In Vitro Ischemia-Reperfusion Injury in Term Human Placenta as a Model for Oxidative Stress in Pathological Pregnancies

Abstract

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. 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 and transferred to a The was flushed with 95% N2/5% CO2 to a gas of oxygen The of oxygen in the culture medium of this hypoxic was with a oxygen and at to After of incubation hypoxic villous tissues were to culture plates with medium that had been saturated at 37°C with N2/5% CO2 or and in flushed with these gas for to Using these the oxygen in the were to and to In placental tissues were hypoxic the A of placentas were After villous samples from individual were snap-frozen in or in liquid and at for or the generation of ROS during the of placental tissues were hypoxic for as then transferred to a This was from a with a in the sealed with a 0 using The medium that was saturated with CO2 at 37°C and of and was with CO2 and at 37°C on a Using a Germany) the of the 2′,7′-dichlorofluorescein as a result of of 2′,7′-dichlorofluorescein by was in a with The was with a to with a of and a rate of was between to Placental tissues in medium with 95% N2/5% as was in were at in for with with in for with normal in at for then with the primary for for with normal in at The were incubated with at for at in and then incubated in to the for the for at was used as the peroxidase and to was were then and of the primary as the of were as normal in was used to for the of and were used as the primary and secondary was used to the of 72, and were at in for with with normal in at for then with the primary as in for and for and a to nitrotyrosine were After in the were incubated with a of and UK) at with at for The were observed by with and of both The of the in the same results in a leading to a in case of 50 mg of placental tissue was in with mmol/L and Ltd., were at for and the concentrations were by the of using a protein and at of protein samples per were with the of After proteins in one were transferred to a Ltd., with in for and with a or a for the of at or secondary were used in with Chemical to the and on Rochester, The were with a mmol/L and mmol/L at for 50 and with Sigma Chemical to for The was with Ltd., UK) to the The of protein was to the and by using the Placental tissues were with scavengers of ROS in at concentrations to test effects on the expression of and the of after H/R of hypoxia then of reoxygenation with These desferrioxamine α-phenyl-N-tert-butylnitrone 10, and superoxide and of and were as the of placental tissues with were used as The effects of these ROS scavengers were as a of was in from are as were tested for the of and first, then with of or were effects were was used to test the between the of that or could and the of was at of the confirmed that ROS are generated in placental tissues on reoxygenation. cells were to during the of the was on the and the between reoxygenation and the first was The of the and was within of reoxygenation with CO2. The was in the villous endothelium, and to a lesser extent in the syncytiotrophoblast and stromal cells a to In was detected in tissues hypoxic of was in placental tissues with After of culture by of incubation in saturated with CO2 or N2/5% was observed principally in the villous and underlying smooth muscle cells, but also within stromal cells, cytotrophoblast cells, and the syncytiotrophoblast to was observed in tissues immediately after or hypoxic This was to the stromal cells a and A generally of was observed for nitrotyrosine was in the villous to a lesser the stromal cells and vascular smooth muscle in from placental tissues H/R to However, there was in villous tissues that were immediately after or hypoxic a and of the toxic was also observed, within the villous after reoxygenation with CO2 or N2/5% CO2 for to there was in villous tissues a and the of 72, and was by the of to be a more for oxidative stress than nitrotyrosine and which primarily to the endothelium, smooth muscle cells, and the syncytiotrophoblast. In there was also a mild increase in the of in the syncytiotrophoblast after H/R that was not detected using the and the response to hypoxia and the of oxygen tensions and at time was by increased expression of was in placental tissues H/R as to those immediately after or hypoxic was between reoxygenation with CO2 or with N2/5% CO2. there was between and of hypoxia or H/R on the expression of and of after H/R were observed in placental tissues with ROS reduced the expression of = = and the of = at a of mmol/L and the levels of = = and = = with the at after H/R However, not provide effects the expression of or of not of on the expression of and of after of A was observed in placental tissues with at a from to mmol/L H = = and H = = for