Departments of 1 Pathology, 2 Radiology, 3 Medicine, and 4 Radiation Oncology, University of Texas Health Science Center, San Antonio, Texas 78229; and 5 Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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We have investigated the role of
inhibitor B
(I
B
) in the activation of nuclear factor
B
(NF-
B) observed in human aortic endothelial cells (HAEC) undergoing
a low shear stress of 2 dynes/cm2. Low shear for 6 h
resulted in a reduction of I
B
levels, an activation of NF-
B,
and an increase in
B-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion.
Overexpression of I
B
in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of
I
B
is the major factor in the low shear-induced activation of
NF-
B in HAEC. We then investigated the role of nitric oxide (NO) in
the regulation of I
B
/NF-
B. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-
B activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 µM) or
sodium nitroprusside (1 mM) before low shear stress significantly
increased cytoplasmic I
B
and concomitantly reduced NF-
B
binding activity and
B-dependent VCAM-1 promoter activity. Together,
these data suggest that NO may play a major role in the regulation of
I
B
levels in HAEC and that the application of low shear flow
increases NF-
B activity by attenuating NO generation and thus
I
B
levels.
low shear stress; inhibitor B; endothelial nitric oxide
synthase; nuclear factor-
B
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INTRODUCTION |
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REGIONS OF THE ARTERIAL
TREE that experience low fluid-imposed shear stress are highly
predisposed to the development of atherosclerosis (1, 10,
15). We have demonstrated in vitro that vascular endothelial
cells subjected to low shear stress for prolonged periods of time show
an enhanced and persistent activation of the early response
transcription factor, nuclear factor B (NF-
B). In contrast, cells
exposed to high shear stress show a transient, less intense activation
of NF-
B. Our results (17, 18) and data from other
investigators (19, 25, 27) have also shown that activation
of NF-
B in endothelial cells by low shear results in the increased
expression of the vascular cell adhesion molecule VCAM-1. Endothelial
cell-derived VCAM-1 mediates increased adhesion of circulating
monocytes (6, 26) and the enhanced recruitment of
monocytes to the subintimal space, both hallmarks of early atherosclerosis.
NF-B, in its active binding form, is a collection of homo- and
heterodimers (13, 21) composed of various combinations of
members of the NF-
B/Rel family. The NF-
B/Rel family of proteins includes NF-
B1 (p50 and its precursor p105), NF-
B2 (p52 and its
precursor p100), c-Rel, RelA (p65), and RelB. The NF-
B complex is
maintained in an inactive form by sequestration in the cytoplasm through interaction with the inhibitory protein inhibitor
B (I
B) that sterically hinders binding of import proteins to the nuclear localization sequence of the NF-
B subunits. The I
B family
includes I
B
, I
B
, I
B
, I
B
, Bcl-3, and the
precursors of NF-
B1 (p105) and NF-
B2 (p100). Members of
the I
B family of proteins are characterized by the presence of six
or more ankyrin repeats, an NH2-terminal regulatory domain,
and a COOH-terminal domain that contains a PEST motif involved in basal
turnover. On stimulation, an activation cascade results in the
phosphorylation of I
B, which leads to polyubiquitination and
degradation by the 26S multicatalytic proteosomes (13).
The released NF-
B translocates to the nucleus where it binds to
B
sites in the promoters and enhancers of target genes. The signal is
eventually terminated through cytoplasmic resequestration of NF-
B,
which depends on I
B
synthesis, a process itself requiring NF-
B
transcriptional activity. Of all inhibitor subunits, I
B
is the
one subunit that is best characterized.
Recently, Bhullar et al. (3) demonstrated the involvement
of upstream kinases in the phosphorylation of IB
and the
activation of NF-
B in high shear stress (12 dynes/cm2)-exposed vascular endothelial cells (EC).
However, the mechanism of activation of these upstream mediators in low
shear-exposed EC where, unlike high shear exposed cells, NF-
B
activation is persistent is unclear. In other studies, it has been
shown that nitric oxide (NO) can inhibit the activation of NF-
B by
stabilizing the NF-
B/I
B
complex (14, 20, 24). It
has been further suggested that NO may also attenuate NF-
B
activation by mediating an as yet unknown signaling pathway that leads
to the increased transcription of the I
B
gene (23).
