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From the Department of Obstetrics and Gynaecology,*
Chang Gung Memorial Hospital, Taipei, Taiwan; and the Department of
Anatomy,
University of Cambridge, Cambridge,
United Kingdom
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Abstract |
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-phenyl-N-tert-butylnitrone reduced
levels of oxidative stress after reoxygenation. These changes are
consistent with an ischemia-reperfusion injury, and mirror
those seen in preeclampsia. Consequently, in
vitro hypoxia/reoxygenation may represent a suitable model
system for investigating the generation of placental oxidative stress
in preeclampsia and other complications of pregnancy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An alternative hypothesis is that the retention of vasoreactivity in the incompletely remodeled arteries results in the maternal blood flow to the intervillous space being more variable than normal. The constancy of the placental perfusion may be a more important factor than the absolute rate of blood flow, for because both the fetus and the placenta extract considerable quantities of oxygen during mid to late gestation the placental tissues will soon become locally hypoxic during periods of vasoconstriction. When the maternal blood flow is re-established there will therefore be a rapid increase in tissue oxygenation, and such fluctuations in oxygen tension could provide the basis for an ischemia-reperfusion type insult. Depending on the severity and frequency of these insults the outcome might range from mild oxidative stress to severe tissue damage and frank infarction.
Ischemia-reperfusion injury is now a well-recognized consequence of malperfusion in many organ systems, and is mediated principally through the generation of cytotoxic reactive oxygen species (ROS).10 "Reactive oxygen species" is a term used to describe a broad category of molecules that includes oxygen-containing radicals, such as superoxide, nitric oxide (NO), and hydroxyl radicals, and nonradical but reactive molecules derived from oxygen, for example hydrogen peroxide (H2O2), hypochlorous acid, and the peroxynitrite anion.11 If the generation of ROS exceeds the capacity of the antioxidant defenses then oxidative stress results, in which there may be indiscriminate damage to lipids, proteins and DNA, leading to cell dysfunction and tissue damage. In the case of the placenta such injuries could account for the oxidative stress observed, and for the increased rates of infarction and syncytial necrosis.12
The aim of the present study was to investigate the ischemia-reperfusion phenomenon in term placental tissues during the periods of hypoxia and reoxygenation (H/R) in vitro, to determine whether H/R may represent a suitable model system for investigating the generation of placental oxidative stress in preeclampsia and other complications of pregnancy. Our hypotheses to be tested were; first, that abundant ROS are generated during reoxygenation of hypoxic placental tissues; second, that there is evidence of oxidative stress in placental tissues after H/R; and third, that preloading of placental tissues with ROS scavengers reduces the levels of oxidative stress in hypoxia/reoxygenation.
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Materials and Methods |
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Except where suppliers are stated individually, the materials and chemicals used in this study were purchased from Sigma Chemical Co., St. Louis, MO.
Tissue Collection
Term placentas (n = 15) were obtained from
normal pregnancies with permission immediately after elective cesarean
deliveries for repeat section before onset of labor. Villous samples
(n 
10, each 
40 to 50 mg wet weight) were
taken midway between the chorionic and basal plates, from five to seven
lobules free of visible infarction, calcification, hematoma, or tears.
After a brief rinse in cold phosphate-buffered saline (PBS), one sample
was snap-frozen in liquid nitrogen as a time 0 control. The remaining
samples were placed into culture medium (Medium-199 with 25 mmol/L
HEPES, Earle’s salts, and L-glutamine; Life
Technologies Ltd., Paisley, UK) equilibrated with 95%
N2/5% CO2 (BOC, Guildford,
UK) in a sealed glass bottle, and transferred to the laboratory on ice
for individual experiments.
Establishment of Hypoxic Conditions
Using a special incubation bag with a moistened Anaerocult IS (Merck KgaA, Darmstadt, Germany) inside, villous samples were incubated in 4-well, flat-bottom culture plates (one sample per well) (Nunclon Delta Multidishes; Nalge Nunc International, Rochester, NY) with fresh medium that had been saturated at 37°C with 95% N2/5% CO2. The bag was flushed with the same gas mix for 1 minute, sealed, and transferred to a separate chamber (Desiccator Cabinet; Scientific Laboratory Supplies Limited, Nottingham, UK). The chamber was continuously flushed with 95% N2/5% CO2 to maintain a gas phase PO2 of <1 mmHg (OM-14 oxygen monitor; SensorMedics Corporation, Yorba Linda, CA). The partial pressure of oxygen in the culture medium of this hypoxic condition was monitored with a portable dissolved oxygen meter (model 9071; Jenway Inc., Princeton, NJ) and kept at 12 to 16 mmHg.
