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Original Article |
in Human Preeclamptic and Growth-Restricted Placentas
The Sir Quinton Hazel Research Centre for Molecular Medicine, Department of Biological Sciences (E.Ka., E.W.H., D.K.G.), University of Warwick, Coventry CV4 7AL, United Kingdom; and University of Crete, Division of Medicine, Department of Obstetrics and Gynecology (A.G., E.Ko.), Heraklion, Crete, Greece 71110
Address all correspondence and requests for reprints to: Dr. D. K. Grammatopoulos, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: dgrammatopoulos{at}bio.warwick.ac.uk.
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Abstract |
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In this study, we sought to investigate the expression of CRH-R1
levels in placentas from women who have undergone normal deliveries (control group) and patients who have been diagnosed as having preeclampsia or IUGR. Results showed that placental CRH-R1 mRNA levels (as shown by quantitative RT-PCR) and protein levels (shown by Western blotting analysis) were significantly (P < 0.05) reduced in all of the complicated pregnancies. In contrast, levels of the angiotensin II receptor were elevated in preeclampsia and reduced in IUGR subjects, as shown by RT-PCR and Western blotting analysis. These findings might suggest that changes in receptor expression may contribute toward dysregulation of the dynamic balance controlling vascular resistance.
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Introduction |
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Similarly, intrauterine growth retardation (IUGR) is an abnormality of fetal growth and development that affects 3–7% of all deliveries, depending on the diagnostic criteria used (3). The growth-retarded fetus is at greater risk for mortality and morbidity (3). There are several causes of IUGR that may be divided into three main categories: maternal, fetal, and uteroplacental. It should be stressed, however, that in almost half of the cases of IUGR, the etiology is unknown.
One of the hormones that plays a major role controlling mechanisms for the maintenance of pregnancy is the CRH. Although the exact functions of placental CRH are uncertain, it has been demonstrated that CRH plasma levels are significantly higher in women delivered preterm, and significantly lower in women delivered post term, than in women delivered at term (4). These differences were evident as early as 10 wk of gestation. It seems, therefore, that the patterns of plasma CRH that are associated with aberrant timing of delivery are established by the end of the first trimester and persist throughout gestation. It has been proposed that this process is analogous to a placental clock that triggers the onset of parturition after a predetermined length of gestation, and that the maternal plasma CRH is an indicator of the rate of progress toward this event (4). Moreover, CRH participates in the maintenance of vascular tone by activating a guanylate cyclase/nitric oxide pathway in the human placenta (5).
Maternal plasma CRH concentrations are significantly elevated in complicated pregnancies (6, 7, 8, 9, 10). The mean umbilical cord plasma CRH in preeclamptic pregnancies was significantly higher (3-fold) than that from normotensive pregnancies (11). This was also reflected by maternal plasma CRH levels that were significantly higher (3-fold) in hypertensive women than those with uncomplicated pregnancies. Similarly, the mean umbilical cord plasma CRH level in the growth-retarded fetuses was higher than that in the normally grown fetuses matched for gestational age, presence or absence of labor, and mode of delivery (11). Moreover, there is differential processing of the pro-CRH in normal and preeclamptic pregnancies (12). Such impaired dishabituation has immediate implications for fetal neurological development (13). Moreover, further in adult life, fetuses that were born prematurely or growth retarded can suffer from diabetes, high blood pressure, and cardiovascular diseases.
CRH exerts its actions by binding to two different families of CRH receptors (CRH-Rs), termed: CRH-R1 and CRH-R2 (14, 15). In the past few years, we have elucidated the expression of CRH-R subtypes and their signaling characteristics in fetomaternal tissues such as human placenta, myometrium, and fetal membranes (16, 17, 18).
The purpose of this study was to investigate and compare expression levels of mRNA and protein of CRH-R1 in placentas from women with preeclampsia or IUGR.
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Subjects and Methods |
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Placental biopsies were obtained from women undergoing elective cesareans (n = 6, control group), deliveries due to IUGR (n = 6), or preeclampsia (n = 7). Table 1
summarizes patient demographic data. None of the IUGR patients were diagnosed with preeclampsia. All 19 patients were nulliparous, undergoing elective cesareans, and were of Caucasian origin.
