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Elevated glucocorticoid metabolism in placental tissue from first trimester pregnancies at increased risk of pre-eclampsia
Placenta 32 (2011) 687e693
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Placenta journal homepage: www.elsevier.com/locate/placenta
Elevated glucocorticoid metabolism in placental tissue from first trimester pregnancies at increased risk of pre-eclampsia S. Mukherjee a, b, *, J.L. James c, B. Thilaganathan a, b, G.St.J. Whitley c, A.E. Michael c, J.E. Cartwright c a
Fetal Medicine Unit, St.George’s Hospital, Blackshaw Road, London SW17 0QT, UK Division of Clinical Sciences, St.George’s, University of London, Cranmer Terrace, London SW17 0RE, UK c Division of Biomedical Sciences, St.George’s, University of London, Cranmer Terrace, London SW17 0RE, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 20 June 2011
Background: The local actions of glucocorticoids in the placenta can be modulated by 11b-hydroxysteroid dehydrogenase (11bHSD) enzymes, which catalyse inter-conversion of cortisol with its inert metabolite, cortisone, and are known to be expressed in the term placenta and decidua. However, the expression and activity of these enzymes have not been well characterised in the first trimester placenta. The aim of this study was to compare 11bHSD2 expression and activity in first trimester placental tissue from pregnancies at either relatively low or high risk of developing pre-eclampsia as determined by Doppler ultrasound. Methods: Enzyme expression was assessed by western blot analysis and immunohistochemistry while 11bHSD enzyme activities were quantified using radiometric conversion of [3H]-cortisol in the presence of NADPþ or NADþ. Results: 11bHSD2 was expressed in syncytiotrophoblast of first trimester placenta, and there was no difference in the level of expression of placental 11bHSD2 protein between 9 high pre-eclampsia risk and 14 low pre-eclampsia risk pregnancies. NADþ-dependent cortisol oxidation was elevated 3-fold in placental tissue from pregnancies at higher risk of pre-eclampsia than in normal pregnancies (50.9 15.9 versus 18.3 1.9 pmol cortisone/mg protein.10 min, n ¼ 11 & 12, respectively; P < 0.05). Conclusions: Expression of 11bHSD2 is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11bHSD2 is down regulated in term pre-eclamptic placentae, our study suggests that there is increased cortisol inactivation in first trimester placenta prior to week 10 of gestation, from pregnancies at higher risk of developing pre-eclampsia. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Glucocorticoid 11bHSD Pre-eclampsia Placenta
1. Introduction Controlling the level of glucocorticoids (GCs) in the fetal circulation is essential for normal fetal organ growth and development. Excessive GC exposure in utero has been shown to disturb the pattern of growth and differentiation leading to fetal maldevelopment and intrauterine growth restriction (IUGR) in humans and may predispose the fetus to hypertension and diabetes in adult life [1]. The local actions of GCs can be modulated by the 11b-hydroxysteroid dehydrogenase (11bHSD) enzymes which catalyse interconversion of cortisol with its inert metabolite, cortisone. 11bHSD1 is a relatively low affinity, bi-directional isoenzyme which
* Corresponding author. Fetal Medicine Unit, St.George’s Hospital, Blackshaw Road, London SW17 0QT, UK. Tel.: þ44 0 447753494948; fax: þ44 0 2087252858. E-mail address: [email protected] (S. Mukherjee). 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.06.014
regenerates the conversion of cortisol from cortisone in the presence of NADPH [2]. In contrast, 11bHSD2 is a high affinity, unidirectional dehydrogenase which inactivates cortisol using NADþ as its pyridine dinucleotide co-substrate (Fig. 1). Thus the relative levels of 11bHSD enzymes are important in determining the intracellular concentration and actions of cortisol [2]. The syncytiotrophoblast covers the placental chorionic villi and plays a critical role in controlling metabolic and endocrine functions throughout pregnancy and regulating nutrient and gas exchange between the mother and the fetus. As mononucleated cytotrophoblast differentiate into the multinucleated syncytiotrophoblast, there is an upregulation in expression of 11bHSD2 [3] which becomes the major placental isoenzyme restricting the passage of active GC across the placenta into the fetal circulation and thus may protect the fetus from the detrimental effects of maternally derived cortisol [4,5]. High cortisol levels in the presence of decreased activity of 11bHSD2 are associated with low birth weight and IUGR [6].
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Fig. 1. Schematic representation of the inter-conversion of active cortisol and inert cortisone by the two cloned 11bHSD isoenzymes.
Defective placental GC metabolism by 11bHSD2 has also been implicated in pre-eclampsia. Specifically, independent studies have shown that at term, both the expression and the oxidative activity of placental 11bHSD2 are decreased in pre-eclamptic relative to normotensive pregnancies [2,7e9]. Pre-eclampsia is a common obstetric complication and although the clinical symptoms of preeclampsia become evident as pregnancy progresses, the pathology is thought to develop in the first trimester. Inadequate or defective trophoblastic invasion of the uterine decidua and the spiral arteries in early pregnancy is generally considered to be one of the major aetiological factors in the development of pre-eclampsia [10]. Measurement of uterine artery resistance indices (RI) by Doppler ultrasound in the first trimester can be used to assess uteroplacental perfusion and the risk of developing pre-eclampsia [11,12]. It has been shown that uterine artery Doppler indices reflect the degree of trophoblast invasion in the first trimester [13,14]. Although 11bHSD isoenzymes are known to be expressed at the maternal-fetal interface at term, their expression and activity have not been well characterised in first trimester placenta and we have not, until now, been able to relate expression of 11bHSD2 in the first trimester to pre-eclampsia. Our ability to use Doppler ultrasound to separate first trimester pregnancies by their risk of developing preeclampsia makes this the first study to investigate glucocorticoid metabolism in first trimester placenta at high risk of developing pre-eclampsia. In this study, we have investigated the 11bHSD enzyme activity and expression in first trimester placental tissue from pregnancies characterised by uterine artery Doppler for their risk of developing pre-eclampsia. 2. Methods 2.1. Determination of uterine artery resistance Doppler ultrasound examination of the maternal uterine arteries was performed on women attending a clinic for termination of pregnancy in the first trimester. Approval for this study was granted by the Wandsworth Local Research Ethics Committee and all women had given written informed consent. Women with preexisting medical conditions such as diabetes mellitus, connective tissue disorders and essential hypertension were excluded from the study. Only singleton pregnancies were included. Gestational age was confirmed by measurement of the crown-rump length on ultrasound. A GE Voluson 730 PRO ultrasound machine equipped with a transvaginal probe was used, as previously described [15]. The RI (Resistance Index) was measured and recorded and the presence or absence of an early diastolic notch was noted. Measurements were made bilaterally and the mean RI was calculated. High resistance/risk cases were defined as those with bilateral uterine artery notches and a mean RI above the 95th percentile, representing less than 5% of the population. Normal resistance/low risk cases were defined as those with a mean RI below the 95th percentile and no uterine artery notches, representing 40% of an unselected population. These groups represent those most (>21%) and least (<1%) likely respectively, to have developed pre-eclampsia if the pregnancy had progressed [12,16]. For each of the following, the mean values were compared for the high and low risk groups by unpaired t tests: maternal age (years), gestational age (days), gestational age at scanning (days) or scan to sample interval (days). The mean gestational age (days SEM) for patients in the 11bHSD2 expression studies was 80 9; n ¼ 14 (low risk), 74 10; n ¼ 9 (high risk; t test, p ¼ 0.99) and for patients in
