Apoptosis 2005; 10: 887894 C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.

Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells

C.-N. Lee, W.-F. Cheng, M.-C. Chang, Y.-N. Su, C.-A. Chen and F.-J. Hsieh
Department of Obstetrics and Gynecology(C.-N. Lee, W.-F. Cheng, M.-C. Chang, C.-A. Chen, F.-J. Hsieh); Department of Medical Genetics (Y.-N. Su) National Taiwan University Hospital, Taipei, Taiwan

Background: To evaluate the inuence of hypoxia and molecular events in endothelial and embryonic stem cells. Materials and Methods: Human umbilical vein endothelial cells (HUVECs) and mouse embryoid body (EB) cells were subjected to hypoxic conditions for different time courses. DNA fragmentation assay, quantication of apoptotic cells by TUNEL assay measured by owcytometry, and Western blot analysis for the molecular events of apoptosis were performed. Results: DNA fragmentation could be identied under hypoxic conditions in HUVECs and mouse EBs. The DNA fragmentation increased when the hypoxic interval was extended. In situ internucleosomal DNA fragmentationTUNEL assay also found that the percentages of apoptotic cells increased gradually in HUVECs and mouse EBs when the hypoxic interval was extended. Furthermore, the levels of expression of p53 and Bax both increased in hypoxic conditions. Conclusions: Hypoxia increases both HUVEC and mouse EB apoptosis, which is associated with increase in p53/Bax expression. Keywords: apoptosis; human umbilical vein endothelial cells; hypoxia; mouse embryoid body.

Introduction
Fetal hypoxia is the major cause of poor fetal outcome and future neonatal and adult handicaps. For early pregnancies, many early spontaneous abortions are associated with chromosomal anomalies. 1,2 However, a denite cause cannot be found for around half of early pregnancy loss. Many abortions have precedent maternal hemorrhage.3 Hemorrhage may be one of the major causes of fetal demise due to hypoxia in early pregnancy. Hypoxia caused by hemorSupported by grant from National Science Committee of Taiwan (NSC90-2314-B-002-189)

rhage or ischemic events in the early stage of pregnancy inuences the gestational tissues and nally induces fetal demise.4,5 Various situations such as maternal hypertension, infection, malnutrition, and drug abuse may be responsible for fetal hypoxia in the late stage of pregnancy.6 Intrauterine hypoxia indeed represents a major form of intrauterine pathology in embryonic and chorionic tissues. Vascular occlusion or injury is a common and important cause of tissue hypoxia. If the hypoxic process progresses, the embryo can die. Recently, several in vivo and in vitro studies have demonstrated that hypoxia can cause cell death in endothelial cells and embryoid bodies.7,8 Apoptosis and necrosis in cellular demise can occur simultaneously in tissues or cultured cells exposed to hypoxia. The intensity and duration of insults may decide the outcome. Hypoxia indeed plays an important role in the early development of endothelial cells within the vessels of the umbilical cord and the embryo. However, the inuence of hypoxia on endothelial cells and embryos and the following events are still unclear. So we utilized an in vitro culture system to elucidate the morphologic and molecular changes in endothelial cells and embryos during hypoxic conditions. The results should give us information on the molecular and morphologic events in the maternal-fetal system in early fetal demise.

Materials and methods

Culture of human umbilical vein endothelial cells (HUVECs) HUVECs were isolated from fresh human umbilical veins after normal spontaneous birth as described, with some modications.9,10 Briey, human umbilical veins were ushed with phosphate-buffered saline (PBS), then lled with PBS containing 0.2% collagenase (Life Technologies, Grand Island, NY, USA) and incubated for 10 min at 37 C. The HUVECs were removed from the vein by PBS wash. The primary isolated HUVECs were maintained in 2% gelatin-coated tissue culture plates in complete
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Correspondence to: Chi-An Chen, No. 7, Chung-Shan South Road, Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan. Tel.: 886-2-23123456, ext. 5166; Fax: 886-2-23365509; e-mail: cachen@ha.mc.ntu.edu.tw

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growth medium (M199, Life Technologies, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria), 10 g/ml endothelial cell growth factor (Upstate, Lake Placid, NY, USA), 10 mg/ml heparin, and penicillin-streptomycin (50mg/ml each; Life Technologies, Grand Island, NY, USA) at 37 C in a 5% CO2 incubator. HUVECs from passages 34 were used in this study.

transferred onto a regular tissue culture dish, where they spread and differentiated and the medium was changed every 2 days. Similar sized EB cells were selected and conrmed under microscopic manipulation.

