Hydrogen Peroxide Induces Up-Regulation of Fas
in Human Endothelial Cells
This information is current as
of June 16, 2017.
Toshimitsu Suhara, Keisuke Fukuo, Tomosada Sugimoto, Shigeto
Morimoto, Takeshi Nakahashi, Shigeki Hata, Masumi Shimizu
and Toshio Ogihara
J Immunol 1998; 160:4042-4047; ;
http://www.jimmunol.org/content/160/8/4042
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References
Hydrogen Peroxide Induces Up-Regulation of Fas in Human
Endothelial Cells
Toshimitsu Suhara,* Keisuke Fukuo,1* Tomosada Sugimoto,† Shigeto Morimoto,*
Takeshi Nakahashi,* Shigeki Hata,* Masumi Shimizu,* and Toshio Ogihara*
F
as, also called APO-1 or CD95, is a type I membrane
protein belonging to the TNF and nerve growth factor receptor family, which mediates a death signal (1). Triggering this pathway requires the cross-linking of Fas with either agonistic Abs to Fas or cells expressing Fas ligand (Fas-L).2 Various
cells express Fas, whereas Fas-L is expressed predominantly in
activated T cells. Malfunction of the Fas system causes lymphoproliferative disorders and accelerates autoimmune diseases,
whereas its overactivity may cause tissue destruction. Therapeutic
uses of the Fas system might include blocking the exacerbated
Fas-based pathologic manifestations with the soluble form of Fas,
neutralizing Abs to Fas or Fas-L, or with inhibitors of Fas-L induction or Fas-mediated apoptosis.
Injury of endothelial cells (ECs) is a critical event in the acute
inflammatory process and the development of atherosclerosis (2,
3). In the genesis of inflammatory lesions, ECs can interact with
macrophages, platelets, and vascular smooth muscle cells (VSMC)
as well as T cells. One form of injury to ECs results from excessive
levels of oxygen radicals released from leukocytes, macrophages,
and ECs themselves (4 –10). Increasing evidence suggests that oxidative stress is a mediator of apoptosis (11). This hypothesis is
based on the findings that many agents that induce apoptosis are
oxidants or stimulators of cellular oxidative metabolism, and that,
conversely, many inhibitors of apoptosis have antioxidative activ*Department of Geriatric Medicine, Osaka University Medical School, Suita, Osaka,
Japan; and †2nd Department of Oral Anatomy, Okayama University Dental School,
Okayama, Japan
Received for publication May 28, 1997. Accepted for publication December 17, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Keisuke Fukuo, Department of
Geriatric Medicine, University Medical School, 2-2 Yamadaoka, Suita, Osaka 565,
Japan. E-mail address: [email protected]
2
Abbreviations used in this paper: Fas-L, Fas ligand; ECs, endothelial cells; NO,
nitric oxide; VSMCs, vascular smooth muscle cells; NF-kB, nuclear factor-kB; AP-1,
activator protein 1; H2O2, hydrogen peroxide.
Copyright © 1998 by The American Association of Immunologists
ities or enhance cellular antioxidant defenses (12–17). Furthermore, there is recent evidence that diffusable reactive oxygen intermediates such as nitric oxide (NO) and hydrogen peroxide
(H2O2) can modulate cellular functions through altering signal
transduction in many cell types, including ECs, VSMC, and T cells
(18 –25). Recently, we demonstrated that NO induces up-regulation of Fas and apoptosis in VSMC (26). In the present study, we
therefore examined whether H2O2 can affect the level of Fas in
cultured ECs. We found that H2O2 up-regulates Fas expression
through the activation of protein tyrosine kinase in ECs.
Materials and Methods
Materials
mAbs against human Fas, clone UB2 (IgG) and clone CH11 (IgM) were
from Medical Biologic Laboratories (Nagoya, Japan). FITC-labeled goat
anti-mouse IgG was obtained from Seikagaku (Tokyo, Japan). Catalase and
genistein were purchased from Sigma (St. Louis, MO). Sodium pervanadate and propidium iodide were from Wako Pure Chemical Industries
(Osaka, Japan).