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For IUGR samples, growth-restricted fetuses were prospectively identified, using ultrasound biometry, and diagnosed by growth being less than the third centile or if the fetus had crossed a centile, plus the absence of umbilical artery end diastolic blood flow. IUGR women had systolic blood pressure of 123 ± 11 mm Hg and diastolic of 70 ± 10 mm Hg. There was no significant difference between IUGR and controls, whereas preeclamptic women had significantly (P < 0.05) higher blood pressure than that of IUGR women.
There was no significant difference in age and gestational age at delivery in the three groups. Birth weight was significantly lower in the IUGR group compared with control (P < 0.01), and IUGR compared with preeclampsia (P < 0.05), but was not significantly different in preeclampsia and control pregnancies. Placental weight was significantly lower in IUGR, compared with control (P < 0.01) and preeclamptic placental weight (P < 0.05), but was not significantly different in preeclampsia and control pregnancies.
Immediately after delivery, the maternal and fetal surfaces of the placenta were dissected off, and fetal membranes were peeled away from the placenta. Placental samples were washed in PBS and immediately snap-frozen in liquid nitrogen. Informed consent was obtained from each woman, and ethical approval from the local ethical authority was granted.
Total RNA preparation and cDNA synthesis
Total RNA was prepared from individual samples using the RNeasy Total RNA Kit (QIAGEN, Crawley, UK) according to the manufacturer’s guidelines. First-strand cDNA synthesis was performed using RNase Reverse Transcriptase (Life Technologies, Inc., Paisley, UK).
Real-time RT-PCR
Quantitative PCR was performed on a Light Cycler system (Roche Molecular Biochemicals, Mannheim, Germany). PCR reactions were carried out in a reaction mixture consisting of 5.0 µl reaction buffer and 2.0 mM MgCl2 (Biogene, Kimbolton, UK), 1.0 µl of each primer (1 ng/µl), 2.5 µl cDNA, and 0.5 µl Light Cycler DNA Master SYBR Green I (Roche Molecular Biochemicals).
Protocol conditions consisted of denaturation at 95 C for 15 sec; followed by 40 cycles at 94 C for 1 sec, 58 C for 12 sec, and 72 C for 15 sec; followed by melting curve analysis. For analysis, quantitative amounts of CRH-R1 gene expression were standardized against the housekeeping gene ß-actin. Quantitative data analysis was made possible through the use of CRH-R1 RNA from serially diluted placental cDNA, using 1-, 10-, 100-, and 1000-fold dilutions. Similarly, angiotensin II receptor (AT1-R) mRNA levels were assessed in the same samples. Ten microliters of the reaction mixture were subsequently electrophoresed on a 1.6% agarose gel and visualized by ethidium bromide, using a 1-kb DNA ladder (Life Technologies, Inc.) to estimate the band sizes. As a negative control for all the reactions, distilled water was used in place of the cDNA.
The primers used were: for CRH-R1/ß, sense 5'-GGCAGCTAGTGGTTCGGCC-3', antisense 5'-TCGCAGGCACCGATGCTC-3'; for CRH-R1c, sense 5'-ACATCTCAGACAATGGCTAG-3', antisense 5'-TCGCAGGCACCGATGCTC-3'; for ß-actin, sense 5'-AAGAGAGGCATCCTCACCCT-3', antisense 5'-TACATGGCTGGGGTGTTGAA-3'; and for AT1-R, sense 5'-ACAGCTTGGTGGTGATAGTC-3', antisense 5'-CACACTAGCGTACAGGTTGA-3'.
Sequence analysis
The PCR products from the placental samples were purified from the 1.6% agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). PCR products were then sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Centre for Biotechnology Information, confirming the identity of our products.