Hypoxic conditions Hypoxic conditions were achieved using a Hypoxia Jar and BBL GasPac (Becton Dickinson, Cockeysville, MD, USA), which can catalytically reduce oxygen levels to less than 1% within 15 min.13

Immunohistochemistry To identify the specicity of the cultured HUVECs, the surface expression marker CD31 in the HUVECs was examined. Rat anti-human CD31 Ab (1:25 dilution; BD Biosciences PharMingen, San Diego, CA, USA), followed by biotinylated goat anti-rat Ab (1:100 dilution; Jackson ImmunoReseach Laboratories Inc., West Grove, PA, USA) and streptavidin-conjugated horseradish peroxidase (HRP) (DAKO Corp., Carpinteria, California, USA) were used in a protocol previously described.11 Samples without primary antibody staining served as negative controls.

DNA fragmentation The HUVECs and mouse EBs cells were subjected to hypoxic conditions as described earlier for different time courses. The DNA fragmentation assay was performed as described with some modications.14 The drifting and adherent cells were collected separately, washed with icecold PBS and lysed with DNA extraction buffer (10 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.5% Triton X-100) for 20 min at 4 C. The samples were then centrifuged at 12,000 g, 4 C for 15 min. The DNA in the drifting or adherent cells was extracted in three steps with equal volumes of phenol, phenol/chloroform, and then chloroform. The DNA was precipitated with 1/10 of the total volume of 3 M sodium acetate (pH 5.2) and 2 times of the total volume of ethanol. The DNA was resuspended in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and treated with 40 g/ml RNase A for 1 h at 37 . Twenty micrograms of DNA from each sample was subjected to agarose gel electrophoresis on a 2 % gel in TAE buffer (40 mM Tris-HCl (pH 8.5), 2 mM EDTA). The gel was stained with 0.5 mg/ml ethidium bromide for 15 min and the fragmented DNA was visualized under UV light and photographed.

Embryonic stem cell culture and differentiation Mouse embryonic stem (ES) cells (CCRC60003), obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan), were maintained on feeder cells, STO broblasts (CCRC 60206), in highglucose DMEM (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal calf serum (PAA Laboratories GmbH, Pasching, Austria), 0.1 mM nonessential amino acids (Life Technologies, Grand Island, NY, USA), 1 mM sodium pyruvate (Life Technologies, Grand Island, NY, USA), penicillin-streptomycin (50 mg/ml each; Life Technologies, Grand Island, NY, USA), and 1000 U/ml leukemia inhibitory factor (LIF) (1.5% conditioned medium, Genetics Institute, Boston, MA, USA). ES cells were maintained in an undifferentiated state by culture on freshly prepared layers of STO broblasts pretreated with radiation at 3,0009,000 rads. Cells were passaged every 2 days, and the medium was changed every day. For in vitro differentiation experiments, the same medium without LIF was used for EB in vitro culture.12 The ES cell suspension was centrifuged at 100 g for 5 min, and the pellet was resuspended in a suitable volume containing EB medium for counting. Thirty micro liter drops, containing 400 cells, were placed on the surface of the Petri dishes and incubated at 37 C. After 2 days of hanging drop culture, the cell aggregates contained in the drops were collected in 10 ml of EB medium and transferred into a bacterial Petri dish, where the aggregates were grown in suspension for 3 days. The aggregates were
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TUNEL assays using ow cytometric analysis Quantication of apoptotic cells was performed by TUNEL assay to determine the percentage of cells undergoing apoptosis using a cell death detection kit (Boehringer Mannheim, Indianapolis, IN, USA). The cells (1 105 ) were washed in Dulbeccos PBS (DPBS) and xed with 4% paraformaldehyde for 30 min at room temperature and resuspended with 80% ethanol. The cells were incubated with FITC-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase enzyme solution for 1 h at 37 C. Following incubation, cells were washed with DPBS, and owcytometry analysis was performed on a Becton Dickinson FACScan with CELLQuest

