In Vitro Ischemia-Reperfusion Injury in Term Human Placenta as a Model for Oxidative Stress in Pathological Pregnancies
2001; Elsevier BV; Volume: 159; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)61778-6
ISSN1525-2191
AutoresTai‐Ho Hung, Jeremy N. Skepper, Graham J. Burton,
Tópico(s)Pregnancy-related medical research
ResumoOxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2′,7′-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and α-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. Oxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2′,7′-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and α-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. Preeclampsia is a major cause of maternal and perinatal morbidity and mortality, yet the cause remains unknown. Recent theories have implicated placental oxidative stress as a key intermediary event in the generation of the syndrome.1Roberts JM Hubel CA Is oxidative stress the link in the two-stage model of preeclampsia?.Lancet. 1999; 354: 788-789Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 2Redman CWG Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (432) Google Scholar The mechanisms underlying the generation of the placental stress are however uncertain. Although the activities of the principal antioxidant enzymes and the expression of other antioxidant systems, such as thioredoxin, are decreased within the placental tissues, it is not clear whether these are primary effects or secondary to depletion through the increased generation of free radicals.3Wang Y Walsh SW Antioxidant activities and mRNA expression of superoxide dismutase, catalase, and glutathione peroxidase in normal and pre-eclamptic placentas.J Soc Gynecol Invest. 1996; 3: 179-184Crossref PubMed Scopus (242) Google Scholar, 4Sahlin L Östlund E Wang H Holmgren A Fried G Decreased expression of thioredoxin and glutaredoxin in placentae from pregnancies with preeclampsia and intrauterine growth restriction.Placenta. 2000; 21: 603-609Abstract Full Text PDF PubMed Scopus (32) Google Scholar The most widely recognized predisposing factor for preeclampsia is deficient invasion of the endometrium by extravillous cytotrophoblast cells during the first trimester of pregnancy.5Brosens I Robertson WB Dixon HG The role of spiral arteries in the pathogenesis of preeclampsia.Obstet Gynecol Ann. 1972; 1: 177-191PubMed Google Scholar This results in incomplete conversion of the spiral arteries, such that the myometrial segments do not dilate and remain contractile. Consequently, these vessels display an abnormally high vascular resistance, and are associated with reduced uteroplacental perfusion as confirmed by Doppler flow velocimetric studies.6Khong TY De Wolf F Robertson WB Brosens I Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants.Br J Obstet Gynaecol. 1986; 93: 1049-1059Crossref PubMed Scopus (1408) Google Scholar, 7Meekins JW Pijnenborg R Hanssens M McFadyen IR van Asshe A A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies.Br J Obstet Gynaecol. 1994; 101: 669-674Crossref PubMed Scopus (783) Google Scholar, 8Aquilina J Harrington K Pregnancy hypertension and uterine artery Doppler ultrasound.Curr Opin Obstet Gynecol. 1996; 8: 435-440Crossref PubMed Scopus (22) Google Scholar Hence, in the past it has generally been believed that the changes that characterize the preeclamptic placenta are the result of chronic hypoxia. However, comparison with morphological findings in other situations associated with low oxygen tensions suggests that hypoxia alone is insufficient to account for these changes.9Burton GJ Jauniaux E Watson AL Influence of oxygen supply on placental structure.in: PMS O'Brien T Wheeler DJP Barker Fetal programming: influences on development and disease in later life. RCOG Press, London1999: 326-341Google Scholar 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).10Schachter M Foulds S Free radicals and the xanthine oxidase pathway.in: Grace PA Mathie RT Ischaemia-Reperfusion Injury. Blackwell Science, London1999: 137-147Google Scholar "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.11Halliwell B Gutteridge MC Oxygen is a toxic gas—an introduction to oxygen toxicity and reactive oxygen species.in: Halliwell B Gutteridge MC Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: 1-35Google Scholar 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.12Jones CJ Fox H An ultrastructural and ultrahistochemical study of the human placenta in maternal preeclampsia.Placenta. 1980; 1: 61-76Abstract Full Text PDF PubMed Scopus (180) Google Scholar 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. Except where suppliers are stated individually, the materials and chemicals used in this study were purchased from Sigma Chemical Co., St. Louis, MO. 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. 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. 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. 