© 2000 American Heart Association, Inc.
Integrative Physiology |
Hypoxia-Induced Endothelial Apoptosis Through Nuclear Factor-
B (NF-B)–Mediated bcl-2 Suppression
In Vivo Evidence of the Importance of NF-B in Endothelial Cell Regulation
From the Department of Geriatric Medicine (H.M., R.M., M.A., H.N., Y.T., K. Yamamoto, J.H., T.O.) and Division of Gene Therapy Science (R.M., T.N., K. Yasafumi), Osaka University Medical School, Suita 565, Japan.
Correspondence to Ryuichi Morishita, Associate Professor, Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. E-mail morishit{at}geriat.med.osaka-u.ac.jp
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Abstract |
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Abstract—The transcription factor nuclear factor-
B (NF-B) plays a pivotal role in the coordinated
transactivation of cytokine and adhesion molecule genes
involved in endothelial activation. Although recent
reports have documented the contribution of NF-B to
apoptosis, it is still controversial. Especially, the role of
NF-B in endothelial apoptosis is largely
unknown. Hypoxia significantly induced human aortic
endothelial cell death and apoptosis in a
time-dependent manner (P<0.01), accompanied by NF-B
activation. Decrease in total cell number and increase in
apoptotic cells induced by hypoxia were significantly
attenuated by NF-B decoy, but not by scrambled decoy,
oligodeoxynucleotides (ODNs) (P<0.01).
Increase in DNA fragmentation induced by hypoxia was also
significantly inhibited by NF-B decoy ODNs as compared with
scrambled decoy ODNs (P<0.01). Moreover, transfection
of NF-B decoy ODNs resulted in a significant decrease in
caspase-3–like activity, which is a common pathway for
apoptosis, compared with scrambled decoy ODNs. Importantly,
transfection of NF-B decoy ODNs significantly increased protein of
bcl-2, an inhibitor of apoptosis, and did not alter
bax, a promoter of apoptosis, thereby resulting in a
significant increase in the ratio of bcl-2 to bax
(P<0.01). bcl-2 mRNA was also decreased by
hypoxia, whereas transfection of NF-B decoy ODNs
significantly attenuated decrease in bcl-2 mRNA. These results
demonstrate that activation of NF-B by hypoxia induced
endothelial apoptosis in a bcl-2–dependent
manner. The importance of NF-B in endothelial
apoptosis was confirmed by the observation that pyrrolidine
dithiocarbamate, a potent NF-B inhibitor,
prevented endothelial apoptosis, caspase
3–like activity, and bcl-2 downregulation induced by hypoxia.
To test this hypothesis in vivo, we transfected NF-B decoy ODNs into
rat intact carotid artery after reperfusion injury. Reperfusion injury
was associated with a significant increase in
endothelial apoptosis at 24 hours, whereas
NF-B decoy ODN treatment markedly decreased terminal
deoxynucleotidyltransferase–mediated
dUTP nick end labeling (TUNEL)–positive endothelial
cells at 24 hours after reperfusion (P<0.01). Here,
using synthetic double-stranded DNA with high affinity for NF-B as a
decoy approach, we demonstrated that activation of NF-B by
hypoxia caused aortic endothelial cell death
and apoptosis through the suppression of bcl-2.
NF-B–mediated endothelial apoptosis induced
by hypoxia may be involved in the pathogenesis of
endothelial dysfunction observed in
cardiovascular ischemic diseases.
