(Circulation. 1998;98:2255-2261.)
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports |
-Tocopherol Enrichment of Monocytes Decreases Agonist-Induced Adhesion to Human Endothelial Cells
From the Departments of Pathology (K.N.I., S.D., I.J.) and Internal Medicine (I.J.), University of Texas Southwestern Medical Center at Dallas.
Correspondence to Ishwarlal Jialal, MD, PhD, Department of Pathology and Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9073. E-mail jialal.i{at}pathology.swmed.edu
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Abstract |
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Background—Monocyte-endothelium adhesion is a crucial early event in atherogenesis. Several reports indicate that
-tocopherol (AT) is a potent antioxidant
in plasma and LDL and also has intracellular effects that are
antiatherogenic. Recently, it has been shown that AT supplementation
results in decreased monocyte–endothelial cell
adhesion. However, there is a paucity of data on the mechanisms by
which AT inhibits adhesion of monocytes. We studied the effect of AT
enrichment of a human monocytic cell line, U937, on adhesion to human
umbilical vein endothelial cells (HUVECs).
Methods and Results—Both lipopolysaccharide (LPS)– and
N-formyl-methionyl-leucyl-phenylalanine
(FMLP)–stimulated U937 adhesion to HUVECs were studied. AT (50 and
100 µmol/L) significantly decreased adhesion of both LPS- and
FMLP-stimulated U937 cells to HUVECs (LPS-treated cells,
P<0.0125; FMLP-treated cells, P<0.05).
Expression of the adhesion molecules CD11a, CD11b, CD11c, very late
antigen-4 (VLA-4), and L-selectin, as assessed by flow cytometry, was
increased in the stimulated U937 cells, and AT resulted in significant
reduction in the expression of CD11b and VLA-4. In addition, activation
of the transcription factor nuclear factor-
B (NF-B), as assessed
by gel shift assays, was inhibited by pretreatment with AT in
LPS-treated U937 cells.
Conclusions—AT significantly decreases adhesion of
activated monocytes to endothelial cells by
decreasing expression of CD11b and VLA-4 on monocytes, possibly by
inhibiting the activation of NF-B.
Key Words: cell adhesion molecules • antioxidants • endothelium • NF-kappa B
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Introduction |
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The monocyte-macrophage is a critical cell in atherogenesis.1 2 An early event in the genesis of the fatty streak lesion is the attachment of monocytes to vascular endothelium. After monocytes have adhered to the endothelium, they migrate into the intima, imbibe lipid, and become foam cells. The exact mechanism by which monocyte-endothelium adhesion occurs in vivo remains to be elucidated. Recent work has identified specific adhesion molecules on endothelial cells (ECs) and monocytes.3 These adhesion molecules include E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) on ECs and members of the ß-2 integrin family (CD11a, CD11b, and CD11c/18) and the ß-1 integrin VLA-4 (very late antigen-4 [CD49d/29]) on monocytes. Modulation of monocyte-EC adhesion could be an important target in the treatment of atherosclerosis. It has also been shown that agonist-induced adhesion of monocytes to the endothelium is mediated by activation of the transcription factor nuclear factor-
B
(NF-B).4 Recent findings implicate reactive
oxygen species in NF-B activation.5 Thus, the
modulation of these processes, ie, activation of NF-B, expression of
adhesion molecules, and monocyte-EC adhesion by antioxidants, assumes
great significance. Recently, Weber et al6 showed
that increased adhesion of monocytes to ECs in smokers with increased
expression of CD11b could be prevented by vitamin C
supplementation.
