HO in pregnancy

Free Radical Biology & Medicine 38 (2005) 979 – 988 www.elsevier.com/locate/freeradbiomed

Serial Review: Heme oxygenase in human diseases Serial Review Editor: Phyllis A. Dennery

HO in pregnancy

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Shannon A. Bainbridgea,*, Graeme N. Smitha,b,c,1 a

Department of Anatomy and Cell Biology, Faculty of Health Sciences, Botterell Hall, Queen’s University, Kingston, Ontario, Canada K7L 3N6 b Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada c Department of Obstetrics and Gynecology, Faculty of Health Sciences, Queen’s University, Kingston General Hospital, 76 Stuart Street, Kingston, Ontario, Canada, K7L 2V7 Received 22 June 2004; revised 20 October 2004; accepted 1 November 2004 Available online 31 December 2004

Abstract The enzyme heme oxygenase (HO) has been implicated in several physiological functions throughout the body including control of vascular tone and regulation of the inflammatory and apoptotic cascades as well as contributing to the antioxidant capabilities in several organ systems. These various properties attributed to HO are carried out through the catalytic products of heme degradation, namely carbon monoxide (CO), biliverdin, and free iron (Fe2+). As the newly emerging roles of HO in normal organ function have come to light, researchers in several disciplines have assessed the role of this enzyme in various physiological and pathological changes taking place in the human body over a lifetime. Included in this new wave of interest is the involvement of HO, and its by-products, in the normal function of the vital organ of pregnancy, the placenta. In this review the role of HO, and its catalytic products, will be examined in the context of pregnancy. The different isoforms of the HO enzyme (HO-1, HO-2, HO-3) have been localized throughout placental tissue, and have been shown to be physiologically active. The HO protein and more specifically its catalytic by-products (CO, biliverdin, and Fe2+) have been postulated to be involved in the maintenance of uterine quiescence throughout gestation, regulation of hemodynamic control within the uterus and placenta, regulation of the apoptotic and inflammatory cascades in trophoblast cells, and the maintenance of a balance of the oxidant-antioxidant status within the placental tissues. The association between this enzyme system, and its above-noted roles throughout pregnancy, with the hypertensive disorder of pregnancy preeclampsia (PET), will also be examined. It is hypothesized that a decrease in HO expression and/or activity throughout gestation would be capable of initiating several pathological processes involved in the etiology of PET. This hypothesis has led to further discussion emphasizing the possibility of novel therapeutic designs targeting this enzyme system for the treatment of PET. D 2004 Elsevier Inc. All rights reserved. Keywords: Heme oxygenase; HO; HO-1; HO-2; Carbon monoxide; CO; Bilirubin; Biliverdin; Ferritin; Pregnancy; Placenta; Placental perfusion; Antioxidants; Preeclampsia; Free radicals

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The HO enzyme and pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carbon monoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: HO, heme oxygenase; CO, carbon monoxide; NO, nitric oxide; ROS, reactive oxygen species; RT-PCR, real-time polymerase chain reaction; ODQ, 1H-(1,2,4)oxadiazole(4,3-1)quinoxalin-1-one; YC-1, 3-(5-hydroxymethyl-2V-furyl)-1-benzylindazole; NOS, nitric oxide synthase; PET, preeclampsia. $ Sources of financial support: Strategic Training Initiative in Research in Reproductive Health Sciences (STIRRHS), CIHR. $$ This article is part of a series of reviews on bHeme oxygenase in human disease.Q The full list of papers may be found on the home page of the journal. * Corresponding author. Fax: +1 613 548 1330. E-mail addresses: [email protected] (S.A. Bainbridge)8 [email protected] (G.N. Smith). 1 Fax: +1 613 548 1330. 0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.11.002

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The antioxidant capabilities of biliverdin/bilirubin . . . . . . . . Heme oxygenase and preeclampsia . . . . . . . . . . . . . . . . Targeting the heme oxygenase signaling pathway therapeutically . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction The enzyme heme oxygenase (HO) was originally characterized in 1968 by Tenhunen, and was described as the only mediator of heme metabolism in a cell [1]. This led to the notion that this enzyme was a housekeeping protein, strictly involved in maintaining homeostasis of the heme pool. Since then, great strides have been made in the areas of research surrounding heme metabolism in general and HO in particular. It is now recognized that HO is involved in

