© 1999 American Heart Association, Inc.
Vascular Biology |
Gallates Inhibit Cytokine-Induced Nuclear Translocation of NF-
B and Expression of Leukocyte Adhesion Molecules in Vascular Endothelial Cells
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
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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
B
(NF-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
(IL-1)– or tumor necrosis
factor- (TNF-)– 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-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-B consensus sequence and the
luciferase reporter gene. Immunoblot analysis of
nuclear extracts and whole-cell lysates demonstrated the decreased
amounts of NF-B p65 in nuclei but equal amounts of
inhibitor-B (I-B) 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-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-B p65 by way of a mechanism independent of I-B
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-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
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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
(IL-1) and tumor necrosis factor- (TNF-).
Nuclear factor B (NF-B) has been implicated in the
transcriptional activation of numerous genes, including those relevant
to atherogenesis and inflammatory responses.12 13 14 15 NF-B
activity is mediated by homodimeric or heterodimeric combinations of
NF-B family proteins, such as p50 (NF-B1), p52 (NF-B2), p65
(RelA), c-Rel, and RelB. NF-B is found in an inactive form in the
cytoplasm and appears associated with its inhibitor
molecule (I-B). On stimulation, I-B is
phosphorylated,16 and thereby NF-B is
released from I-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-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-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-B in cultured vascular
endothelial cells.
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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-
, IL-1, and FCS were obtained from
Boehringer Mannheim. Antibodies directed to the NF-B subunit
p65 and I-B were obtained from Santa Cruz Biotechnology. A
double-stranded oligonucleotide having the NF-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]
-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- (1.25
ng/mL) or IL-1 (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-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-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-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-B, 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-B p65 or a
rabbit antibody directed to I-B, 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-B binding sequence adjacent to the SV40 promoter, which was
designated (NF-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-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-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- (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).
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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
and TNF-. 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
(Figure 1A
) or TNF- (Figure 1B)
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
Table).
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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 (Table
).
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
or TNF- for 5 hours induced cell-surface expression of VCAM-1,
ICAM-1, and E-selectin (Figure 2A).
Pretreatment of HUVECs with ethyl gallate markedly suppressed IL-1–
and TNF-–induced expression of VCAM-1, ICAM-1, and E-selectin.
Ethyl gallate, at a concentration of 10 µmol/L, inhibited
IL-1–induced expression of VCAM-1, ICAM-1, and E-selectin by 58%,
54%, and 85%, respectively. TNF-–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 2B).
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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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 or
TNF- for 3 hours resulted in increased amounts of mRNA for VCAM-1,
ICAM-1, and E-selectin (Figure 3), as
previously reported. Pretreatment of HUVECs with ethyl gallate markedly
suppressed the IL-1– and TNF-–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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Ethyl Gallate Inhibits Cytokine-Induced Activation of
NF-B
Transcriptional regulation involving activation of NF-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-B activation, we performed gel
shift assays with the use of a 32P-labeled
oligonucleotide with the consensus NF-B binding
sequence. HUVECs were preincubated with different concentrations of
ethyl gallate and subsequently stimulated with IL-1 or TNF- for 1
hour at 37°C. Gel shift assays showed that treatment with IL-1 or
TNF- resulted in the appearance of shifted bands, as previously
reported (Figure 4). These shifted bands
are specific for NF-B binding, because they were undetectable when a
100-fold excess of unlabeled NF-B oligonucleotide
was included (data not shown). Pretreatment with ethyl gallate reduced
the densities of the NF-B shifted bands induced by IL-1 or
TNF- (Figure 4). Pretreatment with ethyl gallate (1 to
20 µmol/L) for 15 hours dose-dependently reduced the densities
of the NF-B shifted bands induced by IL-1 (Figure 5A). As shown in Figure 5B, preincubation of HUVECs with ethyl gallate time-dependently inhibited
NF-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-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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Ethyl Gallate Inhibits Nuclear Translocation of NF-B
p65
Inflammatory cytokines, such as IL-1 and TNF-, have
been shown to induce rapid phosphorylation and
proteolytic degradation of I-B, a cytoplasmic
inhibitor of NF-B activation, resulting in translocation
of the activated p50/p65 heterodimer of NF-B from the
cytoplasm to the nucleus. Herbimycin A43 and caffeic acid
phenethyl ester (CAPE)44 have been shown to inhibit
activation of NF-B by blocking the binding of NF-B to DNA. To
determine whether the inhibitory effects of gallates on
NF-B activation were due to the inhibition of NF-B binding to DNA
or the inhibition of nuclear translocation of NF-B, we examined the
effect of ethyl gallate on NF-B and DNA binding. Nuclear extracts
from IL-1–stimulated HUVECs were incubated with 10 µmol/L
ethyl gallate. Figure 5C shows that ethyl gallate was not able
to prevent NF-B binding to DNA. We further examined the NF-B p65
protein levels in the nuclei of IL-1– and TNF-–stimulated
HUVECs by Western blot analysis. As shown in Figure 6, IL-1– and TNF-–dependent
translocation of NF-B p65 to the nucleus was suppressed by
pretreatment with ethyl gallate. In contrast, time-dependent decreases
in I-B protein in whole-cell lysates of IL-1– and
TNF-–activated HUVECs were not significantly altered by
ethyl gallate pretreatment (Figure 6). These results demonstrate
that ethyl gallate inhibits the translocation of NF-B p65 to the
nucleus but not by blocking the binding of NF-B to DNA and without
affecting degradation of the inhibitor protein
I-B.
