-60 adhesion to cytokine-activated HUVECs, suggesting that the galloyl group is an active component of galloyl compounds, and differences in ester parts of gallate molecules do not significantly affect their inhibitory activities (the TableDown).



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  		Figure 1. Effects of ethyl (Et) gallate on HL-60 cell adhesion to IL-1{alpha}– (A) and TNF-{alpha}– (B) activated HUVECs. Confluent HUVECs were preincubated with the indicated concentration of ethyl gallate and subsequently incubated with either IL-1{alpha} (1.25 ng/mL) or TNF-{alpha} (1.25 ng/mL) for 5 hours. After HUVECs were washed, BCECF-AM–labeled HL-60 cells were then added to each well and incubated for an additional 30 minutes at 37°C. Numbers of HL-60 cells bound to HUVECs were measured as described in "Methods." Experiments were performed in triplicate wells, and data are shown as mean±SD.


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  		Table 1. Gallates Inhibit HL-60 Cell Adhesion to IL-1{alpha}– or TNF-{alpha}–Activated HUVEC

To further characterize structure-activity relationships in the gallate-induced inhibition of leukocyte adhesion to cytokine-activated endothelium, the effects of a series of structural analogues were examined (TableUp). Deletion of a hydroxyl group at the 5 position of ethyl gallate, yielding the 3,4-dihydroxybenzoic acid ethyl ester (compound 8), showed reduced inhibitory activity. Deletion of a hydroxyl group at the 4 position of methyl gallate (compound 10) resulted in the loss of activity. Disruption of hydroxyl groups at both positions 3 and 5 of ethyl gallate (compound 12) or its isomer (compound 11) did not show inhibitory activity. 1,2,3-Trihydroxybenzene (compound 13), which lacks a carboxyl group of gallates, was also without activity. These results suggest that the 3,4,5-trihydroxybenzoate structure (the galloyl structure) is a minimum component required for activity and that the positions of hydroxyl groups in gallate compounds are critical determinants. Furthermore, an ester bond of gallic acid appears to be important in the inhibitory actions of gallates.

Ethyl Gallate Inhibits Cell-Surface Expression of ELAMs
Previous studies have revealed that adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, play significant roles in HL-60 adhesion to cytokine-activated HUVECs. Therefore, to examine whether gallate-induced decreases in the adhesion of HL-60 cells to cytokine-activated HUVECs depend on reduced expression of ICAM-1, E-selectin, and VCAM-1, we conducted FACS analysis with the use of specific monoclonal antibodies. In accordance with previous studies, pretreatment of HUVECs with IL-1{alpha} or TNF-{alpha} for 5 hours induced cell-surface expression of VCAM-1, ICAM-1, and E-selectin (Figure 2ADown). Pretreatment of HUVECs with ethyl gallate markedly suppressed IL-1{alpha}– and TNF-{alpha}–induced expression of VCAM-1, ICAM-1, and E-selectin. Ethyl gallate, at a concentration of 10 µmol/L, inhibited IL-1{alpha}–induced expression of VCAM-1, ICAM-1, and E-selectin by 58%, 54%, and 85%, respectively. TNF-{alpha}–induced expression of VCAM-1, ICAM-1, and E-selectin was blocked by ethyl gallate by 87%, 56%, and 91%, respectively. Constitutive expression of ICAM-1 in unstimulated HUVECs, in contrast, was not significantly altered by the same treatment (10 µmol/L) with ethyl gallate (Figure 2BDown). Ethyl gallate did not affect total cellular protein synthesis, which was assessed by uptake of tritiated leucine, or cell morphology, as assessed by microscopic observation (data not shown). Taken together, these findings indicate that ethyl gallate does not cause general cytotoxicity but specifically inhibits cytokine-induced expression of ELAMs.



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  		Figure 2. Effects of ethyl gallate on IL-1{alpha}– and TNF-{alpha}–induced cell-surface expression of E-selectin, VCAM-1, and ICAM-1 (A) and the constitutive expression of ICAM-1 (B). A, Confluent HUVECs were pretreated with 10 µmol/L ethyl gallate and then exposed to either TNF-{alpha} (1.25 ng/mL) or IL-1{alpha} (1.25 ng/mL) for 5 hours. Cell-surface expression of adhesion molecules was determined by indirect immunofluorescence and FACS analysis as described in "Methods." Representative histograms are shown. The x axis indicates relative fluorescence on a logarithmic scale; the y axis shows the number of cells on a linear scale. Cells (104)were analyzed for each histogram. B, Confluent HUVECs were pretreated with 10 µmol/L ethyl gallate without cytokine stimulation for 20 hours. Cell-surface adhesion molecule expression was then determined by indirect immunofluorescence and FACS analysis as described in "Methods."

