Hyperbaric oxygen downregulates ICAM-1 expression induced by
hypoxia and hypoglycemia: the role of NOS
Jon A.
Buras1,
Gregory L.
Stahl2,
Kathy K. H.
Svoboda3, and
Wende R.
Reenstra4
1 Department of Emergency Medicine and
2 Center for Experimental Therapeutics and
Reperfusion Injury, Department of Anesthesiology, Brigham and Women's
Hospital, Boston 02115; 4 Department of
Pathology, Boston University School of Medicine, Boston, Massachusetts
02118; and 3 Department of Biomedical
Sciences, Baylor College of Dentistry, Dallas, Texas 75246
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ABSTRACT |
Hyperbaric oxygen (HBO) is being
studied as a therapeutic intervention for ischemia/reperfusion
(I/R) injury. We have developed an in vitro endothelial cell model of
I/R injury to study the impact of HBO on the expression of
intercellular adhesion molecule-1 (ICAM-1) and polymorphonuclear
leukocyte (PMN) adhesion. Human umbilical vein endothelial cell (HUVEC)
and bovine aortic endothelial cell (BAEC) induction of ICAM-1 required
simultaneous exposure to both hypoxia and hypoglycemia as determined by
confocal laser scanning microscopy, ELISA, and Western blot. HBO
treatment reduced the expression of ICAM-1 to control levels. Adhesion
of PMNs to BAECs was increased following hypoxia/hypoglycemia exposure
(3.4-fold, P < 0.01) and was reduced to control levels with
exposure to HBO (P = 0.67). Exposure of HUVECs and BAECs to HBO
induced the synthesis of endothelial cell nitric oxide synthase (eNOS).
The NOS inhibitor nitro-L-arginine methyl ester attenuated
HBO-mediated inhibition of ICAM-1 expression. Our findings suggest that
the beneficial effects of HBO in treating I/R injury may be mediated in
part by inhibition of ICAM-1 expression through the induction of eNOS.
cell adhesion molecules; ischemia; reperfusion injury; hyperoxia; neutrophils
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INTRODUCTION |
ISCHEMIA/REPERFUSION (I/R) injury
represents a final common pathway in many disease states including
myocardial infarction, stroke, compartment syndrome, and acute
peripheral extremity ischemia (4, 12). Reperfusion injury is
mediated in part through the effects of neutrophil infiltration into
damaged tissue and free radical production (12, 19). Intercellular
adhesion molecule-1 (ICAM-1) has been implicated in I/R injury by
recruiting CD11/CD18-expressing polymorphonuclear leukocytes (PMNs)
(23, 38). Inhibition of ICAM-1 function through blocking peptides,
monoclonal antibodies, and an ICAM-1-null transgenic mouse model has
shown a significant reduction in reperfusion injury (8, 17, 30, 39,
52). Nitric oxide (NO) also plays a role in I/R injury (reviewed in Ref. 22). Inhibition of endothelial cell-derived NO synthesis promotes
the expression of ICAM-1 and the adhesion of PMNs in several models
(10, 20). Also, infusion of organic NO donors decreases adhesion of
PMNs to damaged endothelium (4a, 37).
Hyperbaric oxygen (HBO), exposure to oxygen at a pressure greater than
one atmosphere absolute (ATA), has been used as an adjunctive therapy
for several I/R injuries, including acute peripheral ischemia
and myocardial infarction (4). Given the general association of high
PO2 with formation of reactive oxygen
species (ROS) during reperfusion, the use of HBO in treating I/R injury appears counterintuitive. However, recent in vivo studies have shown
that HBO treatment improves outcome following experimental I/R injury.
A rodent raised pedicle muscle flap model demonstrated improved
survivability of grafts following I/R with HBO treatment (41). Studies
to date suggest that HBO may interfere with the destructive PMN
infiltration response following I/R. Real-time videomicroscopy of
HBO-treated muscle flaps following I/R have demonstrated decreased PMN
adhesion to the endothelium and greater microvessel diameter (50).
Also, absolute PMN content of HBO-treated muscle flaps following I/R
injury is decreased vs. nontreated flaps (51). HBO pretreatment has
been shown to decrease leukocyte adhesion and improve postischemic
microvascular flow velocity and tissue ATP levels following I/R in a
rodent liver model (5).
The molecular mechanisms responsible for the beneficial effect of HBO
on PMN adhesion and infiltration are not well defined. This study
sought to determine whether HBO affects the adhesion of PMNs to
endothelium through modulation of ICAM-1 protein expression. We found
that endothelial cell expression of ICAM-1 in vitro required exposure
to both hypoxia and hypoglycemia. HBO treatment of endothelial cells
following hypoxia/hypoglycemia stimulation abolished the induction of
ICAM-1 protein expression and also the adhesion of PMNs to endothelial
monolayers. HBO exposure also induced the production of endothelial
nitric oxide synthase (eNOS). Inhibition of eNOS interfered with the
HBO-mediated downregulation of ICAM-1 in our model. Our findings
suggest that the beneficial effect of HBO in treating I/R injury may be
mediated through decreased expression of ICAM-1, which in part may be
related to an upregulation of eNOS activity.
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MATERIALS AND METHODS |
Reagents.
