Hypoxia enhances induction of endothelial ICAM-1: role for
metabolic acidosis and proteasomes
Gregor
Zünd1,
Shoichi
Uezono2,
Gregory L.
Stahl1,
Andrea L.
Dzus1,
Francis X.
McGowan2,
Paul R.
Hickey2, and
Sean P.
Colgan1
1 Center for Experimental
Therapeutics and Reperfusion Injury, Department of Anesthesia,
Brigham and Women's Hospital, and
2 Department of Anesthesia,
Children's Hospital and Harvard Medical School, Boston,
Massachusetts 02115
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ABSTRACT |
Intercellular
adhesion molecule 1 (ICAM-1) is an important molecule in promotion of
polymorphonuclear neutrophil transendothelial migration during
inflammation. Coincident with many inflammatory diseases is tissue
hypoxia. Thus we hypothesized that combinations of hypoxia and
inflammatory stimuli may differentially regulate expression of
endothelial ICAM-1. Human endothelial cells were exposed to hypoxia in
the presence or absence of added lipopolysaccharide (LPS) and examined
for expression of functional ICAM-1. Although hypoxia alone did not
induce ICAM-1, the combination of LPS and hypoxia enhanced (3 ± 0.4-fold over normoxia) ICAM-1 expression. Combinations of hypoxia and
LPS significantly increased lymphocyte binding, and such increases were
inhibited by addition of anti-ICAM-1 antibodies or antisense
oligonucleotides. Hypoxic endothelia showed a >10-fold increase in
sensitivity to inhibitors of proteasome activation, and combinations of
hypoxia and LPS enhanced proteasome-dependent cytoplasmic-to-nuclear
localization of the nuclear transcription factor-
B p65
(Rel A) subunit. Such proteasome
activation correlated with hypoxia-evoked decreases in both
extracellular and intracellular pH. We conclude from these studies that
endothelial hypoxia provides a novel, proteasome-dependent stimulus for
ICAM-1 induction.
leukocyte; endothelium; inflammation; sepsis; intercellular
adhesion molecule 1
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INTRODUCTION |
IN SETTINGS OF inflammation, tissue hypoxia often
occurs. For instance, the hallmark of septic shock is vascular
hypotension, leading to widespread cell and tissue hypoxia (reviewed in
Ref. 24, 33). Endothelial cells that line blood vessels bear activable receptors for many inflammatory stimuli, including
lipopolysaccharides (LPS). Because endothelia are anatomically
positioned at the interface of the blood and tissue exchange,
endothelia are especially influenced by conditions of hypoxemia (22). A
number of studies have examined the influence of hypoxia on endothelial
cell structure/function (recently reviewed in Ref. 30). Hypoxia-induced
changes are complex and include changes in energy metabolism,
alterations in gene expression, and induction of specific cell surface
proteins (reviewed in Ref. 30).
Tissue injury resulting from reperfusion of hypoxic tissue has been
shown to be mediated, at least in part, by recruitment of leukocytes
(32). Leukocyte recruitment across the intact endothelium occurs
through a concerted series of adhesion and deadhesion events involving
a number of cell surface adhesion proteins (28). Previous studies have
demonstrated that short-term hypoxia/anoxia induces increased adhesion
of leukocytes to vascular endothelial cells, and such adhesion can be
blocked, at least in part, by antibodies directed at intercellular
adhesion molecule 1 (ICAM-1, CD54) (26, 35), a 90- to 110-kDa
glycoprotein found on the surface of endothelial cells that mediates
the firm adherence of leukocytes to the endothelium (28). ICAM-1
expression is readily induced by stimulation of cell surface receptors
for a number of inflammatory mediators, including LPS (28), and is regulated by the nuclear transcription factor (NF)-
B, a pleiotropic activator of a number of genes related to inflammation (9). Previous
studies have defined a role for the ubiquitin-mediated proteasome
pathway in NF-
B induction, specifically through degradation of p50
and inhibitory (I)-
B (20). At present, it is unclear whether molecules such as ICAM-1 are differentially regulated by a
combination of hypoxia and mediators found within the microenvironment of inflamed tissue.
Thus we hypothesized that hypoxia may augment cell surface expression
of leukocyte adhesion receptors, such as ICAM-1. We report here that,
whereas hypoxia alone does not induce ICAM-1 expression over baseline,
the combination of hypoxia and LPS enhances expression of ICAM-1. Such
enhancement was not universal for other extracellular membrane proteins
and correlated with hypoxia-elicited increases in nuclear content of
the NF-
B p65 subunit. We speculate that in the setting of hypoxia,
inflammatory mediators such as LPS may perpetuate expression of
molecules important in leukocyte trafficking, such as ICAM-1.
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MATERIALS AND METHODS |
Cell culture.
Human umbilical vein endothelial cells (HUVECs) were obtained
and harvested as described elsewhere (13) using 0.1% collagenase (Worthington Biochemical, Freehold, NJ). Human pulmonary microvascular endothelial cells (HPMVECs) were purchased from Clonetics (San Diego,
CA) as first passage cells and, when used, were cultured under similar
conditions as for HUVECs. Endothelial monolayers were established,
maintained, and subcultured with Dulbecco's modified Eagle's medium
(GIBCO, Grand Island, NY) containing 10% heat-inactivated fetal calf
serum, glucose, pyruvate, glutamine, penicillin, and streptomycin (13).
Confluent endothelial monolayers exhibited typical cobblestone
appearance and uptake of acetylated low-density
lipoprotein (Biomedical Technology, Stoughton, MA; data
not shown). Where indicated, endothelial monolayers were exposed to LPS
(from Escherichia coli; Sigma, St. Louis, MO) at indicated
concentrations.
Confluent endothelial monolayers were exposed to hypoxia as follows:
growth medium was replaced with fresh medium equilibrated with hypoxic
gas mixture, and cells were placed in a hypoxic chamber (Coy Laboratory
Products, Ann Arbor, MI). This hypoxic chamber consists of an airtight
glove box, with the atmosphere continuously monitored by an oxygen
analyzer interfaced with oxygen and nitrogen flow adapters. Oxygen
concentrations are as indicated, with the balance made up of nitrogen,
carbon dioxide (ambient 5%
CO2), and water vapor from the
humidified chamber. Media
PO2 and pH were measured in a Ciba-Corning 238 blood gas analyzer (Chiron Diagnostics, Norwood, MA).
Cell surface immunoassay.
ICAM-1 cell surface expression was quantified with a cell surface
enzyme-linked immunosorbent assay (ELISA), as previously described (7).