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the control of vascular tone [2–4], regulating anti-inflammatory [5–7] and antiapoptotic [6–9] responses as well as reducing oxidative stress and subsequent tissue damage in several organ systems [10–12]. These various properties attributed to HO are carried out through the catalytic products of heme degradation, namely carbon monoxide (CO), biliverdin, and free iron (Fe2+) (Fig. 1). Three isoforms of the HO protein have been identified. HO-1 is a 32-kDA inducible form of the enzyme; HO-2 is a 36-kDa constitutive form of the enzyme while HO-3 is the

Fig. 1. Heme degradation pathway and physiological roles of its breakdown products CO, Fe2+, and biliverdin during a healthy and PET pregnancy.

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least characterized and least active of the isoforms [13,14]. HO-1 is ubiquitously distributed in mammalian tissues but is expressed in high concentrations in the spleen and liver, areas of high erythrocyte turnover [13,15]. HO-1 protein expression and activity have been demonstrated to be induced by several stimuli such as heme, metalloporphyrins, transition metals, cytokines, endotoxins, hyperthermia, hypoxia, and oxidants [13,15] and as such it has been coined a stress protein. HO-2 is found in numerous tissues throughout the body, and appears to be involved in the maintenance of basal heme metabolism [13,15]. This vast array of activating stimuli coupled with HO’s antioxidant, anti-inflammatory, antiapoptotic, and regulation of vascular tone properties has led to questions about the functional significance and role of this enzyme under both physiological and pathological circumstances. All three catalytic products of heme degradation, CO, biliverdin, and Fe2+, were originally deemed to be toxic compounds that were quickly excreted; at high enough concentrations, all three of these compounds do possess cytotoxic properties [16–18]. However, it has now been demonstrated that at the concentrations produced through the actions of HO, all three compounds instead display fairly potent cytoprotective properties. Carbon monoxide has been demonstrated to display several of the same physiological functions as its diatomic cousin nitric oxide (NO). These functions include decreasing vascular tone via vasodilation [3,19], inhibition of platelet aggregation [20], as well as inhibition of the apoptotic and inflammatory cascades [6,7,9]. Promising results are now emerging concerning the potential benefits that CO may provide to increase the success of transplanted organs. Free heme can undergo autooxidation to produce superoxide (O2 ) and hydrogen peroxide (H2O2), which in turn may promote the formation of other highly toxic reactive oxygen species (ROS). The enzyme HO is thus recognized as an antioxidant, by removing free heme. In addition, the second and third catalytic products of heme degradation (biliverdin and Fe2+) contribute to HO’s antioxidant effect. Biliverdin and its subsequent breakdown product bilirubin demonstrate potent antioxidant characteristics [21]. A recent description of the cycle in which biliverdin is reduced to bilirubin, via biliverdin reductase, and then is subsequently recycled back into biliverdin suggests a mechanism through which the antioxidant properties of these two molecules may be amplified in vivo [22]. Free iron, released from the core of the heme molecule, is capable of extensive cellular damage. Through the Fenton reaction it is capable of promoting the generation of damaging free radicals [23,24]. The enzyme HO, however, is capable of interactions with intracellular iron pumps [25] as well as upregulating the generation of ferritin [26], a potent iron-chelating molecule. Therefore, while heme catabolism will initially increase levels of free iron, these additional actions of HO have the net effect of

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decreasing intracellular free iron and thus limit the generation of ROS. The antioxidant properties of both biliverdin/bilirubin and ferritin have been used therapeutically to ameliorate hepatic injury in several models, including ischemia/reperfusion injury [27,28].

