Endothelial permeability and IL-6 production during hypoxia:
role of ROS in signal transduction
Mir H.
Ali1,
Scott A.
Schlidt2,
Navdeep S.
Chandel3,
Karen L.
Hynes2,
Paul T.
Schumacker3, and
Bruce L.
Gewertz2
1 Pritzker School of Medicine,
2 Section of Vascular Surgery,
Department of Surgery, and
3 Section of Pulmonary and
Critical Care, Department of Medicine, The University of Chicago,
Chicago, Illinois 60637
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ABSTRACT |
Prolonged
hypoxia produces reversible changes in endothelial permeability, but
the mechanisms involved are not fully known. Previous studies have
implicated reactive oxygen species (ROS) and cytokines in the
regulation of permeability. We tested whether prolonged hypoxia alters
permeability to increasing ROS generation, which amplifies cytokine
production. Human umbilical vein endothelial cell (HUVEC) monolayers
were exposed to hypoxia while secretion of tumor necrosis factor-
(TNF-
), interleukin (IL)-1
, IL-6, and IL-8 was measured. IL-6 and
IL-8 secretion increased fourfold over 24 h in a pattern corresponding
to changes in HUVEC permeability measured by transendothelial
electrical resistance (TEER). Addition of exogenous IL-6 to normoxic
HUVEC monolayers caused time-dependent changes in TEER that mimicked
the hypoxic response. An antibody to IL-6 significantly attenuated the
hypoxia-induced changes in TEER (86 ± 4 vs. 63 ± 3% with
hypoxia alone at 18 h), whereas treatment with anti-IL-8 had no effect.
To determine the role of hypoxia-induced ROS on this response, HUVEC
monolayers were incubated with the antioxidants ebselen (50 µM) and
N-acetyl-L-cysteine (NAC, 1 mM) before hypoxia. Antioxidants attenuated hypoxia-induced IL-6 secretion (13 ± 2 pg/ml with ebselen and 19 ± 3 pg/ml with NAC vs. 140 ± 15 pg/ml with hypoxia). Ebselen and NAC prevented changes in TEER during hypoxia (94 ± 2% with ebselen and 90 ± 6% with NAC vs. 63 ± 3% with hypoxia at 18 h).
N-nitro-L-arginine (500 µM) did not decrease hypoxia-induced changes in
dichlorofluorescin fluorescence, IL-6 secretion, or TEER. Thus ROS
generated during hypoxia act as signaling elements, regulating
secretion of the proinflammatory cytokines that lead to alterations of
endothelial permeability.
superoxide; hydrogen peroxide; antioxidants; ischemia; cytokines; human umbilical vein endothelial cells; reactive oxygen species
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INTRODUCTION |
THE VASCULAR ENDOTHELIUM plays a vital role in the
regulation of the passage of fluids, solutes, and cells from blood into tissues. Disruption of vascular permeability contributes to the pathogenesis of a wide range of diseases, including atherosclerosis, inflammatory tissue injury, and acute respiratory distress syndrome. A
better understanding of the basic mechanisms underlying alterations in
endothelial permeability may lead to interventions that could preserve
barrier function with therapeutic benefits.
Three cytokines have been implicated in the regulation of barrier
function in inflammatory states. Tumor necrosis factor-
(TNF-
),
interleukin (IL)-1, and IL-6 are increased in blood (2, 11) and edema
fluid (31) after tissue injury. Addition of these cytokines to
uninjured human umbilical vein endothelial cell (HUVEC) monolayers
causes reversible alterations in permeability (9). Furthermore, albumin
clearance studies have demonstrated that antibody blockade is effective
in preventing changes in endothelial permeability caused by
inflammatory cytokines and lipopolysaccharide in vivo (19, 36).
Although many studies have focused on neutrophils as the sole source of
proinflammatory cytokines, endothelium-derived cytokines may also
contribute to the alteration of endothelial permeability via an
autocrine or a paracrine mechanism.