Conditions for Hypoxia-Reoxygenation
After 20 minutes of incubation under hypoxic conditions, villous tissues were randomly assigned to culture plates with medium that had been saturated at 37°C with either 5% O2/90% N2/5% CO2 or air/5% CO2, and maintained in separate humidified chambers continuously flushed with these respective gas mixes for up to 2 hours. Using these settings, the measured dissolved oxygen pressures in the media were 45 to 62 mmHg and 143 to 160 mmHg, respectively. In comparative experiments, placental tissues were cultured under hypoxic conditions throughout the 2-hour period. A total of six separate placentas were studied.
After culture, villous samples from individual experiments were snap-frozen in either precooled isopentane or directly in liquid nitrogen, and stored at -80°C for further immunohistochemistry or Western blot analysis.
Detection of ROS Generation with 2',7'-Dichlorofluorescein Diacetate (DCFH-DA)
To detect the generation of ROS during the period of reoxygenation, placental tissues were cultured under hypoxic conditions for 20 minutes as previously described, then transferred to a microscope chamber. This was created from a 30-mm Petri dish with a 25-mm hole in the bottom, sealed with a 27 mm, no. 0 thickness coverslip using elastomeric glue (Sylgard; Dow Corning Corporation, Wiesbaden, Germany). The chamber contained medium that was saturated with air/5% CO2 at 37°C and 20 µmol/L of DCFH-DA, and was continuously gassed with air/5% CO2 and kept at 37°C on a heating stage. Using a Leica TCS SP-MP confocal microscope (Leica Microsystems, Heidelberg, Germany) the formation of the fluorescent product 2',7'-dichlorofluorescein (DCF), as a result of oxidation of 2',7'-dichlorofluorescein (DCFH) by ROS, was recorded in a time-lapse series with 30-second intervals. The dye was excited with a Tsunami T1/Sapphire laser tuned to 780 nm with a pulse width of 1.3 picoseconds and a repetition rate of 82 MHz. Emitted light was captured between 500 to 560 nm. Placental tissues kept in medium continuously gassed with 95% N2/5% CO2 served as controls. Each experiment was repeated in two separate placentas.
Immunohistochemical Staining of Inducible Heat Shock Protein 72 (HSP 72), Nitrotyrosine, and 4-Hydroxy-2-Nonenal (4-HNE)
Serial sections were cut at 10 µm, fixed in acetone (-20°C) for 10 minutes, washed with PBS, quenched with 3% H2O2 in methanol for 10 minutes, blocked with 10% normal goat serum, 0.2% Tween-20 in PBS (PBS-T) at room temperature for 1 hour, then reacted with the primary antibodies [1:2000 for rabbit polyclonal anti-HSP 72 antibody (Stressgen Biotechnologies Corp., Victoria, BC, Canada) 1:1000 for rabbit polyclonal anti-nitrotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY)] diluted with 5% normal goat serum in PBS-T at 4°C overnight. The sections were incubated with biotinylated goat anti-rabbit IgG at 1:200 dilution for 30 minutes at room temperature, washed in PBS, and then incubated in avidin-biotin-peroxidase solution according to the instructions for the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) for another 30 minutes. Diaminobenzidine tetrahydrochloride at 0.5 mg/ml was used as the peroxidase substrate, and allowed to develop until optimal staining was achieved. Slides were then counterstained, dehydrated, and coverslipped. Omission of the primary antibodies served as the negative controls.
For detection of 4-HNE, sections were processed as described above except 10% normal horse serum in PBS-T was used to block nonspecific binding. Mouse monoclonal anti-4-HNE antibody (1:10; Japan Institute for the Control of Ageing, Shizuoka, Japan) and biotinylated horse anti-mouse IgG (Vector Laboratories) were used as the primary and secondary antibodies, respectively.