The HO enzyme and pregnancy As the newly emerging roles of HO in normal organ function have come to light, researchers have studied this enzyme’s role in various physiologic and pathologic processes taking place in the human body. Included is the involvement of HO, and its by-products, in pregnancy and specifically its role in placental function. In order for this enzyme to be recognized as a significant contributor to normal placental function three criteria must be fulfilled: ! HO must be localized in placental tissue, ! HO must be functional within the placenta, and ! HO by-products must be capable of exerting physiological effects on the various placental and/or fetal tissues. The identification and localization of the HO proteins within the placenta have been studied extensively. The mRNA products for both the HO-1 and the HO-2 isoforms have been measured in placental tissue homogenate using RT-PCR. These studies have demonstrated elevated concentrations of HO-2 mRNA compared to that of HO-1 [29,30]. They also indicate that both placental HO-1 and HO-2 mRNA increase with advancing gestation [5,30]. The HO proteins have been identified using Western blot analysis in chorionic villi, chorionic plate, basal plate, and fetal membranes. HO-2 protein content was again found to be higher than HO-1 in all areas examined [29,31,32]. The differential expression of the various HO isoforms throughout the placenta has also been characterized using immunohistochemical staining. A wide distribution of placental HO was observed in the syncytiotrophoblasts, endothelium, and smooth muscle cells of umbilico-placental blood vessels as well as all fetal membranes. Early in pregnancy HO-2 appeared in abundance in the syncytiotrophoblast cells, cytotrophoblast cell columns, and vascular endothelium [30,32], while at term it was reported in the endothelial and vascular smooth muscles cells of placental blood vessels [30], cytotrophoblast shell, and cell islands as well as intravascular and extravillous interstitial trophoblast cells [32]. In all studies, the HO-1 staining was significantly less for all areas examined. However the HO-1 enzyme was identified in syncytiotrophoblast and cytotrophoblast cells as well as the vascular endothelium [29,30,32]. With the presence of the HO protein in the placenta, the activity of HO under physiological conditions was examined. Based on the rate of CO formation from exogenous

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heme, HO activity was found to be the highest in the chorionic plate, chorionic villi, and basal plate [33]. Enzymatic activity was also measurable in the umbilical artery and vein [34]. Recently the effects of both oxygen and glucose availability on HO activity in the placenta were examined and it was determined that although an increase in HO-1 mRNA and protein expression was seen in response to hypoxia, the overall HO enzymatic activity decreased under hypoxic conditions [35]. The opposite was true in the case of glucose, where a 24-h preincubation in a glucosedeficient medium resulted both in an upregulation of HO protein content and in enzymatic activity [36]. These two studies indicate that the regulation of the HO system in the placenta is complex, and in part reliant upon local glucose and oxygen concentrations. With the first two criteria for physiological significance of the HO enzyme within the placenta being met, researchers have now focused their efforts on the various roles that each of the breakdown products of heme metabolism may play during gestation.

Carbon monoxide Attention has focused on the vascular effects of CO in the placenta. It has been noted that at term arterial vessels of the feto-placental circulation are maintained in a state of near-maximal dilation in order to facilitate oxygen and nutrient delivery [37]. The placenta lacks innervation [38] and therefore is reliant solely upon locally produced and circulating vasoactive compounds for hemodynamic control. With the abundance of HO protein found throughout the human placenta, coupled with the extensive knowledge of CO’s vasodilatory properties in other organ systems, it was hypothesized that CO was involved in maintaining placental vascular tone throughout gestation [32,39]. It has been demonstrated, using the dually perfused cotyledon preparation, that inhibition of HO by zinc protoporphyrin increased placental perfusion pressure [32], indicating a role for CO in the basal vascular tone within the placenta. Studies completed in our laboratory, using the same preparation, demonstrated that CO, at concentrations found in umbilical cord blood at the time of delivery, was capable of decreasing placental perfusion pressure [39], indicating CO-induced vasorelaxation of feto-placental resistance blood vessels. The CO-induced decrease in placental perfusion was attenuated by 1H-(1,2,4)oxadiazole(4,31)quinoxalin-1-one (ODQ), an inhibitor of sGC, and augmented by 3-(5-hydroxymethyl-2V-furyl)-1-benzylindazole (YC-1), an activator of sGC, indicating that sGCcGMP formation is the major signal transduction system involved in this circulatory action of CO (Fig. 2) [39]. Further work completed by our group has demonstrated that CO is capable of relaxing preconstricted anchoring villi [40]. This would indicate that CO not only can increase intraplacental fetal perfusion by dilating placental blood