Hypoxia that develops in regions of tissue inflammation may amplify the
biochemical and functional responses of the vascular endothelium in
that state. Proinflammatory cytokines could contribute to the
reversible changes in endothelial permeability observed during periods
of prolonged hypoxia. Recent studies have begun to implicate reactive
oxygen species (ROS) in the cellular responses to inflammatory
cytokines (8, 32), whereas other studies have demonstrated that ROS
participate in the intracellular signaling initiated during
physiological hypoxia (7, 10). The involvement of ROS in both of these
cellular responses suggests that cytokines and hypoxia may interact in
the regulation of endothelial barrier function during inflammation. The
present study sought to clarify the interactions between hypoxia and
cytokines in an in vitro model of cultured HUVEC monolayers.
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METHODS |
HUVEC culture.
Endothelial cells were cultured from human umbilical veins (<24 h
postpartum) by a modification of the method described by Jaffe et al.
(16). After cannulation of both ends, cords were flushed with 120 ml of
HEPES-buffered saline (50 mM) and incubated with 0.2% collagenase
(Sigma Chemical) at 37°C for 15 min. HUVECs were grown to 100%
confluence on gelatin-coated T25 flasks (Becton Dickinson) in medium
199 supplemented with 10% human serum, 10% FCS, penicillin,
streptomycin, and amphotericin B (Sigma Chemical) at pH 7.4 and
37°C. Cells were studied between the first and second passages
after characterization of endothelial phenotype via positive staining
for CD31 (platelet-endothelial cell adhesion molecule-1) and factor
VIII. After treatment with 0.1% collagenase and 0.25% EDTA, cells
were split 3:1 onto 1)
gelatin-coated 25-mm glass coverslips (Fisher Scientific) for ROS
determination-2',7'-dichlorofluorescein fluorescence (DCFH)
studies, 2) gelatin-coated cell
culture inserts (12 mm diameter, 3.0 µm pore size; Costar) for
permeability analysis and PO2
determination, and 3) gelatin-coated
60-mm dishes (Becton Dickinson) for the collection of supernatants for
cytokine measurements.
Induction of hypoxia.
Cultured cells were placed in a modular incubator chamber
(Billups-Rothenberg) flushed with a gas mixture (1%
O2-5%
CO2-94% N2) to purge it of atmospheric
air. The desired level of hypoxia (PO2 = 14 ± 3 Torr) was achieved
within 30 min. Samples of medium were analyzed for
PO2 and pH at 3-h intervals over 24 h. The PO2 was analyzed using an
O2-quenching phosphorescence
method with palladium-meso-tetra(4-carboxyphenyl)porphine (15).
Phosphorescence decay was detected and calculated using an Oxyspot
system (Medical Systems). The pH of aliquots of medium was measured
using a blood gas analyzer (Radiometer).
Measurement of transendothelial electrical resistance as an index
of permeability.
First-passage endothelial cells were split 3:1 to 12-mm Transwell
tissue culture inserts. They were rinsed with HEPES-buffered saline and
fed every other day until confluent by visual inspection. The
resistances of the monolayers were monitored daily until they reached a
steady state. Once stable resistances were obtained (>25
· cm2),
the cells were exposed to hypoxia for 3-24 h. Transendothelial electrical resistance (TEER) was measured with a resistance meter (model EVOM, World Precision Instruments) together with the Endohm-12 chamber (World Precision Instruments). Measurements were taken in
triplicate and reported as percentage (mean ± SE) for each time
point relative to the same insert at time
0.
Cytokine quantification.
HUVEC monolayers cultured on gelatin-coated 60-mm plates were exposed
to hypoxia as described above. Culture medium was collected at 3-h
intervals over 24 h. Hypoxia-induced production of IL-6, IL-8, IL-1
,
and TNF-
was assessed by ELISA (R&D Systems). Each sample was
measured in duplicate and is expressed as an average of these values.
Exogenous cytokine administration.