Preparation of placental proteins
Tissues were weighed and homogenized in 6 ml Dulbecco’s PBS containing 10 mM MgCl2, 2 mM EGTA, 0.15% BSA (wt/vol), 0.15 mM bacitracin, and 1 mM phenylmethylsulfonylfluoride (pH 7.2; extraction buffer) at 22 C for 40 sec. The homogenate was centrifuged at 3,000 rpm for 30 min at 4 C. The resultant pellet was washed, resuspended in extraction buffer, and spun at 19,000 rpm for a further 60 min at 4 C. The final pellet was resuspended in extraction buffer using homogenizer for 20 sec.
Western blotting
Placental proteins (100 µg) were centrifuged at 13,000 rpm for 15 min at 4 C. The supernatant was then discarded, and the resultant pellets were solubilized with Laemmli buffer [5 M urea, 0.17 M SDS, 0.4 M dithiothreitol, and 50 mM Tris-HCl (pH 8.0)], mixed and placed in a boiling water bath for 5 min and allowed to cool at room temperature.
Samples were separated on an SDS-10% polyacrylamide gel, and the proteins were electrophoretically transferred to a nitrocellulose filter at 250 mA for 16–18 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. The filter was then blocked in PBS containing 0.1% Tween-20 and 5% dried milk powder (wt/vol) for 2 h at room temperature. After three washes with PBS-0.1% Tween, the nitrocellulose filters were incubated with primary antibody for the CRH-R1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The primary antiserum was used at a 1:1000 dilution in PBS-0.1% Tween for 1 h at room temperature. As a negative control, the primary antibody was preabsorbed with synthetic receptor peptide (1 µM, Santa Cruz Biotechnology, Inc.). The filters were washed thoroughly for 30 min with PBS-0.1% Tween before incubation with the secondary antirabbit horseradish peroxidase-conjugated Ig (1:2000) for 1 h at room temperature and further washing for 30 min with PBS-0.1% Tween. Antibody complexes were visualized as previously described (19). Nitrocellulose membranes were then stripped by serial washes in H2O for 10 min, NaOH (0.2 M for 5 min), and H2O for another 10 min then reprobed using AT1-R and ß-actin antibodies.
CRH RIA
Blood was collected in tubes containing ethylenedianetetraacetic acid (1.5 mg/ml), chilled immediately, and centrifuged within 8 h of sampling at 4 C. Plasma was frozen at -20 C and thawed only once. CRH was extracted from plasma with Sep Pak C-18 cartridges (Waters Corp., Milford, MA), and CRH concentrations were determined by RIA using an in-house CRH antibody (20). Radiolabeled CRH and CRH standards obtained from Peninsula Laboratories, Inc. (San Carlos, CA). The investigators responsible for the RIA were blinded of the patient classification.
Statistical analysis
Data are shown as the mean ± SEM of each measurement. Statistical ANOVA was performed, measuring the intensity of immunoreactive staining using a scanning densitometer (Scion Image, Scion Corp., Frederick, MD). Student’s t test was employed to calculate the significance of differences in the means between the different groups. A P-value < 0.05 was considered to be significant.
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Results |
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Serial dilutions of CRH-R1 provided the template on which a line of best fit was plotted and used as a standard curve, to demonstrate accuracy and reproducibility of analysis. Melting curve analysis of the PCR products is shown as fluorescence over time, against temperature. The melting curve analysis showed a single melting maximum of 89.70 C for the CRH-R1 gene (Fig. 1A), a single melting maximum of 91.80 C for the human AT1-R gene (Fig. 1B), and a single melting maximum of 90.30 C for the ß-actin gene (Fig. 1C), thus confirming product specificity. The CRH-R1 melting peak corresponded to a 271-bp fragment (Fig. 1D); the AT1-R peak, to a 212-bp fragment (Fig. 1E); and the ß-actin, to a 216-bp fragment (Fig. 1F).
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Using this analysis, and correcting over the ß-actin gene expression, it was shown that preeclamptic or IUGR placentas expressed significantly (P < 0.05) less amount of the CRH-R1 gene at mRNA level (Fig. 2A).
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). These differences were also visible when the PCR products were resolved on a 1.6% agarose gel.