Key Words: hypoxia • nuclear factor-B • bcl-2 • bax • decoy
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Severe damage to endothelial cells induced by a complex interaction of multiple cytokines and adhesion molecules has been postulated to be responsible for cardiovascular disease such as myocardial infarction and atherosclerosis.1 2 3 4 5 Its process would be largely dependent on the coordinated activation of a series of cytokine and adhesion molecule genes that results in the adhesion of leukocytes and release of cytotoxic molecules. Importantly, a critical element in the regulation of these genes involves the complex formed by nuclear factor-
B (NF-B) and
inhibitory B.6 Dissociation of the
transcription factor NF-B from this complex is proposed to play a
pivotal role in the regulation of ischemic changes by inducing
a coordinated transactivation of genes involved in these processes,
including interleukins 1, 6, and 8; intercellular adhesion molecule;
vascular cell adhesion molecule; and endothelial
leukocyte adhesion molecule, to name a few.6 To
investigate the role of NF-B in the pathogenesis of myocardial
injury after reperfusion, we utilized synthetic double-stranded
oligodeoxynucleotides (ODNs) as "decoy" cis
elements that block the binding of nuclear factors to promoter regions
of targeted genes, resulting in the inhibition of gene
transactivation.7 8 9 Indeed, transfection of a
sufficient quantity of decoy ODNs containing the NF-B cis
element into coronary artery effectively prevented
transactivation of the gene expression of essential cytokine
and adhesion molecule proteins and, thereby, protected the
myocardium from infarction in vivo.10
From the previous study, we postulated that prevention of myocardial
infarction by NF-B decoy would be due to the blockade of
transactivation of cytokines and adhesion molecules.
On the other hand, recent studies demonstrated more direct
actions of NF-B in the process of apoptosis in various
cells.11 12 13 Proliferation and cell death are considered
to be two mechanically related phenomena. According to this view, cells
are programmed to commit suicide by default and require specific
extracellular factors to survive.14 A number of studies
suggest that cell survival is regulated by (1) triggering of specific
signaling pathways, (2) modulation of the activity of
antiapoptotic molecules, and (3) inhibition of cell death
effectors.15 Because endothelial cells
work as a biological barrier to prevent the migration of neutrophils
and other blood components that are essential to promote myocardial
injury, it is extremely important to elucidate the molecular mechanisms
of endothelial cell death in response to
hypoxia for understanding the pathogenesis of
cardiovascular disease. In addition, although
hypoxia has been reported to cause apoptosis, its exact
mechanisms are not yet clarified. Thus, it is valuable to investigate
the molecular mechanisms by which human endothelial
cells undergo apoptosis in response to hypoxia. As the
mechanisms responsible for endothelial cell death
induced by hypoxia remain enigmatic, we have addressed the
following specific questions: (1) how endothelial cells
undergo apoptosis in response to hypoxia and (2) how
NF-B acts as a proapoptotic factor against
endothelial cell death induced by hypoxia.
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Materials and Methods |
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In Vitro Experiments
Using human aortic endothelial cells, hypoxia was induced with BBL GasPak (Becton Dickinson), which catalytically reduces O2 to undetectable levels under severe conditions within 90 minutes.16 In preparation for experiments for detection of cell death, endothelial cells were grown to confluence. After reaching confluence, medium was changed to fresh, defined serum-free medium.17 On day 2, an index of cell proliferation was determined using sulfonated tetrazolium salt, WST-1.18 As an assay of cell death by apoptosis, we used fluorescent DNA-binding dyes to define nuclear chromatin morphological features as a quantitative index of apoptosis within the cell culture system.19 20 Using membrane-permeable (H33342) dye in the assay allowed the determination of cell viability and plasma membrane integrity and an accounting of any nonapoptotic toxic or necrotic death induced in the study groups. Also, we used the measurement of cellular DNA fragmentation using a cellular DNA fragmentation ELISA kit (Boehringer Mannheim) to quantity apoptosis.21 Measurement of caspase-3–like activity was also performed.
For in vitro transfection of NF-B or scrambled decoy ODNs,
endothelial cells were treated with hemagglutinating
virus of Japan (HVJ)-cationic liposome complex (100 µL). After
incubation, the medium was changed to fresh medium containing 0.5%
serum, and cells were incubated under hypoxic conditions. Cell counting
and staining for apoptosis were performed on day 3 after
transfection. For analysis of NF-B activity, nuclear extract
was prepared from cultured endothelial cells for gel
mobility shift assay.10 NF-B decoy ODNs were labeled as
a probe at the 3' end by the 3' end-labeling kit. For the competition
assay, unlabeled double-stranded NF-B or scrambled ODNs were
preincubated with a parallel sample 10 minutes before the addition of
the labeled probe. Western blotting was performed for analysis
of bax and bcl-2 proteins at 3 days after transfection. bcl-2 mRNA
level was also examined using Northern blot analysis.