the 11bHSD2 activity studies was 77 8; n ¼ 12 (low risk), 74 10; n ¼ 11 (high risk; t test, p ¼ 0.96). There was no significant difference in the demographic and clinical characteristics between the two groups (data not shown). 2.2. Western blot analysis First trimester chorionic villous tissue was dissected from fetal membranes, rinsed in PBS and snap-frozen in liquid nitrogen at 80 C. Tissue samples from twenty three patients (both high and low risk) was lysed in RIPA buffer (1 PBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) with Complete Protease Inhibitor Cocktail Tablet (Roche, West Sussex, UK) and homogenised using a FastPrepR-24 instrument (MP Biomedicals Inc, Ohio, USA). The lysate was centrifuged at 15,600 g for 5 min at 4 C and a protein assay (Pierce BCA protein assay, Thermo Fisher Scientific, Loughborough, UK) was performed on the supernatant according to the manufacturer’s instructions. A constant amount of protein (30 mg) from each sample was separated by SDS-polyacrylamide gel electrophoresis on a 12% (w/v) polyacrylamide gel and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. Following incubation in blocking buffer (10 mM Tris, pH 8, 150 mM NaCl, 0.05% (v/v) Tween 20, 5% (w/v) milk powder) for 1 h at room temperature, the membrane was incubated with (0.2 mg/ml) rabbit polyclonal antibody to 11bHSD2 (H-145, Santa Cruz Biotechnology, CA, USA) in the blocking buffer overnight at 4 C. After washing, the membrane was incubated with antirabbit IgG-peroxidase conjugate (A6154 Sigma, Dorset, UK) (0.45 mg/ml) in the blocking buffer for 1 h at room temperature. After further washing, membrane bound antibodies were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, UK) according to the manufacturer’s instructions and exposure to x-ray film (Amersham HyperfilmÔ ECL, Amersham, UK). Equal loading was confirmed by stripping the membrane and reprobing with an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (MAB3741, Chemicon, CA, USA) (20 ng/ml) followed by anti-mouse IgG-peroxidase conjugate (A4416, Sigma, Dorset, UK) (0.4 ng/ml). Densitometric analysis was carried out using ImageJ (National Institute of Health-rsbweb.nih.gov/ij/). 2.3. Immunohistochemistry 2.3.1. Cryosections First trimester chorionic villous tissue was snap frozen in embedding compound (Cryo-M-Bed, Bright Instrument Company, Huntingdon, UK). 7 mm sections were cut and mounted on glass slides. Slides were briefly rinsed in dH20, air-dried for 45 min, fixed with ice-cold acetone for 10 min, air-dried again and stored at 20 C until use. At the time of staining, slides were blocked in 10% (v/v) goat serum for 20 min. After washing with PBS, the slides were incubated with rabbit anti-11bHSD2 antibody or normal rabbit immunoglobulin (X0903, DAKO, Denmark; negative control) at 4 mg/ ml in PBS-T (PBS/0.2% (v/v) Tween 20) containing 10% (v/v) goat serum (blocking solution) for 1 h. The slides were washed three times with PBS-T and then incubated with 5 mg/ml biotin-conjugated goat anti-rabbit IgG (Vector Laboratories, Peterborough, UK) diluted in blocking solution for 45 min. Following a further three washes with PBS-T, the slides were incubated with streptavidin-fluorescein (Vector Laboratories, Peterborough, UK) at 15 mg/ml for 15 min in a dark chamber. After extensive washing, the slides were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Peterborough, UK) and examined using a fluorescence (Olympus IX 70) microscope with an attached XC 10 camera (Olympus, Tokyo, Japan). Images were captured using cell^D software (Olympus Tokyo, Japan). 2.3.2. Paraffin sections First trimester chorionic villous tissue was fixed in 4% (v/v) paraformaldehyde and PBS for 24 h, washed in 70% (v/v) ethanol and taken to 100% (v/v) ethanol and then embedded in paraffin. 5 mm sections were cut from paraffin-embedded tissue blocks. Slides were de-waxed by immersing twice in xylene (BDH) for 15 min. Slides were rehydrated by immersing in ethanol at 100%, 90%, 80%, 70%, 50% and 30% (v/v) for 2 min each time and then in PBS for 15 min. Proteinase K (Invitrogen, Loughborough, UK) was added at 20 mg/ml in PBS for 20 min at 37 C and then for 10 min at
S. Mukherjee et al. / Placenta 32 (2011) 687e693 room temperature. The slides were washed once with PBS-T and blocking solution was added for 10 min at room temperature. Slides were washed again in PBS-T and incubated with primary antibodies diluted in blocking solution for 1 h at room temperature at the following concentrations; mouse anti-human cytokeratin-7 at 2.33 mg/ml (OV-TL 12/30, DAKO, Denmark), rabbit anti-11bHSD2 at 2 mg/ml (H-145, Santa Cruz Biotechnology, CA, USA) or rabbit IgG at 4 mg/ml (X0903, DAKO, Denmark). The slides were washed three times in PBS-T and quenched with 3% (v/v) H2O2 in methanol for 5 min. Following a further two washes in PBS-T, a broad spectrum biotin-conjugated secondary antibody (Zymed Lab Inc, CA, USA) was added for 10 min, slides were washed three times in PBS-T, and HRP streptavidin (Zymed Lab Inc, CA, USA) was added for 10 min. The slides were washed three times in PBS-T prior to addition of 3-amino-9-ethylcarbazole (SigmaeAldrich, Dorset, UK). Slides were examined for development of colour after 10e15 min and then washed twice in dH20. Images were captured as described above.
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which the organic extracts (containing the [3H]-steroids) were evaporated to dryness (under N2 at 45 C) and transferred to a silica-60 thin layer chromatography plate that was subsequently developed in an atmosphere of 92:8(v/v) chloroform: 95% (v/v) ethanol. Fractional conversion of [3H]-cortisol to [3H]-cortisone was quantified using an AR 2000 Bioscan radiochromatogramme scanner with inline Lauralite 3.0 software (Lablogic, Sheffield, UK). 2.5. Statistical analysis Having confirmed that each data set conformed to a Gaussian distribution using KolmogoroveSmirnov tests, all quantitative data are presented as mean SEM. All data were analysed using either unpaired t-tests or one-way ANOVA followed by application of the post hoc Bonferroni multiple comparison test, as appropriate, using Graph Pad Prism statistical software, version 4.01 (Graph Pad Inc., San Diego, CA, USA). A P value of <0.05 was accepted as statistically significant in all tests.
2.4. Radiometric conversion assay 11bHSD enzyme activities were assessed in tissue homogenates from a different set of twenty three patients (due to a different tissue preparation methodology), using a radiometric conversion assay as previously described [17]. In brief, homogenates of first trimester placenta, prepared in PBS, were incubated for 10 min at 37 C with 100 nM [1,2,6,7-3H]-cortisol (0.5 mCi/100 pmol) and 4 mM NADPþ or 4 mM NADþ (SigmaeAldrich, Dorset, UK) as preferential co-substrates for 11bHSD1 and 11bHSD2, respectively. The specific activity of steroid substrates was adjusted prior to addition to the assay by mixing of tritiated and non-radioactive steroids. Incubations were terminated by the addition of 2 volumes of chloroform, after
3. Results 3.1. 11bHSD2 enzyme expression does not differ between placental tissues from high risk versus low risk of pre-eclampsia The expression of 11bHSD2 in first trimester placental tissue was determined by immunohistochemistry that localised 11bHSD2 protein to the syncytiotrophoblast and placental vessels (Fig. 2).
Fig. 2. Immunohistochemical localization of 11bHSD enzyme expression in first trimester chorionic villous (CV) tissue. Panels A, B & D: Primary antibodies reactive against 11bHSD2 demonstrate localization of the 11bHSD2 enzyme to the syncytiotrophoblast. Panel C: Primary antibody raised to human cytokeratin-7 identified trophoblast. Panel E: Negative control using normal rabbit immunoglobulin (IgG) in place of primary antibody. For panels A to C inclusive, binding of the primary antibody was detected using a biotin-conjugated secondary antibody and HRP-conjugated streptavidin. For panels D and E, the secondary antibody was goat anti-rabbit IgG visualised with streptavidin-fluorescein conjugate. Each panel is representative of CV tissue from at least 3 different patients.