and are as from with on for and from placentas a at a of and placental tissues with and and and mmol/L was used to for the that desferrioxamine might with leading to a reduced of 72, placental tissues were to heat shock in the of mmol/L Increased expression was observed to with desferrioxamine alone not the of these it was that there was between placentas in the of expression in the samples immediately after levels of stress in determine whether this the of that the ROS scavengers could provide after was The of the for expression associated with the of each was as a of that for the an of the This was then with the of the for the time 0 The was = and = for desferrioxamine and that desferrioxamine and are to provide the H/R injury when levels of stress are Our findings the stated and that the human placenta is to an ischemia-reperfusion injury when in vitro. the results with that ROS are generated in the villous endothelium, and to a lesser extent in the syncytiotrophoblast and stromal cells. superoxide radicals, be by a of after These and a of enzymes such as oxidase and xanthine M Foulds S Free radicals and the xanthine oxidase pathway.in: Grace PA Mathie RT Ischaemia-Reperfusion Injury. Blackwell Science, London1999: 137-147Google Scholar of has been in villous and the syncytiotrophoblast in placentas from A A A JM is present in human 1996; Full Text PDF PubMed Scopus Google Scholar hypoxia both the and of and conversion of the to the is also of xanthine to oxidase in a J Google Scholar, JM free radicals in tissue J PubMed Scopus Google Scholar, AL of endothelial cell xanthine xanthine oxidase expression by oxygen J 1994; Google Scholar This of the to oxygen, superoxide the of hypoxia there is also a of in an increased of Consequently, when oxygen is will the to superoxide and mechanisms of tissue PubMed Scopus Google Scholar The of the generation of toxic hydroxyl radicals from these have that the expression of of oxidative stress is increased after reoxygenation. of the derived from of ischemia-reperfusion injury at other sites are consistent with a role for both superoxide and as in the of 2000; PubMed Scopus Google Scholar, Barker oxide in Grace PA Mathie RT Ischaemia-Reperfusion Injury. Blackwell Science, London1999: Scholar The endothelial of nitric oxide has been in the human and to the villous vessels and L AL of nitric oxide in the human Full Text PDF PubMed Scopus Google Scholar of the of has also been in the villous and to a lesser extent in the syncytiotrophoblast and vascular L AL I F nitric oxide in human placental villous tissue of preeclamptic and intrauterine Full Text PDF PubMed Scopus Google Scholar mRNA the of was in the syncytiotrophoblast and villous smooth muscle A A expression of nitric oxide in and human 1999; PubMed Scopus Google Scholar normal the of exceeds the rate of superoxide However, within after of tissues the between and superoxide is in of superoxide as with than the rate of superoxide S The of with PubMed Scopus Google Scholar the peroxynitrite that is a and R M L R of by 1994; PubMed Scopus Google Scholar R the cytotoxic of superoxide and nitric PubMed Scopus Google Scholar and by such as H L J M by superoxide PubMed Scopus Google Scholar Our of nitrotyrosine residues, a of superoxide, and the the and J 1996; Google Scholar that the villous smooth muscle and the syncytiotrophoblast are the most by peroxynitrite during of has been as the most of free H H and of and PubMed Scopus Google Scholar from acid, and damage by the of H H and of and PubMed Scopus Google Scholar may and the of DNA, and Our results that the principal sites of in placental tissues after H/R are the villous and the consistent with of ROS generation with and of nitrotyrosine The syncytiotrophoblast is to oxidative stress as it is the the intervillous and is the first to the fluctuations in oxygen In in gestation the syncytiotrophoblast concentrations of the principal antioxidant enzymes than other villous AL Jauniaux E GJ in expression of superoxide in villous trophoblast of the human placenta with Full Text PDF PubMed Scopus Google Scholar, AL Jauniaux E GJ in and of within the human placenta during Full Text PDF PubMed Scopus Google Scholar a result it is to oxidative stress at the of the trimester when the oxygen tension in the intervillous space with onset of the maternal to the E Watson AL J GJ of maternal and placental oxidative a possible factor in human J 2000; Full Text Full Text PDF PubMed Scopus Google Scholar The are to as to and of other proteins during periods of stress cell of stress and for and PubMed Scopus Google Scholar In is the major of the and protein levels increase through of factor in response to a of heat oxidative radicals, and stress cell of stress and for and PubMed Scopus Google Scholar, shock