Lipopolysaccharide (LPS), anti-
-tubulin antibody, and
nitro-L-arginine methyl ester (L-NAME) were
purchased from Sigma Chemical (St. Louis, MO) unless otherwise
indicated. The MY-13 monoclonal anti-ICAM-1 antibody was purchased from
Zymed Laboratories (CA). Anti-eNOS and anti-inducible NO synthase
(anti-iNOS) primary antibodies and horseradish peroxidase
(HRP)-conjugated secondary antibodies were purchased from Transduction
Laboratories (Lexington, KY).
Cell culture and in vitro model of I/R.
Briefly, early passage primary bovine aortic endothelial cells (BAECs;
a kind gift from Dr. D. Larson, Boston University), human umbilical
vein endothelial cells (HUVECs), or the RAW 264.7 murine macrophage
cell line (generously provided by Dr. M. J. Fenton, Boston University)
were plated in 60-mm tissue culture dishes or Labtek 4 chamber slides
(Nunc, Marsh Biomedical, NY). Cells were grown in low-glucose (100 mg/dl) DMEM (GIBCO BRL), supplemented with 10% fetal
bovine serum (Hyclone). To simulate ischemia in vitro, cells
were exposed to hypoxia and hypoglycemia (28). Culture media were
replaced with DMEM (containing 0 mg/dl glucose) and cells were placed
in a modular incubator chamber (MIC-101, Billups-Rothenberg, Del Mar,
CA). Room air gas, PO2 ~150 mmHg
(normoxic), in the incubator chamber was then exchanged with gas
containing 95% N2 and 5% CO2 (hypoxic) by
equilibration flow for 30 min. These conditions acutely reduce culture
media PO2 content to ~25-29
mmHg (28). Cells were incubated under mock ischemic conditions for
4 h, a time period chosen to mimic prior in vivo pedicle flap I/R
models (50). After 4 h of mock ischemia, mock reperfusion was
simulated by changing culture medium to low-glucose DMEM
(normoglycemic) and incubating cells under normoxic conditions for an
additional 20 h, for a total time of 24 h. In experiments utilizing
L-NAME, this compound was diluted to final
concentration and added to the cells at the time corresponding to the beginning of the reperfusion period.
HBO exposure.
Experimental treatment with 100% O2 at 1.0, 1.5, 2.0, or
2.5 ATA for 90 min was performed on initiation of the reperfusion phase
after replacing hypoglycemic media with low-glucose DMEM. Access to a Sechrist experimental hyperbaric animal research chamber 1300B was kindly provided by Dr. R. Fabian, Massachusetts Eye and Ear
Infirmary. HBO treatment conditions were selected to mimic in vivo
models and current human treatment protocols for I/R injury (2, 50).
After HBO treatment, cells were incubated under normoxic conditions for
18.5 h (total reperfusion period 20 h) unless otherwise indicated.
Cells from all experimental groups were >95% viable throughout the
studies as determined by trypan blue exclusion.
Confocal laser scanning microscopy.
Membrane expression of ICAM-1 protein was analyzed on nonpermeabilized
cells with a Leica confocal laser scanning microscope using a
monoclonal ICAM-1 antibody (14). Briefly, cells were washed with
1× PBS and fixed with 4% paraformaldehyde. Cells were incubated
with a 1:1,000 dilution of a monoclonal anti-ICAM-1 antibody
recognizing the extracellular portion (Clone MY-13, Zymed) for 2 h at
25°C. Cells were then rinsed with 1× PBS and blocked for 30 min with goat serum, washed with 1× PBS, incubated with a
secondary FITC-conjugated antibody, and rinsed and mounted. Measurement
of intracellular eNOS or iNOS was performed by membrane permeabilization with 0.01% Triton X-100 for 5 min after initial fixation. Cells were washed with 1× PBS four times and processed as above, except that anti-eNOS or anti-iNOS antibodies were used primarily at a 1:1,000 dilution. Negative controls included cells incubated with secondary antibody alone and irrelevant mouse IgG primary antibody. Propidium iodide staining of nucleic acid was performed using a concentration of 2 µg/ml for 10 min before final washes. Confocal laser scanning microscopy (CLSM) quantitation was
determined by analysis of fluorescence intensity. For quantitation of
fluorescence intensity, the background laser intensity was set to
untreated control conditions, and all subsequent samples were scanned
under these conditions so that increases in ICAM-1 signal intensity
relative to controls could be determined. Fluorescence intensity was
measured in 12-20 representative cells from different slides for
each condition by a blinded operator and statistically analyzed as
described below. Each experiment was repeated a minimum of three times.
Cell surface immunoassay.
ICAM-1 cell surface protein was quantified by ELISA as previously
described with the following minor modifications (54). Briefly, cells
were plated at a density of 105 cells per well in 96-well
flat-bottomed culture plates and exposed to experimental conditions.
For ELISA, cells were washed four times with Hanks'
balanced salt solution (HBSS) and incubated with a 1:1,000 dilution of
the MY-13 monoclonal anti-ICAM-1 antibody for 30 min at 37°C. Cells
were then washed four times with HBSS and incubated with a 1:2,000
dilution of HRP-conjugated secondary IgG for 1 h. Cells were washed
four times with HBSS, and a developing solution containing
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), final
concentration 1 mM, was added for 10 min. Sample optical density was
analyzed using a microtiter plate spectrophotometer (Molecular Devices)
at 405 nm. Conditions were replicated with 6-24 wells per
condition in each experiment, and each experiment was repeated a
minimum of three times.