In Vivo Experiments
All surgical procedures were performed according to the
Principles of Laboratory Animal Care and the Guide for
the Care and Use of Laboratory Animals. The left common carotid
artery of male Sprague-Dawley rats was surgically
exposed.22 23 For the reperfusion injury model, the
carotid artery was temporarily ligated. After 30 minutes of occlusion,
the temporary ligature on the common carotid artery was released and
blood flow was restored. Ten minutes before release (20 minutes after
occlusion), transfection of HVJ-liposome complex containing (1) NF-B
decoy ODNs (15 µmol/L) or (2) scrambled decoy ODNs (15
µmol/L) was performed. Then, 200 µL of HVJ-liposome complex was
infused into the segment and incubated for 10 minutes at room
temperature. After 10 minutes of incubation (total 30 minutes of
occlusion), the infusion cannula was removed. The vessel was excised 24
hours after reperfusion. Quantification of apoptotic
endothelial cells was performed by terminal
deoxynucleotidyltransferase–mediated
dUTP nick end labeling (TUNEL) staining. Frequency of apoptotic
cells was expressed as TUNEL index, defined as the ratio of the number
of TUNEL-positive nuclei to that of all the nuclei in the media. All
values are expressed as mean±SEM. ANOVA with subsequent Duncan test
was used to determine the significance of differences in multiple
comparisons.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Vitro Experiments
Effects of NF-
B Decoy ODNs on Hypoxia-Induced Cell
DeathInitially, we examined the effects of hypoxic treatment on human endothelial cell growth. After 12 hours under hypoxic conditions, some cells started to become round and eventually detached from the plate and floated in the medium, leaving many holes in the confluent cells. Cell number under hypoxic conditions decreased significantly in a time-dependent manner (P<0.01), as shown in Figure 1A
. Accordingly,
apoptotic cells also increased significantly in response to
hypoxia as assessed by nuclear staining with Hoechst 33342
(Figure 1B, P<0.01) and DNA fragmentation (Figure 1C, P<0.01). It is noteworthy that the binding
affinity of NF-B was markedly increased in
endothelial cells at 6 hours after the treatment under
hypoxic conditions as compared with normoxic conditions, as assessed by
gel mobility shift assay (Figure 2A),
consistent with our previous report.10
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Therefore, we hypothesized that hypoxia-induced
activation of NF-B would mediate apoptosis and cell death in
endothelial cells. Thus, we examined the
inhibitory effects of transfection of NF-B decoy ODNs
into endothelial cells on hypoxia-induced cell
death. To transfect decoy ODNs, we used the cationic HVJ-liposome
method, which is a novel gene transfection method. Previous reports
have mentioned that this method is more efficient for transfection into
various cells in vitro as compared with the conventional (anionic)
HVJ-liposome complex method.24 25 First, we tested whether
in vitro transfection of decoy ODNs into human
endothelial cells by the cationic HVJ-liposome method
is possible by using FITC-labeled decoy ODNs. Five minutes after
transfection of FITC-labeled ODNs by the cationic HVJ-liposome method
(3 µmol/L), intense nuclear fluorescence was observed.
Nearly 80% to 90% of endothelial cells could be
transfected by this method. Furthermore, the fluorescence could
be detected up to at least 72 hours after transfection (data not
shown). In contrast, no fluorescence was observed in
untransfected (untreated) cells. Using this transfection method, we
elucidated the role of NF-B in endothelial cell
death induced by hypoxia. Expectedly, transfection of NF-B
decoy ODNs resulted in significant inhibition of cell death induced by
hypoxia as compared with transfection of scrambled decoy ODNs
(Figure 2B
, P<0.01). Similarly, apoptotic
cells were also significantly decreased by transfection of NF-B, but
not scrambled decoy, ODNs as assessed by nuclear staining (Figure 1
online [see http://www.circresaha.org] and Figure 4A, P<0.01) and DNA fragmentation (Figure 3B, P<0.01). In
addition, activity of caspase 3, an interleukin-1ß converting enzyme
homologue that cleaves poly(ADP-ribose)polymerase during early
apoptosis,26 was also observed under hypoxic
conditions (Figure 3C, P<0.01). The increase in
caspase-3–like activity induced by hypoxia was also
significantly attenuated by transfection of NF-B decoy, but not
scrambled decoy, ODNs (Figure 3C).
