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Original Article |
Departments of Physiology (N.A., S.G., C.D., I.C., J.R.G.C.) and Obstetrics and Gynecology (I.C., J.R.G.C.), Canadian Institutes for Health Research Group in Development and Fetal Health, and Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital (C.D., I.C., J.R.G.C.), University of Toronto Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Dr. Nadia Alfaidy, Department of Physiology, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: n.alfaidy{at}utoronto.ca.
Abstract
Preeclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality. The genesis of PE is related to deficient trophoblast invasion of maternal spiral arteries, which might result in a reduction of placental (PL) oxygen (O2). An absence of increased O2 that normally occurs around the 10–12th wk of gestation results in aberrant expression of genes that might contribute to the pathophysiology of PE. We examined the expression and regulation of PL 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD) in normal pregnancies and in PE. Two types of 11ß-HSD exist in the placenta, 11ß-HSD1 and 11ß-HSD2. 11ß-HSD2 is thought to protect the fetus from cortisol excess. In PE, both the expression and activity of PL 11ß-HSD2 were reduced significantly compared with those in age-matched controls. As PE is associated with a reduction of PL O2, we next investigated whether in normal pregnancy 11ß-HSD2 expression changes at the time of the increase in O2. 11ß-HSD2 was detected as early as 5 wk, with expression limited to the syncytiotrophoblast (ST). At 10–12 wk, this expression increased and was also found in the cytotrophoblast and extravillous trophoblast. These results were substantiated by Western blot. The ability of O2 to regulate 11ß-HSD2 was determined both in cultures of villous explant from early gestation and in term trophoblast cells after incubation under 3% or 20% O2. Villous explants cultured under 20% O2 showed higher enzyme activity and expression compared with 3% O2. Term trophoblast cells also exhibited higher enzyme activity at 20% vs. 3% O2. No change in 11ß-HSD1 expression was observed in early pregnancy or in PE. This is the first report to suggest that 11ß-HSD2 is O2 dependent in first and third trimester placenta during human gestation.
PREECLAMPSIA (PE) IS a disease that complicates 5–10% of all pregnancies, resulting in significant maternal and fetal morbidity. The disease is manifest in the third trimester of pregnancy with a wide variety of maternal symptoms, of which hypertension, proteinuria, and edema are late manifestations of a multifactorial, multisystemic disorder initiated in early pregnancy (1). The placenta plays a key role in the genesis of PE, as histological examination of placental bed biopsies from PE women has revealed limited trophoblast migration to superficial decidua, reduced trophoblast invasion of maternal spiral arteries, and failure of uterine artery remodeling (2). One of the consequences of impaired remodeling of uterine spiral arterioles in the first trimester of pregnancy is an increase in uteroplacental vascular resistance and impaired placental perfusion; the latter, in turn, leads to localized regions of ischemia and reduced oxygen levels (hypoxia) in the placenta (3, 4). The inadequate perfusion of the placenta often causes fetal growth restriction and can lead to hypoxia/asphyxia and fetal death.
In early pregnancy, placentation occurs in relatively hypoxic conditions critical for proper embryonic development. Before the ninth week of gestation, placental oxygen tension is low (
20 mm Hg), and at 10–12 wk gestation it increases to approximately 55 mm Hg (5). It is at this time that the trophoblast cells transit from a proliferative to an invasive phenotype (6), penetrate endometrial vessels, and gain direct access to maternal blood. At this time, the opening of the intervillous space may expose invasive trophoblast cells to high levels of biologically active maternal glucocorticoids (GCs). In humans, excessive exposure of the fetus to GCs during gestation has been shown to disturb the pattern of growth and differentiation, leading to fetal maldevelopment and intrauterine growth restriction (7, 8); in the rat placenta, excessive GCs increase the rate of apoptosis (9). Thus, the importance of maintaining low levels of GC during early pregnancy becomes critical to ensure optimal development of the embryo.
