1 Division of Cardiovascular
and Special Intervention and
2 Division of Radiation Oncology, We recently reported that prolonged exposure of human aortic
endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the
transcriptional regulator nuclear factor-
transcription regulator; hemodynamics; atherosclerosis; human
aortic endothelial cell; nuclear factor- ENDOTHELIAL CELLS LINING the vascular system are
subjected to different hemodynamic blood flow patterns depending on
arterial geometry. Early lesions of atherosclerosis tend to occur in
regions of the vessel that experience low shear stress and reversing
flow patterns such as arterial bifurcations and curved segments of the
large elastic arteries (2, 11, 29). These lesion-prone areas are
characterized by the enhanced recruitment, adhesion, and
transendothelial migration of monocytes, leading to the development of
foam cells in the subendothelial space (3, 9). Although there are
several reports associating low shear stress areas and early
atherosclerotic lesions (2, 11, 29), the molecular mechanisms involved
in the cellular interactions at the lesion-prone areas have not been
clearly defined. In vitro studies from our laboratory using human
aortic endothelial cells (HAEC) (26) and in vivo studies by others
using animal models (31) have shown that low shear stress enhances the
expression of the vascular cell adhesion molecule (VCAM-1; CD106).
VCAM-1 binds to those cells expressing the integrin very late antigen-4
(VLA-4), such as monocytes, T and B lymphocytes, basophils, and
eosinophils (10). The increased expression of VCAM-1 in the arterial
intima of human atherosclerotic plaques correlates with the specific enhanced recruitment of monocytes that subsequently leads to the progression of atherosclerotic lesions (21). A recent report from our
laboratory demonstrated that exposing HAEC to prolonged low shear
stress also induces a sustained activation of a key transcriptional
regulator, nuclear factor- Cell culture.
HAEC (Clonetics/BioWhittaker, San Diego, CA) were cultured in MCDB-131
medium (Sigma, St. Louis, MO) containing 10% bovine calf serum (BCIS,
HyClone, Kansas City, KS) enriched with 250 ng/ml fibroblast growth
factor (Pepro Tech, Rocky Hill, NJ), 1 mg/ml epidermal growth factor
(Pepro Tech), 1 mg/ml hydrocortisone (Sigma), 100 U/ml
penicillin, and 100 mg/ml streptomycin (Mediatech, Herndon, VA). Cells
from passages
4-7
were used for all the experiments.
Shear stress experiments.
HAEC were seeded on polyester film (10 × 19-cm Mylar sheets;
Regal Plastics, San Antonio, TX) precoated with 2% gelatin and grown
to near confluence, which was attained within 2-3 days. The cells
on the slips were incubated in MCDB-131 medium containing 2% bovine
calf serum in the absence of supplemented growth factors and
hydrocortisone for 20 h before initiation of flow shear. Flow experiments were performed using the closed loop flow system described previously (22). In brief, HAEC-covered sheets were placed within rectangular parallel plate flow chambers, resulting in an available 90-cm2 cell surface area exposed
to flow. In this flow system, the circulating medium flows by
hydrostatic pressure from an upper reservoir through the flow chamber
into a lower reservoir. The height difference between the two
reservoirs determines the flow rate and the shear stress level in the
cell chamber. Regardless of the shear regimen, flow rate was maintained
at the same level through all chambers. Shear stress level was altered
by adjusting the height of separation between the parallel plates. At
any given flow rate, shear stress varies inversely with the square of
chamber channel height. The medium was circulated back to the upper
reservoir using a peristaltic roller pump (Master flex, Cole-Palmer
Instrument, Chicago, IL). The temperature within the flow chamber was
maintained at 37°C. The culture medium was buffered with 15 mM
HEPES without bicarbonate to maintain the medium at a constant pH (pH
7.4). The flow chamber was designed so that it could be positioned on
an inverted light microscope (Optiphot, Nikon, Japan) for continuous
monitoring. Cells were subjected to 6 h of low shear stress (2 dyn/cm2) and harvested for
nuclear protein extraction. As a control, slips with nearly confluent
cells were incubated in the flow medium in a 37°C chamber under
static conditions. Cells grown on 100-mm culture dishes and treated
with 10 ng/ml interleukin-1 Inhibition studies with antioxidants.