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Western blot analysis confirmed the expression of 11bHSD2 in first trimester chorionic villous tissue (Fig. 3). Expression of 11bHSD2 was compared by western blot analysis between placentae categorised as being at low and high risk of developing pre-eclampsia. Densitometric analysis (11bHSD2/ GAPDH ratio) showed no statistically significant difference in expression of 11bHSD2 between the two risk groups (P > 0.05, Fig. 3). 3.2. 11bHSD2 activity is higher in placental tissue from pregnancies at higher risk of pre-eclampsia Irrespective of the uterine arterial resistance and the risk of preeclampsia, enzyme activities were consistently higher in the presence of 4 mM NADþ which increased the net oxidation of [3H]cortisol by up to 51 fold relative to the baseline enzyme activity measured in the absence of added pyridine dinucleotide cosubstrates (Fig. 4). NADþ-dependent 11bHSD activity was 2.8-fold higher in placental tissue from pregnancies at higher risk of pre-eclampsia than in low risk pregnancies (50.9 15.9 versus 18.3 1.9 pmol cortisone/mg protein.10 min, n ¼ 11 and 12, respectively, P < 0.01, Fig. 4). Further to the above result, a sub-analysis of those samples obtained of less than 10 weeks gestation was performed. There was a significant difference between the NADþ-dependent inactivation
Fig. 3. Expression of 11bHSD2 in first trimester placentae from Doppler screened pregnancies. Panel A: Western blot demonstrating expression of 11bHSD2 and GAPDH protein in first trimester chorionic villous tissue from 2 patients at low risk (L) and 2 patients at high risk (H) of developing pre-eclampsia (as assessed by uterine arterial resistance index using Doppler ultrasound in the first trimester). Panel B: Quantification of 11bHSD2 protein expression (expressed as a ratio relative to GAPDH) by densitometric analysis of western blots of homogenized placental tissue samples from low risk (n ¼ 14) and high-risk (n ¼ 9) pregnancies. Horizontal bars for each risk group denote the mean level of 11bHSD2 protein expression for that group; unpaired t test between means, P > 0.05.
Fig. 4. 11bHSD enzyme activities in first trimester placentae from Doppler screened pregnancies. Enzyme activities were measured over 10 min in the absence of any exogenous co-substrates (None), or in the presence of NADPþ or NADþ for each of 12 low risk and 11 high risk placentae (open bars and hatched bars, respectively). 11bHSD activity was significantly greater (10-fold greater) with the addition of NADþ than with the addition of NADPþ in both low and high risk placentae (P < 0.05). NADþ-dependant cortisol oxidation activity was 3-fold higher in high-risk placentae in comparison to low risk placentae (P < 0.01). Mean values were compared by ANOVA followed by Bonferroni’s multiple comparison test.
of cortisol to cortisone in those samples at or before 10 weeks of gestation in placental tissue from pregnancies at higher risk of preeclampsia than in low risk pregnancies (93.8 32.8 versus 12.8 1.5 pmol cortisone/mg protein.10 min, n ¼ 4 and 3, respectively, P < 0.01). However this difference was not observed in pregnancies sampled between 10.1 and 14 weeks of gestation (26.3 8.6 versus 20.1 2.2 pmol cortisone/mg protein.10 min, n ¼ 7 and 9, respectively, P > 0.05, Fig. 5). For 19 of those 23 patients for whom it had been possible to assess 11BHSD enzyme activities (10 low risk; 9 high risk), height and weight data had also been recorded from which it was possible to calculate their body mass index (BMI). There was no significant difference in BMI between those patients at low versus high risk of pre-eclampsia (32.32.5 versus 27.33.2, respectively; t ¼ 1.228, P ¼ 0.236) and across all 19 samples irrespective of pre-eclampsia risk, there was no significant correlation between BMI and the NADþ-dependent oxidation of cortisol (Pearson’s correlation coefficient R ¼ 0.043, P ¼ 0.860).
Fig. 5. 11bHSD enzyme activities in first trimester placentae from Doppler screened pregnancies analysed by gestational age. Enzyme activities in pregnancies at low or high risk of developing pre-eclampsia were measured over 10 min and analysed by gestational age. In pregnancies of 10 weeks of gestation or earlier 11bHSD mediated inactivation of cortisol to cortisone was significantly greater in the high-risk (n ¼ 4) placentae in comparison to the low risk (n ¼ 3) placentae (P < 0.01). In samples greater than 10 weeks of gestation there was no significant difference in the 11bHSD activity between pregnancies at high risk (n ¼ 7) or low risk of pre-eclampsia (n ¼ 9, P > 0.05). Mean values were compared by ANOVA followed by a Bonferroni’s multiple comparison test.
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4. Discussion This study has demonstrated that in early pregnancy, the NADþdependent inactivation of the physiological GC cortisol by 11bHSD2 is enhanced 2.8-fold in those pregnancies at high risk of subsequent pre-eclampsia (as determined by Doppler assessment of uterine artery RI) relative to low risk pregnancies. However, this increased cortisol metabolism in high risk pregnancies is not accompanied by an increase in expression of 11bHSD2 protein in the high-risk group. In the radiometric conversion assay of enzyme activities, NADPþ and NADþ were used as the preferential pyridine dinucleotide cosubstrates for 11bHSD1 and 11bHSD2, respectively. Given that the larger co-substrate binding domain, 11bHSD1 is capable of binding both NADPþ and the smaller co-substrate, NADþ, the use of different nucleotides does not allow for a definitive discrimination between the activities of 11bHSD1 versus 11bHSD2; although NADPþ will only support cortisol oxidation by 11bHSD1, NADþ can be used to facilitate cortisol metabolism by either 11bHSD isoenzyme. However, in the present study, the rates of cortisol oxidation were 10-and 34-fold higher in the presence of 4 mM NADþ than in the presence of 4 mM NADPþ for the high risk and low risk placentae, respectively. These ratios compare favourably with a report [18] which showed human placental cortisol oxidation to be 8-fold higher in the presence of NADþ than NADPþ. Hence, we would surmise that between 90% and 97% of NADþ-dependent cortisol metabolism can be attributed to the placental 11bHSD2 isoenzyme. Previous published studies have suggested that both the expression and activity of 11bHSD2 are decreased in term pre-eclamptic placentae relative to normotensive pregnancies [2,7e9]. Alfaidy et al. (2002) also demonstrated decreased activity and expression of 11bHSD2 in pre-eclamptic patients at 25e36 weeks gestation compared to controls matched for gestational age. In contrast, our study indicates that in the first trimester, increased risk of developing pre-eclampsia is associated with a significant increase in the enzymatic inactivation of cortisol by 11bHSD2. Several studies have shown that the expression of 11bHSD2 mRNA and protein is under local control by glucocorticoids that appear to up-regulate the expression of 11bHSD2 in the placenta, possibly via increased secretion of hCG from the syncytiotrophoblast [1,19,20]. Hence, in the development of pre-eclampsia, it is possible that increased glucocorticoid metabolism by the placental 11bHSD2 enzyme in early pregnancy decreases glucocorticoid exposure of placental cells leading ultimately to a subsequent decline in the expression and activity of placental 11bHSD2 beyond week 25 of gestation through to term. In this study, the 3-fold increase in NADþ-dependent inactivation of cortisol in placentae at high risk of pre-eclampsia was not accompanied by corresponding changes in the levels of expression of the 11bHSD2 protein. Hence, the increase in 11bHSD2 activity appears to result from post-translational regulation of this enzymatic barrier to the trans-placental transfer of cortisol from the maternal circulation. Numerous mechanisms have been identified and reported for the control of 11bHSD2 expression and activity ranging from transcriptional regulation, through post-transcriptional mechanisms and modification of mRNA half-life to posttranslational regulation of enzyme activities by agents such as liquorice and glycyrrhetinic acid [1,21]. The activity of placental 11bHSD2 is known to be sensitive to hormones secreted from the placenta and/or decidua e.g. hCG, progesterone and prostaglandins [4,19,22] which regulate enzyme activity via signal transduction molecules such as cyclic adenosine 30 , 50 -monophosphate (cAMP), calcium and protein kinases [23,24]. As indicated above, placental 11bHSD2 can also be stimulated by cortisol via increasing hCG [1,19,20]. Hence, placental 11bHSD2 activity can be regulated at the post-translational level by a paracrine/autocrine system of