In this study, we have investigated the effect of low shear on the
expression of IB
in human aortic endothelial cells (HAEC) and
whether this is influenced by an altered generation of NO. A reduced
level of I
B
would lead to persistent activation of NF-
B. In
support of this observation, we have also demonstrated downregulation
of NF-
B activation, as well as
B-dependent VCAM-1 expression and
endothelial-monocyte adhesion under conditions of low shear stress by
forced overexpression of I
B
.
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MATERIALS AND METHODS |
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Cell culture. HAEC (Clonetics, San Diego, CA) were cultured in MCDB131 medium (Sigma, St. Louis, MO) containing 10% bovine calf serum (BCS, Hyclone, Kansas City, KS) and enriched with 250 ng/ml fibroblast growth factor (Pepro Tech, Rocky Hill, NJ), 1 mg/ml of epidermal growth factor (Pepro Tech), 1 mg/ml of hydrocortisone (Sigma), 100 units/ml of penicillin, and 100 mg/ml streptomycin (Mediatech, Herndon, VA). Cells from passages 4 to 7 were used in all experiments.
Shear stress. 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 within 2-3 days. The cells were subsequently incubated in complete MCDB-131 medium supplemented with 2% BCS alone for 20 h before the initiation of the flow shear to avoid any influence of growth factors on the induced responses. Flow experiments were performed using the closed loop flow system described previously (17). The cone and the plate model (4) were used in shear stress experiments involving NO measurements to limit the volume of culture medium to be used.
Treatment conditions.
Cells were treated with 300 µM of S-nitrosoglutathione
(GSNO; Sigma) or 1 mM of sodium nitroprusside (SNP; Sigma) or
N-nitro-L-arginine (500 µM
L-NNA) (Sigma) for 30 min before shear stress. NO donors at
the same concentration were added to the circulating medium while the
cells were kept under shear stress. Care was taken to avoid
photosensitization of SNP by performing the experiment under yellow
light. Stock solutions were flushed with nitrogen gas and stored in
amber-colored bottles at
20°C.
Transient transfections.
The VCAM-1 promoter fragment was generated by PCR using human genomic
DNA as the template and the following nested primers: (forward-outer)
5'-TGC GGT TAA ATC TCA CAG CCC-3', (forward-inner) 5'-AGA GAT TTG CCA
CTT CAG ATG G-3', (reverse-outer) 5'-GTA TAT TTG AGG CGC CAA GG-3', and
(reverse-inner) 5'-GAC CAT CTT CCC AGG CAT TTT AAG-3' derived from
published sequences (28). A PCR product of ~900 base
pairs (bp) (VCAM-1 5' flanking sequence from 755 to +143 containing
the two NF-
B binding sites) was obtained and cloned into the pCR
2.1-TOPO vector (Invitrogen, Carlsbad, CA). The insert was then
subcloned into the pGL3-Basic luciferase reporter vector at the
SacI and XhoI sites. For the eNOS overexpression
studies, full-length cDNA of eNOS (provided by Dr. Philip A. Marsden,
Renal Division and Department of Medicine, University of Toronto,
Toronto, ON, Canada) was subcloned into the pCI-neo-expression vector
(Promega, Madison, WI) and used for transient transfections. Plasmid
pcDNA 3.1 containing the full-length cDNA for I
B
provided by Dr.
John Morris (Lineberger Comprehensive Cancer Center, Chapel Hill, NC)
was used for I
B
overexpression studies. The transfection
efficiency was periodically checked by cotransfecting the cells with
pEGFP (enhanced green fluorescent protein) luciferase construct
(Clontech Laboratories), along with other plasmids of interest.
Transfected cells were subjected to FACS analysis. The fluorescent
intensity of pEGFP-positive cells indicated a transfection efficiency
of ~30%.
Electrophoretic mobility shift analysis.
After application of flow shear stress for the indicated time periods,
cells were washed in ice-cold PBS and harvested. Nuclear extracts were
prepared as reported by Mohan et al. (17). The total
protein concentrations were measured using the bicinchonic acid (BCA)
method following the manufacturer's protocol (Pierce, Rockford, IL).
