Scholarly article on topic 'Hypoxia and Reoxygenation: a Possible Mechanism for Placental Oxidative Stress in Preeclampsia'

Hypoxia and Reoxygenation: a Possible Mechanism for Placental Oxidative Stress in Preeclampsia Academic research paper on "Basic medicine"

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{hypoxia / "oxidative stress" / placenta / preeclampsia / "reperfusion injury"}

Abstract of research paper on Basic medicine, author of scientific article — Tai-Ho Hung, Graham J. Burton

Summary Preeclampsia is a human pregnancy-specific disorder that is diagnosed by the new appearance of hypertension and proteinuria after 20 weeks' gestation. It is a leading cause of perinatal morbidity and mortality, and the only intervention that effectively reverses the syndrome is delivery. Oxidative stress of the placenta is considered to be a key intermediary step in the pathogenesis of preeclampsia, but the cause for the stress remains unknown. Hypoxia-reoxygenation (H/R) injury, as a result of intermittent placental perfusion secondary to deficient trophoblast invasion of the endometrial arteries, is a possible mechanism. In this review, we present evidence to show that there is a plausible basis from which to assume that blood flow in the intervillous space will be intermittent in all normal pregnancies. The intermittency will be exacerbated by impaired conversion of the spiral arteries, or by the presence of atherotic changes that reduce their caliber as seen in preeclampsia. Placental oxidative stress can be the consequences of fluctuations in oxygen concentrations after H/R through the actions of reactive oxygen species. On this basis, there will be a complete spectrum of placental changes among the normal, the late onset and the early onset preeclamptic states. Viewing the syndrome as a continuum of H/R insults provides new insight into the pathophysiology of pregnancy that will hopefully lead to improved clinical interventions.

Academic research paper on topic "Hypoxia and Reoxygenation: a Possible Mechanism for Placental Oxidative Stress in Preeclampsia"


Hypoxia and Reoxygenation: A Possible Mechanism for Placental Oxidative Stress in Preeclampsia

Tai-Ho Hung*, Graham J. Burton1 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Taipei, Taiwan and department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.


Preeclampsia is a human pregnancy-specific disorder that is diagnosed by the new appearance of hypertension and proteinuria after 20 weeks' gestation. It is a leading cause of perinatal morbidity and mortality, and the only intervention that effectively reverses the syndrome is delivery. Oxidative stress of the placenta is considered to be a key intermediary step in the pathogenesis of preeclampsia, but the cause for the stress remains unknown. Hypoxia-reoxygenation (H/R) injury, as a result of intermittent placental perfusion secondary to deficient tro-phoblast invasion of the endometrial arteries, is a possible mechanism. In this review, we present evidence to show that there is a plausible basis from which to assume that blood flow in the intervillous space will be intermittent in all normal pregnancies. The intermittency will be exacerbated by impaired conversion of the spiral arteries, or by the presence of atherotic changes that reduce their caliber as seen in preeclampsia. Placental oxidative stress can be the consequences of fluctuations in oxygen concentrations after H/R through the actions of reactive oxygen species. On this basis, there will be a complete spectrum of placental changes among the normal, the late onset and the early onset preeclamptic states. Viewing the syndrome as a continuum ofH/R insults provides new insight into the pathophysiology of pregnancy that will hopefully lead to improved clinical interventions. [TaiwaneseJ Obstet Gynecol 2006;45(3):189-200]

Key Words: hypoxia, oxidative stress, placenta, preeclampsia, reperfusion injury

Definition and Etiology of Preeclampsia

Preeclampsia is a human pregnancy-specific disorder that is diagnosed by the new appearance of hypertension and proteinuria after 20 weeks' gestation [1]. The incidence of preeclampsia is between 2% and 10% of pregnancies, depending on the definition used and population studied [2-5]. It is a leading cause of perinatal morbidity and mortality, and the only intervention that effectively reverses the syndrome is delivery. Consequently, a large proportion of the perinatal

* Correspondence to: Dr Tai-Ho Hung, Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, 199, Dun-Hua North Road, Taipei 105, Taiwan. E-mail: Accepted: May 25, 2006

morbidity is due to iatrogenic prematurity. It is estimated that up to 15% of preterm births are secondary to delivery for preeclampsia [6].

The cause of preeclampsia is still unknown. A completely satisfactory, unifying hypothesis has not emerged. There may be several underlying predispositions with effects that result in the common group of signs and symptoms. In 1989, Roberts et al formally proposed that maternal endothelial cell dysfunction is the key event resulting in the diverse clinical manifestations of preeclampsia [7]. Endothelial cell dysfunction or activation is a term used to define an altered state of endothelial cell differentiation, typically induced as a result of cytokine stimulation. Evidence has since accumulated to support a major role for endothelial dysfunction, including vasospasm and increased vascular reactivity, as the final common pathway of several different pathophysiologic mechanisms in preeclampsia [8-10].