PE is believed to develop in early pregnancy, although clinical symptoms manifest in the third trimester. Trophoblast cells from PE pregnancies show a highly proliferative phenotype and shallow trophoblast invasion. Genbacev et al. (6, 10, 11) provided in vitro evidence to support a role for low oxygen tension in maintaining trophoblasts in a proliferative, noninvasive, and immature phenotype. In vivo studies that measured blood gas values of the fetus using cordocentesis provided evidence for an association of PE with hypoxemia (12, 13, 14). The adaptive response to hypoxia is accompanied by an increase in the expression of a variety of genes, including the vascular endothelial growth factor, glycolytic enzymes, and inducible nitric oxide synthetase (15, 16, 17, 18). Recent studies in the literature have shown that GC metabolism is altered in the placentas of preeclamptic patients through a reduction in the activity of the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) (19). Two distinct isozymes of 11ß-HSD, known as 11ß-HSD1 and 11ß-HSD2, have been characterized and cloned (20). 11ß-HSD1 possesses both oxidase (cortisol to cortisone) and reductase (cortisone to cortisol) activities, has a higher affinity for cortisone than for cortisol (21, 22, 23), and operates predominantly as a reductase. In contrast, 11ß-HSD2 exhibits only oxidase activity under physiological conditions and has a much higher affinity for cortisol (24, 25). In the term human placenta, 11ß-HSD1 is expressed in placental intermediate trophoblast cells and vascular endothelium (26). 11ß-HSD2 has been localized to placental syncytiotrophoblast (27). Its high affinity for cortisol makes it more suited to regulate the amount of maternal glucocorticoid affecting the trophoblast and passing across the placenta into the fetal circulation.
We have therefore hypothesized that 11ß-HSD2 might be regulated by oxygen and that the poorly regulated oxygen tension associated with PE may alter 11ß-HSD2 expression and activity. In the present study we have examined the ontogeny of 11ß-HSD1 and -2 expression in early pregnancy and investigated their expression in PE. Using a human villous explant culture system, we have investigated the relationship between oxygen tension and 11ß-HSD2 expression and activity as a determinant of GC metabolism in early pregnancy. We have also determined the effect of O2 tension at term to examine whether placental 11ß-HSD2 is differentially regulated through pregnancy.
Materials and Methods
Tissue collection
Collection and processing of human placentas were approved by the district and local hospital ethical committees, and collection was performed according to the Mount Sinai Hospital and University of Toronto code of practice. Informed patient consent was obtained in all cases. First to second trimester human placentas (5–17 wk gestation) were obtained from elective terminations of pregnancies by dilatation and curettage. Preeclamptic and age-matched control group (AMC) placentas were obtained from patients between 25–36 wk gestation. The preeclamptic group was selected to represent classic preeclampsia according to both clinical and pathological criteria (28). PE was defined as an increase in blood pressure to at least 140/90 mm Hg after the 20th wk of gestation, an increase in diastolic blood pressure of at least 15 mm Hg from the level measured before the 20th wk, or an increase in systolic blood pressure of at least 30 mm Hg from the level measured before the 20th wk, combined with proteinuria (protein excretion, at least 0.3 g/24 h) or edema. We included as cases of PE all pregnancies with a specified diagnosis of PE and pregnancies with a combination of pregnancy-related hypertension and proteinuria. The AMC groups were primiparous with the following clinical conditions: cervical cancer, multiple pregnancies, and premature rupture of membranes, but did not show clinical or pathological signs of PE or any other placental disease. Term human placentas were obtained from uncomplicated pregnancies after elective cesarean section delivery between 38–40 wk gestation.
Tissue culture
Human chorionic villous explant culture. Villous explant cultures were established from fresh placenta of 5–8 wk gestation as described previously (29). Briefly, placental tissue was placed in ice-cold PBS and processed within 2 h of collection. The tissue was dissected in a sterile manner. Decidual tissue and fetal membranes were removed. Small fragments of placental villi (25–45 mg wet weight) were teased apart and placed on Millicel-CM culture dish inserts (Millipore Corp., Bedford, MA) in a 24-well culture dish. Explants were cultured in serum-free DMEM/Ham’s F-12 (Life Technologies, Inc., Grand Island, NY) supplemented with streptomycin (100 µg/ml). Villous explants were maintained at 37 C under standard tissue culture conditions (5% CO2/95% air) or in an atmosphere of 3% O2/93% N2/5% CO2 for 3 d. Each experiment was performed in triplicate and repeated with at least four different placentas. As previously described, the viability of the explants was assessed by measuring hCG and progesterone production rates in the culture medium (29). In addition, the morphological integrity and viability of villous explants and their extravillous trophoblast (EVT) differentiation were assessed by monitoring EVT outgrowth from the distal end of the villous tip and its migration into the surrounding matrix, which was observed up to 6 d in culture (29, 30).