Ethyl Gallate Decreases mRNA for ELAMs
To determine whether the reduced cell-surface expression of VCAM-1, ICAM-1, and E-selectin by ethyl gallate depends on decreased amounts of mRNA for VCAM-1, ICAM-1, and E-selectin, we conducted Northern blot analysis. Treatment of HUVECs with IL-1{alpha} or TNF-{alpha} for 3 hours resulted in increased amounts of mRNA for VCAM-1, ICAM-1, and E-selectin (Figure 3Down), as previously reported. Pretreatment of HUVECs with ethyl gallate markedly suppressed the IL-1{alpha}– and TNF-{alpha}–induced increases in the amounts of VCAM-1, ICAM-1, and E-selectin mRNA. Levels of GAPDH mRNA, in contrast, were not significantly altered by the same treatment with ethyl gallate.



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  		Figure 3. Effects of ethyl (Et) gallate on IL-1{alpha}– and TNF-{alpha}–induced E-selectin and ICAM-1 mRNA expression in HUVECs. Confluent HUVECs were pretreated with 10 µmol/L ethyl gallate or were left untreated for 15 hours and subsequently incubated with either IL-1{alpha} (1.25 ng/mL) or TNF-{alpha} (1.25 ng/mL) for 3 hours. E-selectin and ICAM-1 mRNA levels were determined by Northern blot analysis with the use of 32P-labeled cDNA probes as described in "Methods." Blots were rehybridized with GAPDH cDNA.

Ethyl Gallate Inhibits Cytokine-Induced Activation of NF-{kappa}B
Transcriptional regulation involving activation of NF-{kappa}B has been implicated in the cytokine-induced expression of VCAM-1, ICAM-1, and E-selectin genes.17 18 19 20 21 22 To examine whether ethyl gallate inhibits NF-{kappa}B activation, we performed gel shift assays with the use of a 32P-labeled oligonucleotide with the consensus NF-{kappa}B binding sequence. HUVECs were preincubated with different concentrations of ethyl gallate and subsequently stimulated with IL-1{alpha} or TNF-{alpha} for 1 hour at 37°C. Gel shift assays showed that treatment with IL-1{alpha} or TNF-{alpha} resulted in the appearance of shifted bands, as previously reported (Figure 4Down). These shifted bands are specific for NF-{kappa}B binding, because they were undetectable when a 100-fold excess of unlabeled NF-{kappa}B oligonucleotide was included (data not shown). Pretreatment with ethyl gallate reduced the densities of the NF-{kappa}B shifted bands induced by IL-1{alpha} or TNF-{alpha} (Figure 4Down). Pretreatment with ethyl gallate (1 to 20 µmol/L) for 15 hours dose-dependently reduced the densities of the NF-{kappa}B shifted bands induced by IL-1{alpha} (Figure 5ADown). As shown in Figure 5BDown, preincubation of HUVECs with ethyl gallate time-dependently inhibited NF-{kappa}B activation. With regard to the structural specificity of gallates, differences in the alkyl chain length of gallate did not significantly influence the inhibitory effects on the cytokine-induced activation of NF-{kappa}B (data not shown). These results appear to be in parallel with the inhibitory effects of ethyl gallate on HL-60 cell adhesion to cytokine-activated HUVECs.



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  		Figure 4. Effects of ethyl (Et) gallate on IL-1{alpha}– and TNF-{alpha}–induced NF-{kappa}B activation in HUVECs. Confluent HUVECs were preincubated for 15 hours at 37°C with 10 µmol/L ethyl gallate, followed by a 1-hour incubation with either 1.25 ng/mL IL-1{alpha} (A) or 1.25 ng/mL TNF-{alpha} (B). Nuclear protein extracts were prepared, and a gel shift assay was performed using radiolabeled oligonucleotides containing consensus NF-{kappa}B binding sequences, as described in "Methods."