Recently, the hypothesis has been extended in two ways. First, it has been proposed that some women are more sensitive to endothelial dysfunction or have preexisting endothelial dysfunction associated with a long-term tendency to diseases such as hypertension, diabetes or collagen vascular diseases [11]. This gives a clear explanation of the long-known association between these medical conditions and preeclampsia. Second, it has been suggested that endothelial dysfunction is one aspect of a generalized systemic maternal inflammatory response that also affects circulating leukocytes [12,13]. In other words, preeclampsia is not a separate condition but simply the extreme end of a range of maternal systemic inflammatory response engendered by pregnancy itself. This continuum theory of preeclampsia implies that any factor that increases the maternal systemic inflammatory response to pregnancy will predispose to preeclampsia [14,15].

Role of Placenta in Preeclampsia

So, what feature of pregnancy results in the profound and reversible alteration of maternal physiologic functions such as the inflammatory response and endo-thelial activation that are characteristic of the preeclampsia syndrome? Diverse evidence shows that the placenta serves as the centerpiece in the patho-genesis of preeclampsia. Preeclampsia can occur with hydatidiform moles, suggesting that the presence of a fetus is not strictly necessary [16,17]. A distended uterus is probably not required as preeclampsia may develop in abdominal pregnancies in which the uterus is minimally enlarged [18,19]. Moreover, the clinical signs and lesions of preeclampsia remit within days after termination of pregnancy [20].

If we accept that the placenta is where the disease originates, then this is where we should initially look for evidence of pathologic changes. The most widely recognized predisposing factor for preeclampsia is poor placentation, revealed as insufficient cytotrophoblast invasion and defective remodeling of the maternal spiral arteries.

Poor placentation in preeclampsia

During the early stages of normal pregnancy, the cytotrophoblasts invade the uterine spiral arteries and progressively displace the vascular endothelial cells. By the end of the second trimester, these vessels have lost their muscular and elastic components that have been replaced by a fibrinoid layer of variable thickness in which trophoblastic cells are embedded. As a result, the spiral arteries are transformed into flaccid tubes

with a diameter at least four times greater than that of nonpregnant vessels, thus providing a low resistance circuit to the intervillous space. Furthermore, the loss of vascular wall components renders the altered vessels unresponsive to vasoreactive stimuli. This process of vascular transformation or remodeling is thought to involve virtually all the 100-150 spiral arteries in the placental bed, and extends as far as the inner third of the myometrium [21].

In contrast, morphologic examinations of placen-tal bed biopsies from women with preeclampsia show that such physiologic changes are restricted, being limited to the superficial region of the endometrium or being absent altogether [22-24]. As a result, the mean external diameters of the spiral arteries in women with preeclampsia are less than half of the diameters of similar vessels from uncomplicated pregnancies. It is to be expected that placental perfusion would be reduced in the context of the spiral artery pathology just described. This is confirmed by Doppler ultrasound assessment of blood flow velocimetry in the uterine arteries [25] and foci of placental infarction in women with preeclampsia [26]. Moreover, surgical reduction of the blood supply to the uterus or placenta in several animal experiments induces a hypertensive state that resembles hypertension in human pregnancy, suggesting a possible relationship between pla-cental ischemia and preeclampsia [27]. Accordingly, a consensus has gradually emerged that the placental lesions associated with preeclampsia arise from a state of chronic hypoxia.

Evidence Against Chronic Placental Hypoxia in Preeclampsia

However, a survey of the impact of oxygen on placental development and structure indicates that the concept of chronic placental hypoxia in preeclampsia may be overly simplistic for a number of reasons. Firstly, hypoxia refers to relatively low oxygen supply with respect to tissue metabolic demand, either as a result of an abnormal inspired oxygen or inadequate blood supply. For instance, a moderate chronic restriction of uterine blood flow (30% reduction in the last one-third of gestation) in sheep results in a significant reduction in fetal and placental weights [28]. However, examination of placental metabolism in preeclampsia shows that there is no reduction in energy supplies as would be expected if there were indeed chronic hypoxia [29]. Similarly, in many cases of preeclampsia, birth and placental weights are within the normal range. A large-scale retrospective cohort study even shows that