Using real-time PCR and the specific primers for CRH-R1c, we were not able to detect this particular receptor variant in any of the cDNAs. This might be attributable to very low copies of the gene present in the human placenta. To test this hypothesis, we applied nested PCR with primers spanning the entire length of CRH-R1 (16). After 70 cycles of amplification, we were able to detect (as a faint band) CRH-R1c, indicating that the gene is expressed in very low copies (Fig. 3A). In addition, the primers used for the amplification of CRH-R1 could also amplify CRH-R1ß. The fact that an additional band was not detected in our preparations suggests that this receptor variant is not present in the human placenta (Fig. 3B), thus confirming our previous observations (16).
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Immunoblotting analysis was used to determine the apparent molecular weight of the membrane-bound CRH-Rs in placental samples and compare protein expression in placentas obtained from normal and abnormal pregnancies. SDS-PAGE of membrane proteins was performed using an antibody raised against a peptide corresponding to amino acids 425–444 in the C terminus of the human CRH-R1/2 precursor. Given the homology of the region, this particular antibody cannot distinguish between the different receptor subtypes. Two bands with apparent molecular masses of 55 and 75 kDa were detected in preparations from all the different samples (Fig. 4B). Previous studies, using cross-linking experiments, have also shown that the placental CRH-Rs have molecular masses of 55 kDa (21) and 75 kDa (22); the higher molecular-mass band could be attributable to posttranslational modifications of the CRH-R1.
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).
Similarly, the anti-AT1-R antibody recognized two bands, of 60 and 45 kDa, that were both significantly (P < 0.05) increased in preeclampsia and reduced in IUGR (Fig. 4, A and D). To ensure homogenous protein content, the same placental membranes were subjected to Western blotting, using anti-ß-actin antibody (Fig. 4C), which showed no apparent difference in expression between the different placental homogenates (Fig. 4D).
RIA
CRH plasma levels were measured from all the patients who participated in the study. The mean maternal plasma CRH concentration in women with preeclampsia was significantly higher (1650 ± 390 pg/ml), when compared with normotensive women (660 ± 130 pg/ml) (P < 0.05). Similarly, levels of CRH in women diagnosed with IUGR were significantly (P < 0.05) higher (1525 ± 440 pg/ml), when compared with the control group. Comparison between the CRH levels of preeclamptic vs. IUGR women showed no apparent differences.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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mRNA and protein levels in placentas associated with abnormalities such as preeclampsia or IUGR. This data are of potential significance because they show inverse correlation between CRH and CRH-R levels.
We previously showed that the human placenta expresses two CRH-R subtypes (namely, CRH-R1 and CRH-R1c). We focused on the expression of CRH-R1 because it has been shown to couple readily to multiple G proteins (17, 19, 23) and transmit CRH actions across the placenta. On the contrary, the splice variant CRH-R1c, which has a 40-amino-acid deletion at the N terminus, is unable to bind CRH and stimulate effector molecules at low CRH concentrations (24). Using the sensitive Light Cycler real-time PCR, this particular transcript has not been detected. It was, however, barely detectable only upon using nested PCR, a finding arguing against any functional role for this receptor variant in the human placenta. It seems, therefore, that the predominant subtype is the CRH-R1, because the other variant CRH-R1ß is not present in the placenta. Another study (25) also has shown that CRH-R1ß is not present in the human placenta and fetal membranes. In addition, it has been reported that the CRH-R2ß subtype is expressed in human placentas (22). However, using RT-PCR, we were unable to detect any CRH-R2 subtypes in our tissue preparations (data not shown).
In this study, we have used a method that is based on real-time analysis of PCR amplification and that has several advantages over the conventional RT-PCR-based quantitative methods, as well as Northern blotting analysis. Quantification with the Light Cycler greatly amplifies the process and makes CRH-R quantitation much more precise and reproducible.