Endothelial cells were grown and assayed for antibody binding following
exposure to normoxia or hypoxia in the presence or absence of LPS, as
indicated. Endothelia were lightly fixed with paraformaldehyde (1%
wt /vol in phosphate-buffered saline) to preserve cell surface
protein. Cells were washed with Hanks' balanced salt solution (HBSS;
Sigma) and blocked with medium for 30 min at 4°C. Anti-ICAM-1
monoclonal antibody (MAb) [clone P2A4 (12), obtained from the
Developmental Studies Hybridoma Bank (Iowa City, IA) and used as
undiluted cell culture supernatant] or R6.5 (27) [a kind
gift from Dr. Robert Rothlein, Boehringer Ingelheim Pharmaceuticals
(Ridgefield, CT), and used as purified MAb at 20 µg/ml] was
added to fixed cells and allowed to incubate for 2 h at 4°C. Where
indicated, MAb to major histocompatibility complex (MHC) class I (5)
(clone W6/32, obtained from the American Type Culture Collection and
used as 1:100 diluted ascitic fluid) was used as a control. After a
wash with HBSS, a peroxidase-conjugated sheep anti-mouse secondary
antibody (Cappel, West Chester, PA) was added. Secondary antibody
(1:1,000 final dilution) was diluted in medium containing 10% fetal
bovine serum (FBS). After washing, plates were developed by addition of
peroxidase substrate
[2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 1 mM final concentration, Sigma] and read on a microtiter plate
spectrophotometer at 405 nm (Molecular Devices). Controls consisted of
medium only and secondary antibody only. Optical density data at
405 nm (OD405,
background subtracted) are presented as means ± SE.
Immunoprecipitation of biotinylated endothelial membranes.
HUVECs were grown to confluence on six-well plates, exposed to
experimental conditions, and washed with HBSS. Extracellular cell
surface proteins were then labeled with biotin (ImmunoPure sulfo-NHS-biotin; 1 mM; Pierce, Rockford, IL) as described previously (18). Unbound biotin was quenched with
NH4Cl (50 mM) in HBSS. Labeled
HUVECs were lysed [in buffer containing 150 mM NaCl, 25 mM
tris(hydroxymethyl)aminomethane (Tris), 1 mM
MgCl2, 1% Triton X-100, 1%
Nonidet P-40, 5 mM EDTA, 5 µg/ml chymostatin, 2 µg/ml aprotinin,
and 1.25 mM phenylmethylsulfonyl fluoride (PMSF), all from
Sigma]. Cell debris was removed by centrifugation (10,000 g, 5 min). HUVEC lysates were
precleared with 50 µl preequilibrated protein G-Sepharose (Pharmacia,
Uppsala, Sweden) for 2 h. For immunoprecipitation of ICAM-1, primary
antibody (500 µl of P2A4 cell culture supernatant) was added
(immunoprecipitated for 2 h), followed by addition of 50 µl of
preequilibrated protein-G Sepharose (immunoprecipitated overnight on an
end-over-end rotator). Washed immunoprecipitates were
boiled in nonreducing sample buffer [2.5% sodium dodecyl sulfate
(SDS), 0.38 M Tris (pH 6.8), 20% glycerol, and 0.1% bromphenol
blue], separated by SDS-polyacrylamide gel electrophoresis (PAGE;
10% linear gel) under nonreducing conditions and transferred to
nitrocellulose using standard protocols. Biotinylated proteins were
labeled with streptavidin-peroxidase and visualized by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL). Resulting
ICAM-1 bands were quantified from scanned images using National
Institutes of Health (NIH) Image software (Bethesda, MD).
Lymphocyte adhesion assay.
Peripheral blood mononuclear cells (PBMC) were obtained by
centrifugation through Ficoll-Hypaque (1.077). Lymphocyte-enriched fractions were obtained by incubating PBMC in 10% FBS-RPMI 1640 on
tissue culture-treated plastic 24-well plates for 1 h at 22°C and
collecting nonadherent cells. For studies of adhesion, enriched lymphocyte populations (>95%) were labeled for 30 min at 37°C with
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM, 5 µM final concentration; Calbiochem, San Diego, CA)
and washed three times in HBSS. Labeled lymphocytes (1 × 105/monolayer) were added to
washed normoxic or hypoxic monolayers, plates were centrifuged at 150 g for 4 min to uniformly settle lymphocytes, and adhesion was allowed for 10 min at 37°C.
Monolayers were gently washed three times with HBSS, and fluorescence
intensity (485-nm excitation, 530-nm emission) was measured on a
fluorescent plate reader (Cytofluor 2300, Millipore, Bedford, MA).
Adherent cell numbers were determined from standard curves generated by serial dilution of known lymphocyte numbers diluted in
HBSS. All data were normalized for background fluorescence
by subtraction of fluorescence intensity of samples collected from
monolayers incubated in buffer only, without addition of lymphocytes.
In subsets of experiments, treated HUVECs were preincubated with MAb
directed against domains specific to the lymphocyte
functions-associated antigen (LFA)-1 (RR1/1, 20 µg/ml; Ref. 29) and
Mac-1 (R6.5, 20 µg/ml; Ref. 11) of ICAM-1 or MHC class I (W6/32,
binding control, 20 µg/ml) before lymphocyte adhesion. Anti-ICAM-1
MAb were a kind gift from Dr. R. Rothlein.
ICAM-1 antisense oligonucleotide treatment of HUVECs.
Antisense oligonucleotide treatment of confluent HUVECs was done
exactly as described previously using the oligonucleotides ISIS-1939
(targets the mRNA 3' untranslated region, sequence
CCCCCACCACTTCCCCTCTC) or ISIS-3067 (targets the mRNA 5'
untranslated region, sequence TCTGAGTAGCAGAGGAGCTC) (6).
Oligonucleotides were a kind gift from Dr. C. F. Bennett, ISIS
Pharmaceuticals (Carlsbad, CA). Confluent HUVECs were washed in
serum-free Opti-MEM (GIBCO) and then in Opti-MEM
containing 10 µg/ml
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (Lipofectin solution at 10 µg/ml, GIBCO), followed by the
indicated concentrations of filter-sterilized oligonucleotides. Cells
were incubated for 4 h at 37°C and then replaced with normal growth
medium in the presence or absence of LPS and incubated in normoxia or
hypoxia as described above. ICAM-1 expression or MHC class I expression
(control) was quantified with the methods described in
Cell surface immunoassay.
Methods for lymphocyte adhesion to such oligonucleotide-treated cells
were carried exactly as described in Lymphocyte
adhesion assay.
Proteasome inhibition experiments.
