Peroxide-induced membrane blebbing in endothelial cells
associated with glutathione oxidation but not apoptosis
Roosje M. A.
van Gorp1,
Jos L. V.
Broers2,
Chris P. M.
Reutelingsperger3,
Nancy M. H. J.
Bronnenberg1,
Gerard
Hornstra1,
Maria C. E.
van
Dam-Mieras3, and
Johan W. M.
Heemskerk1,3
Departments of 1 Human Biology,
2 Molecular Cell Biology and
Genetics, and 3 Biochemistry,
Maastricht University, 6200 MD Maastricht, The Netherlands
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ABSTRACT |
Cells under
oxidative stress induced by peroxides undergo functional and
morphological changes, which often resemble those observed during
apoptosis. Peroxides, however, also cause the oxidation of
intracellular reduced glutathione (GSH). We investigated the relation
between these peroxide-induced effects by using human umbilical vein
endothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 and
ECV304. With HUVEC, tert-butyl
hydroperoxide (tBH) or hydrogen
peroxide application in the presence of serum induced, in a
dose-dependent way, reorganization of the actin cytoskeleton, membrane
blebbing, and nuclear condensation. These processes were accompanied by
transient oxidation of GSH. With ECRF24 cells, this treatment resulted
in less blebbing and a shorter period of GSH oxidation. However,
repeated tBH addition increased the
number of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVEC
and ECRF24 cells, but not in ECV304 cells. Neither membrane blebbing
nor nuclear condensation in any of these cell types was due to
apoptosis, as evidenced by the absence of surface expression of
phosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although high
tBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells,
peroxide-induced cytoskeletal reorganization and bleb formation
correlate with the degree of GSH oxidation but do not represent an
early stage of the apoptotic process.
apoptosis; endothelial cells; glutathione; membrane blebbing
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INTRODUCTION |
BECAUSE OF THEIR LOCATION at the interface of
the vascular system, endothelial cells in the blood vessel
wall are from time to time exposed to peroxides, for instance, during
local inflammatory reactions or contact with oxidized lipoproteins. In
vitro experiments have indicated that endothelial and other cells, when
subjected to oxidative stress with hydrogen peroxide or
tert-butyl hydroperoxide (tBH), undergo remarkable changes in
morphology and in the structure of the actin cytoskeleton, often
resulting in membrane blebbing in a manner resembling that due to
apoptosis (programmed cell death) (4, 14, 15, 19, 28). A
whole variety of intracellular signals have been put forward as
intermediates of these peroxide-evoked changes in cell function and
structure, e.g., elevated levels of intracellular
Ca2+ (9, 38); activation of
phospholipase D (30), mitogen-activated protein kinases (5, 20), or
calpain (28); heat shock protein 27 phosphorylation (12, 19); and
changed concentrations of cGMP (23) or cAMP (16). However, the precise
mechanism by which peroxides, and thus oxidative stress, trigger cells
is still a matter of debate.
Another well-documented direct effect of peroxides, and especially of
tBH, on cells is the oxidation of
cytosolic glutathione, which is normally present in its reduced form
(GSH) (8, 33). Although there are good indications of ties between
oxidative stress, glutathione depletion, and the onset of apoptosis
(reviewed in Ref. 6), these have not yet been studied in endothelial cells. In the present paper, we report on the effects of
tBH on a number of structural and
functional properties of human umbilical vein endothelial cells (HUVEC)
and two HUVEC-derived cell lines, ECV304 (34) and ECRF24 (10). We note
remarkable differences in the capacities of these cells to respond by
actin reorganization, membrane blebbing, and nuclear condensation,
which closely parallel differences in sensitivity to GSH oxidation.
Because these tBH-evoked morphological
changes suggested the commencement of apoptosis, we determined
two markers of this process, phosphatidylserine (PS)
externalization (21, 35) and DNA fragmentation (31). It appeared that
even prolonged incubation with high doses of tBH failed to induce these apoptotic responses.