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hormones and associated signalling mechanisms, several of which might be altered in the early stages of pre-eclampsia. Our data has demonstrated that 11bHSD2 activity varies even within the first trimester. For the first 10e12 weeks of gestation the placenta and embryo develop in a physiologically normal low (w2%) oxygen environment due to the presence of trophoblast plugs in the uterine spiral arteries [25,26]. When these plugs dislodge between 10 and 12 weeks of gestation oxygenated maternal blood is able to reach the placenta for the first time [25,26]. Oxygen tension plays important roles in regulating trophoblast proliferation and invasion, and premature exposure of the placenta to increased oxygen tension may be detrimental for pregnancy [27,28]. Changes in oxygen tension have previously been implicated in the regulation of the placental 11bHSD2 enzyme [3,9,29e31]. Therefore, 11bHSD2 activity was further analyzed after dividing the samples by gestational age within the first trimester. This analysis revealed a significant increase in 11bHSD2 activity in placental tissue from high risk pregnancies in comparison to that from low risk pregnancies in samples less than or equal to 10 weeks of gestation, but not in those over 10 weeks of gestation, and thus this difference corresponds to the time at which the placental tissue is exposed to low oxygen conditions in vivo. There is no consensus as to whether hypoxia decreases or increases the expression of 11bHSD2 in the placenta, but all studies are consistent in finding that any effect of oxygen tension is mediated at the transcriptional level by HIF1a [32]. However, as previously discussed, the observation that 11bHSD2 activity, but not expression, is altered between high and low risk placentae strongly suggests that this is a result of post-translational modification of enzyme activity and not of protein expression. So what could be responsible for the differences in placental 11bHSD2 activity between low and high-risk pregnancies of less than 10 weeks of gestation? The answer may be that aside from its direct effects on 11bHSD2, oxygen is capable of regulating a large number of cytokines and growth factors, in particular hCG, which may in turn have important indirect effects on 11bHSD2 activity. As 11bHSD2 activity is only significantly greater in high risk placentae under 10 weeks of gestation, these tissues appear to be responding aberrantly to the environment generated under these low oxygen conditions, but this aberrant response does not appear to continue after 10 weeks of gestation once the placental environment is exposed to increased oxygen levels. Addition of NADPþ to the radiometric conversion assay had no effect on baseline cortisol metabolism in the low risk placentae and only supported a modest increase in the cortisol oxidation by tissue samples from the high-risk group. Alfaidy et al. (2002) showed that pre-eclampsia was associated with decreased expression of 11bHSD2 protein in the samples obtained in the third trimester (26 weeks to term) of pregnancy [9]. Hence, it would appear that preeclampsia is associated with dysregulation of the high affinity placental 11bHSD2 enzyme, both in the first trimester and at term. Having found a significant difference in NADþ-dependent cortisol metabolism in those pregnancies at high versus low risk of pre-eclampsia, it was important to consider whether this could reflect possible confounding differences between the patients or pregnancies in each group. Previously, obesity has been linked to the risk of pre-eclampsia [33,34] and independently to increased 11BHSD2 activity as assessed by urinary free ratios of cortisol:cortisone [35]. This raises the possibility that the findings of the present study could have been confounded by increased BMI in those patients at higher risk of pre-clampsia. However, within the study series reported herein, there was no significant difference in BMI between those patients at low versus high risk of preeclampsia and there was no significant correlation between BMI and the NADþ-dependent rate of cortisol oxidation ascribed to 11BHSD2. Since Murphy et al. reported an association between fetal
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gender and placental 11BHSD2 activities, and previous publications have shown fetal gender to impact on the risk of pre-eclampsia [36e38], fetal gender could theoretically have been an alternative confounding factor in the present study. Unfortunately, the ethical approval governing the collection of tissues from elective terminations of pregnancy did not permit the assessment of fetal gender in the present study. However, while male fetal gender has been associated with higher risk of pre-eclampsia [37,38], such that we might have expected a preponderance of male fetuses in our high versus low risk groups, Murphy et al., found that placental 11BHSD2 activities were significantly lower in placentas attached to male versus female infants at term, making it improbable that fetal gender could account for the higher placental 11BHSD2 activities in the first trimester in those women assessed as being at high risk of pre-eclampsia in the present study [36]. In summary, while other studies have suggested that 11bHSD2 is decreased in term pre-eclampsia placentae (relative to normotensive pregnancies), our study suggests that there is increased NADþdependent activity of 11bHSD2 in first trimester placentae from pregnancies at higher risk of developing pre-eclampsia. This increase in enzyme activity only occurs prior to week 10 of gestation and is not accompanied by an increase in expression of 11bHSD2, consistent with post-translational regulation of enzyme activity. It remains to be determined if this difference in enzymatic inactivation of physiological glucocorticoids is related to the pathophysiology of pre-eclampsia or is a compensatory response to poor trophoblast development. Further research is needed to ascertain the clinical significance of these observations. Conflict of interest All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. This work was supported by the Fetal Medicine Unit at St George’s Hospital, London, UK. Author contribution statement SM, JEC, AEM, GW and BT were involved in designing the study. SM executed the study with help and guidance from JEC, AEM, JLJ and GW. Data were analysed by SM, JEC and AEM. Manuscript was drafted by SM, JEC, AEM and JLJ. Critical discussion was undertaken by all authors. Acknowledgements The authors would like to thank all staff and patients, without whom, this study would not have been possible. References [1] van Beek JP, Guan H, Julan L, Yang K. Glucocorticoids stimulate the expression of 11 b hydroxysteroid dehydrogenase type 2 in cultured human placental trophoblast cells. J Clin Endocrinol Metab 2004;89:5614e21. [2] McCalla CO, Nacharaju VL, Muneyyirci-Delale O, Glasgow S, Feldman JG. Placental 11 b hydroxysteroid dehydrogenase activity in normotensive and pre-eclamptic pregnancies. Steroids 1998;63:511e5. [3] Hardy DB, Yang K. The expression of 11 b hydroxysteroid dehydrogenase type 2 is induced during trophoblast differentiation: effects of hypoxia. J Clin Endocrinol Metab 2002;87:3696e701.