Placental syncytiotrophoblast cell culture.
Placental trophoblast cells were isolated from term placental cotyledons and cultured using a modification of the technique described by Kliman et al. (31). Briefly, the placenta was dissected aseptically to remove fetal membranes and decidua. Cotyledonary tissue (60 g) was removed randomly from the maternal side and digested three times for 30 min each time with 0.125% trypsin (Sigma, St. Louis, MO) and 0.02% deoxyribonuclease I (Sigma) in DMEM (Life Technologies, Inc., Grand Island, NY) containing 10% FCS. The dispersed placental cells were filtered through a 200-µm pore size nylon gauze and loaded onto a continuous Percoll (Sigma) gradient (5–70% in 5% steps of 3 ml each), then centrifuged at 1200 x g for 20 min at room temperature to separate different cell types. Cytotrophoblast cells between the density markers 1.049 and 1.062 g/ml were collected and plated in 24-well plates (Corning, Costar, Cambridge, MA) at a density of 106 cells/ml. The dispersed trophoblast cells were cultured for 12 h at 37 C in 5% CO2/95% air to allow attachment. The cells were then divided into two groups: half were incubated in standard tissue culture condition (20% O2/95% air/5% CO2), and half were incubated in an atmosphere of 3% O2/93% air/5% CO2 for 60 h. The purity of the cell preparation was assessed at the end of the experiment by histochemical staining for cytokeratin, an epithelial cell lineage marker (DAKO Corp., Glostrup, Denmark), or vimentin, a mesenchymal cell lineage marker (DAKO Corp.); cells were counterstained with Carrazzi’s hematoxylin. After 72 h of culture, placental trophoblast cells aggregated to form syncytial clumps corresponding to syncytiotrophoblast (31). The cultured cells were 98 ± 2% cytokeratin positive and vimentin negative, suggesting the presence of mainly trophoblast cells and few fibroblast or decidual cells. Cell viability, assessed by trypan staining, was 95% before and after incubation.
Immunohistochemistry
Placental tissues from 5–17 wk gestation, normal pregnancies, and pregnancies complicated by preeclampsia at delivery were fixed for 24 h at 4 C in 4% (vol/vol) paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. Tissues from villous explants (7–10 wk) kept in culture for 3 d at either 3% or 20% O2 were fixed for 2–4 h and treated similarly. To verify the tissue integrity and select the most representative sections, every 10th section was stained with hematoxylin and eosin. Neighboring sections were stained using the avidin-biotin immunoperoxidase method. Endogenous peroxidase activity was quenched by pretreatment with 3% (vol/vol) hydrogen peroxide in methanol for 30 min. Tissue sections were then washed in PBS and incubated with normal rabbit serum (10%), which served as a blocking agent for nonspecific binding. Immunoreactive 11ß-HSD2 was detected with a sheep polyclonal antibody (1:500, 12.4 µg/ml; The Binding Site, San Diego, CA). The characterization and specificity of this antibody have been previously reported (32). After incubation at 4 C for 18 h, slides were washed three times with PBS and then incubated with biotinylated donkey antisheep IgG for 1 h at 4 C. After washing three times with PBS buffer, the slides were incubated with an avidin-biotin complex for 1 h. After a final wash, the immunoreactive proteins were visualized after the addition of 3,3'-diaminobenzidine (Sigma) as the chromagen for 2 min. Negative controls were treated in an identical manner, except that 11ßHSD2 antibody was replaced by sheep antimouse IgG (Pierce Chemical Co., Rockford, IL) at 12 µg/ml, the same concentration at which the 11ß-HSD2 antibody was used.