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  		Figure 5. Dose and time dependence of ethyl (Et) gallate on the inhibition of IL-1{alpha}–dependent NF-{kappa}B activation in HUVECs. A, Dose response of inhibition of IL-1{alpha}–induced NF-{kappa}B activation by ethyl gallate in HUVECs. Confluent HUVECs were preincubated for 15 hours at 37°C with different concentrations of ethyl gallate, followed by a 1-hour incubation with IL-1{alpha} (1.25 ng/mL). B, Time-dependent inhibition of IL-1{alpha}–dependent NF-{kappa}B activation by ethyl gallate in HUVECs. Confluent HUVECs were preincubated for the indicated time at 37°C with 10 µmol/L ethyl gallate, followed by a 1-hour incubation with IL-1{alpha} (1.25 ng/mL). After these treatments, nuclear protein extracts were prepared, and gel shift assays were carried out using the NF-{kappa}B consensus binding sequence as described in "Methods." C, Effects of ethyl gallate on NF-{kappa}B/DNA binding in vitro. Equal amounts of nuclear protein extracts from IL-1{alpha}–activated HUVECs were incubated with or without 10 µmol/L ethyl gallate for 10 minutes at room temperature. After incubation, 32P-labeled NF-{kappa}B oligonucleotides were added, and NF-{kappa}B/DNA binding was assayed as described in "Methods."

Ethyl Gallate Inhibits Nuclear Translocation of NF-{kappa}B p65
Inflammatory cytokines, such as IL-1{alpha} and TNF-{alpha}, have been shown to induce rapid phosphorylation and proteolytic degradation of I-{kappa}B{alpha}, a cytoplasmic inhibitor of NF-{kappa}B activation, resulting in translocation of the activated p50/p65 heterodimer of NF-{kappa}B from the cytoplasm to the nucleus. Herbimycin A43 and caffeic acid phenethyl ester (CAPE)44 have been shown to inhibit activation of NF-{kappa}B by blocking the binding of NF-{kappa}B to DNA. To determine whether the inhibitory effects of gallates on NF-{kappa}B activation were due to the inhibition of NF-{kappa}B binding to DNA or the inhibition of nuclear translocation of NF-{kappa}B, we examined the effect of ethyl gallate on NF-{kappa}B and DNA binding. Nuclear extracts from IL-1{alpha}–stimulated HUVECs were incubated with 10 µmol/L ethyl gallate. Figure 5CUp shows that ethyl gallate was not able to prevent NF-{kappa}B binding to DNA. We further examined the NF-{kappa}B p65 protein levels in the nuclei of IL-1{alpha}– and TNF-{alpha}–stimulated HUVECs by Western blot analysis. As shown in Figure 6Down, IL-1{alpha}– and TNF-{alpha}–dependent translocation of NF-{kappa}B p65 to the nucleus was suppressed by pretreatment with ethyl gallate. In contrast, time-dependent decreases in I-{kappa}B{alpha} protein in whole-cell lysates of IL-1{alpha}– and TNF-{alpha}–activated HUVECs were not significantly altered by ethyl gallate pretreatment (Figure 6Down). These results demonstrate that ethyl gallate inhibits the translocation of NF-{kappa}B p65 to the nucleus but not by blocking the binding of NF-{kappa}B to DNA and without affecting degradation of the inhibitor protein I-{kappa}B{alpha}.



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  		Figure 6. Ethyl (Et) gallate inhibits nuclear translocation of NF-{kappa}B p65 in IL-1{alpha}–stimulated HUVECs. Confluent HUVECs pretreated with 10 µmol/L ethyl gallate were incubated for different times with IL-1{alpha} (1.25 ng/mL) or TNF{alpha} (10 ng/mL), and the amounts of NF-{kappa}B p65 in nuclei and I-{kappa}B{alpha} in whole-cell lysates were measured by Western blot analysis as described in "Methods."

