Mechanisms of Hypoxia-induced Endothelial Cell Death
ROLE OF p53 IN APOPTOSIS*
April
Stempien-Otero
§,
Aly
Karsan¶,
Carol J.
Cornejo
,
Hong
Xiang**,
Thomas
Eunson
,
Richard S.
Morrison**,
Mark
Kay
,
Robert
Winn
, and
John
Harlan
From the Departments of
Medicine, ** Neurological
Surgery, and
Surgery, University of Washington,
Seattle, Washington 98195-7710, ¶ Department of Pathology,
University of British Columbia, Vancouver,
British Columbia V6T2B5, Canada, and

Department of Genetics, Stanford
University, Palo Alto, California 94305
 |
ABSTRACT |
Endothelial cell death may contribute to tissue
injury from ischemia. Little is known, however, about the
characteristics of endothelial cell death in response to hypoxia. Using
an in vitro model, we found that human umbilical vein
endothelial cells were resistant to hypoxia-induced cell death with
only a 2% reduction in viability at 24 h and 45% reduction in
viability at 48 h. Overexpression of a mutant, I
B
, via
adenoviral vector did not potentiate cell death in hypoxia, indicating
that nuclear factor-
B activation was not involved in cytoprotection.
Cell death in hypoxia was determined to be apoptotic by 3' labeling of
DNA using terminal deoxynucleotidyl transferase staining and
reversibility of cell death with a caspase inhibitor. Exposure of
endothelial cells to hypoxia did not alter levels of proapoptotic
and antiapoptotic Bcl-2 family members Bax and Bcl-XL by
immunoblot analysis. In contrast, changes in p53 protein levels
correlated with the induction of apoptosis in hypoxic endothelial
cells. Inhibition of the proteasome increased p53 protein levels and
accelerated cell death in hypoxia. Overexpression of p53 by adenoviral
transduction was sufficient to initiate apoptosis of normoxic
endothelial cells. These data provide a framework for the study of
factors regulating endothelial cell survival and death in hypoxia.
 |
INTRODUCTION |
Tissue injury from ischemia and reperfusion causes significant
morbidity and mortality in cardiovascular disease. Endothelial cell
(EC)1 death may contribute to
the hypoxic as well as the reperfusion components of this injury. The
mechanisms of cell death in hypoxia are not known but may involve
calcium influx, derangements in mitochondrial function, or purine
nucleotide depletion (1, 2). Limited studies suggest a role for
apoptosis induced by the tumor suppressor gene p53 in hypoxia-induced
cell death. Graeber et al. (3) found that tumor cells
containing wild-type p53 were more sensitive to hypoxia-induced
apoptosis when compared with tumor cells lacking functional p53. Long
et al. (4) showed a role for p53 in cardiomyocyte
apoptosis in response to hypoxia. In contrast, Amellem et
al. (5) demonstrated that hypoxia-induced apoptosis occurred
independent of p53 protein level in MCF-7 cells. There are few data,
however, on the relative susceptibility of EC to hypoxia-induced cell
death or the molecular mechanisms involved. Because EC are invariably
exposed to hypoxia in ischemic conditions, this question has important
therapeutic implications for the prevention of ischemic tissue damage.
We used an in vitro model to examine mechanisms of EC death
during hypoxia. We found that EC underwent significant cell death with
features of apoptosis only after exposure to 48 h of hypoxia. Inhibition of nuclear factor-
B (NF-
B) activation by
adenoviral-mediated overexpression of a dominant negative I
B
mutant did not potentiate apoptosis in hypoxic EC. There was no
correlation between Bax/Bcl-XL ratios and cell death.
However, there was an increase in p53 protein levels concomitant with
EC death. In addition, overexpression of wild-type p53 protein in EC by
adenoviral gene transduction was sufficient to cause apoptosis. We
conclude that a major component of EC death in response to hypoxia is
attributable to apoptosis. EC survival in hypoxia does not appear to
depend on the activation of an NF-
B-dependent
pathway(s). Apoptosis in hypoxia correlated with p53 protein levels but
not with alterations in Bcl-XL and Bax proteins.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents
Human umbilical vein endothelial cells (HUVEC) were isolated
from umbilical cords with collagenase as described previously (6).