The antioxidants PDTC and NAC were freshly prepared for every
experiment. The stock solution of 1 M NAC or PDTC was prepared by
dissolving NAC or PDTC in 1× PBS and was filter sterilized. For
NAC, the pH was adjusted to pH 7.4 with 3 N NaOH before filter sterilization. Cells were preincubated with 100 µM PDTC for 1.5 h or
with 30 mM NAC for 0.5 h and then subjected to low shear stress for 6 h. To study the dose-dependent regulation of VCAM-1 gene expression,
PDTC concentrations in the range of 50, 75, and 100 µM or NAC
concentrations in the range of 30, 40, and 50 mM were used. The cell
viability at these concentrations of PDTC or NAC was determined by the
trypan blue dye exclusion method. In addition, to ensure the reversible
nature of the inhibitor, HAEC were treated with 100 µM PDTC or 30 mM
NAC, and the cells were left in culture for 24 h. The cells were then
challenged with a 10 ng/ml concentration of IL-1 Electrophoretic mobility shift analysis.
After flow shear or IL-1 Northern analysis.
Total cellular RNA was isolated from HAEC by using the Ultraspec
reagent, following the manufacturer's protocol (Biotecx, Houston, TX).
Total RNA (10-15 µg) was separated on 1% agarose-formaldehyde gels in the presence of ethidium bromide, transferred to a
nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and
immobilized by baking at 80°C for 2 h. Both prehybridization and
hybridization were performed at 42°C for 18 h in 6× SSPE
buffer (pH 7.4; 900 mM NaCl, 50 mM
NaPO4, and 5 mM EDTA). After
prehybridization, the membrane was hybridized with a cDNA probe
encoding human VCAM-1. The VCAM-1 probe was a
Xba
I-Apa I fragment cloned in pRc/CMV vector and generously donated by Dr. Daniel K. Burns (Hoffman-LaRoche, Nutley, NJ). The probe was labeled with
[ Flow cytometry.
VCAM-1 protein expression was analyzed by fluorescence-activated cell
sorting (FACS) analysis. HAEC subjected to low shear stress for 6 h
with or without PDTC or NAC treatments were fixed with 1% formaldehyde
for 10 min. Recombinant human tumor necrosis factor- Monocyte adhesion assay.
Whole blood, collected in vacutainer tubes containing EDTA from healthy
volunteers by vein puncture was used within 4 h of collection. The
monocytes were isolated by standard buoyant density centrifugation
technique using NycoPrep 1.068 (Nycomed Pharma, Oslo, Norway). Cells
obtained were further purified on Optiprep (Nycomed Pharma) to
eliminate platelet contamination. Cell viability was determined by the
trypan blue dye exclusion method. Purity of monocyte preparation was
determined by labeling the cells with mouse monoclonal anti-macrophage
antibodies (Enzo Diagnostics, Farmingdale, NY) followed by Texas
red-conjugated goat anti-mouse antibodies (Calbiochem, San Diego, CA).
Typically, >95% of the cells showed positive staining. For adhesion
studies, the purified monocytes (1.67 × 104 cells/ml) were introduced into
150 ml of circulating medium and allowed to circulate at low shear for
an additional 1 h after completion of the designated
experimental flow regimen. The slips were then washed in PBS solution,
fixed in methanol for 5 min, and stained with Giemsa stain (Sigma),
which allows light microscopic identification of adherent monocytes.
Under high-power light microscopy (×400), total adherent
monocytes per high-power field were visualized and counted. No-shear
control slips were subjected to 1 h of low shear with the monocytes in
the circulation and used for comparison with PDTC- or NAC-incubated and
low shear-subjected HAEC.
Antibody blocking assay.
Endothelial cell-monocyte adhesion assay was also performed under low
shear stress in the presence of 4 µg/ml anti-VCAM-1 antibody obtained
from Endogen. HAEC incubated with or without NAC (30 mM) for 45 min and
subjected to low shear stress for 5 h were further incubated with
anti-VCAM-1 blocking antibodies at a concentration of 4 µg/ml for 45 min at 37°C. After incubation, the cells were further subjected to
low shear stress in the presence of freshly purified monocytes for 1 h,
and the number of monocytes adhering to the endothelial cells after a
wash with PBS were counted as described above.