Ethyl Gallate Inhibits Cytokine-Induced Transcription of an NF-{kappa}B–Dependent Gene
To further examine the roles of ethyl gallate in NF-{kappa}B–dependent gene transcription, we constructed a fusion gene containing the SV40 promoter, 4 repeats of the consensus NF-{kappa}B binding sequence, and the luciferase reporter gene. We transfected BAECs with this promoter-reporter gene construct and measured transcriptional activities by stimulating the cells with inflammatory cytokines, with or without ethyl gallate pretreatment. Treatment of BAECs with TNF-{alpha} resulted in a 4.4-fold increase in NF-{kappa}B–driven luciferase activity. Ethyl gallate pretreatment inhibited the TNF-{alpha}–induced luciferase activity by 47% (Figure 7Down). Taken together, these findings indicate that ethyl gallate inhibits cytokine-induced NF-{kappa}B activation and thereby suppresses expression of ELAMs.



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  		Figure 7. Effects of ethyl (Et) gallate on NF-{kappa}B–dependent gene transcription. BAECs were transfected with (NF-{kappa}B)x4-Luc plasmid by the lipofection method. After transfection, cells were cultured in the absence or presence of 10 µmol/L ethyl gallate for 12 hours and stimulated with TNF-{alpha} (10 ng/mL) for 10 hours. Cell extracts were then prepared and assayed for firefly and Renilla luciferase activity by using the dual-luciferase reporter assay system.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Activation of NF-{kappa}B appears to play crucial roles in the transcription of a variety of genes relevant to atherogenesis as well as to the inflammatory response.1 2 3 4 5 6 7 8 9 10 11 12 These proatherogenic genes include cytokines and chemokines such as TNF-{alpha}, IL-1, macrophage colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage chemotactic protein-1,1 12 13 14 15 and tissue factor.45 46 Furthermore, in addition to ELAMs, a recent report has demonstrated that NF-{kappa}B is activated in human atherosclerotic lesions in vivo.23 NF-{kappa}B, therefore, appears to be a key factor that can modulate inflammatory responses and procoagulant activities in atherosclerotic lesions by inducing multiple genes. The present study provides evidence that gallates, naturally occurring phenolic compounds, can suppress cytokine-induced activation of NF-{kappa}B and subsequent expression of ELAMs, such as VCAM-1, ICAM-1, and E-selectin. Gallates may also inhibit expression of other NF-{kappa}B–dependent genes relevant to atherogenesis and thus may protect against atherosclerotic progression in vivo.

Galloyl compounds are naturally present in plants as free acid (gallic acid), alkyl esters (methyl gallate, ethyl gallate, etc), and galloyl tannins (hamameritannin, galloyl glucose, epicatechin gallate, procyanidin gallate, etc). These compounds have been reported to show anti-inflammatory,47 48 antimutagenic,49 antimicrobial,50 and radical scavenger51 activities. In addition to these biological effects of gallates, the present study provides evidence that gallates can act as inhibitors of NF-{kappa}B. The inhibitory actions of gallates do not appear to result from nonspecific cellular toxicity but rather are specific for NF-{kappa}B–dependent gene transcription, because overall cellular protein synthesis (data not shown), expression of GAPDH (Figure 3Up), and constitutive ICAM-1 expression (Figure 7Up) were not significantly affected by gallates. The structural specificity of galloyl compounds in the inhibition of leukocyte adhesion to cytokine-activated HUVECs shows that a galloyl group appears to be an active component of galloyl compounds (the TableUp and Figure 2BUp).

The roles of reactive oxygen intermediates, protein tyrosine kinase, protein kinase C, protein tyrosine phosphatase, proteases, and ceramide have been documented in the IL-1– and TNF-{alpha}–induced activation of NF-{kappa}B.52 53 54 55 Among these, reactive oxygen intermediates including H2O2 have been suggested as important mediators that can activate NF-{kappa}B. Salicylates, N-acetylcysteine, pyrrolidine dithiocarbamate, and CAPE have been shown to exhibit inhibitory effects on NF-{kappa}B activation, in part through their antioxidant actions. Polyphenolic compounds, including gallates, are known to show antioxidant activities by scavenging free radicals.51 56 57 Gallates, therefore, may also suppress NF-{kappa}B activation by a mechanism depending, at least in part, on reactive oxygen intermediates. In the present study, the time course of ethyl gallate pretreatment required for NF-{kappa}B inactivation was relatively long. This suggests that ethyl gallate may not readily permeate through the plasma membrane or that certain metabolites of ethyl gallate might exhibit inhibitory actions on NF-{kappa}B.