Cells were cultured in RPMI 1640 medium supplemented with 20% bovine
calf serum (Sigma), maintained at 37 °C in 5% CO2, and
used at passage 2-3. Hypoxia was induced by placement of cells in an
anaerobic chamber (Plaslabs, Lansing, MI) filled with 5% CO2, 85% N2, and 10% H2 and
heated to 37 °C. O2 concentration was maintained at <14
torr as measured by a Clark electrode (Yellow Springs Instrument,
Yellow Springs, OH) by the catalytic conversion of O2 and
H2 to H2O by palladium crystals. Human dermal
microvascular endothelial cells (HMEC-1) (7) were a gift of Dr. E Ades
(Centers for Disease Control, Atlanta, GA) and Dr. T. Lawley (Emory
University, Atlanta, GA) and were cultured in RPMI 1640 medium
supplemented with 10% bovine calf serum and endothelial cell growth
factor (25 µg/ml) prepared from bovine hypothalamus. Construction of HMEC-bclx and HMEC-neo has been previously described (8).
Recombinant human tumor necrosis factor-
(TNF-
) was purchased
from R & D systems (Minneapolis, MN).
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT),
calpain inhibitor I (ALLN) and calpain inhibitor II
(N-acetyl-leu-leu-methioninal) were purchased from Sigma.
Anti-p53, anti-Bcl-2, anti-Bcl-XL, and anti-Bax antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was purchased from Kamiya
Bio Co. (Seattle, WA). An Apotag kit was purchased from Oncor
(Gaithersburg, MD).
Construction of Recombinant Adenovirus
Construction of the p53 adenovirus carrying the human wild-type
p53 gene under the control of the cytomegalovirus promotor has been
described previously (9). I
B
mutant (serine to alanine at 32 and
36 residues) cDNA was kindly provided by Drs. M. Karin and J. DiDonato (University of California, San Diego, CA) (10). This cDNA
was inserted into the vector pXCJL1 under the transcriptional control
of the phosphoglycerokinase promoter, upstream of the bovine growth
hormone polyadenylation signal. The E1A-deficient recombinant
adenovirus was generated similar to the previously described generation
of recombinant control adenovirus Ad-Rous sarcoma
virus-
-galactosidase (11). Briefly, E1A-deficient adenovirus was
recombined with the pXCJL.1-I
B
mutant plasmid and pJM17 in 293 cells. Purification of a large batch of the recombinant adenovirus was
done by two consecutive cesium chloride centrifugations with storage at
80 °C in 10% glycerol, 10 mM Tris-HCl, pH 7.4, and 1 mM MgCl2.
Infection of HUVEC
Subconfluent HUVEC were washed once with warmed complete medium
and incubated at a multiplicity of infection (m.o.i.) indicated with
control adenovirus (AdLacZ), p53 adenovirus (Adp53), or mutant I
B
adenovirus (AdIkBm) in complete medium.
Immunoblot Analysis
After experimental treatment of HUVEC in 100-mm plates, cell
monolayers were detached from plastic culture dishes with a cell scraper, washed in cold phosphate-buffered saline, and incubated in 50 µl of lysis buffer (0.5% Nonidet P-40 with 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml
aprotinin) for 30 min at 4 °C. The protein was collected by
microcentrifugation at 12,000 rpm for 15 min. Cytosolic extracts from
treated HUVEC were resolved by SDS-polyacrylamide gel electrophoresis
on 10% gels and transferred to nitrocellulose in 25 mM
Tris, 192 mM glycine, and 5% methanol at 100 V for 1 h at 4 °C. Filters were blocked overnight with 10 mM
Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20 containing 5% skim milk. Immunoblots were analyzed for p53 using
antiserum at a 1:1000 dilution. Immunoreactive proteins were detected
according to the enhanced chemiluminescence protocol (Amersham
Pharmacia Biotech) using 1:10,000 horseradish peroxidase-linked
anti-goat secondary antiserum. Blots were exposed to film for 1-10
min. Immunoblotting for Bcl-2, Bcl-XL, and Bax was
identical except for the use of an anti-hamster second antibody for
Bcl-2 and anti-rabbit second antibody for the others.