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MATERIALS AND METHODS |
Materials.
Cell culture media were purchased from Life Technologies (Breda, The
Netherlands), human serum was provided by the Red Cross Blood Bank
Zuid-Limburg (Maastricht, The Netherlands), bovine brain extract was
from Boehringer (Mannheim, Germany), heparin was from ICN (Zoetermeer,
The Netherlands), and FCS was from Integro (Zaandam, The Netherlands).
Oregon green 488-labeled annexin V (Apoptest) was supplied by
Nexins Research (Hoeven, The Netherlands), and rhodamine-labeled
phalloidin was supplied by Molecular Probes (Leiden, The Netherlands).
tBH was obtained from Aldrich
(Milwaukee, WI). Other reagents, including
4',6-diamidino-2-phenylindole (DAPI) and
L-buthionine-[S,R]-sulfoximine
(BSO), were purchased from Sigma (St. Louis, MO);
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was a gift from
Bristol-Myers Squibb (Evansville, IN).
Cell culturing.
HUVEC were collected by trypsin digestion and cultured on
gelatin-coated culture dishes in 1 vol medium 199 and 1 vol RPMI 1640, supplemented with 20% (vol/vol) human serum, bovine brain extract (5 µg/ml), and heparin (10 U/ml), as described previously (39). Repetitions of experiments were always performed with HUVEC
derived from different umbilical cords
(passage
4). ECRF24 cells, kindly provided by
Dr. H. Pannekoek (Dept. of Biochemistry, Academic Medical Centre,
Amsterdam, The Netherlands), were grown in HUVEC
culture medium, but without bovine brain extract or heparin. ECV304
cells were obtained from the European Collection of Animal Cell
Cultures (Salisbury, UK) and cultured in medium 199 plus 10% (vol/vol)
FCS. All experiments were performed with endothelial cells in confluent monolayers.
Microscopic inspection and staining of
tBH-treated cells.
Confluent monolayers of endothelial cells grown in 12-well plates were
treated with 0.1-1 mM tBH in
serum-containing culture medium. Cells were monitored for membrane
blebbing every 10 min by phase-contrast microscopy. All experiments
were run in triplicate with cells in three different wells. Usually,
three independent experiments were performed in which, for HUVEC, a
different isolation was used for each experiment. The observer was
blind to the experimental condition. For F-actin and nuclear staining,
cells grown on gelatin-coated glass coverslips were washed with HEPES
buffer (pH 7.45), fixed with 1% (wt/vol) paraformaldehyde for 10 min,
washed with PBS, permeabilized with 0.02% (wt/vol) saponin, and
finally stained with rhodamine-labeled phalloidin (2.5 U/ml) for 45 min
in the dark. DAPI (62.5 µg/ml) was used for nuclear counterstaining. For quantification of actin rearrangements and nuclear condensation, two to three independent experiments were performed and for each experiment at least three microscopic fields were inspected. Confocal laser scanning microscopy was performed with a Bio-Rad MRC 600 system
as described elsewhere (24).
Markers of apoptosis and necrosis.
Endothelial cells were treated with
tBH or staurosporine in
serum-containing medium for 1 or 16 h in 12-well plates, after which
the medium (possibly containing floating cells) was removed and stored.
Cells were trypsinized, and then the corresponding medium was added
again. Cells pelleted by centrifugation were resuspended in 500 µl
HEPES buffer, pH 7.45, containing (in mM) 150 NaCl, 10 HEPES, 5 KCl,
1.8 CaCl2, 1 MgCl2,10
D-glucose, and 4 L-glutamic acid and 0.25%
(wt/vol) BSA. The surface expression of negatively charged PS in these
unfixed cells was determined by flow cytometry, by using as a probe
Oregon green 488-labeled annexin V (0.25 µg/ml) (17, 21). The
membrane-impermeable DNA stain propidium iodide (1 µg/ml) was
included in these experiments to determine membrane integrity as a
marker of cell vitality. Cells with a low level of propidium iodide
staining were defined to be vital; cells with a high level of annexin V
binding together with a low level of propidium iodide staining were
defined as apoptotic. Finally, all cells with a high level of propidium
iodide staining were defined as necrotic (36).