Low fluid shear stress-induced NF-
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (NF-
B). Here we
investigate the hypothesis that low shear-induced activation of NF-
B
is responsible for enhanced expression of vascular cell adhesion
molecule (VCAM-1) resulting in augmented endothelial cell-monocyte
(EC-Mn) adhesion and that this activation is dependent on intracellular
oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were
preincubated with or without the antioxidants pyrrolidine
dithiocarbamate (PDTC) or
N-acetyl-L-cysteine (NAC). PDTC
strongly inhibited low shear-induced activation of NF-
B, expression
of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive
effect on low shear-induced VCAM-1 expression and EC-Mn adhesion
and only slightly downregulated NF-
B activation. However, cytokine-induced NF-
B activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-
B plays a key role
in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.
B; vascular cell adhesion
molecule
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (NF-
B) (16). Studies of molecular
structural analysis have shown that the 5' promoter regions of
many adhesion molecules, including VCAM-1, contain one or more binding
sites for NF-
B (5, 6, 13, 14). On induction by a variety of stimuli,
the activated form of NF-
B is translocated to the nucleus and
transactivates genes containing functional NF-
B binding sites. In
the present study, we investigate 1)
whether low shear stress-induced NF-
B in HAEC is responsible for the
enhanced expression of VCAM-1 and 2)
whether the blockade of NF-
B activation by the structurally
different antioxidants pyrrolidine dithiocarbamate (PDTC) and
N-acetyl-L-cysteine
(NAC) inhibits low shear stress-induced VCAM-1 expression and
associated endothelial monocyte adhesion. We demonstrate that the
endothelial monocyte adhesion that is augmented by low shear stress,
through activation of NF-
B and enhanced VCAM-1 expression, is
efficiently inhibited by PDTC. Paradoxically, NAC, a general
antioxidant, had a positive effect on low shear-induced VCAM-1
expression and monocyte adhesion. Monoclonal antibodies to VCAM-1
specifically blocked most of the low shear-induced endothelial
cell-monocyte adhesion. On the other hand, in the presence of NAC,
VCAM-1 antibodies only partially blocked the low shear-induced
endothelial cell-monocyte adhesion, suggesting that low shear in the
presence of NAC induces adhesion molecules other than VCAM-1.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(IL-1
; R&D Systems, Minneapolis, MN)
for 4 h were used as positive controls.
for 4 h and were
harvested for nuclear protein extraction to study the NF-
B DNA
binding activity.
stimulation, cells were washed in ice-cold
PBS and harvested at the indicated time periods. Nuclear proteins were
extracted by methods reported by Mohan et al. (16). In brief, the cell
pellet was resuspended in 400 µl of cold
buffer A [10 mM HEPES-KOH, pH 7.9 at
4°C, 1.5 mM MgCl2, 10 mM KCl,
0.5 mM dithiothreitol (DTT), and a cocktail of protease inhibitors containing aprotinin, antipain, leupeptin, and bestain at final concentrations recommended by the manufacturer (Boehringer Mannheim, Indianapolis, IN)]. The cells were allowed to swell in
buffer A on ice for 10 min, followed by
centrifugation for 10 s at 14,000 rpm in a Microfuge centrifuge at
4°C. The pellet was resuspended in 50 µl of cold
buffer
B (20 mM HEPES-KOH, pH 7.9, 25%
glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, and a cocktail
of protease inhibitors) and further incubated on ice for 20 min for
high-salt extraction. The samples were centrifuged for 10 min at 14,000 rpm at 4°C, and the clear supernatants were transferred to
prechilled tubes. The total protein concentrations were measured using
the bicinchoninic acid method, following the manufacturer's protocol (Pierce, Rockford, IL). For electrophoretic mobility shift analysis (EMSA), a double-stranded oligonucleotide containing a tandem repeat of
the consensus sequences of 5'-GGG-GAC-TTT-CC-3' was end
labeled with T4 polynucleotide kinase (Promega, Madison, WI). Free
unbound radioisotope was separated by a push column device (Stratagene,
La Jolla, CA). The binding reaction was performed by mixing nuclear
extract (8 µg of total protein), 0.1 µg of poly(dI-dC) (Pharmacia
Fine Chemicals), and
[
-32P]ATP-labeled
NF-
B-specific oligonucleotide probe [0.5 ng DNA; ~50,000
counts/min (cpm); Amersham, Arlington Heights, IL] in binding
buffer containing 10 mM Tris · Cl (pH 7.5), 100 mM
NaCl, 1 mM DTT, 1 mM EDTA, and 20% (vol/vol) glycerol. For competition assay, the nuclear extract was preincubated with unlabeled homologous NF-
B oligonucleotide for 5 min on ice. This procedure was followed by addition of labeled NF-
B probe. Incubations were performed at
ambient temperature for 20 min. Subsequently, all samples were electrophoresed using 6% polyacrylamide gels in Tris-glycine buffer. The gels were then dried and autoradiographed. Estimation of NF-
B activation was performed by quantitative analysis using the National Institutes of Health (NIH) 1.58b19 image analysis software package with
an integrated density program.