Detection of Apoptosis
DNA Ladder--
Endothelial cells were removed from tissue
culture plates by a 2-min incubation with 0.05% trypsin and 0.02%
EDTA in Hanks' balanced salt solution and pelleted and resuspended in
a lysis buffer containing 100 mM NaCl, 10 mM
Tris-HCl, pH 8.0, 25 mM EDTA, and 0.5% SDS followed by
incubation with 100 µg/ml proteinase K for 4 h at 37 °C. DNA
was extracted using phenol and chloroform followed by ethanol
precipitation. The pellet was resuspended in Tris-EDTA buffer (10 mM
Tris-HCl and 1 mM EDTA) and treated with DNase-free RNase
for 3 h at 37 °C. The DNA was ethanol precipitated and
resuspended in distilled water. The concentration of DNA was measured
by spectrophotometer, and 4 µg was fractionated by electrophoresis on
a 1.5% agarose gel containing ethidium bromide. We evaluated the
electrophoretic patterns of DNA extracted from cells subjected to
hypoxia as well as normoxia. HUVEC were exposed to hypoxia for 24, 36, or 48 h, whereas hepatoma cells were exposed for 4, 12, or 24 h.
Terminal Deoxynucleotidyl Transferase-mediated Biotinylated UTP
Nick End Labeling Staining--
The 3' ends of DNA were labeled
according to the manufacturer's recommendation. Briefly, endothelial
cells were removed from tissue culture as described above with 0.05%
trypsin and 0.02% EDTA in Hanks' balanced salt solution, isolated by
centrifugation, resuspended in 1% paraformaldehyde in
phosphate-buffered saline, and fixed on ice for 15 min. Cells were
washed twice, resuspended in 70% ice-cold ethanol, and held at
20 °C until ready for use. The 3' ends of DNA had digoxigenin
nucleotides catalytically coupled to them using terminal
deoxynucleotidyl transferase. Digoxigenin was detected with fluorescein
isothiocyanate-labeled anti-digoxigenin antibody, and total DNA was
determined by staining with propridium iodide. Double staining allowed
simultaneous display of viable and apoptotic cells. Cells that stained
positive for both fluorescein isothiocyanate and propridium iodide were
considered to be apoptotic and those that stained only with propridium
iodide were considered normal. The percent apoptotic cells was
determined as the ratio of cells positive for fluorescein
isothiocyanate relative to those positive for propridium iodide.
Specimens were analyzed on an Epic XL cytometer (Coulter, Fullerton,
CA). At least 20,000 events were evaluated.
MTT Assay--
The MTT assay of cell viability was performed as
described previously (8). Briefly, after treatment, medium containing 1 mg/ml MTT was added to cells for a final concentration of 0.5 mg/ml and
incubated at 37 °C for 5 h. The medium was aspirated, and the
formazan product was solubilized with dimethyl sulfoxide. Absorbance at
630 nm (background absorbance) was subtracted from absorbance at 570 nm
for each well.
Nucleosome Enzyme-linked Immunosorbent Assay
(ELISA)--
Nucleosome ELISA (Calbiochem, Cambridge, MA) was
performed according to the manufacturer's instructions. Briefly, cells
were treated with adenoviral constructs and then harvested and lysed at
appropriate time points. After freezing lysates at
20 °C for at
least 18 h, specimens were thawed and pipetted into wells of a
pretreated ELISA plate. The plate was then incubated with detector antibody followed by streptavidin conjugate. After washing, substrate solution was added, and plates were incubated in the dark for 30 min.
The reaction was stopped with stop solution, and absorbance was
measured on a plate reader at dual wavelengths of 450 and 595 nm.