Western blotting analysis
Western analysis of proteins was performed by SDS-PAGE on homogenates from the three different sources of placental tissue described previously. Protein samples (70 µg for placental tissue and 25 µg for explants) were solubilized in Laemmli sample buffer (10% sodium dodecyl sulfate, 0.5 M Tris, 22% glycerol, and 0.2% bromophenol blue; Bio-Rad Laboratories, Inc., Hercules, CA), heated at 55 C for 15 min, and resolved onto 12% bis-acrylamide gels at 100 V for 2 h. They were then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc.) at 110 V for 2 h. Proteins were visualized with Ponceau S solution (Sigma) and digitally scanned before immunoblotting to ensure equal lane loading. Nitrocellulose blots were blocked with 5% nonfat milk (Nestle, Glendale, CA) in PBS with 0.1% (v/v) Tween 20 (Bio-Rad Laboratories, Inc.; PBS-T) at 4 C for 18–24 h with constant agitation. All additional incubations took place at room temperature with constant agitation. The 11ß-HSD2 antibody (1:1000 dilution, 6.2 µg/ml) was mixed with 5% nonfat milk solution in PBS-T, and blots were incubated for 1 h. The primary antibody was removed, and the blots were washed with PBS-T five times for 5 min each time. The blots were then incubated with donkey antisheep IgG coupled to horseradish peroxidase (Amersham Life Science, Baie d’Urfe, Canada) at a 1:3000 dilution for 1 h and then washed with PBS-T five times for 5 min each time. Enhanced chemiluminescence detection reagents (Amersham Life Sciences) were added to the membranes for 1 min, and then the membranes were exposed to X-OMAT blue film (Kodak, Rochester, NY). A single band of 40 kDa corresponding to the known molecular mass of 11ß-HSD2 was clearly visible in all specimens tested. The intensities of the immunoreactive bands were measured by scanning (6200C scanner, Hewlett-Packard Co., Mississauga, Canada) and analyzing the image on a desktop computer using Scion Image software (version 4.0.2, Scion Corp., Frederick, MD). Protein bands were digitized, and the mean pixel density for each band was analyzed to obtain relative OD units for each protein.
Levels of 11ß-HSD1 expression were also examined by Western blotting analysis on homogenates from placental tissues 5–17 wk gestation, normal pregnancies, and pregnancies complicated by preeclampsia. The polyclonal antibody against 11ß-HSD1 (5 µg/ml) was provided by Dr. K. Yang (33).
Determination of 11ß-HSD2 activity
Villous explants. Villous explants from placentas harvested at 5–8 wk gestation were kept in culture for 3 d at either 3% or 20% O2. After this time, the explants were washed with fetal calf serum-free DMEM (pH 7.4) and preincubated in the same medium for 1 h. 11ß-HSD2 oxidase activity was assayed using 10 nM [3H]cortisol (specific activity, 64 Ci/mmol; Amersham Life Science, Little Chalfont, UK) as substrate in a 1-ml total volume. Medium was collected after 1-h incubation at 37 C. At the end of the experiment explants were homogenized, and the protein content was measured by the Bradford method using a protein assay kit (Bio-Rad Laboratories, Inc.) with BSA as standard. To measure the conversion of [3H]cortisol to [3H]cortisone, a mixture of cortisol and cortisone (40 µg each) was added to the collected medium to allow subsequent localization of the steroids during purification by thin layer chromatography (34). Steroids in the media and cells were extracted with ethyl acetate (3 ml). The extract from the medium was dried under air, reconstituted with ethyl acetate (100 µl), and applied to a thin layer chromatography plate (Silica gel GF, Fisher Scientific, Pittsburgh, PA). Cortisol and cortisone were separated in the solvent system chloroform/ethanol (95:5, vol/vol). Steroids were visualized under UV light, scraped off the plate, and extracted with ethyl acetate. The solvent was dried, scintillation fluid was added, and the radioactivity was counted in a liquid scintillation counter. In all cases the background conversion, estimated from radioactivity in duplicate blank wells not containing any explants, was subtracted from that in the experimental wells before analysis. 11ß-HSD2 activity was expressed as femtomoles of cortisone produced per milligram of protein per minute. Data are presented as the percent activity relative to the control value.