Confluent HUVEC monolayers grown on 96-well plates were equilibrated to
hypoxia or normoxia for 1 h in the presence of the proteasome
inhibitors
N-acetyl-Leu-Leu-methioninal (ALLM,
calpain inhibitor II, purchased from Boehringer Mannheim, Indianapolis, IN) or N-acetyl-Leu-Leu-norleucinal
(ALLN, calpain inhibitor I, purchased from Boehringer Mannheim) at
indicated concentrations before addition of LPS. After 18 h in hypoxia
or normoxia, cell surface expression of ICAM-1 and MHC class I was
examined by cell surface ELISA as described in Cell
surface immunoassay.
Measurement of intracellular pH.
Intracellular pH was measured as described previously (1) using HUVECs
grown to confluence on 96-well plates. Briefly, endothelia were exposed
to combinations of hypoxia or normoxia with or without addition of LPS
(range was 1-100 ng/ml) for 18 h. Within the setting of normoxia
or hypoxia, monolayers were washed with preequilibrated HBSS and loaded
with BCECF-AM (10 µM final concentration, Calbiochem) for 30 min at
37°C. Loaded monolayers were washed three times in preequilibrated
HBSS, and fluorescence intensity (485-nm excitation, 530-nm emission)
was immediately measured on a fluorescent plate reader (Cytofluor 2300, Millipore). Similarly loaded cells were also measured at a reference
wavelength (360-nm excitation, 530-nm emission), and the fluorescence
ratio of 485:360 was used to determine intracellular pH. Intracellular
pH was calibrated using nigericin (10 µg/ml) to equilibrate
intracellular and extracellular pH (1), and this calibration was linear
in the pH range of 6.7-7.5. In subsets of experiments, pH of
extracellular medium was adjusted with HCl in the range of 6.8-7.3
before addition to HUVEC monolayers.
NF-
B p65 Western blotting.
After experimental treatment of HUVECs, nuclear extracts were prepared
as described previously (34). Confluent monolayers of HUVECs in 100-mm
petri dishes were washed in ice-cold phosphate-buffered saline and
lysed by incubation in 500 µl of buffer
A [in mM: 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), pH 8.0, 1.5 MgCl2, 10 KCl, 0.5 dithiothreitol
(DTT), 200 sucrose, and 0.5 PMSF and 1 µg/ml of both leupeptin and
aprotinin and 0.5% Nonidet P-40] for 5 min at 4°C. The crude
nuclei released by lysis were collected by microcentrifugation (15 s).
Nuclei were rinsed once in buffer A
and resuspended in 100 µl of buffer
C [in mM: 20 HEPES (pH 7.9), 1.5 MgCl2, 420 NaCl, 0.2 EDTA, 0.5 PMSF, and 1.0 DTT and 1 µg/ml of both leupeptin and aprotinin].
Nuclei were incubated on a rocking platform at 4°C for 30 min and
clarified by microcentrifugation for 5 min. Proteins were measured
(detergent compatible protein assay, Bio-Rad, Hercules, CA). Samples
(25 µg/lane, as indicated) of HUVEC lysates were separated by
nonreducing SDS-PAGE, transferred to nitrocellulose, and blocked
overnight in blocking buffer (250 mM NaCl, 0.02% Tween 20, 5% goat
serum, and 3% bovine serum albumin). Primary antibody (rabbit
polyclonal specific for p65 subunit of NF-
B; Biomol Research
Laboratories, Plymouth Meeting, PA) was added for 3 h, blots were
washed, and species-matched peroxidase-conjugated secondary antibody
was added. Labeled bands from washed blots were detected by ECL.
Resulting 65-kDa NF-
B bands were quantified from scanned images
using NIH Image software. Such 65-kDa bands were specific for NF-
B,
since preincubation of rabbit polyclonal antibody with standard p65 antigen (provided by Biomol as a control) resulted in a diminution of
the 65-kDa band by more than 70% (data not shown).
Data presentation.
Data were compared by analysis of variance (ANOVA) or by Student's
t-test. Values are expressed as means ± SE of n experiments.
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RESULTS |
Hypoxia enhances LPS-induced ICAM-1 expression.
Endothelial monolayers tolerated exposure to hypoxia well (ambient
O2 as low as 1%, for up to 24 h).
No changes in morphology were observed, and no evidence of cell death
was apparent (based on soluble lactate dehydrogenase
measurements from supernatants, data not shown). We first examined the
induction of ICAM-1 surface expression induced by LPS under conditions
of endothelial exposure to hypoxia or to normoxia. In Fig.
1, the dose responses for LPS induction of ICAM-1 on HUVEC and on HPMVEC by cell surface ELISA are
shown. As can be seen, unstimulated expression of ICAM-1 was low, and
hypoxia alone (1% ambient O2)
did not induce cell surface ICAM-1 on either HUVEC or HPMVEC
(P = not significant compared with
normoxic control). However, in the presence of LPS, more than twofold
augmentation in ICAM-1 was evident on both HUVEC (two-way ANOVA,
P < 0.025 compared with normoxia)
and HPMVEC (two-way ANOVA, P < 0.01 compared with normoxia).

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Fig. 1.
Induction of endothelial intercellular adhesion molecule 1 (ICAM-1) by
lipopolysaccharide (LPS) and hypoxia. Confluent human umbilical vein
endothelial cell (HUVEC; A) or human
pulmonary microvascular endothelial cell
(B) monolayers were exposed to hypoxia (ambient O2 of 1%, )
or normoxia (ambient O2 of 21%, ) in the presence or absence of indicated concentrations of LPS for
18 h. Monolayers were washed and lightly fixed (1% paraformaldehyde), followed by addition of monoclonal antibody (MAb) specific for ICAM-1
(clone P2A4), and analyzed for specific expression by enzyme-linked immunosorbent assay (ELISA) as described in MATERIALS
AND METHODS. Results of optical density at 405 nm
(OD405, background subtracted) are
means ± SE of 12 monolayers in each condition from at least 3 experiments.
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Figure 2 demonstrates the time course and
O2 dependence of hypoxia-elicited
increases in ICAM-1 surface expression. Augmentation of ICAM-1
induction by hypoxia was apparent as early as 8 h and as late as 36 h
after addition of LPS (100 ng/ml, two-way ANOVA, P < 0.025 compared with normoxia
with LPS). In addition, graded decreases in
O2 concentrations increased
LPS-stimulated ICAM-1 expression (Fig.
2B, one-way ANOVA,
P < 0.025).

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Fig. 2.