Northern Analysis
HUVEC were subjected to normoxia or hypoxia, and RNA was
extracted at 2, 8, 18, 24, and 36 h with Trizol (Life
Technologies) per the manufacturer. Total cellular RNA was separated on
agarose-fromaldehyde gels, blotted onto nitrocellulose filters, and
hybridized with 32P-labeled probes for A1 (8) or A20 (gift
of V. Dixit, Genentech, South San Francisco, CA).
Statistical Analysis
Analysis for statistical significance was performed on Excel
using Student's paired t test.
 |
RESULTS |
HUVEC were found to be relatively resistant to hypoxia-induced
cell death. The MTT assay revealed 98% viability after 24 h and
55% viability after 48 h of continuous exposure to <14 torr of
oxygen (Fig. 1). HUVEC death was
evaluated by several techniques to determine whether this death was
apoptotic in nature. Significant DNA laddering was present after
48 h of exposure of HUVEC to hypoxia but was not seen in control
HUVEC at the same time point (Fig. 2).
Approximately 35% of cells were apoptotic when evaluated by terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling staining at 48 h (Fig. 3).
In contrast, RH7777 hepatoma cells, previously shown to be sensitive to
hypoxia-induced apoptosis (12), underwent significant apoptosis after
only 12 h of exposure to hypoxia (Fig. 3).

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Fig. 1.
HUVEC death in hypoxia is mediated by
caspases. MTT cell viability assay was performed after 48 h
of exposure of HUVEC to hypoxia with or without pretreatment with the
caspase inhibitor fmk-zVAD (100 µM). Values represent the
mean ± S.E. of five experiments. *, p < 0.05 versus hypoxic control (Con); #,
p < 0.05 versus normoxic control
(Con).
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Fig. 2.
HUVEC death in hypoxia is associated with DNA
laddering. HUVEC exposed to hypoxia or normoxia for 24 and 48 h were lysed, and DNA was extracted. DNA was fractionated by
electrophoresis and stained by ethidium bromide.
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Fig. 3.
HUVEC are more resistant to hypoxia-induced
apoptosis than hepatoma cells. Annexin V binding to HUVEC and rat
hepatoma cells (RH7777) was performed at various time points after
exposure to hypoxia. Approximately 35% of HUVEC were apoptotic at
48 h. Values represent the means ± S.E. of three
experiments.
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The caspase family of proteases is thought to be the final execution
pathway in apoptosis (13). To establish the role of the caspase pathway
in hypoxia-induced apoptosis of EC, we treated HUVEC with the caspase
inhibitor zVAD-fmk (14). Fig. 1 shows that pretreatment of HUVEC with
zVAD-fmk largely prevented cell death seen after 48 h of hypoxia,
further establishing that hypoxia-induced death of EC is apoptotic.
The experiments represented by Figs. 1-3 established the time course
of EC death in hypoxia and confirmed that it was attributable primarily
to apoptosis. Next, we examined the role of two Bcl-2 family members in
the response of EC to hypoxia. Based on studies in other cell types
demonstrating changes in Bcl-2/Bax ratios with apoptosis (15), we
performed immunoblot analysis of Bcl-XL and Bax protein
levels in response to hypoxia. These proteins were chosen because they
have been shown to be the predominant cytoprotective and proapoptotic
Bcl-2 family proteins in HUVEC (16), There was no significant change in
their ratios after 24 h of hypoxia in three successive experiments
(Fig. 4). Northern blot analysis for the
inducible EC cytoprotective molecules A1 and A20 also showed no change
in mRNA levels after exposure to hypoxia (data not shown). As we
have previously shown (16), Bcl-2 protein was barely detectable in
HUVEC and showed no increase by immunoblot analysis in response to
hypoxia (data not shown).

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Fig. 4.
Bax and Bcl-XL protein levels and
ratios do not change during hypoxia. Immunoblot analysis for Bax
and Bcl-XL protein was performed at 6, 12, and 24 h
after exposure to hypoxia.