there is a significant association of preeclampsia with large-for-gestational-age infants [30]. Secondly, not all preeclamptic placentas are associated with utero-placental vascular pathology, and equally the same vascular pathologies may occur in other complications of pregnancy, such as premature delivery and premature rupture of membranes [31,32]. Meekins et al found that endovascular trophoblast does not show an all-or-none invasive phenomenon in normal and preeclamptic pregnancies. Instead, there is a gradient of decrease in the percentage of decidual and myo-metrial arteries invaded from normal pregnancy to preeclampsia [23]. The linkage between poor placen-tation and the development of preeclampsia is not absolute, so other factors must be operating. Thirdly, despite the numerous claims of placental hypoxia in preeclampsia, no direct measurements of dissolved oxygen tension within the intervillous space have been performed in vivo to confirm that this is the case. In contrast, pregnancy at high altitude is one condition where it is known that the oxygen tension of the maternal arterial supply to the placenta is significantly reduced [33]. Examination of placentas from uncomplicated pregnancies at high altitude shows that the organ is remarkably normal, and does not show an increased level of infarction [34]. It would appear, therefore, that a reduced oxygen tension per se does not cause the lesion most characteristically associated with preeclampsia. Finally, it is now widely accepted that there are major changes in maternal blood flow to the placenta at the end of the first trimester [35]. As a result, the oxygen tension rises sharply at 10-12 weeks' gestation. Before then, the trophoblast and other tissues have been proliferating and functioning very adequately at low oxygen concentrations, concentrations that are far lower than those compatible with fetal survival in later pregnancy. The trophoblast is therefore well adapted to low oxygen concentrations.

Consequently, it would appear that the absolute oxygen concentration existing in the intervillous space is not of paramount importance. But then what other features of the intervillous circulation can explain the placental pathology in preeclampsia? Recent observations from the first trimester explant culture show that placental tissues thrive in low oxygen concentrations but become stressed, as indicated by rapid degeneration of the syncytium, when the oxygen tension rises [36]. This suggests that changes in oxygen concentration are more important to trophoblast wellbeing than simply the absolute tension prevailing at the time. This idea leads to the development of the concept that fluctuations in oxygen concentrations resulting from intermittent perfusion within the intervillous

space might be the trigger for the placental changes seen in preeclampsia.

Intermittent Perfusion of the Human Placenta In Vivo

Although decisive proof showing that perfusion of the intervillous space in human pregnancy can be intermittent is unavailable at present, studies of the rhesus monkey, a species in which the uterine vasculature and form ofplacentation is very similar to that of the human, show that this might be the case. Martin et al used repeated radioarteriographic placentograms to study the contribution of individual spiral arteries to inter-villous space blood flow during uterine relaxation on rhesus monkeys at various stages of pregnancy [37]. By monitoring the positions of the arterial spurts into the intervillous space in successive films, the patency of individual spiral arteries was assessed. The proportion of arterial entries showing intermittency varied between animals, and the proportion of vessels demonstrating this behavior did not vary with gestational age. Even vessels that did not undergo complete closure showed variations in the size of their spurts between successive films. As the spurts appeared or disappeared independent of recorded myometrial activity, the authors concluded that this variability in blood flow was most likely the result of vasoconstriction in individual arterial walls, rather than of external compression of the vessels.

There is no doubt that external compression of the spiral arteries does occur during uterine contractions in the rhesus monkey [38,39]. Comparable experiments have also been performed on women at midgestation for therapeutic abortion and at term with malformed fetuses for termination to observe the effects of uterine contractions on the uteroplacental blood flow [40]. Although the uterine contractions in these studies were induced by nonphysiologic methods, similar effects, though to a lesser degree, occurred after Braxton-Hicks contractions in normal pregnancy.

Therefore, it seems highly likely that fluctuations in intervillous blood flow occur during normal human pregnancies, and that these can be on a regional or a more global basis. They probably increase in severity toward the end of gestation as uterine contractions become stronger and more frequent, culminating with delivery. It also seems reasonable to assume that periodic vasoconstriction may be more common in preeclampsia due to the retention of smooth muscle within the endometrial segments of the spiral arteries. The associated reduced or absent dilatation of the proximal end of the spiral arteries would restrict flow still further.

Impact on placental oxygenation

If the spiral arteries are intermittent in their functioning, then what might be the impact of this phenomenon on placental oxygenation? In the human organ, and that of the rhesus monkey, it is well established that each spiral artery delivers its oxygenated blood into the center of a placental lobule [41]. The blood then drains peripherally, exchanging with the fetal circulation as it does so. Because of the dense meshwork of villi, it is unlikely that supply from an adjacent spiral artery would be able to compensate for a transient reduction in arterial inflow into an individual lobule. Therefore, during the period of absent or reduced arterial inflow, the oxygen tension will decrease within the affected lobule. Oxygen will continue to be extracted from the intervillous space by the trophoblast, which is metabolically highly active and accounts for approximately 40% of oxygen consumption by the fetoplacental unit [42] and by the fetus. When the maternal arterial inflow to the lobule is restored, the local oxygen tension will rise sharply. Such fluctuations in oxygen tension could provide the basis for an ischemia-reperfusion (hypoxia-reoxygenation [H/R]) type injury, the effects of which are well documented in other organ systems such as the heart, brain and intestine.