Cell culture
HUVEC were purchased from Kurabo (Osaka, Japan) and maintained in
medium (HuMedia-EG; Kurabo) containing 2% FCS, 10 mg/ml heparin, 5
ng/ml recombinant acidic fibroblast growth factor, 10 ng/ml recombinant
acidic endothelium growth factor, and antibiotics. Cultured cells were identified as ECs on the basis of typical morphology and factor VIII immunofluorescence. ECs (passage 3–7) were grown to confluence on 10-cm
dishes with medium. For experiments, medium was replaced with fresh
medium without serum and growth factors before the addition of
compounds.
Detection of Fas by flow cytometry
After treatment with various compounds, floating cells were removed by
rinsing the cell layers with PBS containing 0.2 mM EDTA, and then adherent cells were harvested with trypsin to analyze Fas expression. ECs
(106) were incubated with PBS containing 5% FCS and 10 mg/ml murine
Ab against Fas (UB2) for 1 h at 4°C, washed with PBS three times, and
then incubated with 10 mg/ml FITC-conjugated goat anti-mouse IgG for 30
min at 4°C. Fas expression on the cell surface was analyzed by a method
using a flow cytometer (FACS) on FL-1 channel.
0022-1767/98/$02.00
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Hydrogen peroxide (H2O2), an oxidant generated by inflammatory cells, is an important mediator of injury of endothelial cells
(ECs). Here we show that H2O2 induces up-regulation of the expression of Fas, a death signal, in human ECs in culture. Flow
cytometric analysis with a mAb against human Fas showed that incubation for 24 h with H2O2 induced a dose-dependent increase
in the level of Fas in ECs. Coincubation with catalase, which rapidly degrades H2O2, inhibited H2O2-induced up-regulation of Fas.
H2O2 also induced a dose-dependent increase in Fas mRNA level. A significant increase in Fas mRNA levels was observed from
6 h after stimulation with H2O2. Vanadate, a protein phosphatase inhibitor, significantly enhanced Fas mRNA and protein levels
in H2O2-treated ECs. On the other hand, genistein, a tyrosine kinase inhibitor, inhibited H2O2-induced Fas mRNA expression.
Furthermore, a flow cytometric method with propidium iodide staining and electron microscopic analysis showed that incubation
with an agonistic Ab against Fas (anti-Fas IgM) induced apoptosis in H2O2-treated cells. These findings suggest that H2O2 induces
up-regulation of Fas in ECs and that activation of protein tyrosine kinase may be involved in the mechanism of H2O2-induced Fas
expression. Therefore, Fas-mediated apoptosis may have a pathologic role in H2O2-induced EC injury and thereby provide a new
therapeutic target. The Journal of Immunology, 1998, 160: 4042– 4047.
The Journal of Immunology
4043
Analysis of Fas mRNA
Total RNA from ECs was extracted by a guanidine isothiocynate/acid phenol method (27). Poly(A)1 RNA was prepared using Oligo(dT)-Latex
(Takara Biomedicals, Japan). Northern blot analysis was performed as previously described (28). The probe DNA of Fas was a 2.5-kbp XhoI fragment containing human Fas cDNA (29). The cDNA probes for human Fas
and human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were labeled with [32P]dCTP (111 TBq/mmol) by using the multiprime DNA labeling kit (Amersham International, Buckinghamshire, U.K.). Hybridization with a G3PDH cDNA probe was used to monitor uniform loading of
RNA on Northern blots.
DNA analysis of apoptosis by flow cytometry
Apoptosis was monitored by measuring the population distribution of DNA
content (30). After treatment with H2O2 and anti-Fas IgM, ECs (106) were
suspended in 100 ml of PBS and fixed with 900 ml of cold ethanol and then
resuspended in staining buffer (1 mg/ml RNaseA, 20 mg/ml propidium
iodide, 0.01% Nonidet P-40). The DNA content of the cells was analyzed
by flow cytometry on FL2 channel.