-Tocopherol (AT) is a potent lipophilic
antioxidant that protects membranes from lipid
peroxidation.7 8 In addition to decreasing the
oxidative susceptibility of LDL, AT has been shown to have direct
antiatherogenic effects on cells.9 Recently, it
has been shown that supplementation of human volunteers with AT (1200
IU/d) significantly decreased adhesion of human monocytes to human
umbilical vein endothelial cells (HUVECs) and decreased
the secretion of interleukin-1ß.10 Faruqi et
al11 showed that AT enrichment of ECs inhibited
agonist-induced adhesion of U937 cells to HUVECs. More recently, Martin
et al12 showed that in vitro enrichment of human
aortic ECs with AT significantly inhibited LDL-induced adhesion
of monocytes to ECs in a dose-dependent manner, with a
concomitant reduction in levels of soluble ICAM-1. However, to
date there are no studies examining the effect of AT enrichment of
monocytes on adhesion. Thus, in the present study, we examined the
effect of AT enrichment of monocytes on subsequent adhesion to ECs
after activation with 2 agonists, lipopolysaccharide (LPS) and
N-formyl-methionyl-leucyl-phenylalanine (FMLP). The effect
of AT on the expression of adhesion molecules on monocytes and on
NF-B activation was also studied to gain insight into the mechanisms
by which AT mediated this effect.
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Methods |
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Reagents
The reagents used for the experiments were as follows: RPMI 1640, endothelial cell growth medium (EGM), antibiotics, leupeptin, PMSF, pyrrolidinedithiocarbamate (PDTC), DTT, EGTA, and Nonidet P-40 were from Sigma Chemical Co; 5- and 6-carboxyfluorescein diacetate succinimidyl ester [5(6)-CFDA SE] were from Molecular Probes Inc; poly (dI-dC) was from Pharmacia; FMLP was from Peninsula Laboratories; and the monoclonal antibodies anti-CD11a-FITC, anti-CD11b-PE, anti-CD11c-PE, VLA-4, and anti-L-selectin were from Becton Dickinson. Antibody to the p65 subunit of NF-
B was purchased from
Boehringer Mannheim.
Cell Cultures
HUVECs were purchased from Clonetics Laboratories and cultured
in EGM as described previously.13 14 HUVECs were
cultured in EGM and were maintained at confluence in 5%
CO2-95% air and passaged according to standard
procedures. HUVECs were used 24 hours after confluence between 3 and 10
passages. Isolated ECs were tested by the vendor (Clonetics) to have an
endotoxin concentration of <0.125 EU (endotoxin units)/mL. The
human monocytic tumor cell line U937 was purchased from American Type
Culture Collection and maintained at 0.5 to 1.0 million cells/mL in
RPMI 1640 media supplemented with 10% heat-inactivated
fetal bovine serum (FBS) containing 100 U/mL penicillin, 100 µg/mL
streptomycin, and 2 mmol/L glutamine. Cells were split with fresh
media 1:5 every 2 to 3 days. Cell counts were performed routinely to
maintain low population density.
Supplementation of U937 Cells With -Tocopherol
For all experiments of monocyte-EC adhesion, expression of
counterreceptors, and activation of the transcription factor NFB, 1
set of U937 cells was incubated overnight with the specified
concentrations of AT as described in the figures and below and another
set was incubated with DMSO as vehicle control. After overnight
incubation, cells were pelleted, washed, and incubated again with DMSO
or AT for 30 minutes at a similar concentration before activation with
LPS or FMLP.