Western blotting.
Cells were analyzed for total ICAM-1 and eNOS protein by Western blot
analysis as follows. Briefly, total protein was extracted in buffer
containing 50 mM Tris · HCl (pH 8.0), 0.15 M NaCl,
0.5% deoxycholate, and 1% Triton X-100 in the presence of 1 µg/ml
aprotinin and 75 µg/ml phenylmethylsulfonyl fluoride. Samples were
sonicated for 1-3 s, and insoluble material was removed by
centrifugation. Twenty micrograms of each protein sample was separated
on 7.5% SDS-PAGE and transferred to nitrocellulose paper at 70 V in a Trans-Blot electrophoretic transfer cell (Bio-Rad, Richmond, CA) while
being cooled to 4°C on an Iso-Temp refrigerated
circulator (Fisher Scientific). Nitrocellulose blots were blocked for 1 h in 1× PBS containing 5% milk and rinsed three times for 10 min with 1× PBS. Blots were then incubated with a primary anti-ICAM-1 monoclonal antibody (Zymed), anti-eNOS (Transduction Laboratories), or
-tubulin (Sigma) at a 1:1,000 dilution at 4°C for 12 h.
Nitrocellulose blots were rinsed three times for 10 min in PBS at
4°C and incubated with HRP-conjugated goat anti-mouse IgG secondary
antibody at 1 µg/ml final concentration for 1 h. Blots were rinsed
three times in 1× PBS and incubated overnight at 4°C with
1× PBS containing 2% milk. Protein was detected using the
enhanced chemiluminescence Western blotting detection system (Amersham,
Arlington Heights, IL). Blots were wrapped in plastic wrap and exposed
to autoradiograph film for various times. Signal intensity
corresponding to ICAM-1 expression was quantified by scanning
densitometry using National Institutes of Health (NIH) Image 1.5 software (NIH, Bethesda, MD). Changes in ICAM protein expression are
expressed relative to baseline control protein expression.
PMN/endothelial cell adhesion assay.
PMN isolation and adhesion to confluent endothelial cell monolayers was
carried out as previously described (6, 54). Adhesion of human PMN to
BAECs has been previously described (11). Briefly, BAECs were cultured
in flat-bottomed 96-well plates at a density of 105
cells/well and exposed to experimental conditions. Plates
were washed four times with HBSS, and 2.5 × 105 PMNs
were added to each well and incubated at 37°C for 30 min. Quantitaion of bound PMNs was determined by measuring total
myeloperoxidase activity by functional assay. Developing solution
containing 1 mM 2,2'-azino-di-3-ethyl dithiazoline sulfonic acid
with 10 mM H2O2 and 0.5% Triton X-100 in 100 mM citrate buffer (pH 4.2) was added to facilitate cell lysis and
provide substrate for assay of myeloperoxidase enzymatic activity.
Plates were incubated at 37°C for 10 min, and sample optical
density was analyzed using a microtiter plate spectrophotometer
(Molecular Devices) at 405 nm. Conditions were replicated with 3-6
individual samples per experiment, and each experiment was repeated a
minimum of three times.
Statistical analysis.
Statistical analysis was performed by ANOVA with Fisher's protected
least-significant differences test using the statistical analysis
software package Statview (Abbacus, Berkeley, CA).
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RESULTS |
Both hypoxia and hypoglycemia are required to induce ICAM-1 protein
expression in endothelial cells: hypoxia/hypoglycemia-induced ICAM-1 is
downregulated by HBO exposure.
Previous studies have suggested that ICAM-1 protein expression is not
induced by hypoxia alone and requires other stimuli such as LPS,
cytokines, or a reperfusion period (13, 29, 49, 53). We sought to
determine whether hypoglycemia was capable of inducing ICAM-1 alone or
modifying ICAM-1 production in combination with hypoxia/reoxygenation.
HUVECs were exposed to 4 h of hypoxia, hypoglycemia, or hypoxia and
hypoglycemia, followed by 20 h of normoxia and normoglycemia. CLSM
analysis was used to determine the induction of ICAM-1. CLSM
demonstrates low levels of ICAM-1 protein in untreated control HUVECs
(Fig. 1A). Four hours of
hypoxia or hypoglycemia alone failed to induce ICAM-1 at 24 h (Fig.
1, compare A, C, and D). The combination of 4 h
of hypoxia and hypoglycemia was sufficient to induce ICAM-1 protein
expression at 24 h (Fig. 1, compare A and E). ICAM-1
expression following hypoxia/hypoglycemia was similar to levels
following LPS stimulation (Fig. 1, compare B and E).

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Fig. 1.