Fig. 2. Percentage decrease in placental perfusion pressure for perfusion with aqueous solutions containing different CO concentrations alone or in the presence of YC-1, 1 AM ODQ, or 10 AM ODQ. The data are presented as group means F SE. *P b 0.05 compared with CO alone. Adapted from Ref. [39] (Bainbridge et al., 2002) with permission from publisher.

vessels but it may also enhance intervillous uteroplacental blood flow. The role that CO plays in the maintenance of uterine quiescence during pregnancy and the onset of labor is one that warrants further investigation. This potential role of CO follows debate regarding the role of NO in the maintenance of uterine quiescence and its potential use as a tocolytic agent [41]. Myometrial quiescence during pregnancy has been characterized by an increase in the concentrations of myometrial cGMP [42]. Nitric oxide, as well as CO, acts to stimulate sGC which augments the intracellular levels of cGMP, which is then capable of smooth muscle cell relaxation (Fig. 1) [43]. Several animal studies have suggested a role for this NO-cGMP pathway in myometrial quiescence during pregnancy and in the onset of labor [44,45]. However debate over the presence of nitric oxide synthase (NOS) within human myometrial tissues has led to controversy over whether NO would even be produced and hence be capable of acting on myometrial smooth muscle cells in vivo [46]. Due to its similar actions on sGC as NO, CO has now come into the spotlight as a potential mediator of this function during pregnancy. Acevedo and co-workers report a 16- to17-fold increase in the expression of the HO-1 and HO-2 proteins in pregnant myometrial tissue compared to nonpregnant myometrium [47]. Expression of these two proteins has also been reported to increase throughout gestation in both the intravascular and the extravillous interstitial trophoblasts within placental bed biopsies [32]. These results are in contrast to findings by Barber et al. who report no measurable HO-1 protein in nonpregnant or pregnant myometrial tissue, with similar expression of

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HO-2 between the two groups [46]. In both of these studies similar HO-1 and HO-2 antibodies were used, the study groups were similar in gestational age, and all myometrial biopsies were reportedly taken from similar portions of the uterus. It is therefore surprising to observe such contrasting results between these two groups. It is important to note that neither study indicated whether external sources of HO upregulation, such as maternal smoking status, were similar between the study and the control groups. Both studies had relatively low sample numbers (n = 4/group in the Acevedo study, n = 10/group in the Barber study); therefore, external confounding factors may play a significant role in the studies outcomes. It is also possible that these varying results were due to technical differences in the methodologies employed by these groups. It is clear that replication of these studies, with clearly delineated study populations, along with widely accepted methodologies must be undertaken to better understand the expression of the HO enzyme system in pregnant myometrial tissue. Regardless of the presence of HO protein expression within the uterus during pregnancy, the potential role of CO as a tocolytic agent still remains. In the Acevedo study they reported that hemin, an HO inducer, was capable of inhibiting both spontaneous and oxytocin-induced myometrial contraction [47]. Further support of CO’s potential uterine quiescent capabilities has been obtained through measurements of maternal end tidal CO concentrations. These results indicated that women who were experiencing either preterm or term uterine contractions had significantly lower levels of end tidal CO, suggesting a possible association between decreases in the intrinsic production of CO and increases in uterine activity [48]. Further studies examining the localization of the HOCO system within the pregnant uterus along with further investigation into CO’s uterine quiescent properties are required. Normal trophoblast migration through the uterine tissue and subsequent spiral arteriole remodeling are essential for the development of a healthy placenta and fetus. It has recently been proposed that CO may be involved in this function. The ability of trophoblast cells to penetrate the spiral arterioles in order to remodel the endothelium may relate to the ability of local autocoids to stimulate NO [49]. Failure to generate adequate amounts of trophoblast NO may predispose women to preeclampsia, a disorder of inadequate placentation [50]. Therefore it is proposed that NO is required for proper trophoblast differentiation and invasion. The localization of HO isoenzymes in extravillous trophoblasts coupled with data indicating that first trimester trophoblast cells in culture are capable of CO production, while keeping in mind the underlying similarities between NO and CO, has led to the suggestion that trophoblast-derived CO may facilitate trophoblast penetration into the spiral arteries in early pregnancy [5]. A healthy pregnancy is characterized by an underlying systemic inflammatory response that must be continually