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% CO2served as controls. Each experiment was repeated in two separate placentas. 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 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. 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 β-mercaptoethanol at 50°C for 50 minutes and reprobed with anti-β-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 β-actin density and quantified by densitometric analysis using the public-domain computer program (Scion Image, Beta Release 4.0.2; Scion Corp., Frederick, MD). 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), α-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. 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. 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). 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). A generally similar pattern of immunolabeling was observed for nitrotyrosine residues. Strong labeling was found in the syncytiotrophoblast, villous endothelium and, to a lesser extent, the stromal cells and vascular smooth muscle layers in stem villi from placental tissues experiencing H/R (Figure 3, c to e). 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). Formation of the toxic lipid peroxidation product, 4-HNE, was also observed, mainly within the villous endothelium after reoxygenation with either air/5% CO2 or 5% O2/90% N2/5% CO2 for 2 hours (Figure 4, c to e). Again, there was virtually no immunoreactivity in control villous tissues (Figure 4, a and b). 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). 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. 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).Figure 8Effects of PBN on the expression of HSP 72 and production of 4-HNE after hypoxia-reoxygenation (2 hours of air/5% CO2). a: A dose-response change was observed in placental tissues preloaded with PBN at a concentration ranging from 1 to 100 mmol/L (Kruskal-Wallis test, H = 11.9, P = 0.0076 and H = 9.7, P = 0.02 for HSP 72 and 4-HNE, respectively). Data are presented as mean ± SEM from three different placentas. *, P < 0.05 compared with vehicle control based on Dunnett's post hoc test. Representative immunoblots for HSP 72 (b) and 4-HNE (c) from two placentas showing a maximal protective effect at a concentration of 100 mmol/L. Lanes 1 and 5, placental tissues preloaded with PBS only (vehicle control); lanes 2 and 6, 1 mmol/L; lanes 3 and 7, 10 mmol/L; lanes 4 and 8, 100 mmol/L PBN, respectively. β-actin shown below was used to normalize for loading variability.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To exclude the possibility that desferrioxamine might adversely interfere with cellular function, leading to a reduced production of HSP 72, placental tissues were subjected to heat shock (45°C, 1 hour) in the presence of 10 mmol/L desferrioxamine. Increased HSP 72 expression was observed compared to controls with desferrioxamine alone (data not shown). 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. Our findings support the three stated hypotheses, and thus confirm that the human placenta is subjected to an ischemia-reperfusion injury when reoxygenated in vitro. First, the results with DCFH-DA show that ROS are generated rapidly in the villous endothelium, and to a lesser extent in the syncytiotrophoblast and stromal cells. Intracellular ROS, mostly superoxide radicals, can be produced by a number of pathways after ischemia-reperfusion. These include mitochondrial electron transfer processes, and a variety of enzymes such as NADPH oxidase and xanthine dehydrogenase/oxidase (XDH/XO).10Schachter M Foulds S Free radicals and the xanthine oxidase pathway.in: Grace PA Mathie RT Ischaemia-Reperfusion Injury. Blackwell Science, London1999: 137-147Google Scholar Immunoreactivity of XDH/XO has been demonstrated in villous endothelium and the syncytiotrophoblast in placentas from uncomplicated pregnancies.13Many A Westerhausen-Larson A Kanbour-Shakir A Roberts JM Xanthine oxidase/dehydrogenase is present in human placenta.Placenta. 1996; 17: 361-365Abstract Full Text PDF PubMed Scopus (53) Google Scholar Under hypoxia both the synthesis and activity of XDH/XO increase, and conversion of the enzyme to the XO form is also enhanced.14Parks DA Williams TK Beckman JS Conversion of xanthine dehydrogenase to oxidase in ischemic rat intestine: a reevaluation.Am J Physiol. 1988; 254: G768-G774PubMed Google Scholar, 15McCord JM Oxygen-derived free radicals in postischemic tissue injury.N Engl J Med. 1985; 312: 159-163Crossref PubMed Scopus (4993) Google Scholar, 16Hassoun PM Yu FS Shedd AL Zulueta JJ Thannickal VJ Lanzillo JJ Fanburg BL Regulation of endothelial cell xanthine dehydrogenase xanthine oxidase gene expression by oxygen tension.Am J Physiol. 1994; 266: L163-L171PubMed Google Scholar This form of the enzyme transfers electrons to molecular oxygen, so generating superoxide radicals. 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