Placentas from preeclamptic pregnancies and AMC. Placental tissue (1–2 g) was homogenized in 4 vol 50 mM Tris-HCl buffer containing 50 mM EDTA. The assay mixture consisted of Tris buffer (300 µl; pH 7.4), 50 µl coenzyme solution (10 mM NAD), 50 µl tritium-labeled cortisol in ethanol (10 µM), and 100 µl placental homogenate. Incubation was carried out for 30 min at 37 C. These conditions were chosen so that the initial velocity was proportional to time for at least 30 min. The velocity was proportional to the protein content of the enzyme preparation. Each assay was performed in duplicate. The reaction was stopped with ethyl acetate (500 µl). 11ß-HSD2 activity was assayed as described above and expressed as femtomoles of cortisone produced per milligram of placental tissue per minute.
Term trophoblast cells. Trophoblast cells from placenta were cultured for 72 h. On the day of an experiment, the cells were washed with fetal calf serum-free culture medium (pH 7.4) and preincubated in the same medium for 1 h. 11ß-HSD2 oxidase activity was assayed using 10 nM [3H]cortisol as substrate. Medium was collected after 30-min incubation at 37 C for determination of 11ß-HSD2 activity as previously described. Activity was expressed as femtomoles of cortisone produced per 106 cells per 30 minutes. Data are presented as the percent activity relative to the control value.
Data analysis
Results are expressed as the mean ± SEM of averages from four to six individual experiments. There were at least three replicates of each condition per experiment. Statistical comparisons were made using paired t test (
= 0.05; SigmaStat, Jandel Scientific Software, San Rafael, CA).
Results
11ß-HSD in preeclampsia
11ß-HSD2 and 11ß-HSD1 expression in preeclampsia.
Both immunohistochemistry and Western blot analysis were used to compare 11ß-HSD2 expression in preeclamptic placentas (n = 14) and AMC (n = 14) ranging from 25–36 wk gestation. At all gestational ages studied there was a significant decrease in 11ß-HSD2 expression in preeclamptic placenta compared with AMC. Figure 1A
shows representative staining for 11ß-HSD2 protein at 26 wk gestation. Strong 11ß-HSD2 immunoreactivity was observed in the syncytiotrophoblast; the intensity of staining was reduced in preeclamptic specimens. Moreover, 11ß-HSD2 protein expression determined by Western blot was reduced significantly in preeclamptic placentas. Figure 1B shows a representative blot of 11ß-HSD2 expression in preeclamptic placentas and AMC at different gestational ages. Densitometric analysis showed a 50% reduction in 11ß-HSD2 expression in preeclamptic placenta (n = 14) compared with AMC (n = 14; P < 0.001; Fig. 1C). Figure 2 shows a representative blot of 11ß-HSD1 expression in preeclamptic placentas and AMC at different gestational ages. Densitometric analysis showed no change in 11ß-HSD1 expression in preeclamptic placenta (n = 14) compared with AMC (n = 14).
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, 11ß-HSD2 activity was significantly reduced in preeclamptic placentas compared with AMC (P < 0.05). 11ß-HSD expression in early pregnancy
Immunohistochemistry and Western blot analysis were used to define the pattern of expression of 11ß-HSD2 isoforms in the human placenta during the first trimester of pregnancy between 5–17 wk of gestation. Immunoreactive 11ß-HSD2 (ir-11ß-HSD2) was observed in all samples. At 5 wk gestation, expression was limited to the syncytiotrophoblast layer of the chorionic villi. As gestational age advanced (12–15 wk), the staining for 11ß-HSD2 expression increased in the syncytiotrophoblast layer and became apparent in the inner cytotrophoblast and EVT forming the anchoring villi (Fig. 3). A similar developmental pattern of expression was obtained by Western blot of total placental homogenates (n = 16 placentas), showing a gradual increase in 11ß-HSD2 protein between 5 and 17 wk gestation (Fig. 4A). Because intervillous blood flow is limited in early placentation, but increases at approximately 10–12 wk gestation, we analyzed these data based on protein values before and after 10 wk gestation, the time when O2 tension changes physiologically in the placenta. The level of 11ß-HSD2 protein expressed per mg placental tissue was 60% greater at 10–17 wk gestation than in placentas obtained at 5–9 wk gestation (Fig. 4B). In contrast, there was no change in 11ß-HSD1 protein expression in placental tissue from 5–17 wk gestation (n = 16 placentas; Fig. 5).