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Having excluded that hypoxia-induced changes in the protein levels of
the predominant Bcl-2 family members expressed in HUVEC accounted for
initiation of apoptosis, we next determined whether p53 protein levels
were altered before cell death. Fig.
5 shows an immunoblot analysis of lysates
of HUVEC exposed to normoxia or progressively longer periods of
hypoxia. Minimal levels of p53 protein were present in normoxia or
early in hypoxia. However, there was a significant increase in p53
protein level by 24 h of hypoxia.

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Fig. 5.
p53 protein levels increase with hypoxia and
are potentiated by a proteasome inhibitor. Immunoblot analysis of
p53 protein was performed at 2, 6, 12, and 24 h after exposure to
hypoxia in the absence or presence of the proteasome inhibitor ALLN
(100 µM). Three separate experiments yielded identical
results.
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The proteasome, a multicatalytic protein involved in intracellular
protein turnover (17), is known to contribute to the normally short
half-life (20 min) of p53 protein (18). The addition of the proteasome
inhibitor ALLN, which inhibits p53 degradation (19) caused an
accelerated accumulation of p53 protein in hypoxic HUVEC (Fig. 5).
Concomitant with its ability to cause an early increase in p53 protein
levels, treatment of hypoxic HUVEC with ALLN resulted in a potentiation
of hypoxia-induced apoptosis (Fig. 6).
There was complete loss of viability of nearly all hypoxic ALLN-treated
HUVEC at 24 h. In contrast, treatment of normoxic HUVEC with ALLN
for 24 h did not cause significant toxicity (Fig. 6). This effect
was not attributable to the inhibitory effects of ALLN on calpain,
because the related calpain inhibitor II, a weak inhibitor of the
proteasome (20, 21), had no effect on HUVEC viability in hypoxia.

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Fig. 6.
Proteasome inhibitor potentiates HUVEC death
in hypoxia. HUVEC were treated with ALLN (100 µM) or
calpain inhibitor II (ALLM, 100 µM) and then
exposed to hypoxia. Viability was determined by MTT assay after 24 h. Values represent the mean ± S.E. of three experiments. *,
p < 0.005 versus control.
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Further experiments were performed to assess whether the effect of ALLN
was attributable to potentiation of an apoptotic pathway versus a nonspecific toxic effect. HMEC-1 overexpressing the
cytoprotective molecule Bcl-XL (HMEC-bclx) were exposed to
ALLN in hypoxic conditions. HMEC-1 expressing Bcl-XL were
completely protected from apoptosis after a 24-h exposure to both
hypoxia and ALLN (Fig. 7).

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Fig. 7.
Overexpression of Bcl-XL protects
EC from death induced by hypoxia. HMEC-1 cells transduced with the
cytoprotective molecule Bcl-XL or neomycin control were
maintained in the presence or absence of the proteasome inhibitor ALLN
(100 µM). Viability was assessed by MTT assay after
24 h of hypoxia. Values represent the means ± S.E. of three
experiments. *, p < 0.002 versus HMEC
Bcl-x.
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To determine whether p53 protein alone was sufficient to initiate
apoptosis in HUVEC, we used a strategy of adenovirus infection to
transduce the p53 gene in HUVEC. Studies with an adenovirus encoding
-galactosidase (AdLacZ) showed near 100% infection efficiency after
48 h of exposure to 500 m.o.i. of AdLacZ (data not shown). These data are comparable to the m.o.i. used by others for adenoviral mediated transduction of EC (22). Immunoblot analysis of normoxic HUVEC
infected with 500 m.o.i. of an adenovirus encoding p53 (Adp53) showed increased levels of p53 protein 24 h after infection
compared with uninfected cells and cells infected with AdLacZ (Fig.
8). There was a marked reduction in HUVEC
viability in normoxia as early as 48 h after infection with Adp53
and near complete loss of viability 72 h after infection (Fig.