Hypoxia-induced apoptosis in HUVECs & ES cells

Figure 1. Microscopic representative gures of HUVECs and ES cells. HUVECs were isolated from human umbilical veins and maintained in 2% gelatin-coated tissue culture plates in complete growth medium at 37 C in 5% CO2 . (A) Isolated HUVECs under low-power eld (40X). (B).To identify their specicity, cultured HUVECs were examined for the surface expression marker CD31 by immuno-staining under low-power eld (100X). (C) Mouse ES cells were maintained on feeder cells, STO broblasts (40X). (D) In vitro differentiation of mouse ES cells, in medium without LIF. The differentiated mouse ES cells are referred to as mouse EBs (40X).

software (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA) as described previously.15,16 Samples without terminal deoxynucleotidyl transferase enzyme served as negative controls.

Results HUVECs were isolated and cultured as described in Materials and Methods.We rst characterized the morphology and phenotype of freshly isolated and cultured HUVECs by light microscopy and immunohistochemical staining. Representative microscopic pictures of the freshly isolated and cultured HUVECs are shown in Figure 1A. As shown in Figure 1B, the cultured HUVECs were positive for CD31. Our data indicated that we had indeed acquired HUVECs which were then used in further experiments. Mouse ES cells were maintained on irradiated feeder cells as described in Materials and Methods (Figure 1C). The LIF was added to keep the ES cells in an undifferentiated state. For in vitro differentiation experiments, LIF was omitted. Representative gures of the differentiated mouse EBs are shown in Figure 1D. DNA Fragmentation could be identied in HUVECs and mouse EBs under hypoxic conditions Drifting and adherent HUVEC cells were collected separately. The DNA of each sample was then extracted and subjected to agarose gel electrophoresis to detect fragmented DNA in HUVECs or mouse EBs cells. As shown in Figure 2, prominent DNA ladders could be found in the drifting HUVECs after 7 days in hypoxic conditions
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Protein isolation and Western blot analysis Cells were lysed with protein extraction reagent (10 mM Tris-HCl, pH7.5/5mM EDTA/0.1% SDS/1% Na-deoxyxholate/1%NP-40). Equal amounts of proteins (50100 g) were loaded and separated by SDS-PAGE using a 12% polyacrylamide gel. The gels were electroblotted on a polyvinylidene diuoride membrane (Bio-Rad, Hercules, CA, USA). Blots were blocked with PBS/0.05% and Tween 20 (PBST) containing 5% nonfat milk for 2 h at room temperature. Membranes were probed with different primary antibodies at a 1:1000 dilution in PBST for 2 h, washed four times with PBST, and then incubated with secondary antibodies IgG and horseradish peroxidase (HRP) conjugate at a 1:7500 dilution in PBST. Membranes were washed four times with PBST and developed using enhanced Hyperlm-enhanced chemiluminescence (Amersham, Piscataway, NJ, USA).

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Figure 2. DNA fragmentation in HUVECs subjected to hypoxia. Cells were incubated in hypoxia jars for different periods of time as described previously. DNA was extracted on agarose gels from HUVECs after hypoxia (from 3 to 7 days) and prepared as described in Materials and Methods. Similar results were obtained in three independent experiments and representative data are shown. (M, marker; D, drifting cells; A, adherent cells). Figure 3. DNA fragmentation in EBs cells subjected to hypoxia. Cells were incubated in hypoxia jars for different periods of time as described previously. DNA extracted on agarose gels from hypoxic EB cells (from 0 and 3 days) was prepared as described in Materials and Methods. Similar results were obtained in three independent experiments and representative data are shown.(M, marker; P, positive control.)

compared to those after 3 or 5 days. However, no definite DNA ladders could be identied in the adherent HUVECs at different time courses of hypoxia. DNA ladders in hypoxic EBs are shown in Figure 3. DNA ladders were noted in EBs after 3 days of hypoxia. Our data indicate that DNA fragmentation could be identied in both the HUVECs and mouse EBs. In addition, DNA ladders could be identied earlier and more prominently in the mouse EBs than in the HUVECs.

at 6 hr, 0.58 0.11% at 12 hr, 1.45 0.24% at 24 hr, and 2.15 0.38% at 48 hr ) (ANOVA, P < 0.05). Our data indicated that apoptosis of HUVECs and mouse EBs increased with the duration of hypoxia and TUNEL assays could detect apoptotic events as early as 48 hours.