-32P]dATP (ICN
Pharmaceuticals, Costa Mesa, CA) using a Megaprime primer labeling kit
(Amersham), yielding a specific activity of ~1 × 109 cpm/µg DNA. Autoradiography
was performed with an intensifying screen at
70°C.
Quantitative analysis of VCAM-1 mRNA expression was performed by using
the NIH 1.58b19 image analysis software package with an integrated
density program.
(TNF-
;
Promega) at a concentration of 10 ng/ml was used as a positive inducer.
The cells were then detached from the plastic slips and probed with
monoclonal anti-VCAM-1 antibody (Endogen, Woburn, MA) and FITC-labeled
anti-mouse IgG antibody (Sigma) after blocking with 1% fetal bovine
serum. In addition to no-shear test controls, cells were also incubated
with isotype-matched (IgG1) immunoglobulin as a negative
control. The labeled cells (~30,000 cells) were analyzed by EPICS
ELITE flow cytometer (Coulter, Miami, FL). Statistical analysis was
performed by the use of the Immuno-4 Analysis program (Coulter).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B activation is
inhibited by PDTC and NAC.
To investigate whether the structurally different antioxidants PDTC and
NAC can attenuate the low shear-induced as well as the cytokine-induced
activation of NF-
B, HAEC were either subjected to 6 h of low shear
stress (2 dyn/cm2) or treated
with 10 ng/ml IL-1
. Cells treated with IL-1
were used as positive
controls in EMSA experiments (Fig.
1C, lane 2). Quantitative analysis of the autoradio-gram, performed using the NIH 1.58b19 image analysis program, showed that nuclear extracts from
cells exposed to 6 h of low shear (Fig.
1A,
lane
2) contained a 5.2-fold higher
NF-
B DNA binding activity than no-shear controls (P < 0.001; Fig.
1D). Preincubation with 100 µM
PDTC strongly inhibited the low shear-induced activation of NF-
B in
HAEC (P < 0.05; Fig.
1A,
lane
3). In contrast, 30 mM NAC only
slightly inhibited this activation (P < 0.001; Fig. 1A,
lane
4). As an internal control, EMSA was
performed with a probe for the ubiquitous factor octamer binding
protein (OCT-1). No change in OCT-1 binding activity was observed,
irrespective of the inhibitor treatment, indicating that the effect on
NF-
B was specific (Fig. 1B). The final concentrations of PDTC (100 µM) and NAC (30 mM) chosen for inhibition studies were found to be nontoxic to the cells. Viability tests performed with various concentrations of PDTC and NAC by the
trypan blue dye exclusion method indicated that cells were >90%
viable after the incubations with either 100 µM PDTC or 30 mM NAC
(Fig. 2, A
and B). In a parallel experiment,
cells treated with the inhibitors were subsequently cultured in
inhibitor-free medium for a further 24 h and challenged with IL-1
(10 ng/ml) for 4 h. EMSA results showed the induction of NF-
B DNA
binding activity after IL-1
treatment (Fig.
2C,
lanes
2 and
4). This clearly indicated that the
effects of the antioxidants are reversible and not cytotoxic.
Therefore, the observed inhibition of NF-
B activation by the
antioxidants is unlikely to have resulted from the effects of cell
death.
View larger version (62K):
[in a new window]
Fig. 1.