9A). Viability of
AdLacZ-infected normoxic cells was similar to control, uninfected
HUVEC. Phase contrast microscopy showed a normal cobblestone appearance
of AdLacZ-infected cells compared with detachment, membrane blebbing, and cellular fragmentation of HUVEC 72 h after infection with 500 m.o.i. of Adp53. Experiments performed with Adp53- and
AdLacZ-infected cells in hypoxic conditions showed complete loss of
viability of Adp53-infected cells within 24 h of exposure to
hypoxia (data not shown). Measurement of free nucleosomes was performed
under identical conditions to evaluate whether this death was
predominantly attributable to apoptosis (Fig. 9B).
Confirmation that Adp53-induced death was dependent in part on caspase
activation was established through the use of the caspase inhibitor
ZVAD-fmk. Pretreatment of HUVEC with ZVAD-fmk partially reversed the
loss of viability seen with Adp53 treatment (Fig.
10).

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Fig. 8.
Infection with Adp53 results in high levels
of p53 protein expression. HUVEC were untreated or infected with
AdLacZ or Adp53 at 500 m.o.i. for 24 h. Cell lysates were
prepared and subjected to immunoblot analysis for p53 protein.
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Fig. 9.
A, overexpression of p53 is
sufficient to cause EC death in the absence of hypoxia. HUVEC
were untreated or infected with AdLacZ or Adp53 at 500 m.o.i.. They were assessed for viability by MTT assay at 24, 48, and
72 h after infection. Compared with uninfected control cells,
there was a significant decrease in viability of Adp53-infected cells
at 48 and 72 h. Values represent means ± S.E. of three
experiments. *, p < 0.05 versus AdLacZ.
B, Adp53 infection results in increase in free nucleosome
levels . HUVEC were infected with AdLacZ, Adp53, or control serum.
Release of nucleosomes was measured by ELISA at 24, 48, and 72 h.
This figure is representative of two separate experiments.
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Fig. 10.
The caspase inhibitor zVAD attenuates
Adp53-induced apoptosis. Cells were treated with fmk-zVAD (100 µM) at the time of adenoviral infection. Viability was
assessed by MTT assay after 48 and 72 h of Adp53 infection
(Ad Rx) at 500 m.o.i. Values represent the means ± S.E. of three experiments. *, p < 0.05 versus untreated Adp53.
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In addition to its ability to block degradation of p53, inhibition of
the proteasome by ALLN has been shown to inhibit activation of NF-
B
(21). To determine whether potentiation of hypoxia-induced apoptosis of
EC by ALLN was in part caused by inhibition of NF-
B, an adenoviral
construct encoding a mutant form of I
B
(IkBm) was used. Serine
residues 33 and 36 are converted to alanine in the mutant protein,
preventing phosphorylation. The overexpressed mutant protein cannot be
degraded by the proteasome but remains bound to NF-
B in the
cytoplasm, inhibiting its translocation to the nucleus (10). To confirm
that this strategy was effective in inhibiting NF-
B-mediated
transcription, assessment of vascular cell adhesion molecule-1 (VCAM-1)
expression in response to TNF-
stimulation was measured by ELISA. As
previously shown in our lab,2
control HUVEC and HUVEC infected with AdLacZ exhibited appropriate increases in VCAM expression after 18-h treatment with TNF-
(10 ng/ml), whereas HUVEC infected with AdI
Bm for 24 h showed
complete inhibition of VCAM-1 protein expression in response to TNF-
(Fig. 11). To ensure maximal inhibition
of NF-
B activity, HUVEC were exposed to AdI
Bm or AdLacZ at
1000 m.o.i. for 72 h before exposure to hypoxia. Although
lower levels of AdI
Bm were sufficient to inhibit TNF-induced VCAM
expression, longer exposures at higher m.o.i. were necessary to
completely abrogate the cytoprotective effect of NF-
B under TNF
stimulation. Overexpression of the mutant I
B did not change the
extent of EC apoptosis in response to hypoxia at 24 h (Fig.