Monocyte-EC Adhesion
HUVECs were seeded in 24-well plates for 3 to 4 days before the
experiment. Only confluent monolayers were used, as confirmed by
microscopic inspection the day before the assay. U937 cells were
incubated in the absence and presence of different concentrations of AT
(25, 50, and 100 µmol/L) overnight in RPMI 1640 medium
containing 10% FBS at 37°C. A stock solution of AT (10 mmol/L)
was prepared in DMSO solution, and 10 µL was added to 1 mL of U937
cells to give a final concentration of 100 µmol/L AT in 1%
DMSO. DMSO was added as vehicle control to cells that were not
incubated with AT. Cells were pelleted and reconstituted in RPMI
containing 2% FBS. After 20 to 30 minutes' preincubation with or
without AT, 1 set of cells was incubated with 100 ng/mL LPS for 2 hours
and another set with 10-7 mol/L FMLP for 30
minutes at 37°C. After a defined incubation period as determined from
preliminary experiments (2 hours for LPS or 30 minutes for FMLP), cells
were again pelleted and reconstituted in RPMI containing 2% FBS and
4 µmol/L of the fluorescent dye 5(6)-CFDA
SE.15 16 Before addition of dye, an aliquot of
cells was kept for fluorescent-activated cell sorter
(FACS) analysis of adhesion molecules. After 30 minutes'
incubation at 37°C, dye loading was stopped by the addition of an
excess of cold RPMI containing 2% FBS. Fluorescence-labeled
cells were pelleted and resuspended (1x106/mL)
in EGM. HUVECs were washed twice with EGM before addition of
fluorescence-loaded U937 cells
(0.5x106/mL) and incubated at 37°C, 5%
CO2 at 90% humidity. After 30 minutes, the cell
supernatant was aspirated, and the bound cells were gently washed twice
with phenol-free RPMI 1640. Cells were lysed with 1 mL of 0.01% Triton
X-100 in 0.1 mol/L Tris buffer per well (pH 8.0), and
fluorescence was measured with excitation and emission
wavelengths of 485 and 535 nm, respectively. Unactivated U937
cells were used to assess basal adhesion. Wells containing HUVECs only
were used as blanks. To simulate the in vivo supplemented state, both
HUVECs and U937 cells were pretreated overnight with AT to determine if
incubation of both types of cells with AT resulted in further
inhibition of adhesion.
FACS Analysis of Adhesion Molecules
Expression of counterreceptors (CD11a/18, CD11b/18, CD11c/18,
CD49d/18, and L-selectin) on LPS- and FMLP-activated monocytes
was assessed by flow cytometry with a FACScan flow cytometer (Becton
Dickinson). LPS- and FMLP-activated monocytes
(1x106 cells/mL), with or without preincubation
with AT, were stained by routine methods with saturating amounts of
directly conjugated, commercially available monoclonal antibodies with
phycoerythrin (PE), FITC, and tricolor (TC) fluorochromes. To avoid
nonspecific binding to Fc receptors, cells were preincubated
with 5% human serum in PBS for 15 minutes on ice, washed, and then
stained. Cells were fixed with 1% paraformaldehyde
before being acquired by the FACScan flow cytometer. Isotype-matched
PE, FITC, and TC controls were run with each sample. Samples were
analyzed with "Paint-a-gate" software (Becton Dickinson)
and results expressed in units of mean fluorescence
intensity.
Electrophoretic Mobility Shift Assay
U937 cells were incubated with or without different
concentrations of AT (25, 50, and 100 µmol/L) overnight in RPMI
1640 medium containing 10% FBS at 37°C as described above. Cells
were pelleted and reconstituted in RPMI containing 10% FBS. After 30
minutes' additional preincubation in the absence or presence of AT,
cells were activated with 100 ng/mL LPS for 2 hours at 37°C.
Preliminary dose-response studies indicated that the optimum dose of
LPS that activated NF-B was 100 ng/mL. This is in accord
with the published literature.11 12 17
Because PDTC is known to inhibit NF-B activation, cells were also
preincubated with PDTC (100 µmol/L) before activation with LPS.