H/R Injury

The detrimental effects of H/R arise mainly from its ability to generate high concentrations of free radicals [43]. A free radical is defined as any species that can exist independently and contains one or more unpaired electrons [44]. Examples include superoxide, nitric oxide (NO) and hydroxyl radicals. Most free radicals in biology fit within the broader category of reactive oxygen species (ROS), which includes oxygen-containing radicals, and nonradical but reactive molecules derived from oxygen, such as hydrogen peroxide and peroxyni-trite anion [44].

Generation of ROS in H/R

ROS can be produced at several sites, but the two principal sources as far as H/R is concerned are the electron leakage from the respiratory chain in mitochondria and the xanthine dehydrogenase/xanthine oxidase (XDH/XO) system. Under normal aerobic conditions, electrons are shuttled along the enzyme respiratory chain on the inner membrane of mitochondria until they are passed on to molecular oxygen, building in turn the proton gradient in the intermembrane space, which drives adenosine triphosphate (ATP) synthesis [44]. The mitochondrial enzymes do not function perfectly,

however, and inevitably a small number of electrons will leak on to oxygen to form superoxide radicals. During the hypoxic period, there is little or no molecular oxygen available to act as the final recipient, and so electrons build up on the respiratory chain. Rather paradoxically, this accumulation leads to increased superoxide formation as it causes increased leakage on to whatever oxygen is around. However, if oxygen is reintroduced before cellular function has been compromised too far, then a much greater burst of superoxide radical formation takes place, as there is suddenly more oxygen available for the electrons accumulated on the respiratory chain to leak on to [45,46].

The second, and probably more major, source of superoxide radicals in H/R is through the transformation ofXDH to XO [47]. Usually, this enzyme is present as the holoenzyme XDH/XO. XDH converts purines to uric acid with the reduction of nicotinamide adenine dinucleotide (NAD) to reduced NAD (NADH), while XO metabolizes xanthine and hypoxanthine to uric acid, using oxygen as the electron recipient, with the production of superoxide radicals. With hypoxia and in response to several cytokines, XDH/XO synthesis increases, and conversion of the enzyme to the XO form is enhanced. Meanwhile, during the hypoxic period, the substrate hypoxanthine builds up due to breakdown of ATP. So when oxygen is reintroduced, a burst of superoxide radicals is generated.

Damaging effects of oxidative stress in H/R

The accumulation of superoxide that occurs after H/R allows for an enhanced generation of other ROS, therefore exerting a broad spectrum of cytotoxicity [48]. A diverse array of cellular and extracellular fluid antioxi-dants has evolved to control and compartmentalize, but not necessarily eliminate, the production of ROS [48]. However, under particular circumstances such as an insult of H/R, the generation of ROS exceeds the capacity of the antioxidant defenses, so oxidative stress results. Not only do they behave as simple reactants that peroxidize membrane lipids, oxidize DNA or denature enzyme proteins, ROS generated by H/R are now recognized as interacting with physiologic signal transducers [49-51]. But, when present at high and/or sustained levels, ROS can cause cellular dysfunction, growth arrest or ultimately cell death.

Although the mechanisms of cell death in H/R injury have not been fully understood, recent studies indicate that dysregulation of calcium (Ca2+) metabolism and formation of the mitochondrial permeability transition pore (PTP) are key intermediary events [52-54]. ROS originating from H/R can damage the endoplasmic reticulum Ca2+-uptake system and interfere with Ca2+

efflux through the plasma membrane, therefore increasing the levels of intracellular free Ca2+ [52] and subsequently leading to the formation of PTP [53,55,56]. As a result of PTP opening, the mitochondrial membrane potential collapses, leading to the loss of ATP synthesis. If mitochondria throughout the cell are affected, then ATP concentrations fall precipitously, ionic homeostasis is lost and the cell undergoes primary necrosis [54,57]. Involvement of a more limited number of organelles or transient opening of the PTP may allow ATP to be maintained at levels sufficient to permit apoptosis to occur instead [58].

The damaging effects ofH/R can be more profound and global. One of the most significant consequences is the development of microvascular dysfunction within the organ experiencing H/R [59]. Under normal conditions, the flux of NO in endothelial cells greatly exceeds the rate of superoxide formation. However, within minutes after reoxygenation, the balance between NO and superoxide is tipped in favor of superoxide. This imbalance results from a sudden increase in the production of superoxide by endothelial cells and a corresponding decline in the synthesis of NO from endothelial NO synthesis. The relatively low levels of bioactive NO reduce its effectiveness to oppose leukocyte- or platelet-endothelial cell interactions or to serve as the secondary messenger when certain endogenous vasodilators such as acetylcholine interact with their endothelial receptors.