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Ethyl Gallate Inhibits Cytokine-Induced Transcription of an
NF-B–Dependent Gene
To further examine the roles of ethyl gallate in
NF-B–dependent gene transcription, we constructed a fusion gene
containing the SV40 promoter, 4 repeats of the consensus NF-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- resulted in a 4.4-fold increase in
NF-B–driven luciferase activity. Ethyl gallate pretreatment
inhibited the TNF-–induced luciferase activity by 47% (Figure 7). Taken together, these findings
indicate that ethyl gallate inhibits cytokine-induced
NF-B activation and thereby suppresses expression of ELAMs.
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Discussion |
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Activation of NF-
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-, 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-B is activated in human
atherosclerotic lesions in vivo.23 NF-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-B and subsequent expression of ELAMs, such as
VCAM-1, ICAM-1, and E-selectin. Gallates may also inhibit expression of
other NF-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-B. The
inhibitory actions of gallates do not appear to result from
nonspecific cellular toxicity but rather are specific for
NF-B–dependent gene transcription, because overall cellular protein
synthesis (data not shown), expression of GAPDH (Figure 3), and
constitutive ICAM-1 expression (Figure 7) 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 Table and
Figure 2B).
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-–induced
activation of NF-B.52 53 54 55 Among these, reactive oxygen
intermediates including
H2O2 have been suggested as
important mediators that can activate NF-B. Salicylates,
N-acetylcysteine, pyrrolidine dithiocarbamate, and CAPE have
been shown to exhibit inhibitory effects on NF-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-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-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-B.
Among inhibitors of NF-B,
salicylates58 59 and curcumin60 appear to
suppress the cytokine-induced activation of NF-B by
inhibiting the degradation of I-B and the nuclear translocation of
NF-B p65 but not by blocking the binding of NF-B to the promoter
DNA. CAPE,44
N-tosyl-L-phenylalanine
chloromethyl ketone,52 and herbimycin
A43 have been shown to block NF-B binding to the
promoter DNA. In addition, CAPE has been shown to inhibit translocation
of NF-B p65 without affecting degradation of I-B. These
molecular mechanisms appear similar to those of ethyl gallate shown in
this study, although ethyl gallate does not directly block NF-B
binding to the target DNA. At present, it remains unclear how ethyl
gallate can inhibit nuclear translocation of NF-B p65 without
altering the degradation of I-B. It might be that ethyl gallate
inhibits activation of other inhibitor proteins, such as
I-Bß, p105/I-B-, p100/I-B-
, and I-B-
, 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-B activation without affecting nuclear
translocation of NF-B p65.61 PD098063 selectively
inhibited VCAM-1 expression by a mechanism independent of NF-B,
without altering cytokine-induced ICAM-1
expression.62 Therefore, antioxidants in general do not
necessarily inhibit NF-B; furthermore, mechanisms other than those
associated with NF-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-.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-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-–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-B in their
transcriptional regulation may be different among the ELAMs and that
mechanisms other than those involving NF-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-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-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-B activation by suppressing the
translocation of NF-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.
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