For electrophoretic mobility shift analysis (EMSA) analysis, a
double-stranded oligonucleotide containing a tandem repeat of the
consensus NF-B binding sequences 5'-GGG GAC TTT CC-3' was
end-labeled with T4 polynucleotide kinase (Promega) and
[
-32P]ATP (Amersham, Arlington Heights, IL). Free
unbound radioisotope was separated on a push column (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-labeled NF-
B probe (0.5 ng DNA;
~50,000 cpm) 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. Estimation
of NF-
B activation was performed by quantitative analysis of
autoradiograms using a phosphor imager, and statistical analysis was
carried out using StatView 5.0 software.
RNase protection assay. Total cellular RNA was isolated from HAEC using the Ultra spec reagent following the manufacturer's protocol (Biotech, Houston, TX). Total RNA (10-15 µg) was used for the RNase protection assay. The antisense RNA probe for VCAM-1 was obtained from Harlingen (San Diego, CA). Following the manufacturer's protocol, the in vitro transcription and the protection assays were performed.
Monocyte adhesion assay. Whole blood from healthy volunteers was collected into a vacutainer tube containing EDTA by vein puncture and used within 4 h of collection. Enriched monocytes were isolated by standard buoyant density centrifugation technique using NycoPrep 1.068 (Nycomed Pharma AS, Oslo, Norway), as reported earlier (18). 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 antimacrophage 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.
Immunoblotting.
The nuclear and cytoplasmic extracts of shear stress-exposed HAEC were
subjected to SDS polyacrylamide gel electrophoresis, and the proteins
were transferred to PVDF membrane by electrotransfer. The membranes
were blocked with 5% blotto (Amersham) and probed with a 1:1,000
dilution of goat anti-human IB
antibody (Santa Cruz Biotech,
Santa Cruz, CA) or 1:2,000 of anti-eNOS antibody (BIOMOL Research
Laboratories, Plymouth Meeting, PA), followed by horseradish
peroxidase-conjugated secondary antibody. For loading control, the
blots were probed with mouse monoclonal anti-
tubulin provided by
Dr. Asok Banerjee (UT Health Science Center, San Antonio, TX). The
blots were developed with ECL reagent (Amersham) following the
manufacturer's protocol. Quantitative analysis of the I
B
and
eNOS protein expression was performed using the NIH 1.58b19 image
analysis software package with an integrated density program.
Measurement of NO in HAEC exposed to fluid shear stress. Confluent monolayers of HAEC grown in 100-mm dishes were exposed to the laminar shear stress of 16 dynes/cm2 (high shear) or 2 dynes/cm2 (low shear) using the cone and plate system, as described previously (4). This system was used in this experiment because it allows accurate measurements of NO levels (as accumulating levels of nitrite) in small volumes of culture medium. Aliquots (200 µl) of shear media were taken from the dishes at different time intervals to measure accumulating levels of nitrite using an NO sensor (in NO-T nitric oxide measurement system; Harvard Apparatus, Holliston, MA).
Statistical analyses. Data were analyzed with the randomized block analysis of variance (ANOVA), treating experiments as blocks and treatments as repeated measures for the experiment. These two factors were considered in each analysis. In addition, both change from control and ratio to control were considered to determine which change score better met the assumptions for a valid analyses. The ratio to control better satisfied the assumptions of the analysis of variance, so the results from that analyses are presented. In fact, with this ratio, the experimental variability is small and insignificant. Both this ratio to control and the use of experiment in the ANOVA helped to provide valid comparison of the treatment conditions adjusting for both between and within experimental variability. After the ANOVA, comparisons of the treatments with each other and with the static control condition were done with means from the ANOVA and t-tests using these ANOVA results, accounting for experimental variability.
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RESULTS |
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Low shear stress stimulates IB
degradation and induces
NF-
B activation.
To determine whether low shear stress-induced activation of NF-
B is
associated with proteolytic degradation of I
B
, HAEC were
subjected to a low shear stress of 2 dynes/cm2 for 1 h
and 6 h. As shown in Fig.