The fragmentation of DNA was measured as DNA hypodiploidy in
permeabilized, fixed cells. After being harvested by trypsinization, cells in suspension were fixed with 1% (wt/vol) paraformaldehyde for
10 min and then permeabilized with 0.02% (wt/vol) saponin for 5 min.
These cells were analyzed after the addition of propidium iodide (1 µg/ml), also with a flow cytometer (31).
Determination of GSH and total glutathione levels.
After incubation of the endothelial cells on six-well culture plates,
cellular proteins were precipitated by adding directly to the monolayer
500 µl of an ice-cold solution of 4% (wt/vol) TCA and 2 mM EDTA. The
culture plates were then incubated at 4°C for 15 min,
after which cell lysates were collected and centrifuged at 9,000 g for 5 min (4°C). The resulting
supernatants were used for the measurement of GSH and total
glutathione levels, as previously described (37). Glutathione levels
are expressed as percentages of the levels from control incubations.
Unstimulated HUVEC and ECRF24 and ECV304 cells each contained ~120
nmol total glutathione/mg cellular protein.
Inhibition of glutathione reductase levels.
Confluent monolayers of endothelial cells were incubated with specific
glutathione reductase inhibitor BCNU (50 µM) (37) in culture
medium. After 30 min the BCNU-containing medium was replaced by
fresh culture medium and the cells were allowed to recover for 2 h,
essentially as described previously (5). The BCNU-treated cells were
then used for other experimentation.
Determination of NADPH in endothelial cell monolayers.
Endothelial cells were cultured on gelatin-coated glass coverslips in
12-well culture plates. The coverslips were mounted in an open chamber
that was placed on the stage of a Diaphot inverted fluorescence
microscope (Nikon, Tokyo, Japan). Changes in autofluorescence, reflecting intracellular levels of NADPH (22a), in individual cells at
37°C were measured with a Quanticell fluorometric system (Applied
Imaging, Sunderland, UK), basically following previously described
procedures (17). Excitation and emission wavelengths were 340 and 510 nm, respectively.
 |
RESULTS |
Changes in actin cytoskeleton and cell morphology.
The effects of tBH on confluent
monolayers of endothelial cells in the continuous presence of
serum-containing culture medium were investigated. HUVEC were treated
with a low dose of 0.1 mM tBH, and a
microscopic inspection showed that this resulted, within 30 min, in the
development of plasma membrane blebs in, on average, 26% of the cells,
although there was considerable variation between cells derived from
different umbilical veins. After the cells were stained for F-actin to
visualize peroxide-induced changes in the actin cytoskeleton, it
appeared that, in the bleb-forming cells, the normal filamentous
pattern of F-actin had been completely changed into a pattern with
F-actin redistributed as a diffuse ring along the cellular border and
blebs (Fig. 1). The interior of the cell body and the membrane blebs were practically devoid of
F-actin. Counterstaining with DAPI indicated that most of the blebbing
cells also had a condensed nucleus (Fig.
2). Quantitative analysis revealed that the
number of cells with nuclear condensation was indeed similar to the
number of bleb-forming cells (Table 1). When a higher dose of 0.4 mM tBH was used, a larger fraction of
cells showed these morphological changes. The same effects on cellular
structure were observed when HUVEC were incubated with hydrogen
peroxide instead of tBH. For instance,
0.5 mM hydrogen peroxide induced membrane blebbing in 14.7 ± 4.6%
of total cells (mean ± SE; n = 5).