The resultant ROS after H/R can rapidly initiate or exacerbate the inflammatory state in endothelial cells by eliciting the production ofleukotriene B4 and platelet-activating factor (PAF). ROS can also help to sustain the neutrophil-endothelial cell adhesion that occurs several hours after reoxygenation by activating genes that encode adhesion molecules such as E-selectin (sustains leukocyte rolling) and intercellular adhesion molecule-1 (ICAM-1; sustains firm adhesion and emigration of leukocytes). Besides, neutrophils infiltrated within the microvasculature following reperfusion can further lead to local tissue injury [60].

Another devastating consequence of H/R injury is the development of damage in organs not involved in the initial hypoxia insult, known as the systemic inflammatory response syndrome and multiple organ dysfunction syndrome if multiple organ failure occurs [59,61]. Several mechanisms have been proposed to explain the remote organ injury induced by H/R; however, most attention has focused on the role of inflammatory mediators [61]. Indeed, ample evidence shows that postischemic tissues generate and release inflammatory mediators such as tumor necrosis factor-a (TNF-a) and PAF that can activate and attract circulating neutrophils,

induce generalized neutrophil and endothelial adhesion molecule expression and enhance the opportunities for neutrophil-endothelial cell interaction at remote sites.

Evidence of H/R Effects in the Placenta

The human placenta is of the hemochorial type and consists of an extensively branched fetal villous tree that is bathed by the maternal blood circulating in the intervillous space. The contact surface between fetal placental tissues and maternal blood is the syn-cytiotrophoblast, which acts as the endothelium for the intervillous space. Therefore, similar consequences and mechanisms of H/R injury just described can be expected to occur within the placenta. So, if we assume that intermittent blood flow to the intervillous space can lead to fluctuations in oxygen concentrations, and such alterations in oxygenation can further cause H/R injury, then the placenta must have the necessary machinery to generate H/R insults. Furthermore, there should be evidence showing footprints of H/R injury in the placenta, particularly in the preeclamp-tic placenta. Indeed, from reviewing the evidence, it would appear that the human placenta is subjected to H/R injury, and there are several lines of evidence signaling the effects of H/R injury on the placenta in normal and preeclamptic pregnancies.

XDH/XO in the placenta

XDH/XO messenger ribonucleic acid (mRNA) and enzyme activity have been demonstrated in term human placenta [62]. Furthermore, the relative activity of XO to the total XDH/XO activity, an index of conversion of XDH/XO to XO, was found to be considerably higher in the placentas of laboring women [63]. Labor is clearly associated with intermittency of intervillous blood flow and, therefore, fluctuating oxygen tensions. In addition, women in labor have higher blood levels of hypoxanthine than women not in labor, and elevated levels of hypoxanthine are observed in the uterine vein relative to peripheral blood in women during labor, indicating that the uteroplacental unit is the major source [64].

In parallel with observations in women who are in labor, studies on preeclamptic placentas show increased immunostaining of XDH/XO in the floating and anchoring villi as compared to that of normal placentas [65]. The staining is mainly in the villous endothelium and trophoblast. Especially noteworthy is that these cellular components are immunohistochemically co-localized with nitrotyrosine residues, a marker for peroxynitrite formation and oxidative stress [65]. Enzyme activity

assays on placental bed currettings also showed that both XDH/XO and XO activities were increased in placentas from preeclamptic pregnancies compared with normal controls. Furthermore, circulating uric acid levels are known to be increased in patients with pre-eclampsia [66]. Uric acid is a by-product of the XDH/XO pathway, which is activated by H/R. So, it is likely that the elevation of uric acid in preeclampsia is a result of H/R and XO stimulation.

Mitochondria as a possible source of placental oxidative stress

Normally, superoxide concentration in the mitochondria is tightly controlled by manganese superoxide dis-mutase (MnSOD) in the mitochondria and by copper/ zinc isoform (Cu/ZnSOD) in the cytosol [48]. However, as mentioned earlier, a burst of superoxide anions is generated when oxygen is reintroduced to hypoxic cells. This may overwhelm local antioxidant defense and lead to oxidative stress.

In preeclamptic placentas, the mitochondria also show swelling with a loss of cristae. Indeed, placental mitochondria have been suggested as a source of oxidative stress in preeclampsia. Studies on isolated mitochondria show that not only the levels of lipid peroxide, as estimated by malondialdehyde, but also the potential for lipid peroxidation is greater in the mitochondria fraction from preeclamptic placentas than in the mitochondria fraction from normal placentas [67]. It is also known that superoxide is the radical responsible for mitochondrial lipid peroxidation in this system.

Can H/R Injury be a Possible Etiologic Factor for Preeclampsia?