Confocal laser scanning microscopy (CLSM) analysis of intercellular
adhesion molecule-1 (ICAM-1) expression in human umbilical vein
endothelial cells (HUVECs) following ischemia/reperfusion
injury (I/R) and hyperbaric oxygen (HBO). A: control treated
cells. B: lipopolysaccharide (LPS) treatment at 1 µg/ml for 4 h followed by 20 h of control media. C: hypoxia for 4 h
followed by normoxia for 20 h. D: hypoglycemia for 4 h followed
by normoglycemia for 20 h. E: hypoxia/hypoglycemia for 4 h
followed by normoxia/normoglycemia for 20 h. F:
hypoxia/hypoglycemia for 4 h followed by HBO at 2.5 atmosphere absolute
(ATA) for 1.5 h, then normoxia/normoglycemia for 18.5 h. G: HBO
at 2.5 ATA for 1.5 h, normoxia for 22.5 h. H: color bar
intensity wedge corresponding to fluorescence intensity scale.
Photomicrographs taken from representative fields.
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Having defined conditions in vitro leading to the induction of ICAM-1,
we sought to determine whether HBO treatment of endothelial cells could
inhibit ICAM-1 protein induction. The mock I/R protocol was modified to
include 90 min of HBO at 2.5 ATA following the hypoxic/hypoglycemic
period and preceding the normoxic/normoglycemic period. HUVECs
subjected to hypoxia/hypoglycemia alone were transferred directly into
normoxic/normoglycemic conditions for an equivalent 90-min period.
After a total time of 24 h, cells were harvested and analyzed for
ICAM-1 protein expression by CLSM. HBO treatment of
hypoxia/hypoglycemia-treated endothelial cells reduced expression of
ICAM-1 protein to control levels (Fig. 1, compare A, E,
and F). HBO exposure alone did not affect ICAM-1 expression
(Fig. 1G). Similar results were obtained using BAECs and an
identical immunohistochemical staining protocol (Fig.
2). Analysis of fluorescence intensity
staining for ICAM-1 under the various experimental conditions is shown
in Table 1.

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Fig. 2.
CLSM analysis of ICAM-1 expression in bovine aortic endothelial cells
(BAECs) following I/R and HBO. A: control-treated cells.
B: LPS treatment at 1 µg/ml for 4 h followed by 20 h of
control media. C: hypoxia for 4 h followed by normoxia for 20 h. D: hypoglycemia for 4 h followed by normoglycemia for 20 h.
E: hypoxia/hypoglycemia for 4 h followed by
normoxia/normoglycemia for 20 h. F: hypoxia/hypoglycemia for 4 h followed by HBO at 2.5 ATA for 1.5 h, then normoxia/normoglycemia for
18.5 h. G: color bar intensity wedge corresponding to
fluorescence intensity scale. Photomicrographs taken from
representative fields.
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Table 1.
Hypoxia and hypoglycemia induce endothelial cell ICAM-1 protein
expression: HBO exposure downregulates
hypoxia/ hypoglycemia-induced ICAM-1
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After exposure to hypoxia/hypoglycemia, treatment of cells with
normobaric 100% oxygen or a N2/O2 gas mixture
equivalent to air at 2.5 ATA did not reduce
hypoxia/hypoglycemia-induced ICAM-1 levels (data not shown). Further
experiments were performed to establish whether a threshold pressure
was required to suppress ICAM-1 induction. After exposure to
hypoxia/hypoglycemia, cells were treated with HBO at increasing
pressures of 1.5, 2.0, and 2.5 ATA followed by normoxia/normoglycemia.
Analysis of ICAM-1 by CLSM in Table 2 shows
that a pressure of 2.5 ATA was required for maximal inhibition of
hypoxia/hypoglycemia-induced ICAM-1. Increasing pressure showed an
incremental suppression of ICAM-1 expression; however, the greatest
reduction occurred between 2.0 and 2.5 ATA.
The kinetics of ICAM-1 induction by hypoxia/hypoglycemia and response
to HBO treatment were also studied. HUVECs were exposed to
hypoxia/hypoglycemia for 4 h with or without treatment with HBO at 2.5 ATA as described in MATERIALS AND METHODS. At increasing intervals following the return to normoxia/normoglycemia, cells were fixed and analyzed by CLSM for ICAM-1 expression. An increase in
ICAM-1 expression was first noted 4 h following hypoxia/hypoglycemia, with levels continuing to rise over a 24-h period (Fig.
3). HUVECs treated with HBO
showed a blunted rise in ICAM-1 expression, with a small but
significant increase noted only at 8 h following hypoxia/hypoglycemia (Fig. 3).

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Fig. 3.
Effect of HBO on ICAM-1 induction following hypoxia/hypoglycemia.
HUVECs were exposed to the following conditions: 4 h of
hypoxia/hypoglycemia and then normoxia/normoglycemia for 20 h ( ); 4 h of hypoxia/hypoglycemia, then single treatment of HBO at 2.5 ATA for
1.5 h followed by normoxia/normoglycemia for 18.5 h ( ). Cells were
analyzed for ICAM-1 protein expression by CLSM as described in text at
varied times beginning after exposure to hypoxia/hypoglycemia (time = 0). Fluorescence intensity is expressed as means ± SE.
* P < 0.05 vs. time = 0 vs. individual time points using
one-way ANOVA with Fisher's protected least-significant differences
test and a significance level of 5%.