Human IL-6 (R&D Systems) was added to normoxic HUVEC monolayers at 50, 100, and 150 pg/ml. These concentrations were chosen because they span
the range of IL-6 levels observed in cell culture medium during 24 h of
prolonged hypoxia. After cytokine administration, TEER was determined
at 3-h intervals over a 24-h normoxic period and is expressed as means ± SE for each time point.
Cytokine antibody blockade.
Monoclonal antibody to IL-6 (R&D Systems) was added at a concentration
sufficient to neutralize >95% of cytokine activity in vitro (3 µg/ml). A polyclonal antibody to IL-8 (R&D Systems) was utilized at 1 µg/ml, which is also sufficient to neutralize >95% of activity. In
a parallel series of experiments, human monoclonal anti-IL-1 (R&D
Systems) was added at neutralizing concentrations (3 µg/ml) to serve
as an irrelevant antibody control. These antibodies were added to the
HUVEC cultures 24 h before hypoxic experimentation. Cytokine levels
were measured in the culture medium by ELISA every 3 h thereafter along
with TEER. Results are expressed as means ± SE for each time point.
Measurement of ROS production-dichlorofluorescin fluorescence.
An inverted microscope was equipped for epifluorescent illumination and
included a xenon light source (75 W), a 12-bit digital cooled
charge-coupled device camera (Princeton Instruments), a shutter and a
filter wheel (Sutter), and appropriate excitation and emission filters.
Fluorescent cell images were obtained using a ×40 oil-immersion
objective (Nikon Plan Fluor). Data were acquired and analyzed using
Metamorph software (Universal Imaging). ROS generation in cells was
assessed using the probe 2,7-dichlorofluorescein diacetate (DCFH-DA, 10 µM; Molecular Probes). Within the cell, esterases cleave the acetate
groups, thereby trapping the nonfluorescent DCFH probe intracellularly.
Subsequent oxidation by ROS, particularly H2O2
or the hydroxyl radical (OH ·), yields the fluorescent
product dichlorofluorescin (DCF) (4). DCF fluorescence was measured using excitation wavelength of 480 nm, dichroic 505-nm long pass, and
emitter band pass of 535 nm (Chroma Technology). Neutral density filters were used to attenuate the excitation light intensity. Fluorescence intensity was assessed in clusters of cells (<10) identified as regions of interest, and background was identified as an
area without cells or with minimal cellular fluorescence. Intensity
values are reported as percentage of initial values after subtraction
of background.
Statistics.
Data were analyzed with the Minitab II statistical program on the Power
Macintosh 7200. Values are means ± SE and analyzed by
Student's t-tests or ANOVA where
appropriate. Significance was defined as
P < 0.05.
 |
RESULTS |
Effects of hypoxia on permeability.
The effects of hypoxia (1.5%
O2) on TEER were measured in
HUVEC monolayers over 24 h. A blank Transwell insert was used as an
indicator of background effects on TEER and was consistent at 6 ± 1
· cm2. TEER
was measured every 3 h, and values at each time point were reported as
a percentage of the original value at time
0. Normoxic controls maintained in a standard incubator
environment (5% CO2-95% room
air) showed no significant change in TEER over 24 h (data not shown).
During prolonged hypoxia (PO2 = 14 ± 3 Torr, range 11-19 Torr) TEER changed significantly over 24 h (Fig. 1). Under these conditions,
monolayers demonstrated an initial drop in resistance at 9 h (92 ± 2% of original), with the greatest decreases detected at 18 h (63 ± 3% of original, P < 0.01).
Thereafter, resistances began to recover until 24 h, plateauing at 88 ± 3% of their original values. By contrast, TEER failed to change
significantly over 24 h in cells exposed to 25 or 35 Torr. This
suggests that alterations of endothelial permeability are dependent on
the length and severity of hypoxia.

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Fig. 1.