Time and O2 dependence of
hypoxia-elicited increase in ICAM-1 expression. Confluent HUVEC
monolayers were exposed to hypoxia ( ) or normoxia ( ) in the
presence of LPS (100 ng/ml) for indicated periods of time
(A) or at indicated
O2 concentrations
(B). Monolayers were washed and
lightly fixed (1% paraformaldehyde), followed by addition of MAb
specific for ICAM-1 (clone P2A4), and analyzed for specific expression
by ELISA as described in MATERIALS AND METHODS. OD405
(background subtracted) results are means ± SE of 6-8
monolayers in each condition from at least 3 experiments.
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Finally, to demonstrate that our findings were not antibody dependent,
another MAb specific for ICAM-1 (clone R6.5) was used to examine such
hypoxia-elicited increases. Similar to our findings with clone P2A4,
ICAM-1 expression using MAb R6.5 resulted in increased LPS-induced
expression under conditions of hypoxia
(OD405 of 0.12 ± 0.01 and 0.33 ± 0.02 for normoxia and hypoxia, respectively, n = 3, P < 0.01). Thus, whereas hypoxia
alone does not induce ICAM-1 expression, the combination of LPS and
hypoxia quantitatively augments ICAM-1 induction.
Hypoxia increases immunoprecipitable ICAM-1.
To confirm the findings of our whole cell ELISA, we determined the
influence of hypoxia on immunoprecipitable ICAM-1 from biotinylated
HUVEC monolayers. As shown in Fig. 3, HUVEC
exposure to LPS (5 or 50 ng/ml, 18 h) under normoxic conditions
revealed immunoprecipitation of an ~95-kDa biotinylated protein
consistent with endothelial ICAM-1. Some expression was apparent from
control samples not exposed to LPS, consistent with our ELISA findings (Figs. 1 and 2). Confluent HUVECs exposed to a combination of LPS and
hypoxia revealed an immunoprecipitable band of increased density over
normoxia at LPS concentrations of 5 and 50 ng/ml (Fig. 3). Consistent
with our ELISA findings, no observable increase in ICAM-1 was apparent
with hypoxia alone (Fig. 3). Densitometric analysis of these bands
(Fig. 3B) revealed a 330% increase
at 5 ng/ml LPS and an 83% increase at 50 ng/ml LPS under conditions of
hypoxia (Fig. 3B), confirming our
ELISA results.

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Fig. 3.
Immunoprecipitation of ICAM-1 from cell surface biotinylated HUVECs
induced by LPS in normoxia and hypoxia. Confluent HUVEC monolayers were
exposed to medium alone (lanes 2 and
5) or medium containing 5 ng/ml LPS
(lanes 3 and
6) or 50 ng/ml LPS
(lanes 4 and
7) under conditions of normoxia
(ambient O2 of 21%,
lanes 2-4) or hypoxia (ambient
O2 of 1%, lanes
5-7) for 18 h. Cells were cooled to 4°C, and
cell surface proteins were biotinylated, followed by
immunoprecipitation of ICAM-1 (with the exception of
lane 1, negative control, no primary
antibody) as described in MATERIALS AND
METHODS. Blots were probed with streptavidin peroxidase
and developed by enhanced chemiluminescence (ECL). A: resulting blots from
immunoprecipitation of normoxic and hypoxic HUVEC with and without
addition of LPS. MW, molecular weight. B: densitometry tracings from
immunoprecipitated bands shown in A;
representative of 1 of 4 experiments.
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Hypoxia enhances ICAM-1-dependent lymphocyte adhesion.
We next examined whether hypoxic conditions demonstrated here to
enhance ICAM-1 expression on HUVECs were apparent at the functional
level. First, as shown in Fig. 4, a
significant increase in lymphocyte binding was apparent in HUVECs
exposed to a combination of hypoxia and LPS (100 ng/ml) over that of
exposure to LPS alone [see conditions of the binding control (MAb
W6/32), 2.1 ± 0.4-fold increase with hypoxia,
P < 0.001]. Lymphocyte
adhesion to LPS-preexposed HUVECs was significantly diminished by
addition of anti-ICAM-1 MAb directed against the LFA-1-dependent ICAM-1
site (29) (MAb was RR1.1, adhesion decreased by 79 ± 9% and 70 ± 7% for normoxia and hypoxia in the presence of LPS compared with
binding control W6/32, respectively, P < 0.001 for both) but not the Mac-1-dependent ICAM-1 site (11) (MAb
was R6.5, adhesion decreased by 11 ± 7% and 13 ± 5%
for normoxia and hypoxia in the presence of LPS compared with binding
control W6/32, respectively, P = not
significant for both), indicating that a primary contribution of
lymphocyte binding is LFA-1 binding through ICAM-1. However, even in
the presence of saturating concentrations of MAb RR1.1 (20 µg/ml, determined by dilution and cell surface ELISA, data not shown), a
nearly twofold increase in lymphocyte binding was apparent in HUVECs
exposed to both LPS and hypoxia (Fig. 4). No significant differences
existed in lymphocyte binding to HUVECs exposed to hypoxia or normoxia
alone (Fig. 4, P = not significant for
all).

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Fig. 4.
Influence of anti-ICAM-1 MAb on lymphocyte adhesion following
endothelial exposure to LPS and hypoxia. Confluent HUVEC
monolayers were exposed to hypoxia alone (shaded bars), normoxia alone
(hatched bars), hypoxia with LPS (500 ng/ml, open bars), or normoxia
with LPS (500 ng/ml, solid bars) for 18 h. Monolayers were preincubated with anti-ICAM-1 MAb R6.5 (20 µg/ml), anti-ICAM-1 MAb RR1/1 (20 µg/ml), or anti-major histocompatibility complex (MHC) class I MAb
W6/32 (20 µg/ml) and analyzed for lymphocyte adhesion as described in
MATERIALS AND METHODS. Results of
percent lymphocyte bound are means ± SE of 12 monolayers in each
condition from 3 experiments.
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As a second approach to examining the functional role of
hypoxia-elicited ICAM-1, HUVECs were pretreated with antisense
oligonucleotides directed against ICAM-1 mRNA and examined for
lymphocyte binding (Fig. 5). Preexposure of
HUVECs to the oligonucleotides ISIS-1939 (Fig.
5A) or ISIS-3067 (Fig.
5B) resulted in a dose-dependent decrease in LPS-stimulated ICAM-1 expression under conditions of either
hypoxia (Fig. 5, one-way ANOVA, P < 0.01 for both) or normoxia (Fig. 5, one-way ANOVA,
P < 0.01 for both). As a control for
these antisense oligonucleotides, and as shown in Fig. 5, MHC class I
expression was not influenced by HUVEC preexposure to ICAM-1 antisense
oligonucleotides (P = not significant
for all). Finally, as a functional assay, pretreatment of HUVECs with ISIS-1939 (5 µM final concentration) or ISIS-3067 (5 µM final concentration) antisense oligonucleotides inhibited lymphocyte binding
to LPS-stimulated HUVECs exposed to hypoxia (Fig.