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Cell surface ELISA results for ICAM-1 using the hypoxia/hypoglycemia
model paralleled those observed with CLSM (Table
3). Cells were subjected to control media,
LPS at 1 µg/ml, hypoxia, hypoglycemia, or hypoxia/hypoglycemia for 4 h, followed by 20 h of normoxia/normoglycemia. HBO-treated cells were
exposed to 4 h of hypoxia/hypoglycemia for 4 h, transferred to
normoglycemic media, and then exposed to HBO at 2.5 ATA for 1.5 h.
After HBO exposure, cells were incubated in normoxic/normoglycemic
conditions for 18.5 h. Immunoassay of cell surface ICAM-1 demonstrated
significant upregulation of ICAM-1 protein in the LPS (3-fold, P
< 0.01) and hypoxia/hypoglycemia groups (2.6-fold, P < 0.01). A smaller increase in ICAM-1 was seen following hypoxia
(1.5-fold, P < 0.02). Hypoglycemia alone did not lead to
significant increases in ICAM-1 (1.1-fold, P = 0.63). HBO
exposure of cells incubated under hypoxic/hypoglycemic conditions led
to a 28-fold decrease in ICAM-1 expression vs. cells exposed to
hypoxia/hypoglycemia alone (P < 0.01). HBO also decreased
ICAM-1 cell surface protein expression relative to
normoxic/normoglycemic controls by 11-fold (P < 0.01).
HBO treatment decreases total protein expression of ICAM-1 in
hypoxia/hypoglycemia-stimulated endothelial cells.
Western blot analysis of total ICAM-1 protein was performed on both
HUVECs and BAECs subjected to similar conditions as described above for
CLSM and ELISA analysis. Hypoxia or hypoglycemia were insufficient to
induce ICAM-1 protein expression in either HUVECs or BAECs [Fig.
4A: HUVECs, compare lanes 1, 3, and 4 and corresponding optical density (OD) values 7.3, 4.3, and 4.4 for control, hypoglycemic, and hypoxic conditions,
respectively; Fig. 4B: compare lanes 1 and 3 and corresponding OD values 0.0 and 0.2 for control and hypoglycemic
conditions, respectively]. In both HUVECs and BAECs, the
combination of hypoxic and hypoglycemic conditions vs. media controls
induced an ~90-100 kDa protein corresponding to ICAM-1 (Fig.
4A, compare lanes 1 and 5 with corresponding OD
values 0.0 and 20.7; Fig. 4B, compare lanes 1 and
4 with corresponding OD values 6.38 and 10.0). The combination
of hypoxia/hypoglycemia was similar to LPS (0.5 µg/ml) as an inducer
of ICAM-1 in BAECs (Fig. 4B, compare lanes 2 and
4 with corresponding OD values 22.0 and 20.7); however, the
LPS-mediated induction of ICAM-1 in HUVECs was ~4.4-fold greater than
hypoxia/hypoglycemia (Fig. 4A, compare lanes 2 and
5 with OD values 43.5 and 10.0). The structural protein
-tubulin was used to control for equivalent protein loading of gels
(Fig. 4).

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Fig. 4.
Western blot analysis of total protein from HUVECs and BAECs following
mock I/R injury and the effects of HBO on ICAM-1 expression. A:
HUVEC lane 1, control media 24 h; lane 2, LPS (0.5 µg/ml) 4 h followed by 20 h control media; lane 3, hypoxia 4 h followed by 20 h normoxia; lane 4, hypoglycemia 4 h followed
by 20 h normoglycemia; lane 5, hypoxia/hypoglycemia 4 h
followed by normoxia/normoglycemia 20 h; lane 6,
hypoxia/hypoglycemia 4 h followed by HBO at 2.5 ATA for 1.5 h, then
normoxia/normoglycemia 18.5 h; lane 7, HBO at 2.5 ATA for 1.5 h
followed by 22.5 h normoxia. B: BAEC lane 1, control
media 24 h; lane 2, LPS (0.5 µg/ml) 4 h followed by 20 h
control media; lane 3, hypoglycemia 4 h followed by 20 h
normoglycemia; lane 4, hypoxia/hypoglycemia 4 h followed by
normoxia/normoglycemia for 20 h; lane 5, hypoxia/hypoglycemia 4 h followed by HBO at 2.5 ATA for 1.5 h, then normoxia/normoglycemia
18.5 h; lane 6, HBO at 2.5 ATA for 1.5 h, followed by 22.5 h
normoxia. Arrows, positions of ICAM-1 and -tubulin.
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HBO exposure decreases adhesion of PMNs to
hypoxia/hypoglycemia-stimulated endothelial cell monolayers.
We sought to determine whether the observed changes in ICAM-1 protein
expression corresponded to a functional difference in static PMN
binding to endothelial cells. BAECs were exposed to hypoxia/hypoglycemia followed by normoxia/normoglycemia. One group of
cells was treated with HBO as described above. Binding of PMNs was
assessed at 24 h relative to the initiation of hypoxia/hypoglycemia (Table 4). PMN binding to
hypoxia/hypoglycemia-stimulated BAECs was increased 3.4-fold relative
to controls (P < 0.01). Treatment of
hypoxia/hypoglycemia-stimulated BAECs with HBO decreased PMN binding to
control levels (control vs. hypoxia/hypoglycemia/HBO, P =
0.70).