TUNEL assays using FACS detected apoptosis of HUVECs and mouse EBs in hypoxic conditions We further quantitated the number of apoptotic cells in HUVECs and mouse EB cells in hypoxic conditions at different time courses using TUNEL assays and analyzed them using owcytometry as described in Materials and Methods. Representative gures from TUNEL assays using FACS analysis are shown in Figure 4A. As shown in Figure 4B, the percentages of positive cells in the TUNEL assays of the HUVECs increased gradually as the hypoxic duration increased (0.18 0.02% at 0 hr, 0.26 0.05% at 6 hr, 1.98 0.30% at 12 hr, 2.40 0.50% at 24 hr, and 17.04 2.03% at 48 hr) (ANOVA, P < 0.01). We also used TUNEL assays of the mouse EBs in hypoxic conditions. Representative gures from TUNEL assays using FACS analysis are shown in Figure 5A. As shown in Figure 5B, the percentages of positive cells in the TUNEL assays of the EBs also increased gradually during the hypoxic period ( 0.32 0.06% at 0 hr, 0.67 0.12%
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Changes in apoptosis-related protein expression in HUVECs and mouse EB cells under hypoxic conditions We evaluated the protein expression of apoptosis-related molecules in HUVECs and mouse EB cells under hypoxic conditions at different time courses using Western blotting. Cells were subjected to hypoxic conditions, collected and treated as described in the Materials and Methods. Gel electrophoresis and electroblotting were performed. Membranes were probed with different antibodies to evaluate the levels of expression of the proteins of interest. As shown in Figure 6A, the expression level of p53 in HUVECs started to increase as early as 6 hours after hypoxia began. The expression level of Bax also signicantly increased after 24 hours of hypoxia. However, the level of Bad and Bcl-2 did not show a signicant change. In the mouse EBs, only the expression level of Bax and p53 signicantly increased, beginning 6 hours after hypoxia began as shown in Figure 6B. Neither the expression

Hypoxia-induced apoptosis in HUVECs & ES cells

Figure 4. Flow cytometric analysis of DNA content in control and hypoxic HUVECs. Cells were incubated with or without hypoxia for different time periods and analyzed after TUNEL staining. The histograms (A) of the HUVECs are shown in the upper panel. The results are quantitated and presented in the lower panel as mean SEM from three independent experiments.

level of Bad nor Bcl-2 showed signicant change. Our data indicates that changes in apoptosis-related molecules are different in HUVECs and mouse EBs in hypoxic conditions.

Discussion
In this study, we rst noted that DNA fragmentation could be identied in HUVECS and mouse EBs under hypoxic conditions. DNA fragmentation increased when the hypoxic interval was extended. The in situ internucleosomal DNA fragmentation-TUNEL assay, which quantitates apoptotic cells in HUVECs and mouse EBs on a cell-by-cell basis, showed that the percentages of apoptotic cells increased gradually when the hypoxic interval was extended. HUVECs and mouse EBs went through the apoptotic pathway in the hypoxic environment. This suggested that the cell death occurred via apoptosis. Intrinsic molecular events were also observed in the hypoxic HUVECs and EBs. The levels of expression of both p53 and Bax increased. Although cell death by hypoxia as a well-known oxidative stress has been generally believed to be manifested

as necrosis,17 recent biochemical observations have suggested the possibility of hypoxia-induced apoptosis.18,19 In this study, DNA fragmentation and TUNEL apoptotic assays demonstrated apoptosis in HUVECs in hypoxic conditions. DNA fragmentation and the percentages of positively stained HUVEC cells in the TUNEL assay increased gradually during hypoxia. The percentage of positively stained HUVEC cells increased from around 5% within 24 hours of the beginning of hypoxia to 17% after 48 hours of hypoxia. However, prominent DNA fragmentation could not be observed in these cells until after 7 days of hypoxia. The TUNEL assay is a sensitive method for evaluating apoptosis of HUVECs in hypoxia. Pluripotent mouse embryonic stem cells are derived from the inner cell mass of mouse blastocysts and can be propagated in vitro indenitely without any genetic manipulation.20,21 In the absence of leukemia inhibitory factor, normal mouse ES cells differentiate spontaneously into three-dimensional structures termed embryoid bodies.22,23 We rst depleted the LIF to induce the mouse ES cells to differentiate into EBs, and then we observed the changes in EBs in chronic hypoxic conditions. Prominent DNA fragmentation could be identied in the EBs 3 days after hypoxia began (Figure 3). The
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Figure 5. Flow cytometric analysis of DNA content in control and hypoxic EB cells. Cells were incubated with or without hypoxia for different time periods and analyzed after TUNEL staining. The histograms (A) for the EB cells are shown in the upper panel. The results are quantitated and presented in the lower panel as mean SEM from three independent experiments.