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Effect of oxygen on 11ß-HSD2 expression and activity in villous explants.
To determine whether oxygen tension regulates 11ß-HSD2 expression in early pregnancy, we first compared 11ß-HSD2 expression in villous explants from 5–8 wk gestation after incubation under 20% O2 or 3% O2. Explants cultured under 20% O2 had significantly greater 11ß-HSD2 protein expression than explants cultured at 3% O2. At 20% O2, explants showed strong immunoreactive 11ß-HSD2 in the syncytiotrophoblast layer with some staining in the EVT of the cell columns (Fig. 6A). A similar increase in 11ß-HSD2 expression was observed by Western blot analysis (Fig. 6B). The amount of 11ß-HSD2 in four placentas was determined by densitometry and was increased 2.5-fold under 20% O2 incubation (Fig. 6C). We next compared the enzyme activity in villous explants from 5–8 wk after incubation under 20% O2 or 3% O2. Incubation under 20% O2 led to a significant (50%) increase in 11ß-HSD2 activity (P < 0.05; Fig. 7A).
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). Moreover, 11ß-HSD2 activity was 106% and 114% greater than the control value at 6% and 10% O2, respectively (means of triplicates from experiments with trophoblast cells from two term placentas), showing the dose dependency of this relationship. Discussion
The present report is the first to demonstrate that changes in oxygen tension affect both the expression and activity of placental 11ß-HSD2, raising the possibility that sustained low placental oxygenation after the first trimester of pregnancy might be the cause of reduced placental 11ß-HSD2 expression and activity in PE. These conclusions are based on three observations. First, the temporal increase in 11ß-HSD2 expression demonstrated by immunohistochemistry and Western blot analysis coincides with the changes in oxygen tension at about 10–12 wk of gestation. Second, both placental explants from 5–8 wk gestation and isolated term trophoblast cells showed an increase in 11ß-HSD2 activity and expression when cultured under standard oxygen tension (20%) compared with explants or cells from the same patient cultured under low oxygen tension (3%). Third, both the expression and activity of 11ß-HSD2 are down-regulated in preeclamptic placentas compared with AMC. Taken together, these data show that 11ß-HSD2 activity and expression can be regulated by oxygen tension throughout pregnancy.
Our study demonstrated that 11ß-HSD2 was expressed as early as 5 wk gestation in syncytiotrophoblast cells lining the placental villi. However, no staining was observed in the chorionic stem cytotrophoblast cells, mesenchymal cells, or the vasculature of villous tissues. This observation agrees with a recent report showing 11ß-HSD2 expression in placentas from early pregnancy (35). However, no differential expression of 11ß-HSD2 has been reported. Our data confirm the presence of 11ß-HSD2 in early pregnancy and show a temporal increase in 11ß-HSD2 expression within the first trimester at about 10–12 wk. At that time, the amount of ir-11ß-HSD2 in the syncytiotrophoblast increased significantly compared with the levels at 5–9 wk gestation, and immunostaining for the enzyme was also evident in the inner cytotrophoblast and EVT. The increase in 11ß-HSD2 expression at the time of trophoblast invasion of spiral arteries may play a critical role in the success of pregnancy for several reasons. First, in human pregnancy the cortisol to cortisone ratio is approximately 10:1 in the mother and 1:2 in the fetus (36). By inactivating most of the maternal cortisol arriving at the placenta, 11ß-HSD2 will prevent high levels of bioactive GCs crossing to the fetus, thereby diminishing potential deleterious effects of high levels of GC on the developing embryo, effects that have been shown in different animals (37, 38, 39). Second, in early pregnancy, implantation of the human blastocyst in the uterine wall may be influenced by GCs (40). Active GCs exert potent effects on extracellular matrix (ECM)-degrading proteases and their inhibitors (41) and suppress the synthesis of specific ECM components in cultured human trophoblast cells (42). Therefore, adequate GC metabolism and GC responsiveness would favor high levels of placental ECM protein synthesis, thus establishing uterine-placental adherence. The local control of GC levels by 11ß-HSD2 present at the fetal-maternal interface may represent one of the mechanisms involved in controlling trophoblastic invasion in the uterine wall. Third, in vitro studies suggest that GCs increase the rate of apoptosis in trophoblast cells (7). This may limit placental size and function. Appropriate 11ß-HSD2 activity would reduce the levels of bioactive GC acting directly on the trophoblast, thereby contributing to the development of placental villous mass.