Electron microscopy
Cells were fixed with 1% glutaraldehyde, 1% formaldehyde (prepared fresh
from paraformaldehyde), and 0.2 mM CaCl2 in 0.12 M phosphate buffer
(pH 7.3) for 5 to 30 min, osmicated in phosphate-buffered 2% OsO4 for 5
min, dehydrated in a graded series of alcohol, and embedded in epoxy
resin. After polymerization of the resin, the culture dish was removed from
the epoxy resin block. The blocks were cut into thin sections with a diamond knife and examined after contrasting with uranyl acetate and lead
citrate.
increase in Fas expression in ECs. However, catalase (920 U/ml), an
enzyme that hydrolyzes H2O2 to O2 and H2O (31), inhibited the upregulation of Fas induced by 0.5 mM H2O2 (Fig. 1D).
Expression of Fas mRNA in H2O2-treated ECs
Figure 2A shows the time-dependent effect of H2O2 at a concentration of 0.5 mM on the expression of Fas mRNA in ECs. Fas
mRNA was detected in control cells. Two bands of 2.7 and 1.9 kb
were detected in Fas mRNA in ECs. A significant increase in Fas
mRNA level was observed from 6 h after stimulation with H2O2.
As shown in Figure 2B, incubation for 12 h with H2O2 at concentrations from 0.2 to 1.0 mM induced a dose-dependent increase in
Fas mRNA level in ECs. Coincubation with catalase inhibited the
up-regulation of Fas mRNA expression induced by 0.5 mM H2O2.
Vanadate enhances H2O2-induced Fas expression
Next, we examined whether activation of protein tyrosine kinase is
involved in the mechanism of H2O2-induced up-regulation of Fas
expression. As shown in Figure 3A, incubation with 100 mM vanadate, a phosphatase inhibitor, significantly enhanced H2O2-induced Fas expression. However, the basal level of Fas was not
affected by vanadate alone (Fig. 3B). Vanadate also enhanced the
increase in Fas mRNA level induced by H2O2 (Fig. 4A). On the
other hand, incubation with genistein, an inhibitor of protein tyrosine kinase, significantly inhibited H2O2-induced up-regulation
of Fas mRNA expression (Fig. 4B).
Results
H2O2-induced Fas can mediate apoptosis
Induction of Fas in H2O2-treated ECs
We next examined whether H2O2-induced Fas can mediate apoptosis. A flow cytometric method with propidium iodide staining
was used for quantitating endonucleolytic cleavage of DNA in
cells undergoing apoptosis. ECs in control culture demonstrated
Figure 1 shows the effect of H2O2 on Fas expression in HUVEC
determined by flow cytometric analysis. Incubation for 24 h with
H2O2 at concentrations from 0.2 to 1.0 mM induced a dose-dependent
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FIGURE 1. H2O2 induces up-regulation of Fas in ECs. ECs were incubated
for 24 h with H2O2 at concentrations of
0.2 mM (A), 0.5 mM (B), and 1.0 mM
(C). Fas expression was then analyzed
by a flow cytometer (FACS) as described in the text. The histograms obtained from each treatment are shaded.
A solid line denotes a histogram of cells
without H2O2 treatment, which shows
the basal level of Fas in control ECs.
Panel D shows Fas expression in ECs
treated with H2O2 (0.5 mM) and catalase (920 U/ml) for 24 h. A dashed line
represents a histogram of ECs treated
with 0.5 mM H2O2 alone.
4044
normal diploid DNA content with DNA peaks of G1 and G2-M.
The apoptotic population was small (13.4%) (Fig. 5A). Incubation
for 24 h with H2O2 (0.5 mM) alone did not induce a significant
change in the apoptotic population (17.6%) compared with control
cells (Fig. 5B). On the other hand, incubation for 8 h with anti-Fas
IgM induced a large hypodiploid population (41.8%) undergoing
apoptosis with less than 2N DNA in H2O2-pretreated cells (Fig.
5C). However, anti-Fas IgM did not induce a significant change in
the apoptotic population in cells without pretreatment with H2O2
(12.1%) (Fig. 5D). Furthermore, electron microscopic analysis
showed typical morphologic changes of apoptosis such as cellular
shrinkage, membrane blebbing, and chromatin condensation in
most cells treated with H2O2 and anti-Fas IgM (Fig. 6, A and B).
In addition, pretreatment with H2O2 at concentrations from 0.2 to
1 mM induced a dose-dependent enhancement of anti-Fas IgMinduced apoptosis in ECs, although H2O2 at a high concentration
of 1 mM alone induced apoptosis (Fig. 7). These findings indicate
that H2O2-induced Fas is functional in mediating apoptosis in ECs.