Nuclear extracts were prepared from 1x106
treated cells according to Staal et al18 with
some modification. Briefly, cells were harvested, centrifuged
for 5 minutes at 14 000 rpm, and washed in 1 mL of ice-cold PBS. Cells
were pelleted, resuspended in 0.2 mL of buffer A (10 mmol/L HEPES,
pH 7.9; 10 mmol/L KCl; 0.1 mmol/L EDTA; 0.1 mmol/L EGTA;
2.5 mmol/L DTT; 1 mmol/L PMSF; and 5 µg/mL leupeptin) and
incubated on ice for 15 minutes. After addition of a 10% Nonidet P-40
solution (12.5 µL), cells were vigorously mixed for 10 seconds and
centrifuged for 60 seconds at 14 000 rpm at 4°C. Pelleted
nuclei were resuspended in 50 µL of buffer C (20 mmol/L HEPES,
pH 7.9; 25% [vol/vol] glycerol; 0.4 mol/L NaCl; 1 mmol/L EDTA;
1 mmol/L EGTA; 1 mmol/L PMSF; and 5 µg/mL leupeptin), mixed
for 10 minutes, and centrifuged for 5 minutes at 14 000 rpm at
4°C. The supernatant containing the nuclear proteins was harvested,
protein concentration was determined by Bio-Rad Protein assay, and the
supernatant was stored at -80°C until use.
Electrophoretic mobility shift assay (EMSA) was performed essentially
as described previously.19 20 Binding reaction
mixtures (20 µL) containing 5 µg of protein of the nuclear extract,
2 µg of poly (dI-dC), 50 mmol/L NaCl, 4% glycerol, 2.5
mmol/L EDTA, 2.5 mmol/L DTT, 5 mmol/L
MgCl2, 32P-labeled probe,
and 10 mmol/L Tris-HCl (pH 7.5) were incubated for 30 minutes at
room temperature. A double-stranded NF-B
oligonucleotide probe containing HIV-B
enhancer DNA template with a tandem duplicate of an NF-B binding
site (GGGACTTTCC) was used. Incubation was also conducted in the
presence of nonspecific competitor DNA (TTTACTTTCC). Products were
separated by electrophoresis through a native 4%
polyacrylamide gel in a running buffer of 12.5 mmol/L
Tris-borate (pH 8.0) containing 0.25 mmol/L EDTA at 150 V for 2.5
hours, and dried gels were analyzed by
autoradiography. Control reactions with 250-fold molar
excess of unlabeled wild-type and mutant
oligonucleotide probes were performed to demonstrate
the specificity of the shifted DNA-protein complexes for NF-B. In
addition, supershift experiments were conducted in the presence of p65
antibody (2 µg) to demonstrate correspondence of the band to
NF-B/Rel transcription factor.
Statistical Analysis
Results are expressed as mean±SD. Repeated-measures ANOVA was
performed to assess overall differences between the different
treatments, as described previously.10 Fisher's
least significant difference method was applied for multiple
comparisons. Paired t tests were used to compare the
differences between treatments.
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Results |
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There was a significant increase in AT content in U937 cells after pretreatment for 24 hours with 100 µmol/L AT (baseline: 0.09±0.04 versus 2.11±0.99 nmol/mg cell protein in AT-enriched cells; n=4 experiments; P<0.001). As can be seen in Figure 1A
, LPS (100 ng/mL)-induced activation of
U937 cells significantly increased adhesion to HUVECs compared with
DMSO-treated control (P=0.0029). Repeated-measures ANOVA
showed that pretreatment of U937 cells with AT before activation with
LPS or FMLP affected adhesion to HUVECs (P<0.001).
Pretreatment of U937 cells with 50 or 100 µmol/L AT before
activation with LPS decreased adhesion of U937 to HUVECs (LPS, 5.7±2.0
fluorescence units; LPS plus AT 50 µmol/L, 4.5±2.8
fluorescence units; LPS plus AT 100 µmol/L, 3.8±1.3
fluorescence units; P<0.0125 compared with LPS
alone). To determine whether the inhibitory effect of AT
was specific for LPS-induced adhesion, we tested the effect of AT on
another agonist, the chemotactic peptide FMLP (Figure 1B). FMLP also
significantly increased adhesion of U937 cells to HUVECs
(P=0.0151). AT significantly inhibited this increased
adhesion (FMLP, 10.3±3.6 fluorescence units; FMLP plus AT
50 µmol/L, 8.4±2.8 fluorescence units; FMLP plus AT
100 µmol/L, 7.6±2.8 fluorescence units;
P<0.05 compared with FMLP alone).