[4] Sun K, Yang K, Challis JR. Differential expression of 11 b hydroxysteroid dehydrogenase types 1 and 2 in human placenta and fetal membranes. J Clin Endocrinol Metab 1997;82:300e5. [5] Michael AE, Papageorghiou AT. Potential significance of physiological and pharmacological glucocorticoids in early pregnancy. Hum Reprod Update 2008;14:497e517. [6] Dave-Sharma S, Wilson RC, Harbison MD, Newfield R, Azar MR, Krozowski ZS, et al. Examination of genotype and phenotype relationships in 14 patients with apparent mineralocorticoid excess. J Clin Endocrinol Metab 1998;83: 2244e54. [7] Schoof E, Girstl M, Frobenius W, Kirschbaum M, Dorr HG, Rascher W, et al. Decreased gene expression of 11 b hydroxysteroid dehydrogenase type 2 and 15-hydroxyprostaglandin dehydrogenase in human placenta of patients with pre-eclampsia. J Clin Endocrinol Metab 2001;86:1313e7. [8] Schoof E, Girstl M, Frobenius W, Kirschbaum M, Repp R, Knerr I, et al. Course of placental 11 b hydroxysteroid dehydrogenase type 2 and 15hydroxyprostaglandin dehydrogenase mRNA expression during human gestation. Eur J Endocrinol 2001;145:187e92. [9] Alfaidy N, Gupta S, DeMarco C, Caniggia I, Challis JR. Oxygen regulation of placental 11 b hydroxysteroid dehydrogenase 2: physiological and pathological implications. J Clin Endocrinol Metab 2002;87:4797e805. [10] Pijnenborg R, Anthony J, Davey DA, Rees A, Tiltman A, Vercruysse L, et al. Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol 1991;98:648e55. [11] Poon LC, Akolekar R, Lachmann R, Beta J, Nicolaides KH. Hypertensive disorders in pregnancy: screening by biophysical and biochemical markers at 11e13 weeks. Ultrasound Obstet Gynecol 2010;35:662e70. [12] Melchiorre K, Wormald B, Leslie K, Bhide A, Thilaganathan B. First-trimester uterine artery Doppler indices in term and preterm pre-eclampsia. Ultrasound Obstet Gynecol 2008;32:133e7. 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[17] Sharp V, Thurston LM, Fowkes RC, Michael AE. 11 b hydroxysteroid dehydrogenase enzymes in the testis and male reproductive tract of the boar (Sus scrofa domestica) indicate local roles for glucocorticoids in male reproductive physiology. Reproduction 2007;134:473e82. [18] Mericq V, Medina P, Kakarieka E, Márquez L, Johnson MC, Iñiguez G. Differences in expression and activity of 11 b hydroxysteroid dehydrogenase type 1 and 2 in human placentas of term pregnancies according to birth weight and gender. Eur J Endocrinol 2009;161:419e25. [19] Ni XT, Duan T, Yang Z, Guo CM, Li JN, Sun K. Role of human chorionic gonadotropin in maintaining 11 b hydroxysteroid dehydrogenase type 2 expression in human placental syncytiotrophoblasts. Placenta 2009;30: 1023e8. [20] Audette MC, Greenwood SL, Sibley CP, Jones CJP, Challis JRG, Matthews SG, et al. Dexamethasone stimulates placental system A transport and trophoblast differentiation in term villous explants. Placenta 2010;31:97e105. [21] Causevic M, Mohaupt M. 11 b Hydroxysteroid dehydrogenase type 2 in pregnancy and pre-eclampsia. Mol Aspects Med 2007;28:220e6. [22] Hardy DB, Pereria LE, Yang K. Prostaglandins and leukotriene B4 are potent inhibitors of 11 b hydroxysteroid dehydrogenase type 2 activity in human choriocarcinoma JEG-3 cells. Biol Reprod 1999;61:40e5. [23] Sun K, Yang K, Challis JR. Regulation of 11 b hydroxysteroid dehydrogenase type 2 by progesterone, estrogen, and the cyclic adenosine 5’-monophosphate pathway in cultured human placental and chorionic trophoblasts. Biol Reprod 1998;58:1379e84. [24] Hardy DB, Dixon SJ, Narayanan N, Yang K. Calcium inhibits human placental 11 b hydroxysteroid dehydrogenase type 2 activity. Biochem Biophys Res Commun 2001;283:756e61. [25] Hustin J, Schaaps J. Echographic and anatomic studies of the maternotrophoblastic border during the first trimester of pregnancy. Am J Obstet Gynecol 1987;157:162e8. [26] Rodesch F, Simon P, Donner C, Jauniaux E. Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet Gynecol 1992;80:283e5. [27] James JL, Stone PR, Chamley LW. The regulation of trophoblast differentiation by oxygen in the first trimester of pregnancy. Hum Repro Update 2006;12: 137e44. [28] Jauniaux E, Hempstock J, Greenwold N, Burton GJ. Trophoblastic oxidative stress in relation to temporal and regional differences in maternal placental blood flow in normal and abnormal early pregnancies. Am J Pathol 2003;162: 115e25. [29] Homan A, Guan H, Hardy DB, Gratton RJ, Yang K. Hypoxia blocks 11 b hydroxysteroid dehydrogenase type 2 induction in human trophoblast cells
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Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Elevated glucocorticoid metabolism in placental tissue from first trimester pregnancies at increased risk of pre-eclampsia S. Mukherjee a, b, *, J.L. James c, B. Thilaganathan a, b, G.St.J. Whitley c, A.E. Michael c, J.E. Cartwright c a
Fetal Medicine Unit, St.George’s Hospital, Blackshaw Road, London SW17 0QT, UK Division of Clinical Sciences, St.George’s, University of London, Cranmer Terrace, London SW17 0RE, UK c Division of Biomedical Sciences, St.George’s, University of London, Cranmer Terrace, London SW17 0RE, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 20 June 2011
Background: The local actions of glucocorticoids in the placenta can be modulated by 11b-hydroxysteroid dehydrogenase (11bHSD) enzymes, which catalyse inter-conversion of cortisol with its inert metabolite, cortisone, and are known to be expressed in the term placenta and decidua. However, the expression and activity of these enzymes have not been well characterised in the first trimester placenta. The aim of this study was to compare 11bHSD2 expression and activity in first trimester placental tissue from pregnancies at either relatively low or high risk of developing pre-eclampsia as determined by Doppler ultrasound. Methods: Enzyme expression was assessed by western blot analysis and immunohistochemistry while 11bHSD enzyme activities were quantified using radiometric conversion of [3H]-cortisol in the presence of NADPþ or NADþ. Results: 11bHSD2 was expressed in syncytiotrophoblast of first trimester placenta, and there was no difference in the level of expression of placental 11bHSD2 protein between 9 high pre-eclampsia risk and 14 low pre-eclampsia risk pregnancies. NADþ-dependent cortisol oxidation was elevated 3-fold in placental tissue from pregnancies at higher risk of pre-eclampsia than in normal pregnancies (50.9 15.9 versus 18.3 1.9 pmol cortisone/mg protein.10 min, n ¼ 11 & 12, respectively; P < 0.05). Conclusions: Expression of 11bHSD2 is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11bHSD2 is down regulated in term pre-eclamptic placentae, our study suggests that there is increased cortisol inactivation in first trimester placenta prior to week 10 of gestation, from pregnancies at higher risk of developing pre-eclampsia. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Glucocorticoid 11bHSD Pre-eclampsia Placenta
1. Introduction Controlling the level of glucocorticoids (GCs) in the fetal circulation is essential for normal fetal organ growth and development. Excessive GC exposure in utero has been shown to disturb the pattern of growth and differentiation leading to fetal maldevelopment and intrauterine growth restriction (IUGR) in humans and may predispose the fetus to hypertension and diabetes in adult life [1]. The local actions of GCs can be modulated by the 11b-hydroxysteroid dehydrogenase (11bHSD) enzymes which catalyse interconversion of cortisol with its inert metabolite, cortisone. 11bHSD1 is a relatively low affinity, bi-directional isoenzyme which
* Corresponding author. Fetal Medicine Unit, St.George’s Hospital, Blackshaw Road, London SW17 0QT, UK. Tel.: þ44 0 447753494948; fax: þ44 0 2087252858. E-mail address: [email protected] (S. Mukherjee). 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.06.014
regenerates the conversion of cortisol from cortisone in the presence of NADPH [2]. In contrast, 11bHSD2 is a high affinity, unidirectional dehydrogenase which inactivates cortisol using NADþ as its pyridine dinucleotide co-substrate (Fig. 1). Thus the relative levels of 11bHSD enzymes are important in determining the intracellular concentration and actions of cortisol [2]. The syncytiotrophoblast covers the placental chorionic villi and plays a critical role in controlling metabolic and endocrine functions throughout pregnancy and regulating nutrient and gas exchange between the mother and the fetus. As mononucleated cytotrophoblast differentiate into the multinucleated syncytiotrophoblast, there is an upregulation in expression of 11bHSD2 [3] which becomes the major placental isoenzyme restricting the passage of active GC across the placenta into the fetal circulation and thus may protect the fetus from the detrimental effects of maternally derived cortisol [4,5]. High cortisol levels in the presence of decreased activity of 11bHSD2 are associated with low birth weight and IUGR [6].
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Fig. 1. Schematic representation of the inter-conversion of active cortisol and inert cortisone by the two cloned 11bHSD isoenzymes.