5C, 69 ± 6% and 70 ± 5%
decrease compared with control for oligonucleotides ISIS-1939 and
ISIS-3067, respectively, both P < 0.01) or to normoxia (Fig. 5C, 60 ± 7% and 55 ± 5% decrease compared with control for oligonucleotides ISIS-1939 and ISIS-3067, respectively, both
P < 0.01). These data indicate that
a primary component of hypoxia-elicited increases in lymphocyte binding
are attributable to HUVEC expression of ICAM-1.

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Fig. 5.
Influence of ICAM-1 mRNA antisense oligonucleotides on lymphocyte
adhesion following endothelial exposure to LPS and hypoxia. Confluent
HUVEC monolayers were pretreated with indicated concentrations of the
ICAM-1 antisense oligonucleotides (oligo) ISIS-1939
(A) or ISIS-3067
(B) as described in
MATERIALS AND METHODS. Monolayers were
then exposed to exposed to hypoxia (filled symbols) or normoxia (open
symbols) in the presence of LPS (100 ng/ml) for 18 h. Monolayers were
washed and lightly fixed (1% paraformaldehyde), followed by addition
of MAb specific for ICAM-1 (circles, clone P2A4) or MHC class I
(squares, clone W6/32), and analyzed for specific expression by ELISA.
C: lymphocyte adhesion from monolayers
exposed to ISIS-1939 (5 µM) or ISIS-3067 (5 µM) followed by LPS or
no LPS. AS, antisense. Results are means ± SE of 6-10
monolayers in each condition from at least 3 different experiments.
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Evidence for increased proteasome activation in hypoxic endothelia.
The NF-
B/Rel A family of transcription factors are
important for induction and expression of a number of cellular gene
products, including endothelial ICAM-1 (8). In quiescent endothelia, NF-
B is complexed to the inhibitor I-
B. When activation occurs, proteasome-dependent I-
B degradation (9) allows
cytoplasmic-to-nuclear localization of NF-
B. Thus, as a measure of
proteasome activation, we examined cytoplasm-to-nuclear localization of
NF-
B (p65 subunit) in hypoxic and normoxic endothelia. As shown in
Fig. 6, and similar to our findings with
ICAM-1 surface expression (Fig. 1), endothelial exposure to hypoxia
alone failed to induce p65 nuclear localization. However, in the
presence of LPS (concentration range of 1-100 ng/ml), a four- to
sixfold increase in nuclear-to-cytoplasmic p65 ratio was observed in
cells exposed to a combination of hypoxia and LPS (ANOVA,
P < 0.05 compared with normoxia).
Under these conditions, no observable differences between hypoxia or
normoxia were observed in the cytoplasmic fraction of p65 (Fig.
6).

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Fig. 6.
Western blot analysis of HUVEC nuclear transcription factor (NF)- B
p65 subunit in endothelial cytoplasmic and nuclear fractions. Confluent
HUVEC monolayers were exposed to medium or medium containing LPS (50 ng/ml) under conditions of normoxia (ambient
O2 of 21%) or hypoxia (ambient
O2 of 1%) for 4 h. Cells were
cooled to 4°C, and cytoplasmic (cyto) and nuclear fractions were
isolated as described in MATERIALS AND
METHODS. Samples (25 µg/lane) were separated by
SDS-polyacrylamide gel electrophoresis under nonreducing conditions,
and blots were probed with polyclonal rabbit NF- B p65, labeled with
peroxidase-conjugated secondary antibody, and developed by ECL.
A: representative blot from
cytoplasmic and nuclear fractions in normoxia or hypoxia in the
presence of indicated concentrations of LPS (in ng/ml).
B: demonstration of ratio of nuclear
to cytoplasmic p65 from scanning densitometry pooled from 3 separate
experiments. Hatched bars, hypoxia; solid bars, normoxia.
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Recent studies utilizing specific peptidoaldehyde inhibitors revealed
that intracellular proteasomes are required for transcriptional induction of vascular leukocyte adhesion molecules, including ICAM-1
(25). Because hypoxia increases LPS-induced ICAM-1 induction and
cytoplasmic-to-nuclear localization of p65, we used the proteasome inhibitors ALLN and ALLM to examine LPS-induced ICAM-1 expression under
growth conditions of hypoxia and normoxia. As shown in Fig. 7A,
exposure of endothelia to a combination of LPS (50 ng/ml) and ALLN
decreased surface expression of ICAM-1 in both hypoxic and normoxic
endothelia in a concentration-dependent manner. Hypoxic endothelia,
however, were significantly (P < 0.01) more sensitive to ALLN inhibition [50% effective
concentration (EC50) of 8 ± 1.2 µM] than their normoxic counterparts
(EC50 of 95 ± 3.7 µM). As
can be seen, ALLN concentrations of <3 µM did not influence ICAM-1
expression in normoxic cells and effectively normalized hypoxic cell
responses. Similar to previous findings (25), and as shown in Fig.
7B, ALLM did not significantly inhibit
ICAM-1 expression. No significant differences in ICAM-1 expression were observed in unstimulated (i.e., no LPS) hypoxic or normoxic endothelia exposed to either ALLN or ALLM, and neither ALLM nor ALLN influenced surface expression of MHC class I (data not shown). Finally, and as
shown in Fig. 7C, endothelial exposure
to ALLN (100 µM) in combination with LPS (50 ng/ml) significantly
diminished nuclear content of p65 in both hypoxic and normoxic
endothelia. Such data indicate that hypoxia enhances LPS-stimulated
nuclear accumulation of NF-
B and imply a hypoxia-evoked increase in
proteasome activation.

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|
Fig. 7.
Influence of proteasome inhibitors on hypoxia-elicited augmentation in
HUVEC ICAM-1 expression. Confluent HUVEC monolayers were
preequilibrated to hypoxia (ambient
O2 of 1%, filled symbols) or
normoxia (ambient O2 of 21%, open
symbols) for 1 h in the presence of indicated concentrations of
N-acetyl-Leu-Leu-norleucinal (ALLN, calpain inhibitor I, A) or
N-acetyl-Leu-Leu-methioninal (ALLM, calpain inhibitor II, B). Monolayers
were then stimulated for 18 h with LPS (50 ng/ml) and analyzed for
specific ICAM-1 expression by ELISA.
OD405 (background subtracted)
results are means ± SE of 8-10 monolayers in each condition
from at least 3 experiments. C:
influence of ALLN (100 µM) on LPS-stimulated (50 ng/ml) cytoplasmic localization of p65 by Western blot from endothelia exposed to hypoxia
or normoxia.
|
|
Role for metabolic acidosis in hypoxia-elicited increases in ICAM-1.