HBO exposure induces synthesis of eNOS protein.
Previous studies have suggested that hyperoxia leads to increased
production of NO and eNOS protein (1, 32). We wished to determine
whether HBO exposure influenced the expression of eNOS. HUVECs were
subjected to 90 min of HBO at 2.5 ATA, and eNOS protein expression was
analyzed at increasing time intervals using fluorescence intensity
staining as determined by CLSM. A single HBO exposure led to an
increase in eNOS protein production (Fig. 5). iNOS was not detectable
in HUVECs following HBO exposure (Fig. 5). LPS-mediated induction of
iNOS, but not eNOS, was observed in RAW 264.7 macrophages at 24 h in
control studies (Fig. 5). Figure 6
demonstrates the induction of eNOS over 24 h. eNOS expression was
significantly increased 2 h following the initiation of HBO and
continued to rise, with the greatest increase in expression occurring
between 6-8 h. Peak expression was noted at 8 h, and no decrease
in eNOS protein levels was noted at 24 h.

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Fig. 5.
CLSM analysis of endothelial cell nitric oxide synthase (eNOS) and
inducible nitric oxide synthase (iNOS) expression. Cells were
stimulated as follows and analyzed by CLSM as described in text (red
channel indicates propidium iodide staining of nucleic acid; green
channel indicates specific antibody staining). A: eNOS
expression in HUVEC controls. B: eNOS expression in HUVECs
exposed to hypoxia/hypoglycemia for 4 h followed by HBO treatment at
2.5 ATA for 1.5 h, followed by normoxia for 18.5 h. C: iNOS
expression in HUVEC controls. D: iNOS expression in HUVECs
exposed to hypoxia/hypoglycemia for 4 h followed by HBO treatment at
2.5 ATA for 1.5 h, followed by normoxia for 18.5 h. E: eNOS
expression in control RAW 264.7 cells. F: eNOS expression in
RAW 264.7 cells stimulated with LPS at 500 ng/ml for 4 h, followed by
20 h of control media. G: iNOS expression in control RAW 264.7 cells. H: iNOS expression in RAW 264.7 cells stimulated with
LPS at 500 ng/ml for 4 h, followed by 20 h of control media.
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Fig. 6.
Induction of eNOS protein as determined by CLSM analysis. HUVECs were
exposed to a single treatment of HBO at 2.5 ATA for 1.5 h, and eNOS
expression was analyzed at the indicated time points. Fluorescence
intensity is determined as described in MATERIALS AND
METHODS. Time = 0 indicates initiation of HBO treatment.
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eNOS total protein production was similarly upregulated following HBO
exposure as determined by Western blot analysis. eNOS protein was
induced by exposure of HUVECs to HBO (Fig.
7, compare lanes 1, 5, and
6). eNOS protein levels were increased 5.2-fold in the
hypoxia/hypoglycemia/HBO-treated cells vs. controls (Fig. 7, compare
lanes 1 and 5 with respective OD values 1.27 vs. 6.2). HUVECs treated with HBO alone produced a 3.8-fold greater amount of
eNOS protein at 24 h vs. the hypoxia/hypoglycemia/HBO-treated cells
(Fig. 7, compare lanes 5 and 6 with respective OD
values 23.5 vs. 6.2). Hypoxia-, hypoglycemia-, or
hypoxia/hypoglycemia-treated cells did not express detectable levels of
eNOS protein by Western blot analysis (Fig. 7, lanes 2,
3, and 4 with respective OD values 2.2, 2.3, and 2.1).
However, low levels of eNOS were detectable in HUVECs by CLSM.

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Fig. 7.
eNOS and iNOS expression in HUVECs. Lane 1, control media 24 h;
lane 2, hypoxia 4 h, followed by 20 h normoxia; lane 3,
hypoglycemia 4 h, followed by 20 h normoglycemia; lane 4,
hypoxia/hypoglycemia 4 h, followed by normoxia/normoglycemia 20 h;
lane 5, hypoxia/hypoglycemia 4 h, followed by HBO at 2.5 ATA
for 1.5 h, then normoxia/normoglycemia 18.5 h; lane 6, HBO at
2.5 ATA for 1.5 h, followed by 22.5 h normoxia. Arrow, position of
eNOS. * Position of nonspecific protein.
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Inhibition of eNOS with L-NAME attenuates HBO-mediated
downregulation of ICAM-1.
Previous studies have shown that organic NO donors can block the
cytokine-mediated induction of ICAM-1 (10). Therefore, we sought to
determine whether the induction of eNOS protein was related to the
observed downregulation of ICAM-1. HUVECs were subjected to
hypoxia/hypoglycemia as described above, and L-NAME was
added to the culture media on return of the cells to
normoxic/normoglycemic conditions. ICAM-1 expression was analyzed using
CLSM as described in MATERIALS AND METHODS.
L-NAME had minimal effect on ICAM-1 protein expression in
the normoxia/normoglycemia control group, with only a significant
1.96-fold increase noted at 100 µM L-NAME (P < 0.01; Fig. 8). This finding was in keeping
with the low levels of eNOS protein observed in the control cells.