To study the dose dependency of this process, HUVEC were exposed to
various concentrations of tBH in
culture medium and visually checked for the appearance of membrane
blebs. As shown in Fig. 3, the blebbing
response was found to increase in the range of 0.1-1.5 mM
tBH, with almost all cells showing blebs at the highest concentration. Typically, the number of
peroxide-induced blebbing cells decreased considerably after longer
incubation times with lower peroxide concentrations, and the majority
of them regained normal morphology. For hydrogen peroxide stimulation (0.5 mM), 94.6 ± 3.8% (n = 4) of
the blebs had disappeared after 2 h of incubation, whereas with
tBH (0.1 mM) an incubation time of
4-8 h was needed to achieve this level of reduction. These results
strongly suggest that the bleb-forming process is reversible.

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Fig. 1.
Confocal laser scanning micrograph of stained F-actin in human
umbilical vein endothelial cell (HUVEC) treated with
tert-butyl hydroperoxide
(tBH). HUVEC grown to confluence were
incubated with 0.1 mM tBH at 37°C.
After 60 min of treatment, cells were fixed and stained for F-actin
with rhodamine-labeled phalloidin. A representative image of a blebbing
cell is shown. Note that blebbing cell has rounded up to a higher
plateau and is observed on top of surrounding cells. Bar, 8 µm.
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Fig. 2.
Nuclear condensation in blebbing endothelial cells. Photographs are
from 1 microscopic field observed for F-actin
(A) or DNA
(B) staining. HUVEC grown to
confluence were incubated with 0.1 mM
tBH for 60 min, fixed, and stained for
F-actin with rhodamine-labeled phalloidin and then counterstained for
DNA with 4',6-diamidino-2-phenylindole (DAPI). Note condensed DNA
of blebbing cell. Bars, 11 µm.
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Fig. 3.
Dose dependency of tBH-induced
membrane blebbing in HUVEC. Confluent monolayers of HUVEC were
incubated with 0.1-1.5 mM tBH in
culture medium (37°C). Cells were inspected by microscopy for
membrane blebbing for 10-60 min to determine maximal no. of
bleb-forming cells. Data are means ± SE,
n = 3.
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The application of 0.1 mM tBH to
ECRF24 cells had little effect on the cytoskeletal structure. However,
when these cells were subjected to five consecutive treatments with 0.1 mM tBH at 10-min intervals,
significant bleb formation was observed, again with F-actin aligning
along the blebbing structures (Fig. 4).
Under these conditions, ~10% of the cells showed blebbing and
nuclear condensation (Table 1). In contrast, ECV304 cells were unable to respond by bleb formation or nuclear condensation, not even after
five consecutive incubations with a high concentration of 1 mM
tBH (Table 1). Similarly, hydrogen
peroxide (1 mM) did not evoke these morphological responses in ECV304
cells (not shown). Together, these data indicate that HUVEC more
strongly than ECRF24 cells respond to peroxides by changes of cell
actin skeleton and structure, whereas ECV304 cells are remarkably
resistant in this respect.

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Fig. 4.
Confocal laser scanning micrograph of stained F-actin in ECRF24 cells
treated with tBH. Confluent ECRF24
cells were incubated 5 times with 0.1 mM
tBH, with 10-min intervals between
each incubation. After 60 min of treatment, cells were fixed and
stained for F-actin with rhodamine-labeled phalloidin. Representative
image of a blebbing cell is shown. Bar, 5 µm.
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Bleb formation in the absence of apoptosis.
The appearance of circular F-actin, membrane blebbing, and nuclear
condensation in the peroxide-treated HUVEC and ECRF24 cells closely
resembles the alterations in cell morphology in an early stage of
apoptosis (26, 27, 40). We therefore investigated whether
tBH was capable of initiating this
process in these endothelial cells by measuring two characteristic
apoptotic responses: appearance of PS at the plasma membrane outer
surface as an early marker (21, 35) and fragmentation of DNA to mark
more-advanced stages (31). For comparison, the cells were triggered
with the protein kinase inhibitor staurosporine, which is a well-known
inducer of apoptosis in endothelial cells (3).