Defective placental GC metabolism by 11bHSD2 has also been implicated in pre-eclampsia. Specifically, independent studies have shown that at term, both the expression and the oxidative activity of placental 11bHSD2 are decreased in pre-eclamptic relative to normotensive pregnancies [2,7e9]. Pre-eclampsia is a common obstetric complication and although the clinical symptoms of preeclampsia become evident as pregnancy progresses, the pathology is thought to develop in the first trimester. Inadequate or defective trophoblastic invasion of the uterine decidua and the spiral arteries in early pregnancy is generally considered to be one of the major aetiological factors in the development of pre-eclampsia [10]. Measurement of uterine artery resistance indices (RI) by Doppler ultrasound in the first trimester can be used to assess uteroplacental perfusion and the risk of developing pre-eclampsia [11,12]. It has been shown that uterine artery Doppler indices reflect the degree of trophoblast invasion in the first trimester [13,14]. Although 11bHSD isoenzymes are known to be expressed at the maternal-fetal interface at term, their expression and activity have not been well characterised in first trimester placenta and we have not, until now, been able to relate expression of 11bHSD2 in the first trimester to pre-eclampsia. Our ability to use Doppler ultrasound to separate first trimester pregnancies by their risk of developing preeclampsia makes this the first study to investigate glucocorticoid metabolism in first trimester placenta at high risk of developing pre-eclampsia. In this study, we have investigated the 11bHSD enzyme activity and expression in first trimester placental tissue from pregnancies characterised by uterine artery Doppler for their risk of developing pre-eclampsia. 2. Methods 2.1. Determination of uterine artery resistance Doppler ultrasound examination of the maternal uterine arteries was performed on women attending a clinic for termination of pregnancy in the first trimester. Approval for this study was granted by the Wandsworth Local Research Ethics Committee and all women had given written informed consent. Women with preexisting medical conditions such as diabetes mellitus, connective tissue disorders and essential hypertension were excluded from the study. Only singleton pregnancies were included. Gestational age was confirmed by measurement of the crown-rump length on ultrasound. A GE Voluson 730 PRO ultrasound machine equipped with a transvaginal probe was used, as previously described [15]. The RI (Resistance Index) was measured and recorded and the presence or absence of an early diastolic notch was noted. Measurements were made bilaterally and the mean RI was calculated. High resistance/risk cases were defined as those with bilateral uterine artery notches and a mean RI above the 95th percentile, representing less than 5% of the population. Normal resistance/low risk cases were defined as those with a mean RI below the 95th percentile and no uterine artery notches, representing 40% of an unselected population. These groups represent those most (>21%) and least (<1%) likely respectively, to have developed pre-eclampsia if the pregnancy had progressed [12,16]. For each of the following, the mean values were compared for the high and low risk groups by unpaired t tests: maternal age (years), gestational age (days), gestational age at scanning (days) or scan to sample interval (days). The mean gestational age (days SEM) for patients in the 11bHSD2 expression studies was 80 9; n ¼ 14 (low risk), 74 10; n ¼ 9 (high risk; t test, p ¼ 0.99) and for patients in
the 11bHSD2 activity studies was 77 8; n ¼ 12 (low risk), 74 10; n ¼ 11 (high risk; t test, p ¼ 0.96). There was no significant difference in the demographic and clinical characteristics between the two groups (data not shown). 2.2. Western blot analysis First trimester chorionic villous tissue was dissected from fetal membranes, rinsed in PBS and snap-frozen in liquid nitrogen at 80 C. Tissue samples from twenty three patients (both high and low risk) was lysed in RIPA buffer (1 PBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) with Complete Protease Inhibitor Cocktail Tablet (Roche, West Sussex, UK) and homogenised using a FastPrepR-24 instrument (MP Biomedicals Inc, Ohio, USA). The lysate was centrifuged at 15,600 g for 5 min at 4 C and a protein assay (Pierce BCA protein assay, Thermo Fisher Scientific, Loughborough, UK) was performed on the supernatant according to the manufacturer’s instructions. A constant amount of protein (30 mg) from each sample was separated by SDS-polyacrylamide gel electrophoresis on a 12% (w/v) polyacrylamide gel and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. Following incubation in blocking buffer (10 mM Tris, pH 8, 150 mM NaCl, 0.05% (v/v) Tween 20, 5% (w/v) milk powder) for 1 h at room temperature, the membrane was incubated with (0.2 mg/ml) rabbit polyclonal antibody to 11bHSD2 (H-145, Santa Cruz Biotechnology, CA, USA) in the blocking buffer overnight at 4 C. After washing, the membrane was incubated with antirabbit IgG-peroxidase conjugate (A6154 Sigma, Dorset, UK) (0.45 mg/ml) in the blocking buffer for 1 h at room temperature. After further washing, membrane bound antibodies were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, UK) according to the manufacturer’s instructions and exposure to x-ray film (Amersham HyperfilmÔ ECL, Amersham, UK). Equal loading was confirmed by stripping the membrane and reprobing with an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (MAB3741, Chemicon, CA, USA) (20 ng/ml) followed by anti-mouse IgG-peroxidase conjugate (A4416, Sigma, Dorset, UK) (0.4 ng/ml). Densitometric analysis was carried out using ImageJ (National Institute of Health-rsbweb.nih.gov/ij/). 2.3. Immunohistochemistry 2.3.1. Cryosections First trimester chorionic villous tissue was snap frozen in embedding compound (Cryo-M-Bed, Bright Instrument Company, Huntingdon, UK). 7 mm sections were cut and mounted on glass slides. Slides were briefly rinsed in dH20, air-dried for 45 min, fixed with ice-cold acetone for 10 min, air-dried again and stored at 20 C until use. At the time of staining, slides were blocked in 10% (v/v) goat serum for 20 min. After washing with PBS, the slides were incubated with rabbit anti-11bHSD2 antibody or normal rabbit immunoglobulin (X0903, DAKO, Denmark; negative control) at 4 mg/ ml in PBS-T (PBS/0.2% (v/v) Tween 20) containing 10% (v/v) goat serum (blocking solution) for 1 h. The slides were washed three times with PBS-T and then incubated with 5 mg/ml biotin-conjugated goat anti-rabbit IgG (Vector Laboratories, Peterborough, UK) diluted in blocking solution for 45 min. Following a further three washes with PBS-T, the slides were incubated with streptavidin-fluorescein (Vector Laboratories, Peterborough, UK) at 15 mg/ml for 15 min in a dark chamber. After extensive washing, the slides were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Peterborough, UK) and examined using a fluorescence (Olympus IX 70) microscope with an attached XC 10 camera (Olympus, Tokyo, Japan). Images were captured using cell^D software (Olympus Tokyo, Japan). 2.3.2. Paraffin sections First trimester chorionic villous tissue was fixed in 4% (v/v) paraformaldehyde and PBS for 24 h, washed in 70% (v/v) ethanol and taken to 100% (v/v) ethanol and then embedded in paraffin. 5 mm sections were cut from paraffin-embedded tissue blocks. Slides were de-waxed by immersing twice in xylene (BDH) for 15 min. Slides were rehydrated by immersing in ethanol at 100%, 90%, 80%, 70%, 50% and 30% (v/v) for 2 min each time and then in PBS for 15 min. Proteinase K (Invitrogen, Loughborough, UK) was added at 20 mg/ml in PBS for 20 min at 37 C and then for 10 min at
S. Mukherjee et al. / Placenta 32 (2011) 687e693 room temperature. The slides were washed once with PBS-T and blocking solution was added for 10 min at room temperature. Slides were washed again in PBS-T and incubated with primary antibodies diluted in blocking solution for 1 h at room temperature at the following concentrations; mouse anti-human cytokeratin-7 at 2.33 mg/ml (OV-TL 12/30, DAKO, Denmark), rabbit anti-11bHSD2 at 2 mg/ml (H-145, Santa Cruz Biotechnology, CA, USA) or rabbit IgG at 4 mg/ml (X0903, DAKO, Denmark). The slides were washed three times in PBS-T and quenched with 3% (v/v) H2O2 in methanol for 5 min. Following a further two washes in PBS-T, a broad spectrum biotin-conjugated secondary antibody (Zymed Lab Inc, CA, USA) was added for 10 min, slides were washed three times in PBS-T, and HRP streptavidin (Zymed Lab Inc, CA, USA) was added for 10 min. The slides were washed three times in PBS-T prior to addition of 3-amino-9-ethylcarbazole (SigmaeAldrich, Dorset, UK). Slides were examined for development of colour after 10e15 min and then washed twice in dH20. Images were captured as described above.
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which the organic extracts (containing the [3H]-steroids) were evaporated to dryness (under N2 at 45 C) and transferred to a silica-60 thin layer chromatography plate that was subsequently developed in an atmosphere of 92:8(v/v) chloroform: 95% (v/v) ethanol. Fractional conversion of [3H]-cortisol to [3H]-cortisone was quantified using an AR 2000 Bioscan radiochromatogramme scanner with inline Lauralite 3.0 software (Lablogic, Sheffield, UK). 2.5. Statistical analysis Having confirmed that each data set conformed to a Gaussian distribution using KolmogoroveSmirnov tests, all quantitative data are presented as mean SEM. All data were analysed using either unpaired t-tests or one-way ANOVA followed by application of the post hoc Bonferroni multiple comparison test, as appropriate, using Graph Pad Prism statistical software, version 4.01 (Graph Pad Inc., San Diego, CA, USA). A P value of <0.05 was accepted as statistically significant in all tests.