FIGURE 3. Effect of vanadate on Fas expression. Panel A shows the relative expression of
Fas on ECs coincubated with both H2O2 (0.5
mM) and vanadate (0.1 mM) for 24 h. Panel B
shows Fas expression on ECs coincubated with
vanadate alone. Histograms obtained with each
treatment are shown in broad lines. Histograms
of controls are shown in solid lines, and dashed
lines represent histograms of ECs treated with 0.5
mM H2O2.
FIGURE 4. Effect of vanadate on H2O2-induced Fas mRNA in ECs. A,
ECs were incubated with 0.5 mM H2O2 for 12 h in the presence or absence
of 0.1 mM vanadate. B, ECs were incubated for 6 h with 0.5 mM H2O2 in
the presence or absence of 0.1 mM genistein. Northern blot analysis was
performed as described in the text.
Discussion
In this study, we demonstrated that H2O2 induces up-regulation of
Fas in ECs. H2O2 is one of the most important antimicrobial and
antitumor weapons of polymorphonuclear leukocytes (32). H2O2
has also been implicated in cellular and tissue injury in pathologic
conditions, for example, ischemia-reperfusion injury, hyperoxia,
and inflammation (33–38). In ischemia-reperfusion injury, interaction of stimulated polymorphonuclear leukocytes with ECs is
involved in its pathogenesis. Thus, ECs lining blood vessels appear
to be the initial target of activated polymorphonuclear cell-derived
oxidants. In addition, stimulation of various cells with either cytokines, phorbol esters, or growth factors increases the secretion of
H2O2 in the extracellular space in vitro (39 – 43). High concentrations of this diffusable reactive oxygen intermediate exert toxic
effects on susceptible cells. However, low concentrations of H2O2
alter cellular functions by modulating signal transduction in certain
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FIGURE 2. Fas mRNA expression in ECs treated with H2O2. A,
Poly(A)1 RNA was prepared from ECs incubated with H2O2 (0.5 mM) for
the indicated times before harvest. B, Poly(A)1 RNA was prepared from
ECs treated for 12 h with H2O2 at the indicated concentrations in the
presence or absence of catalase (920 U/ml).
Fas UP-REGULATION BY HYDROGEN PEROXIDE
The Journal of Immunology
4045
cells, including ECs, in vitro (20 –25). Although it has been shown
that activated polymorphonuclear leukocytes can generate H2O2
concentrations of up to 0.2 mM in vitro, and cigarette smoke
passed through saline yields H2O2 concentrations of 0.05 to 0.1
mM (9, 44 – 45), it is unknown how much H2O2 is actually produced in inflammatory lesions in vivo. In addition, inactivation of
the antioxidative pathway in a pathologic state may enhance the
effect of H2O2 on ECs. For example, recent evidence has shown
that NO, another diffusable reactive oxygen intermediate, inhibits
glutathione peroxidase, an antioxidative enzyme that scavenges
various peroxides, and increases the levels of intracellular peroxide
(46). Since inducible NO synthase, which produces high levels of
NO, is up-regulated in cytokine-activated cells and hypoxia/reperfusion tissues (47, 48), it may be possible that the concentration of
H2O2 is locally much higher in inflammatory lesions.
Since it has been reported that H2O2 is a potent inducer of many
biologic factors, including platelet-activating factors (49) and vascular endothelial growth factor (50), we examined whether H2O2induced Fas expression is mediated by a new protein synthesis.
However, H2O2-induced up-regulation of Fas mRNA expression
occurred in the presence of the protein synthesis inhibitor cycloheximide (data not shown), indicating that de novo protein synthesis was not required and suggesting a direct involvement of
H2O2. Exogenously added H2O2 can freely diffuse across cell
membranes (31) and induce tyrosine phosphorylation in several
cell types (51, 52). We observed that the tyrosine phosphatase
inhibitor vanadate enhanced H2O2-induced up-regulation of Fas
expression. It should be noted that the combination of H2O2 and
vanadate generates the compound pervanadate, biologic activity of
which has been demonstrated to be far greater than that of vanadate, and which strongly enhances tyrosine phosphorylation (53).