Double-Immunofluorescent Labeling for HSP 72, Nitrotyrosine, and 4-HNE
Double-immunofluorescent labeling was used to demonstrate the relative localization of HSP 72, nitrotyrosine, and 4-HNE. Serial sections were cut at 10 µm, fixed in acetone (-20°C) for 10 minutes, washed with PBS, blocked with 10% normal goat serum in PBS-T at room temperature for 1 hour, then reacted with the primary antibodies as described in immunohistochemistry except different working dilutions (1:1000 for anti-HSP 72 and 1:5 for anti-4-HNE, respectively) and a mouse monoclonal antibody to nitrotyrosine (1:20; Upstate Biotechnology Inc.) were used. After washing in PBS, the sections were incubated with a cocktail of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG (CN Biosciences, Nottingham, UK) diluted at 1:50 with PBS at room temperature for 1 hour. The slides were observed by confocal microscopy (Leica TCS-NT; Leica Microsystems, Heidelberg, Germany), with simultaneous excitation and detection of both dyes. The superimposition of the two chromophores in the same image results in a green/red color scale, leading to a yellow color in case of co-localization.
Western Blot
Approximately 50 mg of placental tissue was homogenized in ice-cold distilled water with 0.05% Triton X-100, 1 mmol/L dithiothreitol, and Complete mini protease inhibitor cocktail (Roche Diagnostics Ltd., East Sussex, UK). Tissue homogenates were centrifuged at 13,000 rpm for 20 minutes and the supernatant removed. Protein concentrations were determined by the Peterson’s modification of microLowry method using a protein determination kit (from Sigma) and absorbance measured at 665 nm. Equal amounts of protein samples (40 µg per lane) were separated with the use of 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, proteins in one gel were transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK), blocked with 5% skimmed milk in PBS-T for 1 hour and subsequently probed with a rabbit polyclonal anti-HSP 72 (1:20,000; Stressgen Biotechnologies Corp.) or a mouse monoclonal anti-4-HNE antibody (1:10; Japan Institute for the Control of Aging) at 4°C overnight. Horseradish peroxidase-linked donkey anti-rabbit or sheep anti-mouse secondary antibodies (1:2000; Amersham Pharmacia Biotech UK Ltd.) were used in conjugation with enhanced chemiluminescence (SuperSignal West Pico, Pierce Chemical Company, Rockford, IL) to visualize the HSP 72 and 4-HNE bands on autoradiography films (BioMax Light; Eastman Kodak Company, Rochester, NY).
The membranes were stripped with a buffer containing 62.5 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, and 100 mmol/L ß-mercaptoethanol at 50°C for 50 minutes and reprobed with anti-ß-actin monoclonal antibody (clone AC-15, 1:2000 dilution; Sigma Chemical Co.) to correct for loading variations. The duplicate gel was directly stained with Coomassie brilliant blue G250 (Bio-Rad Laboratories Ltd., Hertfordshire, UK) to confirm equal loading among the lanes. The relative intensity of protein signals was normalized to the corresponding ß-actin density and quantified by densitometric analysis using the public-domain computer program (Scion Image, Beta Release 4.0.2; Scion Corp., Frederick, MD).
Effects of ROS Scavengers on the Expression HSP 72 and Production of 4-HNE after Hypoxia-Reoxygenation
Placental tissues were preloaded with different scavengers of ROS
dissolved in PBS at various concentrations to test their effects on the
expression of HSP 72 and the production of 4-HNE after H/R (1 hour of
hypoxia then 2 hours of reoxygenation with air/5%
CO2). These included desferrioxamine (0.1, 1, 10
mmol/L), -phenyl-N-tert-butylnitrone (PBN; 1,
10, 100 mmol/L), and superoxide dismutase (SOD; 60 and 600 U/ml).
Immunoblots of HSP 72 and 4-HNE were quantitatively analyzed as the
above described. Densities of placental tissues administrated with PBS
only were used as vehicle controls. The effects of these ROS scavengers
were expressed as a ratio of controls. Each experiment was performed in
triplicate from three separate placentas.
Statistical Analysis
All data are presented as mean ± SEM. They were tested for the homogeneity of variance (Bartlett’s test) and normality (Kolmogorov-Smirnov test) first, then computed with analysis of variance or nonparametric tests (Kruskal-Wallis test). Post hoc tests were performed if significant effects were determined. Pearson correlation analysis was used to test the correlation between the degree of protection that desferrioxamine, PBN, or SOD could offer, and the level of endogenous stress. Statistical significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formation of the fluorescent product DCF confirmed that ROS are
generated rapidly in placental tissues on reoxygenation. Occasional
cells were beginning to fluoresce during the inevitable period of delay
while the chamber was positioned on the microscope stage, and focusing
took place. Overall, the maximum interval between reoxygenation and the
first image capture was 3 minutes. The intensity of the fluorescence
rapidly increased, and saturation was achieved within 15 minutes of
reoxygenation with air/5% CO2. The fluorescence
was localized mainly in the villous endothelium, and to a lesser extent
in the syncytiotrophoblast and stromal cells (Figure 1, a to d)
. In contrast, no fluorescence
was detected in control tissues kept hypoxic throughout (Figure 1e)
.