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We further confirmed the proapoptotic action of NF-B
in endothelial cells using pyrrolidine dithiocarbamate
(PDTC), a potent inhibitor of NF-B. As previous
studies documented the inhibitory effects of PDTC on
NF-B activation,27 28 we used PDTC treatment as an
additional NF-B inhibition. As shown in Figure 4, PDTC treatment with
endothelial cells under hypoxic conditions resulted in
a significant attenuation of endothelial
apoptosis (P<0.01), accompanied by the decrease in
NF-B activity. Moreover, caspase-3–like activity was also
diminished by PDTC treatment (P<0.01, Figure 3C).
Activation of NF-B Downregulated bcl-2 Expression
We next examined the molecular mechanisms by which NF-B
activation in response to hypoxia induces
endothelial apoptosis. In this study, we
focused on the expression of bcl-2 and bax proteins, as bcl-2 and bax
are homologous proteins that have opposing effects on cell life and
death, with bcl-2 serving to prolong cell survival and bax acting as an
accelerator of apoptosis. 29 30 As shown in
Figures 5A and 5B, hypoxic treatment
significantly decreased bcl-2 protein as compared with normoxic
conditions (P<0.01). Interestingly, transfection of NF-B
decoy ODNs resulted in a significant increase in bcl-2 protein that was
downregulated by hypoxia as assessed by Western blotting
(P<0.01). We also examined the effects of PDTC treatment on
bcl-2. Consistent with NF-B decoy experiments, PDTC
treatment significantly attenuated the decrease in bcl-2 induced by
hypoxia (P<0.01, Figures 5C and 5D). In
contrast, no significant change in bax protein was observed in hypoxic
conditions as compared with normoxic conditions (Figure 2 online [see
http://www.circresaha.org]). Moreover, transfection of NF-B decoy
ODNs did not alter bax protein under hypoxic conditions. Thus, the
ratio of bcl-2 to bax was significantly increased in
endothelial cells transfected with NF-B decoy ODNs
as compared with scrambled decoy ODNs (normoxia, 100%;
hypoxia, 42±5%; hypoxia+scrambled decoy, 41±6%;
hypoxia+NF-B decoy, 88±6%; *P<0.01 versus
hypoxia or hypoxia+scrambled decoy; n=6 per group
calculated from 6 independent experiments). In addition, to test
whether the attenuation of the decrease in bcl-2 protein by NF-B
decoy ODNs is due to a change in transcription level, we performed
Northern blot analysis. As shown in Figure 6, bcl-2 mRNA was significantly
decreased in endothelial cells under hypoxic conditions
as compared with normoxic conditions (P<0.01).
Consistent with the results of protein level, transfection of
NF-B decoy ODNs significantly abolished the decrease in bcl-2 mRNA
as compared with scrambled decoy ODNs (Figure 6, P<0.01).
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To confirm whether bcl-2 can act as an antiapoptotic factor in
endothelial cell death induced by hypoxia, we
transfected human bcl-2 expression vector into
endothelial cells. Expectedly, transfection of human
bcl-2 gene resulted in significant inhibition of cell death induced by
hypoxia (Figure 7A, P<0.01). Apoptotic cells were also significantly
decreased by transfection of bcl-2 gene as assessed by DNA
fragmentation ratio (Figure 7B, P<0.01). The degree
of inhibition of endothelial cell death and
apoptosis by bcl-2 gene was similar to that by transfection of
NF-B decoy ODNs.