A number of recent in vivo and ex vivo studies indicate a role for
acidosis in activation of the ubiquitin-proteasome pathway (2, 15, 19).
Thus we determined whether cell culture acidosis, measured as pH of
extracellular medium and intracellular pH, was associated with our
observed increase in ICAM-1 expression. As shown in Fig.
8A,
24-h endothelial exposure to hypoxia alone resulted in a small decrease
in medium pH (P < 0.05). The
combination of hypoxia and LPS enhanced this decrease in medium pH
(ANOVA, P < 0.01) in a
concentration-dependent manner. A small but significant fall in medium
pH was associated with the highest LPS concentration (100 ng/ml) in
normoxia (P < 0.05).
Similar to extracellular medium, parallel decreases in intracellular pH
were observed with endothelial exposure to hypoxia and LPS (Fig.
8B, ANOVA,
P < 0.025).

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|
Fig. 8.
Hypoxia-elicited enhancement is dependent on medium acidification.
A: confluent HUVEC monolayers were
exposed to hypoxia ( ) or normoxia ( ) in the presence of indicated
concentrations of LPS for 18 h. Medium was collected, and pH was
immediately measured. pH results are means ± SE of 4 monolayers in
each condition from at least 3 experiments.
B: intracellular pH from similarly
exposed cells. C: confluent HUVEC
monolayers were exposed to combination of hypoxia and LPS (100 ng/ml)
in the medium with indicated concentrations of bicarbonate for 18 h and
analyzed for medium acidification ( ) and specific expression of
ICAM-1 by ELISA ( ). Results are means ± SE of 4 monolayers in
each condition from at least 3 experiments.
|
|
Using our cell surface ELISA assay, we next determined whether medium
acidification by the hypoxia-LPS combination (100 ng/ml) was associated
with enhanced ICAM-1 expression. As depicted in Fig.
8C, an increase in bicarbonate
concentrations in tissue culture medium (range 3-100 mM) was
associated with increased buffering capacity, since increasing
bicarbonate concentrations increased resulting medium pH (ANOVA,
P < 0.025). Figure
8C also demonstrates that increased
medium buffering capacity results in a decreased ICAM-1 expression
elicited by hypoxia with LPS (ANOVA, P < 0.01), and no significant differences existed between hypoxia and
normoxia at 100 mM bicarbonate
(OD405 of 0.24 ± 0.08 vs. 0.32 ± 0.11 for hypoxia and normoxia with medium containing 100 mM
bicarbonate, respectively, n = 3, P = not significant). Such conditions
were not related to cell toxicity, since 100 mM bicarbonate did not influence MHC class I expression on hypoxic endothelia
(OD405 of 0.37 ± 0.12 vs. 0.39 ± 0.11 for medium containing 3 and 100 mM bicarbonate,
respectively, n = 3, P = not significant) and did not
significantly influence LPS-stimulated ICAM-1 expression on normoxic
endothelia (OD405 of 0.28 ± 0.08 vs. 0.32 ± 0.11 for medium containing 3 and 100 mM
bicarbonate, respectively, n = 3, P = not significant). In addition,
unstimulated basal levels of ICAM-1 expression were not influenced by
increased bicarbonate (OD405 of
0.08 ± 0.01 vs. 0.1 ± 0.03 for medium containing 3 and 100 mM
bicarbonate, respectively, n = 2, P = not significant).
Finally, we determined whether we could recapitulate our observations
with hypoxia by simply decreasing medium pH in the presence of LPS.
Endothelial medium pH was lowered to a measured range of 6.8-7.3
(similar to the extracellular range observed with hypoxia, see Fig. 8)
in the presence of LPS (100 ng/ml) for 24 h and examined for ICAM-1
expression by ELISA. Such conditions resulted in increased ICAM-1
expression with decreased medium pH
(OD405 of 0.36 ± 0.05, 0.31 ± 0.09, 0.19 ± 0.04, 0.23 ± 0.07, 0.11 ± 0.01, and 0.14 ± 0.04 for medium pH of 6.8, 6.9, 7.0, 7.1, 7.2, and 7.3, respectively, P < 0.025, n = 3). Overall, these results
indicate that hypoxia-related increases in LPS-stimulated ICAM-1
expression, which correlate with increased proteasome activation (Figs.
6 and 7), are likely dependent on hypoxia-elicited endothelial
acidification.
 |
DISCUSSION |
In a variety of disease states, tissue hypoxia occurs in conjunction
with other inflammatory events and is associated with accumulation of
leukocytes. Leukocyte recruitment to such sites involves a concerted
series of adhesion and deadhesion events between leukocytes and
vascular endothelial cells. An important adhesion molecule in this
leukocyte recruitment cascade is ICAM-1. For this reason, we
hypothesized that hypoxia may differentially regulate stimulated
endothelial ICAM-1 expression. Our findings indicate that, whereas
hypoxia alone fails to induce endothelial ICAM-1, in the presence of
LPS, hypoxia enhances both expression of ICAM-1 and lymphocyte
adhesion. With the use of specific inhibitors and
cytoplasmic-to-nuclear translocation of NF-
B, these studies implicate a hypoxia-elicited activation of proteasomes. Finally, our
data reveal that conditions that result in enhanced ICAM-1 expression
are associated with metabolic acidosis, the reversal of which
normalizes ICAM-1 expression.
In settings of tissue hypoperfusion in vivo, endothelial surfaces can
be exposed to high concentrations of inflammatory stimuli. A primary
example is the state of sepsis in which profound alterations in tissue
perfusion are coincident with activation of high circulating levels of
LPS and other inflammatory stimuli (33). Thus a relevant in vitro model
that addresses focused questions regarding the role of individual
perturbations should include combinations of cellular hypoxia and
inflammatory stimuli. During development of this model, we consistently
observed that hypoxia alone is unlikely to be a relevant signal for
induction of ICAM-1 and, as we have recently demonstrated, does not
induce endothelial E-selectin (36). Such findings are consistent with
previous reports by others in which short-term (minutes) or relatively long-term (hours) exposure of endothelium to hypoxia resulted in no
change in surface expression of ICAM-1 (21, 26, 35). The presence of
LPS during hypoxia elicited a dose- and time-dependent augmentation of
surface ICAM-1 (Figs. 1-3). Moreover, such enhancement by hypoxia
was verified at the functional level using lymphocyte adhesion assays
(Figs. 4 and 5), and the addition of functionally inhibitable MAb or
ICAM-1 mRNA antisense oligonucleotides reduced such adhesion to nearly
baseline levels. Interestingly, we consistently observed some enhanced
ICAM-1-independent adhesion associated with endothelia exposed to
hypoxia (see Figs. 4 and 5). The source of such residual adhesion is
not known, although others have described similar ICAM-1-independent,
LFA-1-dependent adhesion using endothelial cells exposed to hypoxia
(14). Further work will be necessary to reveal the identity of this
residual lymphocyte adhesion.