12). Longer treatments with either
AdLacZ or AdI
Bm resulted in equivalent cytotoxicity compared with
uninfected cells. As reported by others, addition of TNF-
to both
control and hypoxic EC treated with an inhibitor of NF-
B
translocation resulted in significant loss of viability of these HUVEC
compared with AdLacZ-infected or noninfected cells at 24 h (22).
Therefore, although NF-
B activation is necessary for survival in
response to TNF-
, it does not appear to play a role in survival
during hypoxia.

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Fig. 11.
Inhibition of NF- B abrogates VCAM-1
induction by TNF- . HUVEC were untreated (Control) or
infected with 1000 m.o.i. AdLacz or AdI Bm for 24 h then
treated with 10 ng/ml TNF- for 18 h. VCAM-1 expression was then
assessed by ELISA. This is representative of three separate
experiments.
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Fig. 12.
Inhibition of NF- B potentiates cell death
in response to TNF- but not hypoxia. HUVEC were untreated
(Control) or infected with 1000 m.o.i. AdLacz or
AdI Bm for 72 h and then subjected to hypoxia alone or hypoxia
and TNF- (5 ng/ml). After 24 h viability was assessed by MTT
assay. Values represent the means ± S.E. of six experiments. *,
p = 0.002 versus AdLacZ cells.
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 |
DISCUSSION |
Tissue injury occurs both during hypoxia and after reoxygenation.
In some instances the degree of tissue damage is greater after brief
hypoxia followed by reperfusion than with more prolonged hypoxia alone.
We sought to elucidate the time course and mechanisms of EC death
during hypoxia in an effort to understand tissue resistance to hypoxic
injury. We found that EC were resistant to hypoxia, requiring a
prolonged period of exposure to induce cell death. A major component of
hypoxia-induced death was attributable to apoptosis, as shown by DNA
laddering, increased terminal deoxynucleotidyl transferase-mediated
biotinylated UTP nick end labeling staining, and inhibition of cell
death by a caspase antagonist. These results are consistent with those
found in several other cell lines. However, EC appear to be able to
survive a more prolonged period of hypoxia without significant loss of
viability when compared with other primary cell lines such as
cardiomyocytes, neurons, and renal tubular cells (4, 23-25).
One explanation of EC resistance to hypoxia would be an increased
expression of cytoprotective members of the Bcl-2 family in response to
exposure. Alternatively, a decrease in death-promoting members of the
Bcl-2 family could have a similar beneficial effect on cell survival
(26). However, we found no change in the protein levels of either
Bcl-XL or Bax during hypoxia. Thus, changes in expression
of these two molecules are unlikely to explain early cytoprotection.
Although it is possible that other members of this family may be
modulated by hypoxia, Bcl-XL and Bax appear to be the
dominant members of the Bcl-2 family in HUVEC (16). Alternatively,
phosphorylation or translocation of the Bcl-2 family member to the
mitochondrial membrane may play a role in cytoprotective or
proapoptotic effects independent of changes in protein level (27, 28).
Further experiments to inhibit synthesis or function of these members
will be necessary to define more completely their role in
hypoxia-induced apoptosis of EC.
The tumor suppressor gene p53 is best known for its role in cell cycle
arrest and apoptosis in response to direct genomic stress such as
ionizing radiation and chemotherapeutic agents (29). Recently, it has
been implicated in the death program of cells in response to other
physiological stimuli, including hypoxia and oxidative stress (4, 30).
Also, tumor cells lacking a functional p53 gene have been observed to
have a prolonged survival in hypoxia (3), and p53 is stabilized by
induction of hypoxia-inducible factor 1
(31). However, in some cell
lines p53 protein does not appear to play a role in hypoxia-induced
apoptosis (32). In addition, elevated p53 protein expression may also
be a secondary effect of other apoptotic pathways. Vaziri et
al. (33) showed that p53 protein levels increased as a result of
byproducts of the apoptotic process such as PARP cleavage fragments.