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In addition, pretreatment of both activated U937 cells with AT (100 µmol/L) and HUVECs with AT (50 µmol/L) resulted in additional inhibition of monocyte-EC adhesion compared with LPS-activated cells (U937 cells plus AT 100 µmol/L, 49% inhibition; U937 plus AT 100 µmol/L and ECs plus AT 50 µmol/L, 62% inhibition; P<0.05; n=3 experiments).
The effect of AT enrichment of U937 cells on the expression of
adhesion molecules on activated monocytes as determined by flow
cytometry is shown in Figure 2. After
stimulation with either FMLP or LPS, there was a significant increase
in the expression of CD11a, CD11b, CD11c, VLA-4 and L-selectin compared
with unstimulated cells (P<0.02). There was no significant
change in L-selectin after activation of U937 cells with LPS or FMLP.
Repeated-measures ANOVA showed that pretreatment of U937 cells with AT
before stimulation with LPS or FMLP affected the expression of adhesion
molecules (P<0.0005). AT (100 µmol/L) resulted in a
significant reduction in the expression of CD11b and VLA-4 in both LPS-
and FMLP-stimulated cells (percent reduction by AT in
LPS-activated cells: CD11b, 18%; VLA-4, 17%;
P<0.01; percent reduction by AT in FMLP-stimulated cells:
CD11b, 15%; VLA-4, 15.3%; P<0.01). There was no
significant decrease in CD11a, CD11c, or L-selectin expression with
AT.
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The increase in the agonist-induced adhesion of U937 cells to
HUVECs might be caused by the activation of NF-B in U937 cells.
Therefore, gel shift mobility assays for the treated U937 cells were
performed. Cells were incubated with or without AT and PDTC and then
activated with LPS (100 ng/mL). Nuclear extracts were prepared
from the treated cells and analyzed for the specific DNA
binding of NF-B with EMSAs. As shown in lane 3 in Figure 3, LPS activated the
transcription factor NF-B. This was inhibited by AT (50 and 100
µmol/L) and PDTC, a known inhibitor of NF-B
activation. To establish the specificity of the shifted bands,
competition analysis was performed with nuclear extracts from
cells incubated with 250-fold excess of unlabeled wild-type and mutant
NF-B probes; Although the cold mutant probe had no effect,
incubation with excess cold wild-type probe resulted in complete
elimination of the radioactive NF-B signal (Figure 4A). Incubation of LPS-activated
nuclear extracts with p65 antibody induced a supershifting of the band,
demonstrating its correspondence to the NF-B/Rel transcription
factor (Figure 4B). In addition, when radiolabeled mutant probe was
used instead of radiolabeled wildtype, there was a 10-fold decrease in
NF-B expression in LPS-activated cells (data not shown).
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Discussion |
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There are few data on the effect of antioxidants such as AT on monocyte-EC adhesion. Faruqi et al11 observed that when ECs were pretreated with AT before stimulation with agonists (phorbol ester, interleukin-1, thrombin, and LPS), there was less agonist-induced adhesion of monocytes to ECs. Although Faruqi et al did not show a decrease in NF-
B with E-selectin consensus
sequence in the gel shift assay, they demonstrated a decrease in
E-selectin mRNA and cell surface expression. Recently, Martin et
al12 showed that in vitro enrichment of ECs with
AT significantly inhibited LDL-induced adhesion of monocytes to ECs in
a dose-dependent manner and decreased soluble ICAM-1 release. They did
not study the effect of AT on NF-B activity. Although these studies
suggest that AT enrichment of ECs decreases adhesion of monocytes to
endothelium, there are no studies to date that examine
the effect of AT enrichment of monocytes on monocyte-EC adhesion. In a
recent study10 that tested the effect of AT
supplementation (1200 IU/d) on monocyte function, we showed that AT
significantly decreases adhesion of human monocytes to
endothelium. Like Faruqi et al,11
we could not explain this via an inhibition of protein kinase C by AT.