Hypoxia/hypoglycemia-treated cells incubated with L-NAME at
a concentration of 25 µM did not increase ICAM-1 levels significantly
above those from hypoxia/hypoglycemia-only treated cells (P =
0.93). A small response was observed in hypoxia/hypoglycemia-treated cells incubated with L-NAME at concentrations of 50 and 100 µM, demonstrating a 1.2- and 1.4-fold induction of ICAM-1 vs.
hypoxia/hypoglycemia-treated cells not receiving L-NAME
(P < 0.01). The relative response of control and
hypoxia/hypoglycemia-treated cells to L-NAME was similar with respect to increases in ICAM-1 levels. The greatest effect of eNOS
inhibition was observed in the hypoxia/hypoglycemia/HBO-treated cells.
Addition of 25 µM L-NAME led to a relative threefold
increase in ICAM-1 levels in hypoxia/hypoglycemia/HBO-treated cells
(P < 0.01). Hypoxia/hypoglycemia-stimulated HUVECs
treated with HBO and L-NAME demonstrated a dose-dependent
increase in ICAM-1 protein expression of 3.0-, 4.4-, and 4.7-fold at
concentrations of 25, 50, and 100 µM, respectively (P < 0.01). HBO-only treated cells containing the highest levels of eNOS
demonstrated only a modest increase in ICAM-1 with L-NAME
inhibition (1.6-fold at 100 µM, P < 0.01). Taken together,
these findings suggest that the HBO downregulation of
hypoxia/hypoglycemia-induced ICAM-1 may be mediated, at least in part,
through increased expression of eNOS.

View larger version (33K):
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|
Fig. 8.
nitro-L-arginine methyl ester (L-NAME)
attenuates the HBO-mediated downregulation of ICAM-1 expression in
HUVECs. HUVECs were exposed to normoxia/normoglycemia control
media (open bars); hypoxia/hypoglycemia for 4 h followed by
normoxia/normoglycemia for 20 h (shaded bars); hypoxia/hypoglycemia for
4 h followed by HBO at 2.5 ATA for 1.5 h, then 18.5 h
normoxia/normoglycemia (crosshatched bars); or HBO at 2.5 ATA for 1.5 h
followed by normoxia for 22.5 h (solid bars). L-NAME was
added at the initiation of normoxic/normoglycemic conditions and
following HBO treatment. ICAM-1 protein expression was analyzed by CLSM
as described in MATERIALS AND METHODS.
|
|
 |
DISCUSSION |
The molecular mechanisms of HBO in treating I/R injury are poorly
understood. We have established an in vitro model of I/R injury to
study the effects of HBO on endothelial cell function. Our model of
endothelial cell I/R required both hypoxia and hypoglycemia for
induction of ICAM-1 protein production. The combination of hypoxia/hypoglycemia has been used previously for study of neuronal cell responses to ischemia in vitro (28). We are not aware of any previous reports describing the specific role of hypoxia and hypoglycemia as an inducer of ICAM-1 protein production. Previous studies utilizing BAECs and HUVECs have suggested that
hypoxia alone, or in conjunction with a reoxygenation period, were
insufficient to induce ICAM-1 (29, 49, 53). Similarly, we found that hypoxia or hypoxia/reoxygenation were insufficient to stimulate the
induction of ICAM-1 protein. Hypoxia has been shown to enhance the
production of ICAM-1 in combination with LPS (54). In these studies,
hypoxia promoted increased intracellular acidosis, resulting in an
increase of ICAM-1 expression via a proteasome-dependent pathway (54).
It is possible that the combination of hypoxia and hypoglycemia
exacerbates the hypoxia-induced intracellular acidosis or ATP
depletion, leading to induction of ICAM-1 (25).
The ability of HBO to suppress the production of ICAM-1 in our model
may help explain the beneficial mechanism of HBO in treating I/R
injury. The observation that HBO decreases adhesion of PMNs to the
endothelium and also overall PMN content within previously ischemic
tissue has been well described (50). Investigation of the ICAM-1 ligand
CD11/CD18 on PMNs suggested that there was no long-term alteration of
inducible CD11/CD18 function following HBO exposure (44, 45). One study
suggested a functional inhibition of PMN
2-integrins
using HBO, although this study did not determine the specific
interaction of CD11/CD18 and ICAM-1 (46). Our findings of a reduction
in non-HBO-treated PMN adhesion to HBO-treated endothelial cells
suggests that the underlying mechanism of HBO in treating I/R injury in
vivo may be mediated by effects on both the endothelium and PMNs. It is
possible that other endothelial cellular adhesion molecules are
similarly downregulated by HBO. Finally, the influence of HBO appears
to modulate the expression of endothelial cell proteins specifically;
as ICAM-1 production is decreased, eNOS is increased, and
-tubulin
levels remain constant.
Our finding that HBO was able to reverse the expression of ICAM-1 in
vitro differs from expected results suggested by previous studies of
hyperoxia and ICAM-1 expression. Others have shown that prolonged
hyperoxia is toxic to endothelial cells and lung tissue, inducing the
synthesis of ICAM-1 under both normobaric and prolonged hyperbaric
conditions (3, 36, 43). We similarly found that normobaric
O2 exposure following experimental I/R did not decrease
ICAM-1 production in our model. It is possible that the difference in
the ICAM-1 response to hyperoxia is due to the state of cellular
metabolism (I/R vs. resting) and the
PO2 level and duration of exposure.