There is no doubt that all the conditions necessary for H/R injury seems to be met within the human placenta; however, a systematic study on the causal relationship between H/R and placental changes in preeclampsia has not yet been performed. Can H/R injury be a possible etiologic factor for preeclampsia? There are several features that characterize the preeclamptic placenta and that may be important in the generation of the maternal syndrome. These include increased placental oxidative stress, increased syncytiotrophoblastic apoptosis and increased production ofproinflammatory cytokines such as TNF-a. Each of these placental mechanisms has been the focus of recent investigation. As mentioned already, evidence from other organ systems or in vitro experiments shows that these events can be the consequences of H/R through the actions of ROS.

Oxidative stress in the preeclamptic placenta

Epidemiologic evidence shows that several risk factors for preeclampsia, such as diabetes and obesity, are identical to those that increase the risk of atherosclerosis [68]. The lipid profile associated with preeclampsia is also associated with atherosclerosis: a low concentration of high-density lipoprotein cholesterol, increased concentrations of serum triglycerides and increased formation of small, dense low-density lipo-protein particles. In addition, preeclampsia is associated with a distinct pathologic lesion of the decidual arterioles known as acute atherosis [69]. Acute athero-sis bears a striking resemblance to the atherosclerotic lesions of coronary arteries. These similarities and the generally accepted role of oxidative stress in atherosclerosis lead to the assumption that oxidative stress plays a pivotal role in the cause of the preeclamptic syndrome [68,70,71].

Although the cause of oxidative stress remains unclear, considering the unique role of the placenta in preeclampsia, it is likely that the placenta is the origin or major source of oxidative stress observed in the syndrome. Several lines of evidence support this assumption, including increased lipid peroxidation products, increased nitrotyrosine immunostaining and decreased antioxidant enzyme activities in preeclamptic placentas.

Lipid peroxidation products have been suggested as candidate factors that may mediate disturbance of the maternal vascular endothelium in preeclampsia [70,71]. Studies of women undergoing cesarean section showed significantly higher contents of lipid hydroperoxides, phospholipids, cholesterol and free 8-iso-prostaglandin F2a (8-iso-PGF2a), but not the total (free plus esterified) 8-iso-PGF2a in decidual tissues from women with preeclampsia as compared with tissues from normal pregnancies [72,73]. Moreover, tissue levels of free and total 8-iso-PGF2a are significantly higher in preeclamptic placentas than in normal placentas [74]. Isoprostanes like 8-iso-PGF2a are produced specifically by free radical-catalyzed peroxidation of arachidonic acid [75]. Free 8-iso-PGF2a has activities of relevance to preeclampsia, being a potent vasoconstrictor in kidney [75] and placenta [76], platelet activator [77], and inducer of the release of endothelin from endothelial cells [78].

Increased levels of thromboxane and lipid peroxides associated with a decrease in glutathione peroxide activity have been reported in placentas from pre-eclamptic patients compared with those from normal pregnancies [79]. In parallel, in vitro production of lipid peroxides and thromboxane is increased in both trophoblast cells and villous tissues from women with preeclampsia [80]. Furthermore, production of

8-iso-PGF2a and malondialdehyde (a lipid peroxide metabolite), as measured by the levels in the medium, is higher for preeclamptic placental tissue explants than for normal tissue explants [74]. These data provide convincing evidence that oxidative stress and lipid peroxidation are abnormally increased in the placentas of preeclamptic women.

The endothelial and inducible isoforms of NO synthase have been characterized in the human placenta [81,82]. The NO released may play a role in reducing arteriolar tone, and in preventing platelet and neutrophil adhesion to the endothelial cells and trophoblast surface. However, within minutes after reoxygenation of hypoxic 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, thus producing the peroxynitrite anion, which is a strong and long-lived oxidant [83]. The peroxynitrite anion is capable of modifying proteins by nitration of tyrosine residues to form nitrotyrosine. Therefore, the presence of nitrotyrosine is suggestive of in vivo activity of peroxynitrite and, thus, indicates oxidative damage [84].

Nitrotyrosine immunostaining has been detected around foam cells in human atherosclerotic lesions [85]. In the placenta, Myatt et al found greater nitrotyrosine immunostaining in villous vascular endothelium and its surrounding smooth muscle cells, and in villous stromal cells in preeclampsia compared to normal pregnant controls [86]. Moreover, Many et al found particularly intense immunoreactivity of nitrotyrosine within the invasive cytotrophoblasts in placental biopsies and vascular endothelium in the floating villi obtained from women with preeclampsia [65]. Together, the findings of nitrotyrosine residues in these cellular components of preeclamptic placentas may reflect increased production of superoxide in this setting.

Placental generation of ROS in preeclampsia might be facilitated by a decrease in local antioxidant defense, although it is not clear whether this reduced antioxidant defense is causative or secondary to enhanced depletion. The activity of placental SOD and glucose 6-phosphate-dehydrogenase is decreased in preeclampsia compared to normal pregnancy [87]. Moreover, the activity and mRNA expression of Cu/ZnSOD and glutathione peroxidase, and tissue levels of vitamin E are significantly lower in placental tissues from preeclampsia than from normal pregnancy [88].