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In Vivo Transfection of NF-B Decoy ODNs Into Intact Carotid
Artery After Reperfusion Injury
In vitro studies demonstrated that activation of NF-B in
response to hypoxia induced endothelial cell
death through the suppression of bcl-2. However, none of the reports
have mentioned the role of NF-B in the regulation of
endothelium in vivo. Thus, we transfected NF-B decoy
ODNs into rat intact carotid artery after reperfusion injury. As
previously demonstrated,22 23 ODNs could be transfected
into endothelial cells and vascular smooth muscle cells
by HVJ-liposome method under a higher pressure. In contrast, at
lower perfusion pressure, endothelial cells were mainly
transfected (data not shown). As shown in Figure 8, reperfusion injury was associated with
a significant increase in endothelial apoptosis
at 24 hours after transfection as assessed by TUNEL staining
(P<0.01), whereas few TUNEL-positive cells could be
detected in normal endothelium. Of importance, NF-B
decoy ODNs treatment markedly decreased TUNEL-positive
endothelial cells at 24 hours after reperfusion,
whereas no difference was observed between scrambled decoy ODN-treated
and untransfected rats (Figure 8E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelial cells are known to secrete various vasoactive substances. Recently, it has been hypothesized that endothelial cells may also modulate vascular function as a biological barrier, because many antiproliferative factors such as NO and vascular natriuretic peptides are secreted by endothelial cells.14 However, the molecular mechanisms by which factors can stimulate endothelial cell growth and maintain the endothelium are still unknown. This is of extreme importance, as intact endothelium acts as a sensor and transducer of signals and also provides a nonthrombogenic surface at the blood–vascular wall interface. Hence, the mechanisms that maintain the integrity of the endothelium in physiological and pathological states are of interest. In addition, it is apparent that dysfunction of endothelial cells may promote the migration of neutrophils and other blood-derived components.19 31 32 33 As migration of neutrophils and/or macrophages may be responsible for the development of myocardial reperfusion injury and atherosclerosis, it is critical to maintain the endothelium to prevent cardiovascular disease. Recently, a new concept, that regulation of cell death by apoptosis may be an important determinant of vessel structure, has been postulated. In response to a variety of stimuli and circumstances, cells have an intrinsic capacity to activate a gene-directed program that commits the cell to a suicidal death, described as apoptosis. It has become increasingly clear that the process of cell death by apoptosis is a relatively ubiquitous phenomenon observed in a variety of cell types, including endothelial cells.
Cell death is a major concern in various clinical entities such as
ischemic diseases. Cell death by hypoxia has been
generally believed to be manifested as necrosis.27 In
contrast, recent biochemical observations have suggested the
possibility of hypoxia-induced
apoptosis.34 35 In this study, we demonstrated
that hypoxic treatment induced endothelial cell death
through the induction of apoptosis, consistent with
previous findings.34 35 Of importance, the present
studies revealed a significant decrease in bcl-2, an
antiapoptotic factor, and no change in bax, a
proapoptotic factor, by hypoxic treatment. As our present
data demonstrated marked downregulation of bcl-2 under hypoxic
conditions, the apoptosis induced by hypoxia may be due
to an inappropriate decrease in antiapoptotic factors. These
findings are further supported by the finding that overexpression of
bcl-2 attenuated endothelial cell death and
apoptosis under hypoxic conditions. More importantly,
activation of NF-B binding activity was also clearly observed in
endothelial cells under hypoxic conditions. Numerous
stimuli, including oxidative stress, tumor necrosis factor-
, and
high glucose are well known to induce apoptosis in
endothelial cells.20 35 36 37 Interestingly,
these stimuli also increased NF-B binding activity in
endothelial cells, as previously
demonstrated.38 39 40 Therefore, we hypothesized that
increase in NF-B binding activity might be involved in
hypoxia-induced endothelial cell death.