2.4. Radiometric conversion assay 11bHSD enzyme activities were assessed in tissue homogenates from a different set of twenty three patients (due to a different tissue preparation methodology), using a radiometric conversion assay as previously described [17]. In brief, homogenates of first trimester placenta, prepared in PBS, were incubated for 10 min at 37 C with 100 nM [1,2,6,7-3H]-cortisol (0.5 mCi/100 pmol) and 4 mM NADPþ or 4 mM NADþ (SigmaeAldrich, Dorset, UK) as preferential co-substrates for 11bHSD1 and 11bHSD2, respectively. The specific activity of steroid substrates was adjusted prior to addition to the assay by mixing of tritiated and non-radioactive steroids. Incubations were terminated by the addition of 2 volumes of chloroform, after
3. Results 3.1. 11bHSD2 enzyme expression does not differ between placental tissues from high risk versus low risk of pre-eclampsia The expression of 11bHSD2 in first trimester placental tissue was determined by immunohistochemistry that localised 11bHSD2 protein to the syncytiotrophoblast and placental vessels (Fig. 2).
Fig. 2. Immunohistochemical localization of 11bHSD enzyme expression in first trimester chorionic villous (CV) tissue. Panels A, B & D: Primary antibodies reactive against 11bHSD2 demonstrate localization of the 11bHSD2 enzyme to the syncytiotrophoblast. Panel C: Primary antibody raised to human cytokeratin-7 identified trophoblast. Panel E: Negative control using normal rabbit immunoglobulin (IgG) in place of primary antibody. For panels A to C inclusive, binding of the primary antibody was detected using a biotin-conjugated secondary antibody and HRP-conjugated streptavidin. For panels D and E, the secondary antibody was goat anti-rabbit IgG visualised with streptavidin-fluorescein conjugate. Each panel is representative of CV tissue from at least 3 different patients.
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Western blot analysis confirmed the expression of 11bHSD2 in first trimester chorionic villous tissue (Fig. 3). Expression of 11bHSD2 was compared by western blot analysis between placentae categorised as being at low and high risk of developing pre-eclampsia. Densitometric analysis (11bHSD2/ GAPDH ratio) showed no statistically significant difference in expression of 11bHSD2 between the two risk groups (P > 0.05, Fig. 3). 3.2. 11bHSD2 activity is higher in placental tissue from pregnancies at higher risk of pre-eclampsia Irrespective of the uterine arterial resistance and the risk of preeclampsia, enzyme activities were consistently higher in the presence of 4 mM NADþ which increased the net oxidation of [3H]cortisol by up to 51 fold relative to the baseline enzyme activity measured in the absence of added pyridine dinucleotide cosubstrates (Fig. 4). NADþ-dependent 11bHSD activity was 2.8-fold higher in placental tissue from pregnancies at higher risk of pre-eclampsia than in low risk pregnancies (50.9 15.9 versus 18.3 1.9 pmol cortisone/mg protein.10 min, n ¼ 11 and 12, respectively, P < 0.01, Fig. 4). Further to the above result, a sub-analysis of those samples obtained of less than 10 weeks gestation was performed. There was a significant difference between the NADþ-dependent inactivation
Fig. 3. Expression of 11bHSD2 in first trimester placentae from Doppler screened pregnancies. Panel A: Western blot demonstrating expression of 11bHSD2 and GAPDH protein in first trimester chorionic villous tissue from 2 patients at low risk (L) and 2 patients at high risk (H) of developing pre-eclampsia (as assessed by uterine arterial resistance index using Doppler ultrasound in the first trimester). Panel B: Quantification of 11bHSD2 protein expression (expressed as a ratio relative to GAPDH) by densitometric analysis of western blots of homogenized placental tissue samples from low risk (n ¼ 14) and high-risk (n ¼ 9) pregnancies. Horizontal bars for each risk group denote the mean level of 11bHSD2 protein expression for that group; unpaired t test between means, P > 0.05.
Fig. 4. 11bHSD enzyme activities in first trimester placentae from Doppler screened pregnancies. Enzyme activities were measured over 10 min in the absence of any exogenous co-substrates (None), or in the presence of NADPþ or NADþ for each of 12 low risk and 11 high risk placentae (open bars and hatched bars, respectively). 11bHSD activity was significantly greater (10-fold greater) with the addition of NADþ than with the addition of NADPþ in both low and high risk placentae (P < 0.05). NADþ-dependant cortisol oxidation activity was 3-fold higher in high-risk placentae in comparison to low risk placentae (P < 0.01). Mean values were compared by ANOVA followed by Bonferroni’s multiple comparison test.
of cortisol to cortisone in those samples at or before 10 weeks of gestation in placental tissue from pregnancies at higher risk of preeclampsia than in low risk pregnancies (93.8 32.8 versus 12.8 1.5 pmol cortisone/mg protein.10 min, n ¼ 4 and 3, respectively, P < 0.01). However this difference was not observed in pregnancies sampled between 10.1 and 14 weeks of gestation (26.3 8.6 versus 20.1 2.2 pmol cortisone/mg protein.10 min, n ¼ 7 and 9, respectively, P > 0.05, Fig. 5). For 19 of those 23 patients for whom it had been possible to assess 11BHSD enzyme activities (10 low risk; 9 high risk), height and weight data had also been recorded from which it was possible to calculate their body mass index (BMI). There was no significant difference in BMI between those patients at low versus high risk of pre-eclampsia (32.32.5 versus 27.33.2, respectively; t ¼ 1.228, P ¼ 0.236) and across all 19 samples irrespective of pre-eclampsia risk, there was no significant correlation between BMI and the NADþ-dependent oxidation of cortisol (Pearson’s correlation coefficient R ¼ 0.043, P ¼ 0.860).
Fig. 5. 11bHSD enzyme activities in first trimester placentae from Doppler screened pregnancies analysed by gestational age. Enzyme activities in pregnancies at low or high risk of developing pre-eclampsia were measured over 10 min and analysed by gestational age. In pregnancies of 10 weeks of gestation or earlier 11bHSD mediated inactivation of cortisol to cortisone was significantly greater in the high-risk (n ¼ 4) placentae in comparison to the low risk (n ¼ 3) placentae (P < 0.01). In samples greater than 10 weeks of gestation there was no significant difference in the 11bHSD activity between pregnancies at high risk (n ¼ 7) or low risk of pre-eclampsia (n ¼ 9, P > 0.05). Mean values were compared by ANOVA followed by a Bonferroni’s multiple comparison test.