The analysis of these responses was performed by harvesting the
activated cells from the monolayer by trypsinization and subjecting them to flow cytometry. The externalization of PS was routinely determined by analyzing the capability of unfixed cells to bind the
PS-specific probe Oregon green 488-labeled annexin V
(36). On the other hand, DNA ploidy in fixed,
permeabilized cells was separately measured with the DNA probe
propidium iodide (31). After 60 min of exposure of HUVEC (one
treatment) or ECRF24 cells (five treatments) to 0.1 mM
tBH, essentially no externalization of
PS or fragmentation of DNA was observed (Fig.
5,
A-D).
Nevertheless, microscopic inspection had shown that many of the cells
in the monolayer responded by membrane blebbing. A 60-min incubation with staurosporine (4 µM) resulted in PS externalization in some of
the ECRF24 cells but not in HUVEC, whereas effects on DNA ploidy were
not detected (Fig. 5, E and
F). Because apoptosis is a slowly developing process, the effects of these agonists were also determined after longer incubation periods. However, even after 16 h of treatment with this concentration of tBH,
neither in HUVEC nor in ECRF24 cells was significant PS externalization
or DNA hypodiploidy observed (Fig. 6,
A-D).
As expected, a 16-h incubation with staurosporine led to massive PS
exposure and DNA fragmentation in both HUVEC and ECRF24 cells (Fig. 6,
E and
F).

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Fig. 5.
Short-term effects of tBH on
phosphatidylserine (PS) exposure and DNA fragmentation in endothelial
cells. Confluent monolayers of HUVEC
(left) or ECRF24 cells
(right) remained untreated
(A,
B) or were incubated with
tBH (0.1 mM; 1 treatment for HUVEC and
5 treatments for ECRF24 cells) (C,
D) or 4 µM staurosporine
(E,
F). After 60 min of incubation,
cells were harvested and were either stained with Oregon green (OG)
488-labeled annexin V or, alternatively, fixed and stained with
propidium iodide. Fluorescence of collected cells was analyzed with a
flow cytometer. Lower channel nos. represent autofluorescence of
unlabeled cells (dotted lines); nos. in italics are percentages of
annexin V-positive cells. Results are from typical experiment
representative of 4-6 others.
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Fig. 6.
Long-term effects of tBH on PS
exposure and DNA fragmentation in endothelial cells. HUVEC
(left) or ECRF24 cells
(right) remained untreated
(A,
B) or were treated with
tBH
(C,
D) or staurosporine
(E,
F) during a period of 16 h. Further
conditions and analysis are as described for Fig. 5. Numbers in italics
are percentages of annexin V-positive cells. Results are representative
of those from 4-6 experiments.
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To confirm the suggestion that tBH did
not evoke apoptosis under the conditions employed, HUVEC and ECRF24
cells were subsequently treated with various
tBH concentrations and quantitatively
analyzed for fractions of vital, apoptotic, and necrotic cells, again
by flow cytometry. These experiments were carried out with unfixed cells that were double-stained with Oregon green 488-labeled annexin V
(to detect PS-exposing apoptotic and necrotic cells) and propidium iodide (to detect necrotic cells with increased membrane permeability). As shown in Table 2, in
either cell type, small fractions of apoptotic and necrotic cells were
detected in the absence of stimulation. Such low fractions of
"spontaneously" apoptotic cells have also been observed by others
and are in part due to the cell harvesting procedure applied (36).
Short or prolonged treatment with 0.1 mM
tBH did not influence the apoptotic or
necrotic cell fractions. However, treatment with a higher peroxide dose
of 0.4 mM, sufficient to cause blebbing in ~50% of HUVEC, resulted
in considerable loss in cell vitality (necrosis) but no more than
marginally increased the fraction of apoptotic cells (Table 2). From
these results, it was concluded that the
tBH-induced formation of blebs is not an initial sign of apoptosis.
Effects on glutathione oxidation.
To determine how the cell line-dependent variation in membrane blebbing
was related to the redox or sulfhydryl state of the cells, we compared
the effects of tBH on the
intracellular glutathione levels in HUVEC and ECRF24 and ECV304 cells.