Our results clearly establish that an increase in oxygen levels up-regulates GC metabolism through 11ß-HSD2 activation and suggests that oxygen levels play an important role in placentation and in the pathophysiology of certain complications of pregnancy. The mechanism by which O2 regulates 11ß-HSD2 expression and activity has yet to be investigated. The effect of oxygen tension on 11ß-HSD2 could also be indirect, as other factors, such as cytokines (TNF, interferon-
, and IL-1ß), are increased under hypoxia in the human placenta (43), and in other systems these factors have been shown to decrease 11ß-HSD2 activity (44). In mammalian systems the adaptive response to hypoxia is accompanied by an increase in the expression of a variety of genes, including glycolytic enzymes (45, 46) and alcohol dehydrogenase enzymes (47, 48, 49). The signaling pathways and genetic elements that control the response to hypoxia are complex. In recent reports hypoxia-inducible factor 1 (HIF-1) has been shown to play a critical role in the transcriptional activation of a number of genes in response to hypoxia (50, 51). Caniggia et al. (52) have shown recently that HIF-1 expression in the placenta is high in early pregnancy between 5–8 wk and then dramatically decreases after 10–12 wk, at the time when the intervillous space is perfused and pO2 is believed to increase. Moreover, HIF-1 has been shown to up-regulate TGFß3, a protein that plays a role in the inhibition of trophoblast invasion (53). These genes are up- regulated in placental tissue from preeclamptic pregnancies (52), providing further evidence that in placental tissue this condition may be hypoxic. A number of the genes regulated by hypoxia do not have the enhancer element that binds HIF-1. An additional hypoxia response element distinct from the HIF-1-binding sites has been described in plant alcohol dehydrogenase (47, 54). A promoter element with the core sequence GGGC/T/ACG binds to a number of transcription factors, including members of the SP family, family of transcription factors that bind to cis elements in the promoter regions of various genes (55, 56). SP1 factor is typically a positive acting transcription factor, and its expression is oxygen sensitive. A GenBank screen of the 11ß-HSD2 promoter does not reveal the presence of the HIF-1-binding site, but does show a region corresponding to the SP1 response element, suggesting that the oxygen effect on 11ß-HSD2 expression might be through a pathway distinct from HIF-1.
Mounting evidence suggests that the EVT from preeclamptic placentas are arrested at a relatively immature phenotype, possibly because of failure to undergo complete differentiation along the invasive pathway (57). In the present report we substantiate and extend the decrease in 11ß-HSD2 activity in preeclamptic patients (19) and show that its expression is also altered in PE. We recognized the difficulty in obtaining true control placentas during human pregnancy and studied age-matched material from a variety of pregnancies without diagnosed PE. The consistent pattern across different AMC is supportive of this approach. The cause of the reduction in 11ß-HSD2 might be due to an inadequacy in placentation that occurred in early pregnancy and hence remained throughout gestation. A failure in developmental switching in oxygen tension or a defect in the ability of the trophoblast to respond appropriately to this switch around the 10–12th wk of gestation, as occurs in PE, can result in a sustained reduction in 11ß-HSD2 expression, leading to inadequate protection of the growing fetus from a maternal excess of GC. The changes in placental GC metabolism in PE seem to be specific to 11ß-HSD2, because no change in type 1 was observed in the same patients from this study. Furthermore, there was no change in 11ß-HSD1 expression with increasing gestational age. This differential regulation in 11ß-HSD enzymes emphasizes the physiological importance that 11ß-HSD2 plays in early pregnancy and in pregnancies complicated with PE.