In summary, there appears to be an increase in ROS generation in the placenta of preeclamptic women. There is evidence for increased nitrotyrosine residue formation in the preeclamptic placenta suggestive of peroxynitrite formation, perhaps arising from local

NO production coupled with increased generation of superoxide anions and either regionally decreased or inadequate SOD.

Apoptosis in the preeclamptic placenta Apoptosis is a type of cell death that is accomplished by specialized cellular machinery, which is often programmed as a component of normal development and differentiation in most tissues, or can be induced by exogenous stimuli [89]. In the human placenta, apoptosis has been reported to increase progressively throughout pregnancy [90] and is suggested to play a role in the differentiation, syncytial fusion and degeneration of villous trophoblasts [91,92]. On the other hand, increased apoptosis, particularly in the syncy-tiotrophoblast, has been found in placentas from pregnancies complicated by preeclampsia compared with normal pregnancies [93,94]. The increased pla-cental apoptosis may be a primary pathologic event or, alternatively, a secondary effect of altered placental oxygenation in preeclampsia. In this regard, Redman and Sargent proposed that oxidative stress might stimulate syncytiotrophoblast apoptosis and so increase shedding of microvillous fragments [14]. The resultant debris is thought to activate an enhanced systemic inflammatory response including endothelial cell dysfunction in the mother.

Production of TNF-a in preeclampsia

There is a significant body of evidence suggesting that TNF-a plays an important role in the pathophysiology of preeclampsia [27,70,95-98]. Women with preeclampsia have higher plasma levels of TNF-a compared to normal pregnant women, and elevated levels of TNF-a protein and mRNA have also been demonstrated in their placentas [99,100]. TNF-a can activate the endothelial cells and upregulate the gene expression of numerous molecules such as platelet-derived growth factor, cell adhesion molecules, endothelin-1 and PAI-1. These molecules have been reported to have detrimental effects on the vas-culature and also characterize preeclamptic pregnancy [101-103]. Furthermore, chronic infusion of TNF-a into rats during late pregnancy results in a significant increase in renal vascular resistance and arterial pressure [104,105].

In Vitro H/R as a Model for Placental Oxidative Stress

In an attempt to investigate the effects of H/R on the placenta more systematically, we have developed an

in vitro model in which villi from normal term placentas are subjected to a period of hypoxia followed by reoxy-genation [106]. Abundant ROS are generated rapidly in the villous endothelium, and to a lesser extent in the syncytiotrophoblast and stromal cells when hypoxic villous tissues are reoxygenated in vitro. Furthermore, the expression of several well-characterized markers of oxidative stress including nitrotyrosine residues, 4-hydroxy-2-nonenal (4-HNE) adducts and inducible heat shock protein 72 are greatly increased in villous tissues subjected to H/R compared to the controls maintained under constant hypoxia. In contrast, preloading villous tissues with ROS scavengers such as desferoxamine and a-phenyl-N-tert-butylnitrone significantly reduces the level of oxidative stress in H/R. Moreover, the patterns of immunostaining of nitrotyrosine residues and 4-HNE adducts suggest that there are parallels between the resultant oxidative stress and that observed in preeclampsia [65,86,107,108].

Further experiments using this model system have demonstrated that H/R is a potent inducer of the release of cytochrome c from mitochondria, activation of caspase 3 and cleavage of poly(ADP-ribose) polymerase in villous tissues [109]. These events are associated with an increased number of syncytiotrophoblastic nuclei displaying apoptotic changes and increased lactate dehydrogenase release into the medium. The causal relationship between the generation of ROS and these apoptotic changes is revealed by the fact that pre-administration of desferrioxamine attenuates the insult. These results indicate that H/R is a powerful stimulus for apoptotic changes within the syncytiotrophoblast, another characteristic feature of the preeclamptic placenta and one that has been linked with generation of the syndrome.

Similarly, in vitro H/R was found to cause increased placental expression of TNF-a mRNA and production of TNF-a, which was principally secreted into the culture medium, as compared to controls kept normoxic or hypoxic throughout [110]. Further, conditioned medium from villous tissues subjected to H/R caused growth disturbance and expression ofE-selectin on cultured human umbilical vein endothelial cells (HUVECs). It was found that TNF-a in the conditioned medium contributed, at least in part, to the activation of HUVECs.

Three main conclusions can be drawn from these findings. Firstly, as noted before, a variable degree of oxidative stress is present in the normal term placenta before the onset of labor. Secondly, reoxygenation at 5% O2/90% N2/5% CO2 is just as efficient at generating oxidative stress as is reoxygenation at air/5% CO2, which could arguably be considered a hyperoxic challenge. The mean oxygen tension measured in the

intervillous space at term in vivo is approximately 30-45 mmHg [111,112], and so 5% O2 is within the physiologic range. Thirdly, although there is clearly overlap in the effects caused by the two insults, H/R appears to be a much more potent stimulus of all the changes than hypoxia alone. It should be noted that the level of hypoxia used to induce these changes was almost certainly incompatible with survival of a fetus in vivo, and so this would suggest that H/R may be the more physiologic insult.