Effects of hypoxia on human umbilical vein endothelial cell (HUVEC)
permeability. Graded levels of hypoxia were produced (15, 25, and 35 Torr). Experiments were conducted on confluent HUVEC monolayers after a
stable transendothelial electrical resistance (TEER; >25
· cm2) was
reached. At each time point, TEER was measured and reported as a
percentage of original TEER at time
0.
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Effects of hypoxia on cytokine production.
Prolonged hypoxia (PO2 = 14 ± 3 Torr, range 11-19 Torr) caused endothelial cells to secrete
several types of cytokines. Over the first 18 h, levels of IL-6 in the
medium reached concentrations of 140 ± 15 pg/ml
(P < 0.01 compared with normoxic
controls; Fig. 2), whereas IL-8 peaked at
1,089 ± 246 pg/ml. However, IL-1
levels remained unchanged (4 ± 6 pg/ml) over a 24-h hypoxic incubation, as did levels of TNF-
(data not shown). These findings suggest a direct temporal correlation
between hypoxia-induced IL-6 and IL-8 production and changes in
endothelial permeability.

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Fig. 2.
Secretion of interleukin (IL)-1 , IL-6, and IL-8 by hypoxic
(PO2 = 14 ± 3 Torr) HUVECs.
Levels of IL-6 and IL-8 increased significantly over first 18 h,
plateauing at 140 pg/ml and 3.3 ng/ml, respectively, at 24 h.
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Effects of cytokine antibodies on hypoxia-induced changes in
permeability.
To determine whether cytokines released by endothelial cells
contributed to the observed changes in TEER during hypoxia, antibodies to IL-1
, IL-8, or IL-6 were added to the cell culture medium before
hypoxic incubation (PO2 = 14 ± 3 Torr, range 11-19 Torr). Monolayers supplemented with anti-IL-1
or anti-IL-8 before hypoxia exhibited a permeability profile similar to
that of cells exposed to hypoxia alone (Fig.
3, A and
B). However, addition of anti-IL-6
monoclonal antibody significantly attenuated hypoxia-induced decreases
in TEER. During prolonged hypoxia, TEER decreased to 63 ± 3% of
original levels in controls. However, in the presence of anti-IL-6, the
minimum TEER reached was 86 ± 4% at 18 h (Fig. 3C; P < 0.05). These findings suggest that IL-6, but not IL-8, participates
as a mediator of hypoxic alteration of endothelial permeability during
prolonged hypoxia.

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Fig. 3.
Effects of cytokine antibody (Ab) pretreatment on endothelial
permeability during hypoxia. A: effect
of IL-1 Ab treatment on TEER profile of HUVECs exposed to hypoxia.
Antibody was added 24 h before onset of hypoxia
(PO2 = 14 ± 3 Torr) and was
present throughout hypoxic exposure.
B: effect of IL-8 Ab treatment on TEER
of hypoxic HUVECs over 24 h. C: effect
of IL-6 Ab on changes in endothelial TEER during 24 h of hypoxia.
Significant increases in TEER were observed between 12 and 24 h in
cells treated with anti-IL-6 compared with HUVECs exposed to hypoxia
alone.
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Effects of IL-6 on normoxic HUVECs.
To determine the effects of exogenous IL-6 on TEER, recombinant human
IL-6 (50 pg/ml) was added to the medium of normoxic HUVEC monolayers.
This resulted in an increase in endothelial permeability over the first
9 h, reaching a minimum TEER of 72 ± 2% of original (Fig.
4; P < 0.05). However, unlike the typical biphasic permeability profile
observed during hypoxia, no restoration of TEER to baseline values was
observed at 24 h with this concentration of cytokine. Similar results
were obtained with 100 pg/ml of IL-6, with a minimum TEER of 68 ± 3% (P < 0.01) observed at 9 h. Once again, no reversal of permeability was observed. However, when 150 pg/ml of IL-6 were added to the monolayers, TEER dropped to 66 ± 2% within 6 h (Fig. 4; P < 0.01)
and recovered toward baseline by 24 h
(P < 0.01). This suggests somewhat
paradoxically that concentrations of IL-6 >100 pg/ml are required for
the reversal of endothelial permeability.