These different conditions may result in varied quantitative and
qualitative production of ROS. Few studies have assessed the amount of
ROS-induced damage following HBO treatment in the setting of I/R. One
study demonstrated no significant long-term increase in cerebral lipid
peroxidation following transient global ischemia with
subsequent HBO treatment (24). Others have observed a transient,
nonsustained increase in lipid peroxidation following HBO at extreme
pressures of 5 ATA (26). Overall, however, HBO reduces tissue damage
using in vivo I/R injury models, an outcome less likely predicted in a
setting of oxidative stress and free radical damage (16, 41, 42, 50).
Media PO2 was not measured directly
in our model; however, an extensive previous study has documented the
PO2 dissolved in solution in vitro
under similar hyperbaric conditions compared with the
PO2 of human arterial blood from
subjects undergoing HBO exposure (47). In these previous experiments, the PO2 of saline reached 1,509 and
1,842 mmHg at 2.0 and 2.5 ATA, respectively, after 20 min, whereas the
approximate equivalent PO2 of human
arterial blood required exposure to 2.2 and 2.9 ATA, respectively (47).
Our results suggest that the threshold dose for inhibition of ICAM-1
expression in vitro exists between the
PO2 generated in solution between 2.0 and 2.5 ATA. Because current human treatment protocols include HBO
exposure at up to 3.0 ATA, the PO2
required to achieve a reduction in ICAM-1 production observed in vitro
should be obtainable in the clinical setting, especially in the
microcirculation of the myocardium. Although our findings suggest that
a threshold dose for inhibition of ICAM-1 expression should exist in
vivo, it is not possible to extrapolate our findings directly into
clinical treatment protocols without appropriate further in vivo studies.
Data demonstrating eNOS induction with HBO exposure are consistent with
the literature. Other reports have shown that increases in oxygen
tension at 1 ATA lead to an induction of eNOS mRNA and protein
production (1, 27). Increases in NO production were noted in bovine
cerebellum following HBO exposure by direct measurement; however,
direct analyses of NOS protein levels or subtype were not performed
(32). We have demonstrated that eNOS protein levels are upregulated
following HBO. Our findings of low constitutive eNOS levels have been
similarly described (21). The sustained increase in eNOS following a
single HBO exposure may explain the previous observation that HBO
treatment results in greater vessel diameter of the microvasculature
following I/R injury (50). Previous study of porcine coronary
resistance arterioles showed a similar phenomenon of hyperoxia-induced
NO production with vasodilation (15). The sustained HBO-mediated
increase in eNOS protein levels may also help explain the protective
effect of HBO treatment given before I/R injury in a rodent liver model
(5). Although we have not directly measured NO production, it is likely
that our results follow from an increase in NO. This is supported by
experiments demonstrating that L-NAME competitive
inhibition of NOS resulted in enhanced production of ICAM-1. The lack
of iNOS expression noted in our model system by CLSM suggests that eNOS
is primarily responsible for the observed results; however, we have not
characterized the enzymatic requirements of NOS for calcium dependence.
The inability of L-NAME to completely reverse the
HBO-mediated suppression of ICAM-1 allows for the existence of other
undefined pathways for HBO regulation of ICAM-1 expression.
The regulation of cellular adhesion molecules by NO has been well
described (10, 20, 22). Recent studies suggest that the NO-mediated
downregulation of ICAM-1 may occur via a reduction in activated
NF-
B, which may involve increased production of I
B-
(21, 31,
40). One hypothesis to explain our data is that HBO reduces the
activation of transcription factors such as NF-
B involved in the
transcriptional regulation of ICAM-1 through a NO-mediated mechanism
(18). NO may combine with other ROS formed during states of oxidative
stress to prevent the ROS from initiating other stress responses. Other
studies have shown that NF-
B may be activated in endothelial cells
following hypoxia/reoxygenation and also under states of oxidative
stress induced by H2O2 or chronic O2 exposure at 1 ATA (7, 31, 33-35). If the effect of
HBO is mediated through NO and NF-
B, then other similarly regulated adhesion molecules may be downregulated following HBO exposure.
In summary, we have shown that HBO effectively downregulates ICAM-1
expression in an in vitro model of endothelial cell I/R injury. The
corresponding increase in eNOS and inhibitor studies suggest that the
mechanism of downregulation may be mediated in part by NO. These
findings further define the mechanisms behind the observed paradoxical
benefit of HBO-induced hyperoxia in the setting of I/R injury.
Verification of these findings in vivo would further support the
clinical use of HBO as a therapeutic intervention in I/R disease states.
 |
ACKNOWLEDGEMENTS |
We thank S. P. Colgan for helpful discussions and review of the
manuscript. We also thank M. Morrisey, D. Orlow, and A. Campbell for
technical expertise.
 |
FOOTNOTES |
This work was supported by an institutional award from the Department
of Emergency Medicine, Harvard University.
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: J. A. Buras,
Dept. of Emergency Medicine, Brigham and Women's Hospital, 75 Francis
St., Boston, MA 02115 (E-mail: jburas{at}massmed.org).
Received 30 October 1998; accepted in final form 24 August 1999.
 |
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