Inhibition of nuclear factor- B (NF-
B) DNA binding activity by
pyrrolidine dithiocarbamate (PDTC) and
N-acetyl-L-cysteine
(NAC) in low shear-subjected human aortic endothelial cells (HAEC).
Cells, incubated for 20 h in complete medium containing 2% serum
without growth factors, were treated with PDTC (100 µM) for 90 min or
NAC (30 mM) for 30 min. Cells were then subjected to low shear for 6 h.
Nuclear proteins were extracted, and electrophoretic mobility shift
assay analyses were performed as described in
MATERIALS AND METHODS.
A: electrophoretic mobility shift
analysis (EMSA) using 32P-labeled
NF-
B-specific oligonucleotide probe showing NF-
B DNA binding
activity. Lane
1, no shear (control);
lane
2, low shear (2 dyn/cm2);
lane
3, PDTC-pretreated cells subjected to
low shear; lane
4, NAC-pretreated cells subjected to
low shear. Autoradiogram is representative of at least 3 independent
experiments. B: EMSA using
32P-labeled octamer binding
protein (OCT-1)-specific oligonucleotide probe showing OCT-1 DNA
binding activity. Lane
1, no shear (control);
lane
2, low shear (2 dyn/cm2);
lane
3, PDTC-pretreated cells subjected to
low shear; lane
4, NAC-pretreated cells subjected to
low shear. C: HAEC treated with 10 ng/ml interleukin-1
(IL-1
) for 4 h used as positive control.
Lane
1, untreated control;
lane
2, IL-1
-treated cells;
lane
3, PDTC-pretreated cells subjected to
IL-1
induction. Arrows, specific bands of NF-
B DNA binding.
D: total amount of NF-
B activation
in nuclei from at least 3 independent experiments (means ± SE) was
determined by quantitative analysis using NIH 1.58b19 image analysis
software package with integrated density program. Background density on
an autoradiogram was subtracted from densitometric data of each band.
View larger version (18K):
[in a new window]
Fig. 2.
Viability of HAEC treated with PDTC or NAC at selected concentrations
for 24 h. A and
B: viability of HAEC in presence or
absence of various concentrations of PDTC
(A) and NAC
(B). No. of viable cells was
determined after 24 h by trypan blue dye exclusion method. Each point
is arithmetic mean ± SD of 2 cell counts from 3 independent
experiments and is expressed as a percentage of viability of untreated
controls. C: NF- B DNA binding
activity in PDTC- or NAC-pretreated HAEC after IL-1
induction. HAEC
were pretreated with 100 µM PDTC or 30 mM NAC, left in culture for 24 h, and further induced with 10 ng/ml IL-1
for 4 h. Cells were
subjected to nuclear protein extraction, and EMSA was
performed with
32P-labeled NF-
B
probe. Autoradiogram shows untreated controls
(lanes
1 and
3), cells pretreated with PDTC
(lane
2), and cells pretreated with NAC
(lane
4).
Inhibition of VCAM-1 mRNA expression.
The effect of the antioxidants on the low shear-induced expression of
VCAM-1 was investigated by Northern blot analysis (Fig. 3, A and
B). HAEC subjected to 6 h of low
shear exhibited expression of VCAM-1 mRNA
(P < 0.01; Fig.
3A,
lane
2) in contrast to the no-shear
(static) controls in which the expression of VCAM-1 mRNA was
undetectable (Fig. 3A,
lane
1). Quantitative analyses of the autoradiograms from two independent experiments were performed using
the NIH 1.58b19 image analysis program (Fig.
3B). PDTC at a concentration of 100 µM (Fig. 3A,
lane
3) clearly inhibited the low
shear-induced expression of VCAM-1 mRNA expression, as did lower
concentrations of PDTC (50 and 75 µM; Fig.
3C,
lanes 6 and
7). On the other hand, 30 mM NAC had
minimal effect on the low shear-induced VCAM-1 mRNA gene expression
(P > 0.01; Fig. 3A,
lane
4). Furthermore, increased
concentrations (40 and 50 mM) of NAC not only failed to inhibit VCAM-1
gene expression (Fig. 3C,
lanes
2 and
3) but, in contrast, resulted in an
increase in the level of expression of VCAM-1 mRNA. On the basis of
these results, all subsequent studies were performed using 100 µM
PDTC and 30 mM NAC.
|
Inhibition of VCAM-1 protein expression.