These studies demonstrate that hypoxia augments proteasome activation
in the presence of LPS. First, whereas the calpain inhibitor I (ALLN)
reduced LPS-activated ICAM-1 in both normoxic and hypoxic endothelia,
hypoxic cells showed a greater than one log increase in sensitivity to
this inhibitor (Fig. 7). Second, because proteasome activation is
required for induction of ICAM-1 (9) and cytoplasmic-to-nuclear translocation of NF-
B p65 directly reflects proteasome activity (25), Western blot analysis revealed that hypoxia significantly enhanced the LPS-stimulated nuclear p65 (Fig. 6). The mechanisms that
elicit increased proteasome activation by hypoxia are not known at this
time, but evidence is provided that metabolic acidification (reflected
as a decrease in both extracellular and intracellular pH in our system)
may play a role (Fig. 8). Namely, decreased medium and intracellular pH
by hypoxia correlated with increases in LPS-stimulated ICAM-1, and such
responses were normalized by increasing the buffering capacity of the
extracellular medium; a similar ICAM-1 induction pattern was observed
by adjusting medium pH in the presence of LPS (see
RESULTS). Our results are reflective of recent ex vivo (16, 31) and in vitro (15) studies indicating a
distinct role for both metabolic acidosis and proteasome activation during sepsis. Metabolic acidosis, as reflected by circulating lactate
levels, has in fact been shown to be an excellent clinical indicator
for outcome of septic patients (3, 4). In our model, it is possible
that conditions of metabolic acidification activate proteasomes and
result in persistent decreases in cytoplasmic I-
B, since conditions
that maintain low levels of cytoplasmic I-
B-
have been recently
demonstrated to sustain increased surface expression of ICAM-1,
vascular cell adhesion molecule 1 (CD106), and E-selectin (CD62E) (17).
Our recent studies demonstrated that endothelial exposure to hypoxia
mediates increased induction of transcription and translation of
E-selectin (36). Such observations correlated with decreased endothelial generation of adenosine 3',5'-cyclic
monophosphate (cAMP) under conditions of hypoxia, and this effect is
likely mediated by the cAMP-responsive element/activating transcription factor (CRE/ATF) within the E-selectin gene (10). It is unlikely that
similar mechanisms explain our present observations with ICAM-1, since
no CRE/ATF binding domain has been demonstrated in the ICAM-1 gene (9)
and previous observations have shown that agents that influence
intracellular cAMP levels modulate surface expression of endothelial
E-selectin but not ICAM-1 (23).
At present, it is unclear how universal our findings might be with
regard to other molecules, additional agonists, and ICAM-1 expression
on different cell types. Because E-selectin induction occurs through
activation of NF-
B (9), it is likely that proteasome activation
plays a role in our previous findings with hypoxia and E-selectin (36).
Overall, these findings indicate that tissue hypoxia serves as a
relevant pathophysiological condition during inflammation. Therapeutic
strategies aimed at minimizing tissue hypoxia, and by association
metabolic acidosis, may dampen such responses.
 |
ACKNOWLEDGEMENTS |
We acknowledge the valuable technical assistance of Margaret Morrissey.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-50189 (to S. P. Colgan) and by National
Heart, Lung, and Blood Institute Grants HL-52886 and HL-56086
(to G. L. Stahl), HL-52589 (to F. X. McGowan) and HL-48675 (to P. R. Hickey). G. Zünd is supported by a grant from the Swiss National
Foundation.
Address for reprint requests: S. P. Colgan, Center for Experimental
Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
Received 14 March 1997; accepted in final form 15 July 1997.
 |
REFERENCES |
1.
Alpern, R. J.
Mechanism of basolateral membrane H+/OH
/HCO3 transport in the rat proximal convoluted tubule: a sodium-coupled electrogenic process.
J. Gen. Physiol.
86:
613-636,
1985[Abstract].
2.
Bailey, J. L.,
X. Wang,
B. K. England,
S. R. Price,
X. Ding,
and
W. E. Mitch.
The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway.
J. Clin. Invest.
97:
1447-1453,
1996[Abstract/Free Full Text].
3.
Bakker, J.,
P. Gris,
M. Coffernils,
R. J. Kahn,
and
J. L. Vincent.
Serial blood lactate levels can predict development of multiple organ failure following septic shock.
Am. J. Surg.
171:
221-226,
1996[Medline].
4.
Bakker, J.,
M. Coffernils,
M. Leon,
P. Gris,
and
J. L. Vincent.
Blood lactate levels are superior to oxygen-derived variables in predicting outcome in human septic shock.
Chest
99:
956-962,
1991[Abstract].
5.
Barnstable, C. J.,
W. F. Bodmer,
G. Brown,
G. Galfre,
C. Milstein,
A. F. Williams,
and
A. Ziegler.
Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens
new tools for genetic analysis.
Cell
14:
9-20,
1978[Medline].
6.
Bennett, C. F.,
T. P. Condon,
S. Grimm,
H. Chan,
and
M.-Y. Chiang.
Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.
J. Immunol.
152:
3530-3540,
1994[Abstract/Free Full Text].
7.
Bevilacqua, M. P.,
J. S. Pober,
D. L. Mendrick,
R. S. Cotran,
and
M. A. Gimbrone.
Identification of an inducible endothelial-leukocyte adhesion molecule.
Proc. Natl. Acad. Sci. USA
84:
9238-9242,
1987[Abstract].
8.
Collins, T.
Biology of disease; endothelial nuclear factor-
B and the initiation of the atherosclerotic lesion.
Lab. Invest.
68:
499-508,
1993[Medline].
9.
Collins, T.,
M. A. Read,
A. S. Neish,
M. Z. Whitley,
D. Thanos,
and
T. Maniatis.
Transcriptional regulation of endothelial cell adhesion molecules: NK-
B and cytokine-inducible enhancers.
FASEB J.
9:
899-909,
1995[Abstract/Free Full Text].
10.
DeLuca, L. G.,
D. R. Johnson,
M. Z. Whitley,
T. Collins,
and
J. S. Pober.
cAMP and tumor necrosis factor competitively regulate transcriptional activation through and nuclear factor binding to the cAMP responsive element/activating transcription factor element of the endothelial leukocyte adhesion molecule-1.