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Expression of HSP 72 was greatly enhanced in placental tissues
treated with H/R. After 20 minutes of culture under hypoxia, followed
by 2 hours of incubation in media saturated with either air/5%
CO2 or 5% O2/90%
N2/5% CO2, intense
immunostaining was observed principally in the villous endothelium and
underlying smooth muscle cells, but also within stromal cells,
cytotrophoblast cells, and the syncytiotrophoblast (Figure 2, c to e)
. By contrast, only minimal
staining was observed in control tissues frozen immediately after
delivery or kept hypoxic throughout. This was mainly localized to the
stromal cells (Figure 2, a and b)
.
|
. However, there was virtually
no labeling in control villous tissues that were frozen immediately
after delivery or cultured under hypoxic conditions (Figure 3, a and b)
.
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. Again, there was virtually no
immunoreactivity in control villous tissues (Figure 4, a and b)
.
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Information about the relative localization of HSP 72,
nitrotyrosine, and 4-HNE was revealed by the use of
double-immunofluorescent labeling (Figure 5)
. HSP 72 seemed to be a more general
marker for oxidative stress than nitrotyrosine and 4-HNE, which
appeared primarily confined to the endothelium, adjacent smooth muscle
cells, and the syncytiotrophoblast. In addition, there was also a mild
increase in the fluorescent signal of 4-HNE in the syncytiotrophoblast
after H/R that was not detected using the chromogenic technique (Figure 5, e and f)
.
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To quantitate the cellular response to hypoxia and H/R, the induction of HSP 72 under different oxygen tensions and at different time intervals was monitored by immunoblotting.
Significantly increased expression of HSP 72 was noted in placental
tissues experiencing H/R as compared to those sampled immediately after
delivery or kept under hypoxic conditions throughout (Figure 6)
. There was no difference, however,
between reoxygenation with air/5% CO2 or with
5% O2/90% N2/5%
CO2. Equally, there was no difference between 1
hour and 2 hours of hypoxia or H/R treatment.
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Concentration-dependent responses on the
expression of HSP 72 and production of 4-HNE after H/R were observed in
placental tissues preloaded with ROS scavengers. Desferrioxamine
significantly reduced the expression of HSP 72
(H = 15.3, P = 0.0016) and the
production of 4-HNE (H = 8.9, P
= 0.03), particularly at a concentration of 10 mmol/L
(P < 0.05 and < 0.01, respectively)
(Figure 7)
. Similarly, PBN attenuated the
levels of HSP 72 (H = 11.9, P =
0.0076) and 4-HNE (H = 9.7, P =
0.02), with the maximal effect at 100 mmol/L, after H/R (Figure 8)
. However, SOD did not provide
significant protective effects against the expression of HSP 72 or
production of 4-HNE (data not shown).
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Correlation between the Degree of Protection Provided by Desferrioxamine and PBN and the Level of Endogenous Stress
During the course of these experiments it was noticed
that there was variation between placentas in the level of HSP 72
expression in the samples frozen immediately after delivery, indicating
differing levels of endogenous stress in vivo. To determine
whether this influenced the degree of protection that the ROS
scavengers could provide after H/R, correlation analysis was performed.