FACS analysis was performed to determine low shear stress-induced
VCAM-1 protein expression in HAEC. As shown in Table
1, 100 µM PDTC eliminated
induced VCAM-1 protein expression. However, HAEC incubated with 30 mM
NAC and then subjected to 6 h of low shear stress exhibited increased
expression of VCAM-1 protein expression. As shown in Table 1, 5.3 ± 0.7% of cells stained positive, significantly higher than in the
controls (P < 0.01). HAEC incubated
with NAC without shear showed only background staining. TNF- (10 ng/ml)-induced HAEC were used as positive controls (50.6 ± 6.8%).
Both NAC and PDTC efficiently inhibited TNF-
-induced VCAM-1
expression.
|
Effect of PDTC and NAC on endothelial monocyte adhesion.
The influence of the antioxidants on low shear-induced enhancement of
endothelial monocyte adhesion was also studied. In concordance with
results obtained in NF-B activation and VCAM-1 mRNA and protein
expression, PDTC at a final concentration of 100 µM inhibited the
adhesion of monocytes to HAEC close to the levels observed with
controls, as shown in Fig. 4. Again,
similar to the effect on VCAM-1 mRNA and protein expression, treatment
of low shear-subjected cells with 30 mM NAC enhanced the adhesion of
monocytes significantly (P < 0.05)
compared with the controls and also compared with cells subjected to
low shear alone (P < 0.1).
|
Blocking of monocyte adhesion by anti-VCAM-1 antibodies. Antibodies were used to investigate the role of VCAM-1 antigen in low shear stress-induced monocyte adhesion to HAEC. Anti-VCAM-1 antibodies inhibited 89.7% of the monocyte adhesion to HAEC by blocking VCAM-1 expression induced by low shear stress. On the other hand, they inhibited only 43% of the monocyte adhesion when HAEC were incubated with NAC and further subjected to low shear stress.
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DISCUSSION |
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Areas of the human vascular tree that experience low shear stress are
known to be predisposed to atherosclerosis (25). The results of our
studies reported here indicate that treatment of HAEC with low shear (2 dyn/cm2) induces the activation
of the transcription factor complex NF-B, the enhanced expression of
VCAM-1 mRNA and protein, and the increased adhesion of peripheral blood
monocytes mediated by VCAM-1. The differential effects of the
antioxidants PDTC and NAC on this low shear-induced response suggest
that the mechanism of low shear-induced VCAM-1 differs in some way from
cytokine-induced VCAM-1 expression.
Several studies using cytokines or mitogens as stimuli have shown
VCAM-1 induction to be mediated by the activation of transcription factor NF-B (6, 13, 14, 27). NF-
B, a multisubunit transcription factor activated in response to proinflammatory stimuli, plays a
pivotal role in the development of the cellular immune and inflammatory response (4). Two functional NF-
B binding sites have been identified
in the promoter regions of the VCAM-1 gene, and these together with two
GATA sites in the core promoter of the gene appear to be important in
cytokine-induced VCAM-1 expression (20). Although previous studies in
our laboratory had demonstrated that exposure of HAEC to low shear was
associated with a sustained elevation in both NF-
B activation and
VCAM-1 expression, it was not known whether the low shear-induced
activation of NF-
B was involved in the upregulation of VCAM-1
expression and the enhanced monocyte adhesion. Because several studies
have demonstrated that NF-
B can be induced primarily by
oxidant-sensitive mechanisms and that antioxidants can selectively
inhibit NF-
B activation in many cell lines (12, 17, 23, 24,
30), the antioxidants PDTC and NAC were used in this
current study to define the role of NF-
B in low shear-induced VCAM-1
expression. The antioxidant PDTC has been shown to efficiently and
specifically block NF-
B (8, 24, 32). PDTC may exert its effects by
scavenging superoxide anions and thus prevent the generation of
H2O2.
PDTC may also chelate Fe2+ and
thereby inhibit the Fenton reaction that generates hydroxyl radicals
from
H2O2
(8). Furthermore, PDTC may directly block NF-
B activation by
reversibly inhibiting the release of the inhibitory subunit I
-B
from the active NF-
B complex. NAC has antioxidant activity
comparable to PDTC and acts on · HOCl and OH · radicals, but it has been reported to have no effect on superoxide
radical (1).