J. Biol. Chem.
269:
19193-19196,
1994[Abstract/Free Full Text].
11.
Diamond, M. S.,
D. E. Staunton,
S. D. Marlin,
and
T. A. Springer.
Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation.
Cell
65:
961-971,
1991[Medline].
12.
Dittel, B. N.,
E. A. Wayner,
J. B. McCarthy,
and
T. W. LeBien.
Regulation of human B cell precursor adhesion to bone marrow stromal cells by cytokines which exert opposing effects on the expression of vascular cell adhesion molecule-1.
Blood
81:
2272-2282,
1993[Abstract].
13.
Gimbrone, M., Jr.,
E. J. Shefton,
and
S. A. Cruise.
Isolation and primary culture of endothelial cells from human umbilical vessels.
Tissue Cult. Assoc. Man.
4:
813-818,
1978.
14.
Ginis, I.,
S. J. Mentzer,
X. Li,
and
D. V. Faller.
Characterization of a hypoxia-responsive adhesion molecule for leukocytes on human endothelial cells.
J. Immunol.
155:
802-810,
1995[Abstract].
15.
Isozaki, U.,
W. E. Mitch,
B. K. England,
and
S. R. Price.
Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.
Proc. Natl. Acad. Sci. USA
93:
1967-1971,
1996[Abstract/Free Full Text].
16.
James, J. H.,
C. H. Fang,
S. J. Schrantz,
P. O. Hasselgren,
R. J. Paul,
and
J. E. Fischer.
Linkage of aerobic glycolysis to sodium-potassium transport in rat skeletal muscle. Implications for increased lactate production in sepsis.
J. Clin. Invest.
98:
2388-2397,
1996[Abstract/Free Full Text].
17.
Johnson, D. R.,
I. Douglas,
A. Jahnke,
S. Ghosh,
and
J. Pober.
A sustained reduction in I
B-
may contribute to persistent NF-
B activation in human endothelial cells.
J. Biol. Chem.
271:
16317-16322,
1996[Abstract/Free Full Text].
18.
LeBivic, A.,
X. R. Francisco,
and
E. Rodriguez-Boulan.
Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line.
Proc. Natl. Acad. Sci. USA
86:
9313-9317,
1989[Abstract].
19.
Mitch, W. E.,
R. Medina,
S. Greiber,
R. C. May,
B. K. England,
S. R. Price,
J. L. Bailey,
and
A. L. Goldberg.
Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes.
J. Clin. Invest.
93:
2127-2133,
1994[Medline].
20.
Palombella, V.,
O. Rando,
A. Goldberg,
and
T. Maniatis.
The ubiquitin-proteasome pathway is required for processing the NK-
B1 precursor protein and the activation of NK-
B.
Cell
78:
773-785,
1994[Medline].
21.
Pietersma, A.,
N. DeJong,
J. F. Koster,
and
W. Sluiter.
Effect of hypoxia on adherence of granulocytes to endothelial cells in vitro.
Am. J. Physiol.
267 (Heart Circ. Physiol. 36):
H874-H879,
1994[Abstract/Free Full Text].
22.
Pober, J. S.,
and
R. S. Cotran.
Overview: the role of endothelial cells in inflammation.
Transplantation
50:
537-541,
1990[Medline].
23.
Pober, J. S.,
M. R. Slowik,
L. G. DeLuca,
and
A. J. Ritchie.
Elevated cyclic AMP inhibits endothelial cell synthesis and expression of TNF-induced endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1, but not intercellular adhesion molecule-1.
J. Immunol.
150:
5114-5123,
1993[Abstract/Free Full Text].
24.
Raetz, C. R.
Biochemistry of endotoxins.
Annu. Rev. Biochem.
59:
129-170,
1990[Medline].
25.
Read, M. A.,
A. S. Neish,
F. W. Luscinskas,
V. J. Palomella,
T. Maniatis,
and
T. Collins.
The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression.
Immunity
2:
493-506,
1995[Medline].
26.
Shreeniwas, R.,
S. Koga,
M. Karakurum,
D. Pinsky,
E. Kaiser,
J. Brett,
B. A. Wolitzky,
C. Norton,
J. Plocinski,
W. Benjamin,
D. K. Burns,
A. Goldstein,
and
D. Stern.
Hypoxia-mediated induction of endothelial cell interleukin-1-
: an autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface.
J. Clin. Invest.
90:
2333-2339,
1992[Medline].
27.
Smith, C. W.,
S. D. Marlin,
R. Rothlein,
C. Toman,
and
D. C. Anderson.
Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro.
J. Clin. Invest.
83:
2008-2017,
1989[Medline].
28.
Springer, T. A.
Adhesion receptors of the immune system.
Nature
346:
425-430,
1990[Medline].
29.
Staunton, D. E.,
M. L. Dustin,
H. P. Erickson,
and
T. A. Springer.
The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus.
Cell
61:
243-254,
1990[Medline].
30.
Stevens, T.,
and
D. M. Rodman.
The effect of hypoxia on endothelial cell function.
Endothelium
3:
1-11,
1995.
31.
Tiao, G.,
S. Hobler,
J. J. Wand,
T. A. Meyer,
F. A. Luchette,
J. E. Fischer,
and
P.-O. Hasslegren.
Sepsis is associated with increased mRNA's of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.
J. Clin. Invest.
99:
163-168,
1997[Abstract/Free Full Text].
32.
Welbourne, C. R. B.,
G. Goldman,
C. R. Valeri,
D. Shepro,
and
H. B. Hechtman.
Pathophysiology of ischaemia reperfusion injury: central role of the neutrophil.
Br. J. Surg.
78:
651-655,
1991[Medline].
33.
West, M. A.,
and
C. Wilson.
Hypoxic alterations in cellular signal transduction in shock and sepsis.
New Horizons
4:
168-178,
1996[Medline].
34.
Whitley, M. Z.,
D. Thanos,
M. A. Read,
T. Maniatis,
and
T. Collins.
A striking similarity in the organization of the E-selectin and beta interferon gene promoters.
Mol. Cell. Biol.
14:
6464-6475,
1994[Abstract].
35.
Yoshida, N.,
D. N. Granger,
D. C. Anderson,
R. Rothlein,
C. Lane,
and
P. R. Kvietys.
Anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H699-H703,
1992.
36.
Zünd, G.,
D. P. Nelson,
E. J. Neufeld,
A. L. Dzus,
J. Bischoff,
J. E. Mayer,
and
S. P. Colgan.
Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.
Proc. Natl. Acad. Sci. USA
93:
7075-7080,
1996[Abstract/Free Full Text].
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