1A, immunoblotting of the
cytoplasmic extracts with polyclonal anti-I
B
antibody revealed
reduction of I
B
levels after exposure of HAEC to low shear
stress. Compared with the static control maintained at 1 h, a
decrease (to 83% of control) in the amount of cytoplasmic I
B
could be seen within 1 h of exposure of HAEC to low shear stress.
The levels reduced to 70% of control after 6 h of exposure to low
shear stress compared with static control maintained for 6 h. No
changes were seen in the corresponding levels of
-tubulin used as
the loading control. As shown in Fig. 1B, quantitative
analysis of the autoradiograms from three independent experiments
clearly demonstrated a significant reduction (P = 0.003) of I
B
expression observed at 6 h of low shear stress.
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Overexpression of IB
attenuates low shear-induced NF-
B
activation, mRNA expression, and subsequent endothelial-monocyte
adhesion.
The above data indicate that the degradation of I
B
is a critical
intermediate step in the early low shear-induced activation of NF-
B.
To obtain further evidence for this important role of I
B
, we
examined low shear-induced NF-
B activation in HAEC that were
transfected with an I
B
expression plasmid. Immunoblotting performed in the cytoplasmic extracts of the transfected HAEC confirmed
that the cells overexpressed I
B
compared with cells transfected
with vector alone (Fig. 2A).
The I
B
-transfected cells, when subjected to low shear stress for
6 h, showed a complete attenuation of shear-induced NF-
B
activation (Fig. 2, B and C). Compared with
untransfected HAEC subjected to low shear stress, overexpression of
I
B
significantly blocked (63.4%; P < 0.05) the
NF-
B DNA binding activity. Both control untransfected cells and
cells transfected with empty vector exhibited higher levels of low
shear-induced NF-
B activity, indicating the functional specificity
of the insert.
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|
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Exogenous NO donors inhibit low shear-induced NF-B activation
and VCAM-1 promoter expression by increasing I
B
levels.
NO has been shown to induce synthesis of I
B
in TNF-
-stimulated
human umbilical vein endothelial cells (20, 24). To investigate the effect of NO on low shear-induced NF-
B activation, HAEC were subjected to 6 h of low shear stress in the absence or
presence of SNP (1 mM), an NO donor. As shown in Fig.
5A, EMSA performed with
nuclear extracts of HAEC demonstrated a high induction of NF-
B
activation after 6 h of low shear stress, as we have shown
previously. This low shear-induced NF-
B activation was inhibited by
the presence of SNP (1 mM). Furthermore, to confirm the effect of NO on
low shear-induced NF-
B DNA binding activity, HAEC were exposed to
low shear stress in the presence of L-NNA (500 µM), an
eNOS inhibitor that is known to prevent endogenous generation of NO. As
demonstrated in Fig. 5A, lane 4,
L-NNA enhanced the NF-
B DNA binding activity compared
with HAEC exposed to low shear stress in the absence of the inhibitor.
These results confirm the involvement of NO in regulating the pathway
responsible for low shear-induced NF-
B induction.
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Overexpression of eNOS in HAEC inhibits low shear-induced
activation of NF-B.
Because it is possible that exogenous NO donors may interact with
unrelated cellular components and produce an indirect inhibitory effect
on NF-
B activity, we sought supporting evidence that the observed
inhibitory activity was due to NO. Thus we examined the effect of
increasing endogenous levels of NO by overexpressing full-length eNOS
by transiently transfecting the cells with an eNOS expression vector.
Overexpression of eNOS in HAEC was confirmed by immunoblotting using a
specific anti-eNOS antibody (Fig.
7A). At 24 h
posttransfection, the cells were subjected to 6 h of low shear
stress. Mobility shift analysis of the nuclear extracts (Fig.
7B) and quantitation by image analysis (Fig. 7C)
clearly indicated that overexpression of eNOS significantly
downregulated low shear-induced NF-
B DNA-binding activity. The
untransfected HAEC subjected to low shear stress for 6 h exhibited
a 18.5 ± 6.7-fold increase of NF-
B DNA binding activity
compared with static controls (P < 0.005). Cells
overexpressing eNOS, on the other hand, exhibited only a 0.9-fold
increase when subjected to low shear stress. Thus, consistent with our
observations with the NO donors, overexpression of eNOS dramatically
inhibited low shear-induced NF-
B activity. Compared with no shear
static controls, eNOS-overexpressed HAEC showed only a slight increase
of NF-
B DNA binding activity (4.8%), which was not statistically
significant.