Figure 6. Hypoxia induces p53 and Bax protein expression in a time-dependent manner. HUVECs (A) and EBs (B) were incubated in hypoxic conditions for various periods of time as indicated after treatment. Cell lysates were prepared and analyzed by Western blotting as described in Materials and Methods

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Hypoxia-induced apoptosis in HUVECs & ES cells

percentages of positively stained cells in the TUNEL assay also increased when the hypoxic condition was extended compared to those in the normoxic conditions (Figure 5). However, the percentages of positively stained mouse EB cells (2.15%) were signicantly lower than those of the HUVEC cells (17.04%) after 48 hours of hypoxia. Our explanation is that cells have several differentiated cell components such as the ectoderm, mesoderm and endoderm and it might result in different sensitivities to the hypoxic environment. Some cells can survive longer in hypoxic conditions than the single cell component HUVECs do. Intrinsic molecular events could be observed in hypoxic conditions in both HUVECs and mouse EBs. It is well accepted that apoptosis is a gene-directed process.24,25 The expression of tumor suppressor genes and oncogenes has been shown to play a role in this process in various cell types.26 In this study, we have shown that the product of the tumor suppressor gene, p53 and one of its downstream targets, Bax, were both increased in cultured HUVECs and EBs in response to hypoxia. The expression levels of p53 and Bax in HUVECs and EBs increased with increasing durations of hypoxia, and this increase correlated with the rise in the percentages of HUVECs and EBs with in situ DNA fragmentation. In response to hypoxia, p53 mediates apoptosis through a linear pathway involving Bax transactivation.27,28 Bax translocates from the cytosol to the membranes, which induces cytochrome c release from mitochondria and caspase-9 activation, followed by the activation of caspase-3, -6, and -7.29,30 p53-mediated apoptosis can be blocked at multiple death checkpoints, including reduction of p53 activity directly, elimination of Bcl-2 family members regulating mitochondrial function, termination of E1B 19K activity by blocking caspase-9 activation, and the addition of caspase inhibitors.30 Accumulation of functional p53 protein followed by p53-dependent apoptosis has been described in cultured cells exposed to hypoxia. p53 and Bax play important roles in the apoptosis of HUVECs and mouse EBs in hypoxic conditions. Further studies will be designed to elucidate the roles of these two molecules in the apoptotic mechanisms of HUVECs and mouse EBs in oxygen-deprived conditions. Cells undergo a variety of biological responses when placed in hypoxic conditions, including activation of signaling pathways that regulate proliferation, angiogenesis and death.31 The responses of HUVEC and EBs to oxygen deprivation are highly complex. It is important to detail how p53 inuences apoptotic process in hypoxic cells so that we could well understand the possible pathogenic processes of apoptosis in hypoxia. Take together, our ndings suggest that it is important in basic research and future implications in the design of therapeutics.32 We conclude that HUVECs and mouse EBs exposed to hypoxia show an increased rate of apoptosis. The induction

of apoptosis in HUVECs and mouse EBs correlates with increases in p53 & Bax expression.

Conclusion
Fetal hypoxia is the major cause of poor fetal outcome and future neonatal and adult handicaps. Many abortions have precedent maternal hemorrhage.3 Hypoxia caused by hemorrhage or ischemic events in the early stage of pregnancy inuences the gestational tissues and nally induces fetal demise.4,5 In this study, morphologic changes, in situ internucleosomal DNA fragmentation, and DNA laddering were observed in both HUVECs and embryoid bodies exposed to hypoxia. The results of this study show that hypoxia-induced apoptosis in cultured HUVECs and embryoid bodies is inuenced by both the duration and severity of hypoxia. We observed that hypoxia alone induces apoptosis and it also increases intracellular protein levels of both p53 and Bax. The increase in p53 is an important process in hypoxia-induced HUVEC and EB apoptosis.

References
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