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Sir Quinton Hazell Molecular Medicine Research Centre (E.K., D.K.G.), Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Division of Clinical Sciences (M.V., D.K.G.), Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom; and The Leeds Institute of Health, Genetics and Therapeutics (E.W.H.), University of Leeds, Leeds LS2 9NL, United Kingdom
Address all correspondence and requests for reprints to: Dr. D. K. Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.
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Abstract |
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(CRH-R1) expression. In this study, we investigated the actions of CRH/CRH-related peptides on the NO/cGMP system in normal and PE placentas (n = 8 for each group). Fluorescent in situ hybridization, RT-PCR, and immunofluorescence experiments in human term placenta detected mRNAs expression for both R1 and R2 types of CRH-R, as well as urocortin (UCN) II and UCN III and showed CRH-R protein expression mainly in syncytiotrophoblast, whereas the endothelial NO synthase (eNOS) expression was confined within the cytoplasm of the chorionic villi. In placental explants, CRH and UCN induced mRNA and protein expression of eNOS, but not inducible NOS, and also caused an acute increase in cGMP levels (maximal stimulation, 80–90% above basal; P < 0.05). UCN II also induced a modest induction of cGMP (42% above basal; P < 0.05). These responses were attenuated by the NOS and soluble guanylyl cyclase inhibitors, L-NG-nitro-L-arginine methyl ester and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. In PE placental explants there was a significant reduction in CRH/CRH-related peptide-induced cGMP response; however, changes in the mRNA content of eNOS, inducible NOS, and soluble guanylyl cyclase (assessed by quantitative RT-PCR) between normal and PE placentas were not altered.
In conclusion, we demonstrated that CRH and CRH-related peptides can positively regulate the placental NO/cGMP system. This pathway appears to be impaired in PE and may contribute toward dysregulation of the balance controlling vascular resistance.
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Introduction |
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One of the signaling pathways mediating CRH actions during pregnancy appears to be the nitric oxide (NO)/cGMP pathway (2). In the human pregnant myometrium, CRH modulates the NO/cGMP via a complex pathway involving a short-term effect predominantly mediated by protein kinase A, leading to acute stimulation of membrane-bound guanylyl cyclase (mGC) and a long-term effect increasing constitutive, but not inducible, NO synthase (NOS) expression, mediated by a protein kinase A-independent mechanism. Other studies have shown similar interactions in other intrauterine tissues, including the human placenta, where CRH can induce vasodilation in a paired artery/vein placental system, by activating the NO/cGMP pathway (3). These mechanisms may have important pathophysiological consequences because in pregnancies complicated by preeclampsia (PE) the plasma levels of CRH are elevated (4), together with a concomitant reduction in CRH type 1 receptor (CRH-R1) expression (5). The net effect of these changes could lead to abnormal vascular resistance and the clinical sequelae of PE.
PE remains a leading cause of maternal and perinatal morbidity and mortality (6). The disease is characterized by maternal hypertension, proteinuria, and endothelial dysfunction, as well as fetal growth restriction, and may rapidly progress to eclampsia resulting in maternal cerebral hemorrhage, liver rupture, pulmonary edema, renal failure, and disseminated intravascular coagulopathy (7). A recent Confidential Enquiry into Stillbirths and Deaths in Infancy report cites one in six stillbirths and one in six sudden infant deaths as occurring in pregnancies complicated by maternal hypertension (8). There is accumulating evidence for a pathogenic model of PE, whereby deficiencies in trophoblast invasion of placental bed spiral arteries result in a poorly perfused fetoplacental unit. This deficient placental perfusion, and consequent aberrant tissue oxygenation, results in secretion of factor(s) by the placenta into the maternal circulation that causes "activation" of the vascular endothelium, with the clinical syndrome resulting from widespread changes in endothelial cell function in both small and large vessels leading to increased sensitivity to vasopressor agents (9).
The observation that NOS inhibition can stimulate a PE-like syndrome in rats (10) suggests that abnormalities of the pathways regulating placental NOS expression and/or activity might be important in the pathogenesis of PE. Interestingly, there is conflicting evidence about the level of placental NOS expression in preeclamptic compared with normal patients (11, 12, 13).
In this study, we sought to investigate the regulation of placental NO/cGMP-dependent pathways by CRH and CRH-related peptides by assessing the effects of these peptides on placental NOS and cGMP expression.
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Subjects and Methods |
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Placental biopsies were obtained from women either undergoing normal uncomplicated elective cesarean sections at term (n = 8, normal group) or due to PE (n = 8). All subjects studied were matched for stage of gestation and age; additional patient details are summarized in Table 1
. Patients with PE were defined by hypertension (systolic blood pressure greater than 140 mm Hg, together with either a single diastolic blood pressure reading of at least 110 mm Hg or consecutive readings of at least 90 mm Hg on more than one occasion at least 4 h apart) and proteinuria (>300 mg/24 h or >2+ of proteinuria on two clean-catch urine specimens at least 6 h apart), after wk 20 of pregnancy. All patients were normotensive at booking. Patients with chronic hypertension, renal disease, or multiple pregnancies were not included in our study. Of the eight uncomplicated elective cesarean sections at term, two were for fetal breech presentation, and six were following maternal request. Of the eight elective cesarean sections at term due to PE, three were for worsening of maternal symptoms, four for fetal distress, and one for fetal breech presentation. 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 was granted from the local ethical authority.