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monitored and regulated by various enzyme systems in both maternal and feto-placental tissues [51]. Recently, CO has been found to have anti-inflammatory properties in various organ systems. Little work has been completed examining the role that the HO-CO system may play in maintaining a healthy balance of inflammatory/anti-inflammatory responses throughout gestation. The role(s) CO may play in pathological disorders associated with an increased inflammatory response have been considered and the HO-CO system will be discussed in relation to preeclampsia below.

The antioxidant capabilities of biliverdin/bilirubin The placenta is a site of increased oxidative stress as cells of fetal origin (trophoblast cells) are in direct communication with the highly oxygenated maternal circulation. The growing fetus is susceptible to increased levels of oxidative stress as most reactive oxygen species are membrane permeable and are capable of crossing the placenta [52]. In addition, while total lipids in the fetal circulation are lower than that of an adult, a higher proportion of polyunsaturated fatty acids has been measured in umbilical cord plasma compared to adult plasma [53]. This type of fatty acid is particularly susceptible to oxidation, placing the fetus at potentially higher risk of oxidative injury. Despite the increased risk for oxidative damage in the feto-placental unit, in healthy pregnancies this damage does not in fact occur. This would indicate that antioxidant system(s) must be in place to protect both placental and fetal tissues from this sort of insult. Various enzymatic antioxidant systems, such as superoxide dismutase, catalase, and glutathione peroxidase, have been characterized within the placenta [54–57]. Currently the roles of nonenzymatic scavengers, such as a-tocopherol (vitamin E), ascorbic acid (vitamin C), bilirubin, and ferritin, are now being examined as important antioxidants within the placenta. These nonenzymatic antioxidants prevent lipid peroxidation by trapping oxygen free radicals and breaking the peroxidation chain reactions [58]. A few studies have examined biliverdin/bilirubin in human placental and fetal tissues demonstrating that the concentration of this nonenzymatic antioxidant appears to increase with advancing gestation [52]. This is consistent with the increasing HO expression and activity reported in late gestation; one of the products of HO activity is biliverdin. It was also noted that there was a concentration gradient of biliverdin/bilirubin increasing from maternal circulation to feto-placental circulation. This would indicate a shift of the antioxidant’s protective effects in the direction of the fetus, thus protecting the growing fetus against oxygen toxicity [59]. A further study by Gopinathan et al. indicates that in neonates a very strong correlation between bilirubin levels and total plasma antioxidant activity exists [60], further solidifying the notion that biliverdin/bilirubin plays a

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pivotal role in maintaining a homeostatic redox balance during pregnancy.