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Fig. 4.
Effects of exogenous administration of IL-6 on TEER during normoxia.
IL-6 (50, 100, and 150 pg/ml) was added to medium of confluent HUVECs
under normoxic conditions (PO2 = 150 Torr). Although all concentrations were capable of producing changes in
TEER, only highest concentration mimicked TEER response to hypoxia.
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Effects of hypoxia on DCF fluorescence.
ROS generation in HUVECs exposed to hypoxia was investigated using DCFH
dye. During hypoxia (PO2 = 14 Torr),
the DCF fluorescence signal increased, reaching ~300% of normoxic baseline values at 120 min (Fig. 5;
P < 0.01). The increase in fluorescence was reversed rapidly when the monolayers were reoxygenated at 150 min (PO2 = 150 Torr), and
within 60 min, the DCF fluorescence had returned to its baseline level.
To verify that ROS were responsible for these results, a series of
experiments was performed using antioxidants to reduce ROS
accumulation. Ebselen [2-phenyl-1,2-benzisoselenazol-3-(2H)-one]
is a selenium-containing compound that functions as a glutathione
peroxidase mimetic (23). A second compound,
N-acetyl-L-cysteine (NAC),
enhances the scavenging of ROS by enhancing intracellular pools of
reduced glutathione (26). Initial experiments were performed with these
agents at various concentrations (10-100 µM ebselen and 100 µM
to 1 mM NAC) to determine their effects on ROS levels in HUVECs exposed
to PO2 of 14 Torr. Determination of a
dose-response relationship for these compounds provided insight into
the optimal concentration to be used for subsequent experiments. DCF
fluorescence was measured at different concentrations of ebselen or
NAC. Addition of 50 µM ebselen abolished the hypoxia-induced
increases in DCF fluorescence within 60 min (Fig.
6A). The
antioxidant effects of NAC were evident at 1 mM (Fig.
6B). Because of the possible toxic
or nonspecific actions of ebselen and NAC, the lowest concentrations of
the compounds that would return DCF fluorescence to normoxic levels
within 60 min were used in subsequent experiments.

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Fig. 5.
Effects of hypoxia (2% O2) and
reoxygenation on dichlorofluorescin (DCF) fluorescence in HUVEC
monolayers. Control cells were superfused with normoxic
(PO2 = 150 Torr) solution throughout.
HUVECs were superfused with hypoxic medium for 210 min
(PO2 = 14 Torr) or 150 min, then
subjected to reoxygenation for 60 min
(PO2 = 150 Torr).
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Fig. 6.
Effects of hypoxia (2% O2) on
DCF fluorescence in HUVEC monolayers treated with antioxidants.
A: addition of ebselen (10, 25, or 50 µM) during hypoxia significantly reduced DCF fluorescence. However,
return of DCF fluorescence was achieved only at 50 µM.
B: addition of
N-acetyl-L-cysteine
(NAC; 100 µM, 500 µM, and 1 mM) during hypoxia significantly
reduced DCF fluorescence. However, return of DCF fluorescence was
achieved only at 1 mM. C: addition of
N-nitro-L-arginine
(L-NNA; 100 µM, 500 µM, and
1 mM) did not return DCF fluorescence to normoxic levels. However,
return of 21% O2 to HUVEC
monolayers caused a brisk attenuation of DCF signal.
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To determine whether reactive nitrogen species, particularly nitric
oxide, are involved in the hypoxia-induced changes in DCF fluorescence,
HUVECs were exposed to hypoxia in the presence of
N-nitro-L-arginine
(L-NNA, 100 µM to 1 mM).
Because the DCFH dye can be oxidized by nitric oxide and by
H2O2,
these experiments were included to distinguish between ROS and reactive
nitrogen species generated during hypoxia. In the presence of 500 µM
and 1 mM L-NNA, the DCF signal
was mildly reduced (16%) but never abolished (Fig.