protein and PubMed Scopus Google Scholar Oxidative stress cause proteins to become and at the of the to the heat shock and of protein a consequence of protein 1999; PubMed Scopus Google Scholar of has been in of it a of ischemia-reperfusion R H J J expression of heat shock proteins and mRNA after ischemia-reperfusion injury of 1999; PubMed Scopus Google Scholar, M M of heat shock proteins in after 1986; PubMed Scopus Google Scholar, J A T M S K T K of heat shock factor in 1996; PubMed Scopus Google Scholar Furthermore, evidence that increased expression of is of cells or tissues from the hypoxia and stress cell of stress and for and PubMed Scopus Google Scholar, shock protein and PubMed Scopus Google Scholar the expression of not as a of oxidative but more an response at the of proteins within the The of was with the of or the role of in these or from oxidative damage. may therefore important in vascular muscle and syncytiotrophoblast in have that the ROS scavengers desferrioxamine and the expression of and of after is a that and the generation of hydroxyl radicals from superoxide and in the of B Gutteridge MC disease and a role for B Halliwell MC Gutteridge Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: Scholar The of desferrioxamine therefore an important role for in the generation of hydroxyl radicals in the H/R a has been to ischemia-reperfusion injury in A H Y T T of on during intrauterine and in 2000; PubMed Scopus Google Scholar, J T M of in free generation by with during in PubMed Scopus Google Scholar However, in vitro of it to be a antioxidant and of and of is B Gutteridge MC disease and a role for B Halliwell MC Gutteridge Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: Scholar were to by the expression of or of after at a than that in endothelial M of oxidative stress in injury to endothelial 2000; PubMed Scopus Google Scholar This may be because not and is therefore not to the effects of superoxide which are and remain I and superoxide PubMed Scopus Google Scholar these that the key for an ischemia-reperfusion injury are present within human placental tissues, and that the are to such an in vitro. Furthermore, the of for and nitrotyrosine there are between the oxidative stress and that observed in complications of pregnancy, such as preeclampsia and growth A Hubel CA JM Y evidence of oxidative stress in J 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, L AL I F in of peroxynitrite and 1996; PubMed Scopus Google Scholar, SW Wang Y Placental is increased in 2000; PubMed Google Scholar Although evidence that an ischemia-reperfusion injury in the preeclamptic placenta is at the hypothesis is consistent with the that maternal with the antioxidant and E has a AL R K L of on the of preeclampsia in at increased a 1999; 354: Full Text Full Text PDF PubMed Scopus Google Scholar The of preeclampsia with of spiral artery also a underlying mechanism. confirmed in the present hypoxia alone is not to cause placental oxidative However, retention of vasoreactivity in the spiral arteries may to perfusion of the intervillous and to oxygen concentrations within the placental perfusion be in of maternal and in pregnancy, in which the but effects of both and on the uterine have been to cause decreased uteroplacental perfusion and growth A G and in in to and 1999; Full Text PDF PubMed Scopus Google Scholar Ischemia-reperfusion type injury may therefore provide a for the generation of placental oxidative stress in a of The of the stress may be between the study villous with underlying smooth muscle cells and the syncytiotrophoblast are the most where peroxynitrite and ROS generated from this H/R may as of the endothelial cells and of the vascular smooth muscle K in vascular cell and a of the of reactive oxygen species in smooth muscle and endothelial cell and 2000; PubMed Scopus Google Scholar This is by on the of arteries from preeclamptic placentas more prominent of endothelial and smooth muscle cells as to placentas from normal of arteries of human placenta in of 3: Scholar If the of H/R and the of ROS may the capacity of the placental antioxidant and dysfunction and tissue damage will This may be in the syncytiotrophoblast because of antioxidant is to determine whether the is to the changes in the syncytiotrophoblast that have been to of is increased in and the is to an response in the leading to endothelial cell CWG Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (432) Google Scholar In have that changes consistent with an ischemia-reperfusion injury when hypoxic placental tissues are in vitro. the results have for of the generation of placental oxidative stress in complications of such as preeclampsia and the of the of the for in the placental and the of the of University of which was with from the and where the was