A defect in renal GC metabolism in preeclamptic patients has been suggested as a cause of the sodium retention and hypertension associated with PE. However, maternal plasma cortisol concentrations are unchanged in PE (58), and deficient inactivation of cortisol to cortisone, as shown by urinary steroid profiles, does not contribute to the sodium retention of pregnancy (59). Therefore, a defect in placental 11ß-HSD2 in PE appears to represent a tissue-specific change. A local defect in GC metabolism in PE may explain the intrauterine growth restriction associated with this disease and contribute to the fetal complications of PE (60).
In conclusion, the results of this study have demonstrated that placental 11ß-HSD2 is reduced in PE, and that both the activity and expression of 11ß-HSD2 in placental tissue are oxygen dependent throughout gestation. We speculate that this change in placental 11ß-HSD2 expression and activity occurs in response to the vascular changes of PE and results in increased bioactive GC present in the placenta and crossing to the fetus, thereby contributing to altered placental development and fetal outcome.
Acknowledgments
We thank Dr. Ljiljana Petkovic for providing the placental samples.
Footnotes
This work was supported by the Canadian Institutes for Health Research in Human Development, Child and Youth Health (Grant MOP-42378, to J.R.G.C.) and the Department of Obstetrics and Gynecology, Canadian Institutes for Health Research (Grant MT-14096, to I.C.).
Abbreviations: AMC, Age-matched control; ECM, extracellular matrix; EVT, extravillous trophoblast; GC, glucocorticoid; HIF-1, hypoxia-inducible factor 1; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase 2; PBS-T, PBS with 0.1% (v/v) Tween 20; PE, preeclampsia; PL, placental.
Received February 27, 2002.
Accepted July 17, 2002.
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 C. J McNeil, M. O Nwagwu, A. M Finch, K. R Page, A. Thain, H. J McArdle, and C. J Ashworth Glucocorticoid exposure and tissue gene expression of 11{beta} HSD-1, 11{beta} HSD-2, and glucocorticoid receptor in a porcine model of differential fetal growth Reproduction, March 1, 2007; 133(3): 653 - 661. [Abstract] [Full Text] [PDF]
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 V. E. Murphy, R. Smith, W. B. Giles, and V. L. Clifton Endocrine Regulation of Human Fetal Growth: The Role of the Mother, Placenta, and Fetus Endocr. Rev., April 1, 2006; 27(2): 141 - 169. [Abstract] [Full Text] [PDF]
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P. Hoffmann, J.-J. Feige, and N. Alfaidy Expression and Oxygen Regulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Human Placenta during Early Pregnancy Endocrinology, April 1, 2006; 147(4): 1675 - 1684. [Abstract] [Full Text] [PDF]
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 L. Myatt Placental adaptive responses and fetal programming J. Physiol., April 1, 2006; 572(1): 25 - 30. [Abstract] [Full Text] [PDF]
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H. Chisaka, J. F. Johnstone, M. Premyslova, Z. Manduch, and J. R.G. Challis Effect of Pro-inflammatory Cytokines on Expression and Activity of 11{beta}-Hydroxysteroid Dehydrogenase Type 2 in Cultured Human Term Placental Trophoblast and Human Choriocarcinoma JEG-3 Cells Reproductive Sciences, July 1, 2005; 12(5): 303 - 309. [Abstract] [PDF]
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L. Julan, H. Guan, J. P. van Beek, and K. Yang Peroxisome Proliferator-Activated Receptor {delta} Suppresses 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Human Placental Trophoblast Cells Endocrinology, March 1, 2005; 146(3): 1482 - 1490. [Abstract] [Full Text] [PDF]
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 N. Alfaidy, W. Li, T. MacIntosh, K. Yang, and J. Challis Late Gestation Increase in 11{beta}-Hydroxysteroid Dehydrogenase 1 Expression in Human Fetal Membranes: A Novel Intrauterine Source of Cortisol J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 5033 - 5038. [Abstract] [Full Text] [PDF]
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