Furthermore, genistein, an inhibitor of tyrosine kinase activity, inhibited H2O2-induced up-regulation of Fas mRNA expression.
These findings suggest that H2O2 induces up-regulation of Fas
expression by increasing tyrosine kinase activity in ECs. Many of
the oxidant-sensitive genes have nuclear factor (NF)-kB or activator protein 1 (AP-1) regulatory elements in their promoter regions, and increased binding of endothelial proteins to both types
of elements has been reported after oxidant stimuli (54 –56). Furthermore, H2O2 increases nuclear levels of NF-kB and AP-1
through a tyrosine kinase-dependent mechanism in ECs (57) and
lymphocytes (58). In this respect, it is of interest to find regulatory
elements matching the reported consensus sequences for NF-kB
and AP-1 within the 59 flanking sequence of the human Fas
gene (59 – 61).
Although EC apoptosis is important in normal tissue homeostasis and during development (62– 64), it is also present during remodeling of damaged tissues (65– 67). Sgonc et al. (68) recently
reported that EC apoptosis is a primary pathogenic event underlying skin lesions in avian and human scleroderma. Furthermore,
Laurence et al. (69) also reported that Fas-mediated apoptosis in
ECs may be of pathophysiologic importance in thrombotic thrombocytopenic purpura. In our studies, pretreatment with H2O2 induced a dose-dependent enhancement of apoptosis induced by an
agonistic Ab to Fas (anti-Fas IgM). These results suggest that
H2O2-induced Fas is functional to induce apoptosis in EC. On the
other hand, it should be noted that Richardson et al. reported that
IFN-g-treated ECs did not undergo apoptosis upon Fas ligation
(70). We observed that anti-Fas IgM did not induce apoptosis in
control ECs. The lack of anti-Fas IgM-induced apoptosis in control
ECs that express some baseline Fas mRNA may relate to threshold
levels of Fas protein required for cross-linking. However, it is also
possible that H2O2 treatment may sensitize ECs against Fas-induced apoptosis. In this regard, it is noteworthy that c-myc-induced
apoptosis was recently shown to require interaction of Fas and
Fas-L by sensitizing cells to the Fas death signal in 3T3 fibroblasts
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FIGURE 5. Flow cytometric DNA analysis
of the effects of H2O2 and anti-Fas IgM on ECs.
After incubation with anti-Fas IgM, ECs were
fixed with ethanol and stained with 20 mg/ml of
propidium iodide. A, ECs were incubated in serum-free medium for 24 h. B, ECs were incubated for 24 h with 0.5 mM H2O2, rinsed with
fresh medium, and then incubated for a further
8 h with vehicle without H2O2. C, ECs were
preincubated for 24 h with 0.5 mM H2O2,
rinsed with fresh medium, and then incubated
for a further 8 h with 1.0 mg/ml of anti-Fas IgM
without H2O2. D, ECs were incubated for 24 h
with serum-free medium and then incubated for
a further 8 h with 1.0 mg/ml of anti-Fas IgM.
4046
Fas UP-REGULATION BY HYDROGEN PEROXIDE
(71). In addition, we also observed that H2O2 at a high concentration of 1 mM alone induced apoptosis in ECs. Our preliminary
experiments showed that apoptosis induced by 1 mM H2O2 was
partially inhibited by coincubation with a neutralizing Ab to Fas-L
(data not shown), suggesting that up-regulation of Fas-L may also
participate in the mechanism of H2O2-induced apoptosis in ECs
(72). Taken together, these findings suggest that Fas-mediated apoptosis in ECs may contribute to the mechanism of H2O2-induced
tissue injury and the extravasation of inflammatory cells. Therefore, it will be of interest to examine whether inhibitors of Fasmediated apoptosis (73) may be clinically useful in preventing
inflammatory cell-mediated tissue injury.
Acknowledgments
We thank Dr. S. Nagata for providing human Fas cDNA, Taeko Kaimoto
for technical assistance, and Tomoko Adachi for secretarial assistance.
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FIGURE 6. Electron microscopic appearance of ECs after treatment
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without pretreatment with H2O2. B, ECs were incubated for 24 h with H2O2
(0.5 mM) and then treated with anti-Fas IgM for 8 h.
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