Among inhibitors of NF-{kappa}B, salicylates58 59 and curcumin60 appear to suppress the cytokine-induced activation of NF-{kappa}B by inhibiting the degradation of I-{kappa}B and the nuclear translocation of NF-{kappa}B p65 but not by blocking the binding of NF-{kappa}B to the promoter DNA. CAPE,44 N-tosyl-L-phenylalanine chloromethyl ketone,52 and herbimycin A43 have been shown to block NF-{kappa}B binding to the promoter DNA. In addition, CAPE has been shown to inhibit translocation of NF-{kappa}B p65 without affecting degradation of I-{kappa}B{alpha}. These molecular mechanisms appear similar to those of ethyl gallate shown in this study, although ethyl gallate does not directly block NF-{kappa}B binding to the target DNA. At present, it remains unclear how ethyl gallate can inhibit nuclear translocation of NF-{kappa}B p65 without altering the degradation of I-{kappa}B{alpha}. It might be that ethyl gallate inhibits activation of other inhibitor proteins, such as I-{kappa}Bß, p105/I-{kappa}B-{gamma}, p100/I-{kappa}B-{delta}, and I-{kappa}B-{epsilon}, although the roles of these molecules in vascular endothelial cells have not been clarified. In addition, ethyl gallates might also inhibit nuclear translocation of other transcription factors, thus modulating a variety of endothelial functions.

Flavonoids, including apigenin61 and PD098063,62 belong to another group of phenolic compounds that have antioxidant properties. Apigenin suppressed cytokine-induced VCAM-1, ICAM-1, and E-selectin expression by inhibiting NF-{kappa}B activation without affecting nuclear translocation of NF-{kappa}B p65.61 PD098063 selectively inhibited VCAM-1 expression by a mechanism independent of NF-{kappa}B, without altering cytokine-induced ICAM-1 expression.62 Therefore, antioxidants in general do not necessarily inhibit NF-{kappa}B; furthermore, mechanisms other than those associated with NF-{kappa}B appear to be involved in the expression of ELAMs. In fact, transcriptional regulation depending on c-Jun and activating transcription factor-2 are also involved in E-selectin expression induced by TNF-{alpha}.63 It might be that gallates can also inhibit the c-Jun/activating transcription factor-2 pathway.

In addition, previous studies have also reported that different NF-{kappa}B inhibitors can differently inhibit the expression of ELAMs. As reported by Marui et al,64 50 µmol/L pyrrolidine dithiocarbamate inhibited cytokine-induced expression of VCAM-1, but not of E-selectin or ICAM-1, in HUVECs. Pierce et al58 reported that 10 mmol/L sodium salicylate selectively inhibited cell-surface expression of VCAM-1 and ICAM-1 with negligible effects on E-selectin. Aspirin also inhibited TNF-{alpha}–induced expression of VCAM-1 and E-selectin but did not significantly affect ICAM-1 expression. In the present study, gallates inhibited cytokine-induced expression of VCAM-1, ICAM-1, and E-selectin, suggesting that dependence on NF-{kappa}B in their transcriptional regulation may be different among the ELAMs and that mechanisms other than those involving NF-{kappa}B may be operative. It remains to be fully clarified how gallates affect other transcriptional regulatory mechanisms, as well as signal transduction cascades upstream from the NF-{kappa}B activation and expression of ELAMs.

Oxidative modification of lipoproteins has been implicated in atherogenesis, and the efficacy of antioxidants has been suggested in the prevention of atherosclerotic progression. Because phenolic compounds, including flavonoids (catechins, anthocyanins, tannins, etc) and galloyl compounds,30 31 32 33 34 are plentiful in red wine, consumption of red wine may have beneficial effects on the prevention of atherosclerosis by inhibiting NF-{kappa}B–dependent proatherogenic gene expression, as well as suppressing the oxidative modification of lipoproteins.35 36 37 38 In fact, consumption of red wine by hypercholesterolemic mice was able to reduce atherosclerotic progression in these animals.39 Further studies on the effects of gallates in vivo may extend the possible actions of gallates and may provide a novel therapeutic approach.

In summary, the present study demonstrates that gallates, natural phenolic compounds, can inhibit NF-{kappa}B activation by suppressing the translocation of NF-{kappa}B p65. Further studies related to the anti-inflammatory actions of gallates, especially in vivo, may provide fresh insights into the pathogenesis of atherosclerosis.

Received March 18, 1998; accepted December 1, 1998.


*    References
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