Several reports have shown that cytokine induction of VCAM-1 gene
expression is specifically blocked by preincubation of cultured cells
with 100 µM PDTC and 30 mM NAC (8, 32, 15). From our data presented
here, it is clear that low shear stress-induced NF-B activation,
VCAM-1 mRNA and protein expression, and monocyte adhesion are
effectively inhibited by PDTC at a concentration of 100 µM. NAC (30 mM), on the other hand, partially inhibited shear-induced NF-
B
activation and, remarkably, did not suppress either mRNA or protein
expression of VCAM-1. In fact, the data obtained from flow cytometer
analysis clearly showed increased expression of VCAM-1 after the cells
had been treated with the combination of 30 mM NAC and low shear stress
of 2 dyn/cm2. In contrast, the
TNF-
-induced VCAM-1 expression was efficiently downregulated by 30 mM NAC, as observed by others (15, 32). These results clearly indicate
that NAC acts as a costimulator of low shear-induced VCAM-1 expression.
This effect of NAC is supported by several studies (18, 19, 28) reporting that low-molecular-weight thiols can act as prooxidants as well as antioxidants. This prooxidant effect of NAC has been reported in in vivo animal models: animals receiving low concentrations of NAC survived lipopolysaccharide-induced septic shock, whereas the mortality was very high among animals receiving high doses of NAC (28). The enhanced expression of VCAM-1 seen with low shear stress and NAC treatment correlated well with the augmented endothelial monocyte adhesion. The partial involvement of VCAM-1 in this increased adhesion was confirmed by blocking with monoclonal anti-VCAM-1 antibodies that inhibited the induced monocyte adhesion by 43%. The observed uninhibited portion of adhesion in these NAC-pretreated and low shear-subjected cells may have been due to the induction of adhesion molecules other than VCAM-1.
One possible explanation for the enhanced monocyte adhesion observed in
the presence of low shear and 30 mM NAC may be that VCAM-1 expression
is induced in HAEC by reactive oxygen species, especially superoxide
radicals, which are not scavenged by NAC. It is also possible that PDTC
may have inhibited the low shear-induced NF-B activation by a
mechanism other than its antioxidant attributes, such as its ability to
prevent the uncoupling of I
B
from the NF-
B complex. The
prolonged time frame in which the cells are continuously exposed to low
fluid shear stress and are also generating the superoxide radicals may
induce the cells to express adhesion factors including VCAM-1 and
soluble chemokine-like molecules that favor the binding of the
monocytes. De Keulenaer et al. (7) recently showed in human endothelial
cells exposed to oscillatory flow patterns that there is a sustained
activation of prooxidant processes resulting in redox-sensitive gene
expression, although the maximum levels of shear stress (±5
dyn/cm2) employed in this study
exceeded the levels used in the present work.
In conclusion, our results indicate that effective blocking of NF-B
activation, as observed with PDTC pretreatment of HAEC, also blocks
subsequent VCAM-1 and enhanced monocyte adhesion associated with low
shear stress exposure. This further indicates that NF-
B activation
plays a key role in low shear stress-induced VCAM-1 expression and
monocyte adhesion. The paradoxical activation of VCAM-1 expression and
monocyte adhesion by the antioxidant NAC provides a basis for further
investigation into the low shear-induced endothelial cell-monocyte
adhesion and signaling mechanisms relevant to NF-
B pathway and
suggests that low shear-induced responses may be differentially
mediated by oxidant-sensitive mechanisms compared with cytokine responses.
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ACKNOWLEDGEMENTS |
---|
We thank Jian Luo and Julie Meier for technical assistance in cell culture and shear experiments and Richard A. Salinas (Flow Cytometry Laboratory, Cancer Therapy and Research Center, San Antonio, TX) for performing FACS analysis. We also appreciate the photographic services of Cono Farias (Dept. of Radiology, University of Texas Health Science Center at San Antonio).
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants F32-HL-09694-01A1 (National Research Service Award to S. Mohan) and HL-52218.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. A. Sprague, Dept. of Radiology, Div. of Cardiovascular Interventions, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7800 (E-mail: sprague{at}uthscsa.edu).
Received 16 October 1998; accepted in final form 29 January 1999.
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