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Low shear stress downregulates NO production in HAEC.
The above observations suggested that a reduction in NO release by HAEC
undergoing low shear stress for 6 h might be one mechanism responsible for some of the changes observed in the cellular levels of
NF-B and I
B
. Studies were undertaken to measure NO release in
HAEC exposed to shear stress. Because high shear stress is known to be
the potent inducer of eNOS activity and the subsequent release of NO
(5), HAEC exposed to high shear stress of 16 dynes/cm2 were used as a positive control. The NO
production was significantly reduced in low shear-exposed HAEC,
especially during the earlier time point (measured at 5 min;
P < 0.01) compared with high shear stress. Although it
did not reach a statistical significance, cells exposed to 6 h of
low shear stress showed an ~40% decrease in NO release compared with
cells subjected to high shear stress of 16 dynes/cm2.
However, as shown in Fig. 8, compared
with no-shear controls, both low and high shear stress significantly
increased NO release (P < 0.001). Cells incubated with
nitro-L-arginine methyl ester (L-NAME; 2 mM)
prevented production of NO induced by both low and high shear stress,
indicating the specificity of the assay. These experiments were
conducted using the cone and plate model (4) because it
uses a smaller volume of culture medium and permits the accurate
measurement of NO as accumulating concentrations of nitrite.
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DISCUSSION |
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In this study, we have demonstrated that in HAECs,
low-shear flow-induced activation of NF-B, VCAM-1 mRNA expression,
and endothelial-monocyte adhesion can be inhibited by I
B
overexpression. We also provide evidence for the involvement of
intracellular NO levels in the regulation of low shear-induced I
B
degradation and subsequent activation of NF-
B. Our data suggest that
the relative inhibition of NO production during the early phase of low
shear stress exposure may be partly responsible for the induced activation of NF-
B.
Most studies exploring the mechanism of NF-B activation in
endothelial cells have focused on the cytokines TNF-
and
interleukin-1
(IL-1
) as the stimulating agents (8,
11). However, the mechanisms responsible for NF-
B activation
in response to shear stress have not been extensively investigated and
may differ from those induced by cytokines. Bhullar et al.
(3) demonstrated that a high shear of 12 dynes/cm2 induced activation of I
B kinases and the
transient degradation of I
B
. The degradation of I
B
could be
detected as early as 10 min, and the degradation was complete within 30 min after the onset of high shear stress. However, the I
B
levels
reappeared again within 60 min and thereafter were restored to control
levels. This sequence of degradation and resynthesis of I
B
correlated with the transient NF-
B activation in high shear-exposed
cells (17). In contrast to high shear stress, in which
there was a rapid rebounce of I
B
expression, in the present study
in which a shear of 2 dynes/cm2 was employed, we observed a
prolonged (6 h) absence of cytoplasmic I
B
. This most likely was
responsible for the persistent NF-
B activation observed in the HAEC.
The transient overexpression of I
B
, which blocked the 6 h
low shear-induced NF-
B activity, as well as the
B-dependent
VCAM-1 expression and endothelial-monocyte adhesion, confirmed the role
of I
B
in the regulation of NF-
B activation.
We investigated the possible role of NO to identify the mediators
responsible for prolonged absence of IB
in low shear
stress-exposed HAEC on the basis of the following published reports
providing supportive evidence. Inhibition of endogenous NO production
has been shown to increase NF-
B DNA binding activity in in vitro conditions (20) and promote endothelium-leukocyte
interactions, probably through the enhanced expression of
NF-
B-dependent adhesion molecules in vivo (2, 7). NO
donors have been shown to efficiently block TNF-
-induced adhesion
molecule expression and enhanced monocyte adhesion in human umbilical
and saphenous vein EC (14, 20, 23, 24). Additionally,
Spiecker et al. (24) have reported that NO inhibits
cytokine-induced NF-
B activation, as well as VCAM-1 expression, by
increasing cytoplasmic and nuclear levels of I
B
.