Overview and Conclusion

The cause of increased placental oxidative stress in preeclampsia remains unclear. There is no doubt that both hypoxia and H/R lead to ROS production, and so a large overlap exists between the pathologies that the two insults can create. Both may also arise from the same underlying problem of impaired conversion of the spiral arteries, and so are difficult to separate on a clinical basis. We must therefore speculate as to which is the more likely scenario. From the evidence presented in the literature, it can be seen that there is a plausible basis from which to assume that blood flow in the intervillous space will be intermittent. It is also well established that in many cases of late onset preeclampsia, fetal and placental weights are normal. This argues against chronic hypoxia being the causative agent, as does the constancy of energy levels within the placental tissues. Furthermore, our work has demonstrated that of the two insults, H/R is by far the most potent in inducing the placental changes seen in pre-eclampsia. Although hypoxia can induce some of the same changes, the levels required are not compatible with normal fetal and placental weights, and with normal placental energy levels. Finally, the demonstration of increased XO expression in the preeclamptic placenta provides arguably the strongest evidence for the potential of a H/R injury. All these points reinforce the general concept that at physiologic levels, it is fluctuations in the oxygen concentration that are more important than the absolute level.

The most credible strength of the "H/R injury" theory lies in its ability to explain why preeclampsia can occur in both women with abnormal and normal placenta-tion. From the evidence presented here, it is clear that intermittent perfusion of the intervillous space occurs in all normal pregnancies, and may provide the basis for the baseline level of oxidative stress seen in normal placentas. The variability in blood flow will likely increase towards term as the uterine contractions become more frequent and stronger, and may also be increased

by endogenous or exogenous vasostimulants. Oxygen concentrations in the intervillous space will therefore fluctuate on a regional basis, although overall oxygenation to the fetus is preserved, allowing for normal growth. The magnitude of these fluctuations in oxygen concentration will depend on the severity and duration of the vasoconstriction, but will generally tend to increase towards term as fetal oxygen extraction rises. Increasing fetal extraction will mean that deoxygena-tion of the intervillous space blood occurs more rapidly and more completely during the periods of stasis. When the oxygen returns as blood flow is reestablished, a H/R type insult will occur, the severity of which will depend on the degree of deoxygenation and the level of tissue antioxidant defences. It is likely to be mild in early to mid pregnancy, but will increase towards term as the balance between oxygen delivery to the intervil-lous space and fetal extraction becomes less favorable, resulting in placental oxidative stress.

The effects of the reduced trophoblast invasion associated with complicated pregnancies can easily be superimposed on this basic model. Reduced invasion will leave the spiral arteries vasoreactive, and thus more likely to undergo spontaneous transient vasoconstriction. They will thus be more responsive to endogenous and exogenous vasoactive stimuli. Partial obliteration of their lumens by atherotic changes will also impair flow, and may exacerbate the effects of uterine contractions by reducing the closing dimensions. Hence, the magnitude of oxygen fluctuations for a given gestational age will be increased, so increasing the oxidative stress within the placenta. Excessive production of inflammatory cytokines, deportation of apoptotic microvillous placental fragments, activation of maternal leukocytes and platelets, or depletion of NO production may then cause or contribute to the maternal endothelial response. The degree of the oxidative stress will likely reflect the extent of the maternal vascular pathology. If the latter is sufficiently severe, then some chronic impairment of placental perfusion might also be expected, leading to associated fetal growth restriction. Such cases will probably display clinical symptoms earlier in pregnancy due to the increased placental oxidative stress, and will represent early onset of preeclampsia.

On this basis, there will be a complete spectrum of placental changes among the normal, the late onset and the early onset preeclamptic states. This view fits with the spectrum of vascular changes that has been reported in the spiral arteries [23]. The continuum theory is also supported by the clinical observation that there is a high degree of overlap between the values of almost any parameter in preeclampsia and

normal pregnancy [12]. How the placental changes induce the syndrome is beyond the scope of this work, but broadly preeclampsia may be the result of excessive homeostatic signals or toxic products arising from the placenta, or an abnormally sensitive maternal response to physiologic levels of these same signals and products [8]. However, viewing the syndrome as a continuum of H/R insults provides new insight into the pathophysiology of pregnancy that may hopefully lead to improved clinical interventions.


This work was partly supported by the National Science Council, Taiwan (grant no. NSC94-2314-B182A-142). Dr Tai-Ho Hung is supported by Chang Gung Memorial Hospital as a Physician Scientist.


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