For HUVEC, a single incubation with 0.1 mM
tBH immediately decreased the level of GSH to ~30% of the resting value, after which it was
slowly restored. After 60 min, a considerable part of the oxidized
glutathione still had not been regenerated as GSH (Table
3). Higher
tBH concentrations resulted in more
severe GSH oxidation (data not shown). When ECRF24 cells were used,
treatment with 0.1 mM tBH also
resulted in a pronounced decrease in GSH level but the decrease was
followed by a more rapid recovery, so that all glutathione had regained the reduced form within 60 min (Table 3). The repeated addition of 0.1 mM tBH to ECRF24 cells considerably
delayed this recovery process. At 60 min after five consecutive
tBH incubations, the GSH level still
reached only 52.9 ± 11.8% (mean ± SE;
n = 4) of the total glutathione
concentration. These data suggest that HUVEC differ from ECRF24 cells,
not so much in their GSH peroxidase reactivities (i.e., the enzymatic
reaction inactivating peroxides at the expense of GSH) but more in
their glutathione reductase capacities (by which oxidized glutathione
is reformed to GSH). It should be noted that, in both HUVEC and ECRF24
cells, the total glutathione concentration decreased gradually during
the incubation period (Table 3), possibly because of the efflux of
oxidized glutathione out of the cells, as has been reported for
hepatocytes (8).
In marked contrast to the two other cell types, incubation of ECV304
cells with 0.1 (not shown) or 1 mM tBH
(Table 3) was without significant effect on the total glutathione or
GSH concentration. Even after five additions of 1 mM
tBH, the GSH level was still 98.2 ± 4.9% of the total glutathione level (mean ± SE;
n = 6). Treatment of ECV304 cells with
the glutathione synthesis inhibitor BSO (25 µM for 20 h) resulted in
the expected depletion of the total glutathione concentration to 21.4 ± 2.4% (n = 4) of the control
value. Triggering the BSO-treated cells with 1 mM
tBH decreased the GSH level
insignificantly from 21.4% to 17.8 ± 4.9%. Thus ECV304 cells
appear to be remarkably resistant to GSH oxidation by peroxides. This
was confirmed by performing incubations with various concentrations of
hydrogen peroxide. For instance, 0.5 mM hydrogen peroxide led to a
level of GSH oxidation similar to that produced by 0.1 mM
tBH in HUVEC but did not change the
GSH level in ECV304 cells.
When we compare the data for the various endothelial cell
lines, we note a marked parallel in the effects of
tBH and hydrogen peroxide on
cytoskeleton-dependent changes in morphology (bleb formation and
nuclear condensation) and in their effects on the magnitude and
duration of GSH oxidation. Responsiveness to the peroxides was always
in the order HUVEC > ECRF24 cells > ECV304 cells. Further
experimentation was designed to better define the relationship between
the GSH-oxidizing and morphological effects of the peroxides.
Relation between glutathione oxidation and bleb formation.
To further investigate the role of GSH oxidation in
tBH-induced membrane blebbing, cells
were pretreated with the specific glutathione reductase inhibitor BCNU
(8, 37). When applied to monolayers of HUVEC or the cell lines, this
treatment was without effect on the total glutathione concentration.
For HUVEC and ECRF24 cells, the subsequent addition of
tBH caused a considerable reduction in
the GSH redox state, as expected. At the same time, the BCNU treatment
more than doubled the peroxide-induced blebbing response (Table
4). However, in ECV304 cells treated with
BCNU, tBH still did not lead to
notable GSH oxidation and membrane blebbing remained absent.
Significant GSH oxidation and bleb formation were also not detected in
ECV304 cells that were pretreated with the glutathione synthesis
inhibitor BSO, although the level of total glutathione was considerably
reduced in the cells (see above). In another series of experiments,
HUVEC and other cells were treated with the membrane-permeable
sulfhydryl agent N-ethylmaleimide (30 µM), an agent that oxidizes GSH and protein thiols in endothelial
cells (29). When applied to HUVEC, this treatment resulted in massive F-actin rearrangement and in membrane blebbing in 10.0 ± 1.0% of
the cells (mean ± SE; n = 3).