6C). That this return toward
baseline was achieved with the addition of 1 mM NAC, 50 µM ebselen,
or normoxia suggests that nitric oxide is minimally involved in
hypoxia-induced changes in DCF fluorescence.
Effects of ROS on IL-6 production during hypoxia.
To assess the effects of ROS generated during hypoxia on the release of
cytokines from endothelial cells, the concentrations of cytokines
present in the medium of hypoxic cells were compared with the
concentrations in cells pretreated with 50 µM ebselen or 1 mM NAC.
Ebselen reduced the hypoxia-induced IL-6 production from a maximum of
140 pg/ml at 18 h to 13 pg/ml (Fig.
7A;
P < 0.01). The maximum concentration
detected in the ebselen-treated cells was not different from that in
normoxic controls at 24 h (25 vs. 20 pg/ml,
P > 0.2). Similar attenuation of
cytokine release was seen in cells pretreated with 1 mM NAC before
hypoxia (Fig. 7B). Pretreatment with
500 µM L-NNA did not
significantly alter hypoxia-induced IL-6 secretion (Fig.
7C). These findings suggest that
ROS, not nitric oxide, are involved in the enhanced release of IL-6
during hypoxia.

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Fig. 7.
Effects of antioxidants on HUVEC IL-6 secretion during hypoxia.
A: pretreatment of HUVECs with 50 µM
ebselen significantly decreased hypoxia-induced IL-6 secretion.
B: pretreatment with 1 mM NAC also
significantly attenuated IL-6 secretion during hypoxia.
C: treatment of HUVEC monolayers with
500 µM L-NNA had no
significant effect on hypoxia-induced IL-6 secretion.
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Effects of antioxidants on endothelial permeability.
To determine the effects of ROS generation on the changes in TEER
during hypoxia, HUVEC monolayers were treated with 50 µM ebselen
before hypoxia. Ebselen attenuated the hypoxia-induced changes in TEER
(Fig. 8A;
P < 0.01). The minimum TEER level at 18 h increased from 63 ± 3 to 95 ± 3% of original, and the
typical decrease in TEER between 6 and 18 h was not observed. After 24 h, the ebselen-treated cells exhibited higher resistances than untreated controls (94 ± 2 vs. 88 ± 3%). Similar results were obtained from cells pretreated with 1 mM NAC; the minimum TEER at 18 h
was 90 ± 6% of control (Fig. 8B;
P < 0.05), whereas the value at 24 h
was 95 ± 4% of original TEER. The effects of nitric oxide on these
changes were tested by pretreating HUVECs with 500 µM
L-NNA; these results indicate
that inhibiting nitric oxide production during hypoxia does not alter
changes in TEER (Fig. 8C;
P > 0.2).

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Fig. 8.
Effects of hypoxia on endothelial permeability in presence of
antioxidants. A: 50 µM ebselen added
to confluent HUVEC monolayer cultures before hypoxia
(PO2 = 14 ± 3 Torr) prevented
hypoxia-induced changes in endothelial permeability, as shown by TEER.
B: treatment with 1 mM NAC also
attenuated changes in TEER during 24 h of hypoxia.
C: treatment with 500 µM
L-NNA did not significantly
affect hypoxia-induced changes in TEER over 24 h.
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DISCUSSION |
Many investigators have studied the effects of hypoxia on the vascular
endothelium. Ogawa et al. (25) reported that prolonged hypoxia caused
reversible, selective changes in barrier and coagulant functions that
were not lethal to endothelial cells. Others (3, 22, 27) have reported
that prolonged hypoxia alters monolayer permeability and cell surface
properties. However, the mechanisms linking the decrease in cellular
PO2 to the functional response have
not been established. This investigation focused on the role of
endothelium-derived ROS and the manner in which they mediate
alterations in endothelial permeability during periods of prolonged
hypoxia. Our findings demonstrate that hypoxia induces the generation
of ROS in endothelial cells, which increases cytokine secretion and
leads to changes in permeability.