To resolve this important question, we used the "decoy" approach in
this study, because the decoy strategy is believed to be useful in
analyzing the function of endogenous transcription
factors.11 12 13 Expectedly, transfection of NF-B decoy
ODNs into endothelial cells attenuated
endothelial cell death and apoptosis, which is
similar to the effects of overexpression of bcl-2. More importantly, a
significant decrease in bcl-2 mRNA and protein was also attenuated by
the transfection of NF-B decoy, but not scrambled decoy, ODNs. These
findings revealed that activation of NF-B induced by hypoxia
caused endothelial cell death through bcl-2–dependent
apoptosis. These findings are supported by the observation that
another NF-B inhibitor, PDTC, also inhibited
endothelial apoptosis, increase in
caspase-3–like activity, and bcl-2 downregulation induced by
hypoxia. Moreover, the hypothesis that activation of NF-B
induces endothelial apoptosis was further
confirmed by in vivo experiments. In this study, we failed to discover
how NF-B decreased bcl-2 expression. Although several reports
documented that bcl-2 decreased NF-B activity,41 42
there are publications showing that bcl-2 overexpression
activates NF-B by promoting degradation of
inhibitory B.43 44 In contrast, none
of the studies revealed the effect of NF-B on bcl-2 expression.
Further studies about the promoter of bcl-2 gene would elucidate the
role of NF-B in regulation of bcl-2. Overall, the exact role of
NF-B in the apoptotic pathways is still controversial. In
general, NF-B has been believed to inhibit apoptosis in the
caspase-dependent pathways, because numerous reports documented
antiapoptotic actions of NF-B.13 45 46 In
contrast, in other cells, including endothelial cells,
NF-B has been reported to promote
apoptosis.11 12 20 35 36 37 Probably, cell fate
controlled by NF-B might be dependent on cell type or death
stimulators. Unfortunately, the present study cannot explain the
discrepancy in the role of NF-B among various cells.
The fact that hypoxia induced endothelial cell
death through NF-B activation is extremely important, given that
migration of leukocytes/neutrophils plays a pivotal role in the
development of myocardial reperfusion injury and
atherosclerosis.19 31 32 33 Disruption of
endothelium in response to hypoxia may be
responsible for the pathogenesis of cardiovascular
disease. Indeed, the effective blockade of a series of cytokine
and adhesion molecule genes during the initial acute phase of
reperfusion by in vivo transfection of NF-B decoy ODNs results in a
subsequent inhibitory effect on the development of
myocardial infarction.10 The functional role of
endothelium as a biological barrier was supported by
the observation that migration and/or accumulation of neutrophils into
the infarcted area could be blocked by NF-B decoy, but not scrambled
decoy, ODN treatment, as assessed by cytohistochemical
technique.10 A similar idea was also proposed by Sata and
Walsh.47 They demonstrated that tumor necrosis factor-,
a well-known stimulator of NF-B, decreased the function of
endothelium as a barrier against the migration of
macrophages and leukocytes through the suppression of
Fas-ligand expression. The findings that two important factors related
to apoptosis (Fas ligand and bcl-2) were downregulated in a
NF-B–dependent manner in response to hypoxia might be
critical to understanding the pathophysiology of
endothelial dysfunction in various diseases. Further
studies are necessary to clarify the role of NF-B in the expression
of other apoptosis-related molecules, such as Bcl-xL.
Here, using a decoy approach, we demonstrated that activation of
NF-B by hypoxia induced human aortic
endothelial cell death and apoptosis through
the suppression of an antiapoptotic molecule, bcl-2.
NF-B–mediated endothelial cell apoptosis
induced by hypoxia may be involved in the pathogenesis of
ischemic cardiovascular diseases such as
reperfusion injury and atherosclerosis.
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Acknowledgments |
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This work was partially supported by grants from Japan Health Sciences Foundation, the Hoan-sya Foundation, and the Japan Cardiovascular Research Foundation; a Japan Heart Foundation Research Grant; a Grant-in-Aid from the Tokyo Biochemical Research Foundation; and a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture. We are grateful to Prof Y. Tsujimoto (Osaka University) for the kind gift of the human bcl-2 gene. We thank Rie Kosai and Michiko Tamakoshi for their excellent technical assistance.
Received February 25, 2000; accepted March 21, 2000.
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