The density of the immunoblot for HSP 72 expression associated with the
maximal concentration of each scavenger was expressed as a ratio of
that for the vehicle control, generating an index of the protection
provided. This value was then correlated with the density of the
immunoblot for the time 0 sample. The correlation coefficient was 0.91
(P = 0.001) and 0.86 (P =
0.006) for desferrioxamine and PBN, respectively (Figure 9)
, showing that desferrioxamine and PBN
are able to provide the greatest protection against H/R injury when
levels of endogenous stress are low.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Second, we have demonstrated that the expression of several well-characterized markers of oxidative stress is increased after reoxygenation. Much of the data derived from studies of ischemia-reperfusion injury at other sites are consistent with a role for both superoxide and NO as participants in the altered endothelium-dependent response.18,19 The endothelial isoform of nitric oxide synthase (NOS) has been characterized in the human placenta, and immunohistochemically localized to the stem villous vessels and syncytiotrophoblast.20 Immunoreactivity of the inducible isoform of NOS has also been detected, mainly in the villous stroma and to a lesser extent in the syncytiotrophoblast and vascular endothelium.21 Recently, mRNA encoding the inducible isoform of NOS was found in the syncytiotrophoblast and villous arteriolar smooth muscle cells.22 Under normal conditions, the flux of NO greatly exceeds the rate of superoxide production. However, within minutes after reperfusion of ischemic tissues the balance between NO and superoxide production is tipped in favor of superoxide as previously described. Superoxide reacts with NO much faster than the rate of superoxide dismutation,23 thus producing the peroxynitrite anion that is a strong and long-lived oxidant. Peroxynitrite can inhibit mitochondrial electron transport,24 oxidize proteins, initiate lipid peroxidation,25 and affect signal transduction by nitrating aromatic amino acids such as tyrosine.26 Our immunostaining of nitrotyrosine residues, a fingerprint of peroxynitrite,27 shows that the stem villous vessels, including their smooth muscle layers, and the syncytiotrophoblast are the most vulnerable areas attacked by peroxynitrite during H/R.
Because of its stability, 4-HNE has been suggested as the most reliable index of free radical-induced lipid peroxidation.28 4-HNE originates almost exclusively from phospholipid-bound arachidonic acid, and causes cellular damage mainly by the modification of intracellular proteins.28 It may thus inhibit enzyme reactions and the synthesis of DNA, RNA, and proteins. Our results reveal that the principal sites of lipid peroxidation in placental tissues after H/R are the villous endothelium and the syncytiotrophoblast, consistent with our observations of ROS generation with DCFH-DA and immunostaining of nitrotyrosine residues. The syncytiotrophoblast is particularly vulnerable to oxidative stress as it is the endothelium-like surface layer lining the intervillous space, and so is the first to confront the fluctuations in oxygen tension. In addition, particularly in early gestation the syncytiotrophoblast contains much lower concentrations of the principal antioxidant enzymes than other villous tissues.29,30 As a result it is selectively subjected to oxidative stress at the start of the second trimester when the oxygen tension in the intervillous space rises with onset of the maternal arterial circulation to the placenta.31
The HSPs are known to serve as molecular chaperones to facilitate folding and prevent denaturation of other proteins during periods of stress.32 In particular, HSP 72 is the major inducible member of the family and protein levels increase through activation of heat-shock transcription factor in response to a variety of exogenous stressors including heat shock, oxidative radicals, and inflammatory cytokines.32,33 Oxidative stress can cause thermal stable proteins to become destabilized and undergo denaturation at physiological temperature, thus inducing the synthesis of HSP.34 Up-regulation of HSP 72 has been found in conditions of H/R, rendering it a sensitive marker of ischemia-reperfusion injury.35-37 Furthermore, substantial evidence indicates that increased expression of HSP 72 is capable of protecting cells or tissues from the subsequent noxious conditions, including hypoxia and ischemia-reperfusion.32,33 Therefore, the expression of HSP 72 not only serves as a marker of oxidative stress, but more importantly represents an adaptive response aimed at preventing the aggregation of denatured proteins within the cytosol. The immunostaining of HSP 72 was compatible with the localization of nitrosylated or 4-HNE modified proteins, indicating the potential role of HSP 72 in helping these areas resist or recover from oxidative damage. HSP 72 may therefore play important roles in maintaining fetal vascular muscle tone and syncytiotrophoblast function in vivo.
Third, we have demonstrated that the ROS scavengers desferrioxamine and PBN significantly reduce the expression of HSP 72 and production of 4-HNE after H/R. Desferrioxamine is a membrane-permeable iron chelator that inhibits lipid peroxidation, and the generation of hydroxyl radicals from superoxide and H2O2 in the presence of iron ions.38 The protective efficacy of desferrioxamine we demonstrated therefore implicates an important role for iron in the generation of hydroxyl radicals in the H/R injury.