Similar blebbing morphology was observed when these cells were
incubated with another sulfhydryl reagent, diamide.
To explain why only part of the
tBH-treated cells in a monolayer
exhibit membrane blebbing under conditions of GSH oxidation, the effect
of peroxide on the autofluorescence of endothelial cells in the
monolayer was measured, the level of which largely reflects the
intracellular NADPH concentration (22a). The oxidation of NADPH is
indeed considered to be an adequate measure of the cellular redox state
(8, 33). Monitoring the fluorescence microscopic images from a
monolayer of HUVEC that were exposed to 0.1 mM
tBH revealed considerable cell-to-cell
variety in the peroxide-evoked decrease in autofluorescence. Within a
period of 15 min, the fluorescence of some, but not of all, cells
started to return to the original level (Fig.
7). These data thus point to a significant
intercellular variation in the degree and duration of NADPH (and thus
GSH) oxidation. Taken together, these results suggest that a change in
the redox state and/or the oxidation or thiolation of GSH is important
for the bleb-forming process, not the absolute level of
GSH.

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Fig. 7.
NADPH-oxidizing effect of tBH on
endothelial cells in monolayer. HUVEC on glass coverslips were
incubated with 0.1 mM tBH at 37°C.
NADPH autofluorescence was determined by fluorescence microscopic
imaging at excitation and emission wavelengths of 340 and 510 nm,
respectively. Shown are traces of autofluorescence of 2 individual
cells within 1 microscopic field. Later visual inspection showed
blebbing in ~15% of cells in this particular experiment. Data are
representative of those from 4 independent measurements.
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DISCUSSION |
In this paper, we contrast the responses of HUVEC and two HUVEC-derived
cell lines (ECRF24 and ECV304) to the glutathione-oxidizing compounds
tBH and hydrogen peroxide with regard
to membrane blebbing, actin cytoskeleton rearrangement, nuclear
condensation, and GSH oxidation. For HUVEC, the peroxides increased in
a dose-dependent way the number of cells undergoing bleb formation and
the other morphological changes. As far as we could detect, many or all of the blebbing cells contained an actin cytoskeleton located on the
surface and also a condensed nucleus (Table 1, Fig. 2). The time scale
of these changes was quite similar to that of the transient,
peroxide-induced oxidation of GSH (Table 3). Compared with HUVEC, the
cell lines, ECV304 more so than ECRF24, were more resistant to both the
morphological changes and GSH oxidation. Further confirmation of this
relation between peroxide-induced glutathione-oxidizing capacity and
membrane blebbing could be obtained by treating the cells with the
glutathione reductase inhibitor BCNU, which increased the bleb
formation and the degree of GSH oxidation in HUVEC and ECRF24 cells but
was unable to induce either response in ECV304 cells (Table
4).
Although the oxidative stress imposed by
tBH and hydrogen peroxide may evoke
apoptosis in various cells (4, 14, 32), the morphological changes
observed in the present study are unlikely to result from an apoptotic
process, because they were not accompanied by early markers of
apoptosis such as surface expression of PS or fragmentation of DNA.
This holds for HUVEC as well as for ECRF24 cells (Figs. 5 and 6). More
precisely, at lower peroxide concentrations, the number of blebbing
cells was found to decrease in time (suggesting reversibility of the
process), without indications of apoptosis (Table 2). On the other
hand, at higher peroxide doses, at which many of the cells showed
blebs, a considerable fraction of the cells became necrotic but not
apoptotic. Apparently, depending on the peroxide concentration,
blebbing endothelial cells either recover or die a nonapoptotic death.
We therefore conclude that, under the present experimental conditions,
1) the peroxide-induced morphological changes correlate with the degree of GSH oxidation but
are not early indications of apoptosis and
2) HUVEC and the HUVEC-derived cell
lines are fairly resistant to apoptosis.