The present study was undertaken to elucidate other potential
mechanisms.
In the present study, we have demonstrated that preincubation
of U937 monocytic cells with AT resulted in significant enrichment of
monocytes with AT. In addition, at doses between 25 and 100
µmol/L, AT was not cytotoxic. Furthermore, after enrichment, there
was significant inhibition of both LPS- and FMLP-induced U937 cell
adhesion to HUVECs at doses 
50 µmol/L AT. These levels can be
attained in plasma after AT supplementation in
vivo.21 22 Faruqi et al11
observed in ECs that AT inhibited agonist-induced monocyte adhesion to
ECs, with an IC50 of 45 µmol/L. Martin et
al12 also reported a significant reduction in
LDL-induced monocyte-EC adhesion after preincubation of ECs with
42 µmol/L AT. Because we have shown that AT enrichment of
monocytes decreases adhesion to endothelium, and
previous workers have shown that AT enrichment of HUVECs decreases
adhesion, one can speculate that AT supplementation in vivo will result
in a greater inhibition of monocyte-EC adhesion because AT will
partition in both cells. We confirmed this by demonstrating that
preincubation of both U937 cells and HUVECs with AT results in a
greater inhibition of adhesion than preincubation of U937 cells
alone.
To delineate mechanisms through which AT may be acting to reduce
adhesion, we studied the effect of AT on the expression of adhesion
molecules on monocytes and the role of AT in the activation of NF-B.
Adhesion of monocytes to ECs is mediated by integrins that bind to
counterreceptors on ECs. The most important counterreceptors expressed
on monocytes include CD11a/18 (LFA-1), CD11b/18 (Mac-1), and CD49d/29
(VLA-4), which bind to ICAM-1 and ICAM-2, ICAM-1, and VCAM-1 on the
ECs, respectively. VCAM-1 and ICAM-1 expression has been demonstrated
in human atherosclerotic lesions.23 24 25 26
In the present study, we have shown that the expression of
CD11a, CD11b, CD11c, VLA-4, and L-selectin on U937 cells is increased
after stimulation with LPS or FMLP. In addition, preincubation of U937
cells with AT significantly decreased the expression of CD11b and VLA-4
from these cells. Weber et al6 showed that
long-term cigarette smoking was associated with a CD11b-dependent
increase in adhesiveness of isolated human monocytes to
endothelium, which was decreased after supplementation
with ascorbate for 10 days. In previous studies, Weber et
al27 showed that enhancement of monocyte adhesion
to ECs by modified LDL was mediated via activation of CD11b. They also
showed that enhancement of adhesion was prevented by an anti-CD11b
monoclonal antibody. In addition, Ragab et al28
reported that oxidized lipoprotein(a) and LDL increased CD11b
expression by 
60% in U937 cells, with a concomitant increase in the
adhesion of U937 cells to cultured ECs. Martin et
al12 demonstrated that vitamin E inhibits
LDL-induced monocyte-EC adhesion in part by decreasing expression of
soluble ICAM-1, which is a counterreceptor for CD11b. Although there
are few studies on the role of VLA-4 in monocyte-EC adhesion,
Cominacini et al29 showed that pretreatment of
HUVECs or LDL with antioxidants such as AT and probucol significantly
reduced the expression of both ICAM-1 and VCAM-1 on HUVECs induced by
oxidized LDL. In addition, Marui et al30 reported
that VCAM-1 gene transcription and expression in human vascular ECs are
increased in the presence of interleukin-1ß, LPS, and tumor necrosis
factor- (TNF) and can be repressed by 90% by the antioxidants
PDTC and N-acetyl cysteine. This is supported by the recent
finding that the ability of ECs to express VCAM-1 in response to
cytokine stimulation may be modulated by oxidized LDL by a
redox-sensitive mode of regulation.31
These studies suggest that oxidative stress is an important
regulatory signal in the pathogenesis of
atherosclerosis.