Heme oxygenase and preeclampsia Heme oxygenase and its catalytic products may play a significant role in the progression of a healthy pregnancy to term. With this enzyme being linked to several vital tasks of pregnancy such as placentation, placental hemodynamic control, and antioxidant protection, it is no surprise that the activity or dysfunction of this enzyme is under investigation in several disorders of pregnancy. In this review, the role of HO in the pathophysiology of preeclampsia (PET) will be highlighted as it has been most extensively studied with respect to its association with the HO-CO system. Preeclampsia is a hypertensive disorder of pregnancy, affecting between 5 and 10% of all pregnancies. It is hypothesized that the progression of PET begins with shallow trophoblast invasion and inadequate spiral arteriole remodeling [61–64]. Consequently the placenta is not effectively perfused and localized areas of hypoxia are established [61,62]. Hung et al. have demonstrated that the placental hypoxia/reperfusion injury, characteristic of this disorder, results in increased apoptosis and necrosis of the syncytiotrophoblast layer lining the intervillous space [65]. This placental debris is then transported into the maternal circulation where it is thought to initiate a maternal inflammatory response with subsequent endothelial dysfunction leading to the observed pathologies of the disorder, namely elevated maternal blood pressure, edema, and proteinurea [61,62,66]. Several groups have attempted to profile the HO expression, localization, and activity in the PET placenta with conflicting results to date. One study examined levels of HO mRNA using RT-PCR and found no significant difference in levels of transcript for either isoform of the enzyme between PET and normotensive placentae [5]. However, this same study reported a reduced expression of HO-1 protein in PET placentae using Western blot analysis, suggesting a translational blockage of this isoform [5]. This is in contrast to two other studies examining HO-1 protein content in the PET placenta, one of which reported an increase in protein expression in chorionic villi and fetal membranes [31], while the other indicated that the HO-1 protein was undetectable in the placental homogenates collected from both the PET and the normotensive placentae [67]. The results between the two groups were similar when examining protein expression of HO-2, with no significant changes in HO-2 protein expression being reported in the placenta of PET [31,67]. However, Barber et al. did report a reduction in HO-2 immunohistochemical staining in the endothelial cells of the PET placentae [67]. McLaughlin et al. characterized HO activity in different placental regions (chorionic plate,

basal plate, chorionic villi, and fetal membranes) and found no difference in the enzymatic activities between PET and normotensive placentae [31]. These contradicting results add confusion to HO’s potential role in pathogenesis of PET. However, it has been proposed that subject, disease state, and experimental variability may explain some of these differences. All groups noted large variability in HO protein expression, localization, and activity between patients. Larger sample sizes are required in order to adequately address this question. There were also differences in the HO antibodies used in the various experiments, some being targeted to rat HO protein [5,67] while others were targeted to human HO protein [31] which could result in varying specificity of these antibodies to human placental tissues and hence varying results. In addition, while all of the studies investigating HO protein expression in the placental tissue used the same criteria for the diagnosis of PET, the studies did not report gestational age or disease severity at the time of tissue collection. Furthermore, HO enzyme expression and activity may vary across the placenta. Consequently, it is critical that the sampling site be identified and considered when comparing contradictory data. As previously noted, while HO expression has been shown to be upregulated under hypoxic conditions, HO enzymatic activity appears to decrease with decreasing pO2 [35]. Preeclamptic placentae are known to have increased areas of hypoxia, leading to areas of necrosis or infarct. Infarct regions are present in placentae from uncomplicated pregnancies as well; however, infarcts in placentae from PET pregnancies tend to be larger and more numerous [68]. Lash et al. examined the effect of placental tissue damage on HO expression and activity in both PET and normotensive placentae. They reported no significant differences in HO-1 protein levels in healthy and infarcted tissue for both normotensive and PET placentae; however, they did report that HO-2 protein levels were decreased in the peri-infarct and infarcted villi of PET placentae. Using immunohistochemical analysis they detected decreases in both HO-1 and HO-2 protein expression in all damaged tissue. They further compared HO enzymatic activity in microsomes isolated from morphologically normal and infarcted chorionic villi and found decreased HO activity in the damaged tissue under optimized conditions [69]. The results of this study indicate that the ability of chorionic villi to oxidize heme into CO, biliverdin, and Fe2+ may be compromised in areas of tissue damage in the placentae of women with PET. Considering that the proportion of damaged or infarcted tissue is higher in a PET placenta compared to a healthy placenta, it can be postulated that the PET placental unit would have lower overall HO enzymatic activity and hence produce lower levels of its three catalytic products compared to a placenta from a healthy woman. If one were to imagine a disorder that would present following a reduction in HO enzymatic activity