We found that endogenous p53 protein levels correlated with the
initiation of cell death in hypoxic HUVEC and that elevation of
endogenous p53 protein levels via inhibition of the proteasome potentiated apoptosis in hypoxic HUVEC. We also showed that an EC line
transduced with Bcl-XL was resistant to apoptosis at
24 h after exposure to ALLN and hypoxia. This finding is
consistent with results in other cells showing that p53-induced death
can be inhibited by antiapoptotic members of the Bcl-2 family (34-36). In addition, overexpression of p53 protein via an adenoviral vector was
sufficient to induce apoptosis in normoxic HUVEC. These data are
similar to those seen in the terminally differentiated cells, cardiomyocytes, and primary neurons (4, 9). Although the increase in
p53 protein before cell death suggests a causal role, definitive
experiments in which endogenous p53 function is inhibited are necessary
to establish firmly that it mediates hypoxia-induced apoptosis of EC.
In addition, Adp53 death was only partially reduced by the addition of
the caspase inhibitor fmk-zVAD. The lack of complete protection may
have been attributable to excessive activation of the death program in
the absence of a stimulus (e.g. hypoxia) that up-regulates
cytoprotective pathways. This hypothesis is supported by the finding
that inhibition of transcription with actinomycin D treatment before
the hypoxic insult markedly potentiates EC death (data not shown). This
phenomenon is similar to the response of actinomycin D-treated EC to
TNF-
, interleukin-1, or lipopolysaccharide. In these cases
actinomycin D inhibits the synthesis of cytoprotective proteins,
thereby provoking apoptotic cell death (8, 37, 38). Alternatively,
recent data suggest that some death pathways in fact may be potentiated
by caspase inhibition (39).
Studies have shown that hypoxia induces NF-
B activation in HUVEC
(40). Activation of NF-
B is necessary for survival after exposure to TNF-
in multiple cell types, including HUVEC (41, 42). In addition, NF-
B activation protects against death induced by
ionizing radiation and daunorubicin in tumor cell lines and inhibits
antibody-mediated apoptosis of murine B cells (43, 44). However,
activation of the transcription factor NF-
B does not appear to be
necessary for EC survival during hypoxic stress. Although the
potentiation of hypoxia-induced apoptosis by the proteasome inhibitor
may potentially have been secondary to its ability to inhibit
translocation of NF-
B to the nucleus, inhibition of NF-
B
translocation by an adenovirus encoding a mutant I
B
super-repressor did not potentiate HUVEC apoptosis in hypoxia. Consistent with other studies, inhibition of NF-
B did potentiate apoptosis of both normoxic and hypoxic EC treated with TNF-
(22).
These findings are of potential clinical relevance. Activation of
NF-
B in hypoxia has been implicated in the induction of chemokines
and adhesion molecules (45-47). These products are known to potentiate
the inflammatory response after reperfusion. Activation of endothelium
may be responsible for clinical events such as reperfusion injury and
graft failure (48). Our in vitro results suggest that
therapies targeted at inhibition of NF-
B in response to hypoxia and
reoxygenation should not provoke or potentiate EC apoptosis.
Conversely, antagonism of p53 may potentially reduce EC damage during ischemia.
 |
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. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Medicine,
University of Washington Medical Center, Box 357710, Seattle, WA
98195-7710. Tel.: 206-685-3054; Fax: 206-543-3560; E-mail: april{at}.u.washington.edu.
2
K. Zen, A. Karsan, A. Stempien-Otero, E. Yee,
M. A. Kay, C. B. Wilson, and J. M. Harlan, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
EC, endothelial
cell;
HUVEC, human umbilical vein endothelial cell, NF-
B, nuclear
factor-
B;
TNF, tumor necrosis factor;
VCAM, vascular cell adhesion
molecule;
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide;
HMEC, human microvascular endothelial cell;
ALLN, N-acetyl-leu-leu-norleucinal;
zVAD-fmk, Z-Val-Ala-Asp-fluoromethylketone;
Ad, adenovirus;
m.o.i., multiplicity
of infection;
ELISA, enzyme-linked immunosorbent assay.
 |
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