In our HUVEC studies, prolonged hypoxia
(PO2 = 14 Torr) resulted in a
transient increase in endothelial permeability between 6 and 24 h,
reaching maximum levels at 18 h. These changes corresponded to
increases in the levels of IL-6 and IL-8 secreted by the endothelial
monolayers, suggesting an autocrine effect of these proinflammatory
cytokines on permeability. The addition of a polyclonal antibody to
IL-8 did not alter the permeability profile of cells subsequently
exposed to hypoxia, but the permeability increase was substantially
attenuated in cells pretreated with an anti-IL-6 monoclonal antibody.
These findings demonstrate that the hypoxic alteration of endothelial
permeability is mediated, at least in part, by IL-6. The addition of
exogenous IL-6 at concentrations observed during 24 h of hypoxia
produced time-dependent changes in permeability, supporting this
assertion. Although lower concentrations of IL-6 increased
permeability, only 150 pg/ml mimicked the biphasic changes in
endothelial permeability seen during hypoxia. These findings suggest
that a threshold level of IL-6 is required for the permeability
reversal, with the critical level in the range of 100-150 pg/ml.
Further studies are required to determine the specific mechanisms by
which higher levels of IL-6 mediate the temporal changes in endothelial permeability.
These data also demonstrate that hypoxia induces the generation of
cellular ROS, which appear to participate in the signaling responsible
for cytokine secretion. Many studies have demonstrated that ROS
participate in the signaling cascades induced by lipopolysaccharide (30) and cytokines such as TNF-
(29). Increased ROS levels have also
been shown during periods of physiological hypoxia (5, 6, 21),
suggesting that ROS signaling may be a common factor involved in
cellular hypoxic detection and cytokine responses. Our DCFH studies
revealed a threefold increase in ROS levels during hypoxia. On the
basis of the attenuation of the DCF signal by ebselen or NAC and the
inability to decrease this signal with L-NNA, we conclude that
H2O2
generation is required for the cytokine response. Pretreatment of HUVEC
monolayers with ebselen or NAC also attenuated the changes in
permeability observed during hypoxia. These findings lead to the
conclusion that ROS generated during hypoxia act as intracellular
signals responsible for the secretion of IL-6, an important modulator
of endothelial permeability.
The effects of cytokines on endothelial cells have been extensively
studied in ischemia-reperfusion models, and these molecules have been implicated as mediators of the hypoxic alteration of endothelial permeability. A host of cytokines, particularly TNF-
and
IL-1
, have been shown to increase permeability in a variety of cell
types (28). When determining which of these mediators might be
responsible for the observed changes in permeability of HUVEC
monolayers during hypoxia, the list of candidates becomes much shorter.
Our studies demonstrate that HUVECs secrete very little TNF-
and
IL-1
during prolonged hypoxia; this is consistent with previous
studies demonstrating that the major source of those cytokines in the
microcirculation is the polymorphonuclear leukocytes (17). Our model
consisted of only HUVECs exposed to hypoxia, so it is still possible
that TNF-
and/or IL-1
may contribute to changes in endothelial
permeability in vivo. However, the increase in IL-6 levels in our HUVEC
system mirrored increases in vascular permeability during prolonged
hypoxia, suggesting that it may play an important role in these changes.
An increase in IL-6 secretion in response to hypoxia has been
demonstrated in a variety of cell types ranging from vascular smooth
muscle cells (33) to mononuclear cells (24). Although IL-6 secretion
does increase in hypoxic conditions, it is clear that IL-6 is only one
of the downstream mediators of the hypoxic cell response. Previous
studies by Wenger and colleagues (35) demonstrate in hepatocytes that
the hypoxia-induced acute-phase reaction overlaps with, but is not
identical to, the IL-6-induced acute-phase reaction. This is consistent
with our observation that blocking the effects of IL-6 with a
monoclonal antibody does not completely eliminate hypoxia-induced
changes in permeability. Thus IL-6, although a major mediator, is not
the only participant in the hypoxia-induced response. It follows that
any attempt to completely abolish changes in permeability during
hypoxia must act upstream of the cytokine response, preferably at the
level of the O2 sensor. Indeed,
our studies showed that the permeability changes could be abolished by
antioxidants, which appear to act proximal to the cytokine step in the
signaling response to hypoxia.