CRH seems to exert a number of effects in intrauterine tissues during pregnancy and labor. It has been shown to stimulate the production of ACTH, in a dose-dependent manner, from primary cultures of human placental cells (26). Several reports indicate that CRH stimulates prostaglandin production in placenta, amnion, chorion, and decidua in vitro (27). CRH has also been reported to increase the release of immunoreactive oxytocin from placental cells (28). Moreover, CRH is a potent vasodilator of the human fetoplacental circulation and may act as a local regulator of placental vascular tone, acting via a nitric oxide/cGMP-mediated pathway (5). Interestingly, the vasodilatory response of CRH seems to be impaired in placentas from women with increased vascular resistance (5). Because the signal transduction machinery seems to be intact in preeclamptic pregnancies (29, 30, 31), the dampening of CRH-induced vasodilation could be attributable to the loss of CRH-Rs in placentas from preeclamptic or growth-restricted pregnancies.
Pregnancy-induced hypertension is associated with altered vascular responsiveness, and one of the contributing factors seems to be angiotensin acting via its receptors, the expression of which is altered in abnormal pregnancies (32, 33). In our study, we have shown that AT1-R levels are increased in preeclamptic placentas, both at mRNA and protein levels, which is in agreement with recently published findings (33). Regulation of AT1-R expression seems to be central to the altered intracellular mechanisms found in preeclamptic placenta. Very recently, it has been shown that in preeclampsia, there is a significant increase in heterodimerization of the AT1-R and the bradykinin B2 (B2) receptor, which enhances angiotensin II responsiveness (34).
Taking these findings together, it is possible that in normal placenta, there is a balance between the vasodilatory action of CRH and the opposing actions of vasopressor agents such as angiotensin II. The net result will be a maintenance of a normal vascular resistance to blood flow. However, in preeclampsia, up-regulation of AT1-receptors and formation of heterodimers with B2-receptors alter this balance and potentiate further the vasoconstriction caused by angiotensin II. Interestingly, it has been shown that angiotensin II induces secretion of placental CRH, which, in turn, might down-regulate its own receptors, resulting in a shift toward vasoconstriction (Fig. 5).
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gene expression seems attractive. The fact that maternal plasma CRH concentrations are further elevated in pregnancy-induced hypertension (9) makes this suggestion more plausible. Other studies have demonstrated that long-term CRH stimulation can down-regulate its own CRH-R1 receptor in the pituitary by decreasing mRNA levels (35). These effects were time- and dose-dependent. We have also shown that incubation with CRH (100 nM), over 16 h, caused a significant decrease in CRH-R1 mRNA levels in human myometrial cells (unpublished observations). Future studies are required to elucidate further whether CRH can exert similar effects in placental cell cultures. At the present, however, no direct comparison can be made, given that the expression and signaling characteristics of CRH-Rs in human myometrium differ from those of the placenta (17, 18, 36, 37). Alternatively, unidentified placental defects in preeclampsia might affect the expression of CRH-Rs, causing, as a result, an increase in CRH production as a counter regulatory mechanism to compensate for the reduced signaling efficiency.
In IUGR placentas, a different pattern of expression emerged for CRH-R1 and AT1-R. Both receptors’ levels were decreased, but no obvious conclusions can be drawn, because the etiology of IUGR is far more complicated; and in almost 50% of the cases, the etiology is unknown (38). In addition, in spite of the placenta’s role in intrauterine nutrition, there is no single placental abnormality that is common to all IUGR fetuses.
Both preeclampsia and IUGR are multifactorial diseases associated with abnormal placentation and further complications for the fetuses. In this present study, we have demonstrated a reduction of CRH-R1 levels in placentas from complicated pregnancies. Collectively, this variation in placental CRH-R expression, during normal and abnormal pregnancies, argues for an important role of CRH-Rs in human placental function that remains to be investigated further.
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Acknowledgments |
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Footnotes |
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Abbreviations: AT1-R, Angiotensin II receptor; B2, bradykinin B2; CRH-R, CRH receptor; SDS, sodium dodecyl sulfate; IUGR, intrauterine growth retardation.
Received March 11, 2002.
Accepted September 26, 2002.
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(CRH-1) and the CRH-C variant receptor. J Clin Endocrinol Metab 83:1376–1379 increase immunoreactive oxytocin release from cultured human placental cells. Placenta 17:307–311[CrossRef][Medline]
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