There is only limited literature directly comparing the properties of
ECRF24 and ECV304 cells with those of the parental cells. ECRF24 was
produced by transfection of HUVEC with the E6/E7 open reading frame of
human papilloma virus 16 (10), whereas ECV304 is the result of a
spontaneous HUVEC transformation (34). The ECRF24 line closely
resembles HUVEC with respect to cell morphology, growth
characteristics, and expression of von Willebrand factor and selective
surface adhesion molecules (10). Similarly, the ECV304 line possesses
many HUVEC-like characteristics in terms of morphology and signal
transduction mechanisms, although a certain degree of dedifferentiation
may have occurred (1, 11, 18). From the present results, it is likely
that these cell lines differ from HUVEC in their capabilities to
detoxify peroxides by using the redox equivalents from intracellular
GSH. We hypothesize that this difference is responsible for the
decreased blebbing response. However, the possibility that the cell
lines lack one or more of the other intracellular signaling modules
that are unrelated to glutathione metabolism and that participate in
peroxide-induced cytoskeleton rearrangement and the blebbing response
cannot be excluded. Indeed, a wide variety of peroxide-induced
signaling pathways that may or may not be influenced by the glutathione state of the cells have been described (see
INTRODUCTION). It is thus still
possible that the processes of bleb formation and GSH oxidation are
epiphenomena without a causal relationship. This might also explain why
not all cells start to bleb in response to low peroxide concentrations,
whereas most (or all) of them will undergo a change in their GSH
levels. However, it is also possible that both the degree and time
period of GSH oxidation may determine the blebbing phenomenon. In
agreement with this is the observation that within a single endothelial
monolayer, considerable cell-to-cell heterogeneity in the degree of
peroxide-induced NADPH oxidation was observed (Fig. 7). Because the
peroxide-induced oxidation of GSH and NADPH points to a changed
intracellular redox state, it might well be that such an altered state,
rather than a direct effect of oxidized GSH, is affecting the cell morphology.
The present results differ from those of others, who report that mild
oxidative stress such as that evoked by peroxide triggers the apoptotic
process in endothelial cells (7, 22, 32). It should be noted that our
experiments were performed with cells that were treated with
tBH or hydrogen peroxide in the
continuous presence of serum with growth factors; most of the other
reported studies have been performed with cells activated in serum
(albumin)-free media. There are good indications that serum deprivation
induces apoptosis in endothelial cells (41), whereas growth factor
supplementation protects the cells from entering this process (13). At
the molecular level, it has been proposed that the antiapoptotic effect
of serum (albumin) and growth factors results from a cross talk between stress-stimulated protein kinase pathways (proapoptotic) and parallel growth factor-activated survival pathways (antiapoptotic) (2, 20). In
this model, the final effect of stress activators such as peroxide
would be influenced by the activity of the survival pathways. Thus
peroxide application to endothelial cells that are maintained on serum
might cause a mild stress response such as membrane blebbing (possibly
by a changed GSH oxidation state) without trapping the cells into an
apoptotic program.
Although the endothelial cells in the inner walls of blood vessels can
easily become exposed to peroxides under stress conditions, such as
those produced by activated neutrophils (25), the present data suggest that apoptosis is not a likely result under such in vivo
conditions. On the other hand, the peroxide-evoked bleb formation may
seriously impede the interface function of endothelial cells in the
vessel wall, because it is necessarily accompanied by cell retraction
and thus by the increased permeability of the endothelium.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H. Pannekoek for a gift of the endothelial cell line
ECRF24 and Dr. W. M. J. Vuist for stimulating discussions.
 |
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"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. M. A. van
Gorp, Dept. of Human Biology, Maastricht Univ., PO Box 616, 6200 MD
Maastricht, The Netherlands (E-mail:
R.vanGorp{at}hb.unimaas.nl).
Received 14 July 1998; accepted in final form 19 March 1999.
 |
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