An interesting aspect of this cascade of events is the cellular
detection of hypoxia and subsequent changes in signaling, which lead to
the increase in IL-6 secretion. The responses to hypoxia in the present
study are not likely to reflect cell injury inasmuch as they were
reversible and not associated with loss of viability. Indeed, Farber
and Rounds (12) showed that endothelial cells can withstand long-term
exposure to anoxic conditions without loss of viability or apparent
loss of function. We therefore propose that the results in the present
study represent physiological responses rather than pathophysiological
effects of cellular injury. Recent work has identified a role for ROS
in mediating other hypoxia-induced changes in cell physiology (14). For
example, ROS have been shown to be necessary for the upregulation of
hypoxic genes [vascular endothelial growth factor (VEGF) (20) and
erythropoietin (13)] and the activation of
hypoxia-induced transcription factor-1 (7). Kuroki et al. (18) used
antioxidants to demonstrate that ROS control is critical to the
expression of VEGF in vivo and in vitro. Chandel et al. (7) found that
the activation of mRNA for erythropoietin, glycolytic enzymes, and VEGF
depends on the ROS generated by the mitochondria during hypoxia.
Moreover, in mitochondria-deficient cells, the increase in the
transcription for these hypoxic genes was not observed during prolonged
hypoxia. In addition, Vanden Hoek et al. (34) demonstrated that ROS are
produced by mitochondria during hypoxia and are critical to the
induction of cardiac preconditioning in cardiomyocytes. Duranteau and
colleagues (10) found that ROS generation in cardiomyocytes was
increased during physiological hypoxia and that these ROS mediate a
decrease in contractile activity, a response that was prevented by
pretreatment with antioxidants. Collectively, these results underscore
a growing awareness that low levels of ROS generated during hypoxia
function as signaling molecules affecting gene expression and cell
function during hypoxia. The findings of the present study indicate
that ROS are also responsible for changes in endothelial permeability,
although the specific source of these molecules in HUVECs was not demonstrated.
A major functional consequence of ROS production during hypoxia is the
increase in IL-6 secretion, which contributes to the changes in
endothelial permeability. Ala et al. (1) demonstrated that
hypoxia-reoxygenation increases IL-6 production via ROS; this trend was
prevented by pretreatment with superoxide dismutase or glutathione
peroxidase. In hypoxia-reoxygenation models, the increase in ROS is
thought to occur at reoxygenation and may be responsible for increases
in monolayer permeability. In our study, changes occurred during
continuous hypoxia before reoxygenation. Our DCFH studies help clarify
this theory by demonstrating that an increase in ROS generation occurs
during hypoxia before reoxygenation. We suggest that ROS generated in
response to physiological hypoxia contribute to the regulation of
endothelial permeability and cytokine release, whereas ischemia
and reperfusion may affect permeability by enhancing the generation of
ROS at reoxygenation. Regardless of the site of production of ROS
during hypoxia or reoxygenation, changes in permeability are attenuated
by the use of antioxidants. Future studies are required to fully
clarify the mechanisms by which hypoxia augments intracellular ROS
generation and the specific signaling system by which these ROS augment
cytokine release.
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FOOTNOTES |
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"
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Address for reprint requests and other correspondence: B. L. Gewertz, Dept. of Surgery, The University of Chicago, 5841 S. Maryland
Ave., MC 5029, Chicago, IL 60637.
Received 19 April 1999; accepted in final form 29 June 1999.
 |
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