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during gestation, based on the arguments provided above for the regulatory and cytoprotective roles of HO’s catalytic products, a resemblance to the disorder now recognized as PET becomes apparent. It has been proposed that an initial immune response or rejection of fetal tissue may be responsible for the shallow invasiveness and impaired remodeling of spiral arterioles by trophoblast cells in PET [61,70]. Several studies have demonstrated an infiltration of activated macrophages or foam cells into the maternal spiral arterioles of the PET placenta [71,72]. These activated macrophages release a number of compounds (i.e., TNFa, TGF-h, IDO) that are capable of decreasing trophoblast cell invasion and possibly initiating the apoptotic cascade within these cells [71,72]. This inflammatory environment of the PET placenta has been compared to that established with allograft rejections [61]. The relevance of this similarity comes from publications in the areas of transplantation and allograft rejection research. These studies demonstrated increased success in organ transplantation when the organs were preincubated with CO or had been genetically modified to display an upregulation in HO [7,8,73–75]. They indicate that CO is capable of reducing the inflammatory response and apoptosis characteristic of an allograft rejection [76]. These transplanted organs appear to be perfused to a much higher degree than the nontreated organs, and the survival of the recipients was greatly increased [8,74,75]. This may also be the case in the placenta. It is suggested that inadequate HO enzymatic activity, and subsequently decreased CO availability, would result in the invading trophoblast cells not being provided with sufficient cytoprotection from the anti-invasive and proapoptotic signals initiated by the activated macrophages. Consequently the trophoblast cells would display shallow invasion of the decidua and inadequate remodeling of the spiral arterioles. The decreased CO availability, resulting from the decreased HO activity, would also hinder normal placental hemodynamic control. As vascular control in the placenta is dependent in large part on locally produced vasoactive compounds, the loss of a key vasodilator in this circulatory system could have significant effects on intraplacental perfusion, possibly exacerbating localized areas of hypoxia established via inadequate blood flow through the constricted spiral arterioles. This hypoxic insult, coupled with the reduced expression of the antioxidant bilirubin, could potentially tilt the balance of the oxidant-antioxidant capabilities of the placenta in favor of increased localized oxidative stress. As previously discussed this oxidative insult would then be capable of initiating the apoptotic cascade in the syncitial lining of the intervillous space [65], setting in motion the transport of a placental dysfunction into the maternal compartment, as is observed in the placentae of women with PET [61,62].

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There have been no studies examining the levels of HO expression and activity in the circulatory system of women who develop PET. The results of such studies may provide further insight into the progression of PET from a disorder of placental dysfunction into a disorder of maternal endothelial dysfunction. Preeclamptics have been shown to have increased levels of platelet and neutrophil activation which promotes vascular damage and obstruction, leading to tissue ischemia and further tissue damage [77]. In the human case of HO-1 deficiency, the most pronounced pathological phenotype is also severe and persistent endothelial damage [78]. While there are certainly several key players in the progression of this disorder, we would argue that a reduction in the HO/CO-bilirubin system may be of significant importance in the development and progression of PET.

Targeting the heme oxygenase signaling pathway therapeutically Preeclampsia remains one of the leading causes of maternal and fetal/neonatal morbidity and mortality worldwide [61]; currently no therapeutic intervention to prevent or attenuate the disease process is known. The epidemiological studies showing that women who smoke cigarettes have a 33% reduced risk of developing PET [79] provides supporting evidence for the role that the HO-CO system may play in the development of this disorder and a direction for therapeutic development [80]. A recent study reported that women who used snuff, a form of smokeless tobacco, did not demonstrate the reduced incidence of PET compared to those women who smoked cigarettes [81]. This suggests that one (or several) of the combustible byproducts of cigarette smoke is responsible for reducing the incidence of PET. We hypothesize that CO, one of the major combustible products of cigarettes smoke, is responsible for the effect. As well, nonsmoking women with PET have lower end-tidal CO concentrations compared to their healthy counterparts [82,83], also supporting the hypothesis. This would further indicate a link between low CO levels and the development, or progression, of this disease. A better understanding of how CO functions within the fetoplacental unit may allow us to target therapy at upregulation of endogenous CO production or to provide compounds that function as CO mimetics or CO donors. Heme oxygenase is an important enzymatic system within the human body. Its various functions in several organs have been extensively examined. More recently the role that this enzyme and its catalytic products play in the maintenance and progression of a healthy pregnancy to term has come to light. It is now understood that the HO-CObiliverdin system is involved in normal placentation, hemodynamic control within the placenta, and regulation of the antioxidant status within the placental and fetal

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