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4. Discussion This study has demonstrated that in early pregnancy, the NADþdependent inactivation of the physiological GC cortisol by 11bHSD2 is enhanced 2.8-fold in those pregnancies at high risk of subsequent pre-eclampsia (as determined by Doppler assessment of uterine artery RI) relative to low risk pregnancies. However, this increased cortisol metabolism in high risk pregnancies is not accompanied by an increase in expression of 11bHSD2 protein in the high-risk group. In the radiometric conversion assay of enzyme activities, NADPþ and NADþ were used as the preferential pyridine dinucleotide cosubstrates for 11bHSD1 and 11bHSD2, respectively. Given that the larger co-substrate binding domain, 11bHSD1 is capable of binding both NADPþ and the smaller co-substrate, NADþ, the use of different nucleotides does not allow for a definitive discrimination between the activities of 11bHSD1 versus 11bHSD2; although NADPþ will only support cortisol oxidation by 11bHSD1, NADþ can be used to facilitate cortisol metabolism by either 11bHSD isoenzyme. However, in the present study, the rates of cortisol oxidation were 10-and 34-fold higher in the presence of 4 mM NADþ than in the presence of 4 mM NADPþ for the high risk and low risk placentae, respectively. These ratios compare favourably with a report [18] which showed human placental cortisol oxidation to be 8-fold higher in the presence of NADþ than NADPþ. Hence, we would surmise that between 90% and 97% of NADþ-dependent cortisol metabolism can be attributed to the placental 11bHSD2 isoenzyme. Previous published studies have suggested that both the expression and activity of 11bHSD2 are decreased in term pre-eclamptic placentae relative to normotensive pregnancies [2,7e9]. Alfaidy et al. (2002) also demonstrated decreased activity and expression of 11bHSD2 in pre-eclamptic patients at 25e36 weeks gestation compared to controls matched for gestational age. In contrast, our study indicates that in the first trimester, increased risk of developing pre-eclampsia is associated with a significant increase in the enzymatic inactivation of cortisol by 11bHSD2. Several studies have shown that the expression of 11bHSD2 mRNA and protein is under local control by glucocorticoids that appear to up-regulate the expression of 11bHSD2 in the placenta, possibly via increased secretion of hCG from the syncytiotrophoblast [1,19,20]. Hence, in the development of pre-eclampsia, it is possible that increased glucocorticoid metabolism by the placental 11bHSD2 enzyme in early pregnancy decreases glucocorticoid exposure of placental cells leading ultimately to a subsequent decline in the expression and activity of placental 11bHSD2 beyond week 25 of gestation through to term. In this study, the 3-fold increase in NADþ-dependent inactivation of cortisol in placentae at high risk of pre-eclampsia was not accompanied by corresponding changes in the levels of expression of the 11bHSD2 protein. Hence, the increase in 11bHSD2 activity appears to result from post-translational regulation of this enzymatic barrier to the trans-placental transfer of cortisol from the maternal circulation. Numerous mechanisms have been identified and reported for the control of 11bHSD2 expression and activity ranging from transcriptional regulation, through post-transcriptional mechanisms and modification of mRNA half-life to posttranslational regulation of enzyme activities by agents such as liquorice and glycyrrhetinic acid [1,21]. The activity of placental 11bHSD2 is known to be sensitive to hormones secreted from the placenta and/or decidua e.g. hCG, progesterone and prostaglandins [4,19,22] which regulate enzyme activity via signal transduction molecules such as cyclic adenosine 30 , 50 -monophosphate (cAMP), calcium and protein kinases [23,24]. As indicated above, placental 11bHSD2 can also be stimulated by cortisol via increasing hCG [1,19,20]. Hence, placental 11bHSD2 activity can be regulated at the post-translational level by a paracrine/autocrine system of
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hormones and associated signalling mechanisms, several of which might be altered in the early stages of pre-eclampsia. Our data has demonstrated that 11bHSD2 activity varies even within the first trimester. For the first 10e12 weeks of gestation the placenta and embryo develop in a physiologically normal low (w2%) oxygen environment due to the presence of trophoblast plugs in the uterine spiral arteries [25,26]. When these plugs dislodge between 10 and 12 weeks of gestation oxygenated maternal blood is able to reach the placenta for the first time [25,26]. Oxygen tension plays important roles in regulating trophoblast proliferation and invasion, and premature exposure of the placenta to increased oxygen tension may be detrimental for pregnancy [27,28]. Changes in oxygen tension have previously been implicated in the regulation of the placental 11bHSD2 enzyme [3,9,29e31]. Therefore, 11bHSD2 activity was further analyzed after dividing the samples by gestational age within the first trimester. This analysis revealed a significant increase in 11bHSD2 activity in placental tissue from high risk pregnancies in comparison to that from low risk pregnancies in samples less than or equal to 10 weeks of gestation, but not in those over 10 weeks of gestation, and thus this difference corresponds to the time at which the placental tissue is exposed to low oxygen conditions in vivo. There is no consensus as to whether hypoxia decreases or increases the expression of 11bHSD2 in the placenta, but all studies are consistent in finding that any effect of oxygen tension is mediated at the transcriptional level by HIF1a [32]. However, as previously discussed, the observation that 11bHSD2 activity, but not expression, is altered between high and low risk placentae strongly suggests that this is a result of post-translational modification of enzyme activity and not of protein expression. So what could be responsible for the differences in placental 11bHSD2 activity between low and high-risk pregnancies of less than 10 weeks of gestation? The answer may be that aside from its direct effects on 11bHSD2, oxygen is capable of regulating a large number of cytokines and growth factors, in particular hCG, which may in turn have important indirect effects on 11bHSD2 activity. As 11bHSD2 activity is only significantly greater in high risk placentae under 10 weeks of gestation, these tissues appear to be responding aberrantly to the environment generated under these low oxygen conditions, but this aberrant response does not appear to continue after 10 weeks of gestation once the placental environment is exposed to increased oxygen levels. Addition of NADPþ to the radiometric conversion assay had no effect on baseline cortisol metabolism in the low risk placentae and only supported a modest increase in the cortisol oxidation by tissue samples from the high-risk group. Alfaidy et al. (2002) showed that pre-eclampsia was associated with decreased expression of 11bHSD2 protein in the samples obtained in the third trimester (26 weeks to term) of pregnancy [9]. Hence, it would appear that preeclampsia is associated with dysregulation of the high affinity placental 11bHSD2 enzyme, both in the first trimester and at term. Having found a significant difference in NADþ-dependent cortisol metabolism in those pregnancies at high versus low risk of pre-eclampsia, it was important to consider whether this could reflect possible confounding differences between the patients or pregnancies in each group. Previously, obesity has been linked to the risk of pre-eclampsia [33,34] and independently to increased 11BHSD2 activity as assessed by urinary free ratios of cortisol:cortisone [35]. This raises the possibility that the findings of the present study could have been confounded by increased BMI in those patients at higher risk of pre-clampsia. However, within the study series reported herein, there was no significant difference in BMI between those patients at low versus high risk of preeclampsia and there was no significant correlation between BMI and the NADþ-dependent rate of cortisol oxidation ascribed to 11BHSD2. Since Murphy et al. reported an association between fetal
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gender and placental 11BHSD2 activities, and previous publications have shown fetal gender to impact on the risk of pre-eclampsia [36e38], fetal gender could theoretically have been an alternative confounding factor in the present study. Unfortunately, the ethical approval governing the collection of tissues from elective terminations of pregnancy did not permit the assessment of fetal gender in the present study. However, while male fetal gender has been associated with higher risk of pre-eclampsia [37,38], such that we might have expected a preponderance of male fetuses in our high versus low risk groups, Murphy et al., found that placental 11BHSD2 activities were significantly lower in placentas attached to male versus female infants at term, making it improbable that fetal gender could account for the higher placental 11BHSD2 activities in the first trimester in those women assessed as being at high risk of pre-eclampsia in the present study [36]. In summary, while other studies have suggested that 11bHSD2 is decreased in term pre-eclampsia placentae (relative to normotensive pregnancies), our study suggests that there is increased NADþdependent activity of 11bHSD2 in first trimester placentae from pregnancies at higher risk of developing pre-eclampsia. This increase in enzyme activity only occurs prior to week 10 of gestation and is not accompanied by an increase in expression of 11bHSD2, consistent with post-translational regulation of enzyme activity. It remains to be determined if this difference in enzymatic inactivation of physiological glucocorticoids is related to the pathophysiology of pre-eclampsia or is a compensatory response to poor trophoblast development. Further research is needed to ascertain the clinical significance of these observations. Conflict of interest All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. This work was supported by the Fetal Medicine Unit at St George’s Hospital, London, UK. Author contribution statement SM, JEC, AEM, GW and BT were involved in designing the study. SM executed the study with help and guidance from JEC, AEM, JLJ and GW. Data were analysed by SM, JEC and AEM. Manuscript was drafted by SM, JEC, AEM and JLJ. Critical discussion was undertaken by all authors. Acknowledgements The authors would like to thank all staff and patients, without whom, this study would not have been possible. References [1] van Beek JP, Guan H, Julan L, Yang K. Glucocorticoids stimulate the expression of 11 b hydroxysteroid dehydrogenase type 2 in cultured human placental trophoblast cells. J Clin Endocrinol Metab 2004;89:5614e21. [2] McCalla CO, Nacharaju VL, Muneyyirci-Delale O, Glasgow S, Feldman JG. Placental 11 b hydroxysteroid dehydrogenase activity in normotensive and pre-eclamptic pregnancies. Steroids 1998;63:511e5. [3] Hardy DB, Yang K. The expression of 11 b hydroxysteroid dehydrogenase type 2 is induced during trophoblast differentiation: effects of hypoxia. J Clin Endocrinol Metab 2002;87:3696e701.
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