In the present study, we have demonstrated the inhibition of low shear
flow-induced NF-B activation by incubating HAEC with exogenous NO
donors and by transiently overexpressing eNOS. Incubation with
exogenous NO donors, GSNO (300 µM), and SNP (1 mM) strongly inhibited
the low shear-induced degradation of I
B
and the concomitant increase in VCAM-1 promoter expression and NF-
B DNA binding
activity. Assays performed using the WST-1 reagent confirmed that this
was not the result of NO donor-induced cell death (data not shown). However, it should be noted that differences were observed in the
potency of these donors in reducing VCAM-1 promoter expression. This
may have been due to differences in their stability. Compared with SNP,
GSNO is shortlived and, therefore, may be less efficient in inhibiting
NF-
B DNA binding activity. Also, the different levels of inhibition
caused by different NO donors, as demonstrated in Fig. 6, may suggest
the possibility that they may also inhibit endogenous promoter
expression with varying efficiency.
Ranjan et al. and Malek et al. (22, 16) and results
presented in this study (Fig. 7, D and E)
independently demonstrated the downregulation of eNOS protein
expression in low shear flow. However, Ranjan et al. (22)
observed induction of eNOS mRNA expression in high arterial levels of
shear stress (25 dynes/cm2) and low shear stress-exposed
cells (4 dynes/cm2) compared with static culture
conditions. Results from Ziegler et al. (29) implied that
regulation of eNOS expression by mechanical factors occurs by both
transcriptional and posttranscriptional mechanisms that still need to
be determined. It would be interesting to speculate, on the basis of
this evidence, as well as our findings, that low shear stress may
initiate degradation of the expressed eNOS protein or attenuate
phosphorylation of eNOS. The requirement for phosphorylation at serine
1179 by phosphatidylinositol 3-kinases (9) and PKA
(4) for eNOS activity has recently been reported. The
disconnect in the levels of eNOS expression and NO release in HAEC
maintained under no-shear controls (shown in Fig. 7, D and
E, and Fig. 8) may be due to the lack of phosphorylation of eNOS, which is only initiated at the onset of shear stress. Therefore, under static culture conditions, even though HAEC expresses elevated levels of eNOS compared with low shear exposed HAEC, the NO release is
significantly lower due to the absence of shear stress induction. In
our attempts to measure NO, a ~40% decrease was observed after 6 h of low shear stress compared with high shear stress, and these results correlate well with the eNOS expression levels that are significantly reduced in low shear stress compared with high shear stress. Because NO can bind superoxide anion with extremely high affinity (12), the bioavailability of NO in low shear
regions will be far less than high shear regions under the assumption that both low and high shear stress generate equivalent levels of
superoxide anions and other reactive oxygen species. Together, one
mechanism by which NO stabilizes IB
and inhibits NF-
B
activation may be through quenching superoxide anion, thereby reducing
its dismutation product, hydrogen peroxide, which is known to activate NF-
B in endothelial cells. Nevertheless, whether there is a
differential generation of superoxide and other oxygen free radicals
occur in low vs. high shear-exposed HAEC remains to be determined. In addition, NO may directly affect upstream protein kinases and phosphatases that regulate I
B
phosphorylation.
In summary, the prolonged absence of IB
, which in turn may be
regulated by changes in NO levels, is responsible for the persistent
activation of NF-
B DNA binding activity observed in HAEC exposed to
6 h of low shear stress. Regulating the levels of I
B
may be
one of the mechanisms by means of which NO exhibits its antiatherogenic properties.
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ACKNOWLEDGEMENTS |
---|
We thank Mr. Javier Chapa, Mr. Gilbert Ortiz, and Dr. Thangasamy Amalraj for technical assistance.
![]() |
FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute 1RO1-HL-63032-01A1) and F32-HL-09694-01A1 (to S. Mohan).
Address for reprint requests and other correspondence: S. Mohan, Assistant Professor, Dept. of Pathology, Univ. of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (E-mail: mohan{at}uthscsa.edu).
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. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00464.2001
Received 1 October 2001; accepted in final form 13 December 2002.
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