PBN, a common spin-trapping compound, has been reported to protect against ischemia-reperfusion injury in animal studies.39,40 However, in vitro characterization of PBN shows it to be a poor chain-breaking antioxidant and inhibitor of lipid peroxidation, and so its mechanism of action is uncertain.38
Unexpectedly, we were unable to show protection by SOD against the expression of HSP 72 or formation of 4-HNE after H/R, even at a concentration 10 times higher than that reported in cultured rat hepatic sinusoidal endothelial cells.41 This may be because SOD does not permeate biological membranes and is therefore not able to reduce the detrimental effects of superoxide anions, which are produced and remain injurious intracellularly.42
Taken together, these data indicate that the key intermediaries required for an ischemia-reperfusion injury are present within human placental tissues, and that the villi are indeed vulnerable to such an insult in vitro. Furthermore, the patterns of immunostaining for 4-HNE and nitrotyrosine residues suggest there are parallels between the resultant oxidative stress and that observed in some complications of pregnancy, such as preeclampsia and fetal growth restriction.43-45 Although conclusive evidence that an ischemia-reperfusion injury occurs in the preeclamptic placenta is lacking at present, the hypothesis is consistent with the recent finding that maternal supplementation with the antioxidant vitamins C and E has a beneficial effect.46 The strong association of preeclampsia with failure of spiral artery remodeling also provides a plausible underlying mechanism. As confirmed in the present study, hypoxia alone is not sufficient to cause placental oxidative stress. However, retention of vasoreactivity in the spiral arteries may lead to intermittent perfusion of the intervillous space, and hence to fluctuating oxygen concentrations within the placenta. Similar assumptions regarding intermittent placental perfusion can be made in cases of maternal cocaine use and cigarette smoking in pregnancy, in which the transient but repetitive vasoconstrictive effects of both cocaine and nicotine on the uterine vasculature have been clearly demonstrated to cause decreased uteroplacental perfusion and fetal growth restriction.47 Ischemia-reperfusion type injury may therefore provide a common pathway for the generation of placental oxidative stress in a number of diverse clinical conditions.
The consequences of the stress may be different between the different sites. As our immunohistochemical study revealed, villous endothelium together with underlying smooth muscle cells and the syncytiotrophoblast are the most vulnerable areas where peroxynitrite formation and lipid peroxidation occurred. ROS generated from this intermittent H/R may initially act as intracellular signaling molecules, mediating proliferation of the endothelial cells and hypertrophy of the vascular smooth muscle cells.48 This is supported by observations on the ultrastructure of fetal stem arteries from preeclamptic placentas showing more prominent proliferation of endothelial and smooth muscle cells as compared to placentas from normal pregnancy.49 If the condition of H/R repeats and persists, the amplitude of ROS production may overwhelm the capacity of the placental antioxidant defenses, and cellular dysfunction and tissue damage will result. This may be critical in the syncytiotrophoblast because of its lower antioxidant defenses. Further work is required to determine whether the insult is sufficient to exacerbate the apoptotic changes in the syncytiotrophoblast that have been proposed to underlie shedding of microvillous fragments. Shedding is increased in preeclampsia, and the resultant debris is thought to activate an enhanced inflammatory response in the mother, leading to generalized endothelial cell activation.2
In summary, we have demonstrated that changes consistent with an ischemia-reperfusion injury occur when hypoxic placental tissues are reoxygenated in vitro. Moreover, the results have implications for our understanding of the generation of placental oxidative stress in complications of pregnancy such as preeclampsia and cigarette smoking.
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Acknowledgements |
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Footnotes |
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Supported by a grant from Chang Gung Memorial Hospital, Taiwan (CMRP 1034) and a Benefactor Scholarship from St John’s College, University of Cambridge (to T.-H. H.).
Accepted for publication June 6, 2001.
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, P < 0.01,
significantly different from levels of placental tissues cultured under
hypoxic conditions (95%
N2/5%
CO2). Filled
circle, cultured under hypoxia; filled triangle,
hypoxia-reoxygenation (5%
O2/90% N2/5%
CO2); open
square, hypoxia-reoxygenation (air/5%
CO2). b: The
bands shown are the representatives of immunoblots performed on five
sets of experiments. Lane 1, time 0; lanes 2 and
3, cultured under hypoxia at 1 hour and 2 hours,
respectively; lanes 4 and 5,
hypoxia-reoxygenation (5%
O2/90% N2/5%
CO2) for 1 hour and 2
hours; lanes 6 and 7, hypoxia-reoxygenation
(air/5%
CO2) for 1 hour and 2
hours, respectively. ß-actin shown below was used to normalize for
loading variability.