NF-B is a mammalian transcription factor that is directly
involved in the activation of genes responsible for
inflammation.32 In cells that have inducible
NF-B activity, the factor comprises a p50-p65 heterodimer bound by
an inhibitory subunit I-B in the cytosol and can be
activated by dissociation of I-B.33 34
The importance of activated NF-B in
atherosclerosis has been demonstrated by its presence
in smooth muscle cells, macrophages, and ECs of human
atherosclerotic lesion tissue but not in normal
vessels.35 36 A variety of genes are induced in
the atherosclerotic lesion that have been shown to be regulated by
NF-B, including genes encoding TNF, interleukin-1, tissue factor,
macrophage colony stimulating factor, VCAM-1, and ICAM-1.
Schreck et al17 have suggested a novel signal
transduction pathway for NF-B activation that involves reactive
oxygen species as second messengers based on studies with Jurkat T
cells that responded to the addition of exogenous hydrogen peroxide.
Some of the antioxidants that have been shown to inhibit NF-B
activation include PDTC, N-acetyl cysteine, and
-lipoate.17 37 38 Suzuki and
Packer39 have shown that NF-B activation can
be inhibited in TNF-–activated Jurkat T cells by treatment
with vitamin E acetate and succinate, resulting in decreased DNA
binding activity. However, they did not observe any inhibition with AT
alone.
In the present study, we showed that pretreatment of U937
cells with AT significantly decreased the LPS-induced activation of
NF-B. PDTC was used as a positive control in all experiments and has
been shown by numerous investigators to effectively inhibit activation
of NF-B. There have been conflicting reports on the effect of AT in
NF-B activation. Whereas Suzuki and Packer39
as well as Faruqi et al11 did not show any effect
of AT on activation of NF-B by pretreatment of Jurkat T cells and
ECs, respectively, Hennig et al40 showed that
pretreatment of ECs for 24 hours with AT or 6 hours with
N-acetyl cysteine significantly inhibited linoleic
acid–induced activation of NF-B. It is possible that AT has
differential effects in different cell lines. In addition, none of
these investigators studied the effect of AT enrichment of monocytes on
subsequent NF-B activation. This is clearly important, because the
monocyte is a pivotal cell in atherogenesis, and its respiratory burst
has been shown to lead to activation of
NF-B.41
In the present study, we have shown that enrichment of
monocytes with AT resulted in a significant decrease in adhesion of
monocytes to ECs, mediated by a concomitant reduction in the expression
of the adhesion molecules CD11b and VLA-4. This inhibition is possibly
mediated by inhibition of the activation of NF-B. Support for this
hypothesis comes from studies by Sokoloski et
al,42 who showed that antisense
oligonucleotides to the p65 subunit of NF-B block
CD11b expression on the surface of FMLP- or phorbol ester–treated
HL-60 human promyelocytic leukemia cells. However, additional studies
in monocytes are required to confirm this.
In conclusion, these studies provide evidence for the beneficial effects of AT on a crucial early event in atherosclerosis, viz, monocyte-EC adhesion. This beneficial effect of AT further strengthens its evolving role as an adjunctive therapy in the management of atherosclerosis because in a recent study, although patients with dyslipidemia had elevated levels of soluble adhesion molecules, aggressive lipid-lowering therapy had only limited effects on their levels.43 These and other studies support the concept that the possible beneficial effects of AT supplementation in reducing coronary artery disease can be attributed to its combined effects on inhibition of the oxidative modification of lipoproteins and its intracellular effects on cells critical in atherogenesis, such as monocytes.9
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Acknowledgments |
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The authors are thankful to Richard Scheuermann, PhD, Associate Professor, Department of Pathology, and his fellow, Zhiyong Wang, PhD, for providing the EMSA probe and allowing them to conduct the EMSA studies in his laboratory.
Received May 19, 1998; revision received July 14, 1998; accepted July 21, 1998.
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