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Total RNA was extracted by using ULTRASPEC (Biotecx Laboratories, Houston, TX), according to the manufacturer’s instructions. RNA concentration was determined by spectrophotometric analysis and agarose gel electrophoresis. A set concentration of RNA (500 ng) was reverse-transcribed into cDNA by using 5 IU/µl RNase H reverse transcriptase (Invitrogen, Paisley, UK).
PCR
All PCRs were carried out using Taq DNA polymerase (Life Technologies, Paisley, UK) with 200 ng of cDNA for each amplification. Placental cDNAs were amplified at 94 C (45 sec), 58 C (45 sec), and 72 C (1 min) in a total of 30 cycles with a final extension step at 72 C for 10 min. The set of primers for the amplification of urocortin (UCN) II, CRH-R2 and ß-actin are shown in Table 2. Ten microliters of the reaction mixture were subsequently electrophoresed on a 1.8% agarose gel and visualized by ethidium bromide, using a 1-kb DNA ladder (Life Technologies) to estimate the band sizes. As a negative control for all of the reactions, distilled water was used in place of the cDNA. The resultant PCR products were sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information.
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Quantitative PCR was performed on a Roche Light Cycler system (Roche Molecular Biochemicals, Manheim, Germany). PCRs 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).
Protocol conditions consisted of denaturation of 95 C for 15 sec, followed by 40 cycles of 94 C for 1 sec, 58 C for 5 sec, and 72 C for 12 sec, followed by melting curve analysis. For analysis, quantitative amounts of genes of interest (Table 2
) were standardized against the house-keeping gene ß-actin. As a negative control for all the reactions, preparations lacking RNA or reverse transcriptase were used in place of the cDNA. RNAs were assayed from two to three independent biological replicates. The RNA levels were expressed as a ratio, using "delta-delta method" for comparing relative expression results between treatments in real-time PCR (14).
The resultant PCR products were directly sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information, thus confirming gene identity.
Fluorescent in situ hybridization (FISH)
Paraffin-embedded sections of human placentas were dewaxed and dehydrated by successive washes through ethanol and air-dried. Specific 40-mer synthetic oligonucleotide probes for UCN II, UCN III, and CRH-R2 with fluorescein conjugated at their 5'-ends were used in this study. Hybridization solution (100 µl) containing 1 ng/µl of the probe was allowed to hybridize at 37 C overnight. Slides were then placed in preheated (45 C) 2x standard saline citrate buffer, in which they were washed twice, followed by another 10-min immersion in 0.1x standard saline citrate (45 C). The tissue sections were rinsed with PBS, and the cell nuclei were visualized by applying the DNA-specific dye 4,6-diamido-2-phenylindole (DAPI) at a final concentration of 1 µg/ml.
Western blotting analysis
Placental membranes (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, 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 20, the nitrocellulose filters were incubated with primary antibodies for CRH-R2 (Santa Cruz Biotechnology, Santa Cruz, CA), endothelial NOS (eNOS), inducible NOS (iNOS) (Calbiochem-Novabiochem Corp., San Diego, CA), or soluble guanylyl cyclase (sGC) (Cayman Chemical, Ann Arbor, MI). The primary antiserum was used at a 1:1000 dilution in PBS-0.1% Tween 20 for 1 h at room temperature. The filters were washed thoroughly for 30 min with PBS-0.1% Tween 20, before incubation with the secondary antirabbit horseradish peroxide-conjugated Ig (1:2000) for 1 h at room temperature and further washing for 30 min with PBS-0.1% Tween 20. Antibody complexes were visualized as previously described (5). 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, and reprobed using ß-actin antibodies to confirm equal protein loading.
Immunofluorescence
Paraffin-embedded sections of human placentas were dewaxed and dehydrated by successive washes through ethanol and were washed in PBS and distilled water before blocking for 30 min with 3% BSA in PBS. The primary rabbit polyclonal eNOS, CRH-R1/2 antibodies (Santa Cruz Biotechnology; Phoenix Peptides, Belmont, CA), were used at a 1/100 dilution. All dilutions were made in 1% BSA in PBS. Specimens were incubated with primary antibody for 60 min and were then washed three times with PBS 5 min each, before incubation with antirabbit IgG-fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology) for 45 min. The tissue sections were thoroughly rinsed with PBS, and the cell nuclei were visualized by applying the DNA-specific dye DAPI at a final concentration of 1 µg/ml.
Measurement of cGMP accumulation in tissue explants
Human normal (n = 4) and PE (n = 4) placental explants were teased into small pieces, carefully removing any obvious blood vessels or clots. The tissues were rewashed several times with PBS and placed afterward in sterile centrifuge tubes with 1.5 ml inositol-free DMEM:Ham’s F12 nutrient mixture (1:1) containing 1% BSA, 50 U/ml penicillin, and 50 µg/ml streptomycin for 2 h at 37 C in a 5% CO2 incubator. After initial incubation, explants were treated with various concentrations of CRH and UCN for 5 min in the presence or absence of either L-NG-nitro-L-arginine methyl ester (L-NAME) (eNOS inhibitor, 100 nM) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (the sGC inhibitor, 100 nM).
Immediately after treatments, explants were snap-frozen in liquid nitrogen, and cGMP was measured using RIA (Amersham Biosciences, Little Chalfont, UK), according to the manufacturer’s instructions. Standard cGMP concentrations, covering the range 2–128 fmol/ml, were used for determination of the standard curve of the RIA.
Statistical analysis
Data are shown as the mean ± SD of each measurement. For the real-time PCR measurements, results were evaluated between groups by using two-tailed Student’s t test, with significance determined at the level of P < 0.05. For the second messenger measurements, a one-way ANOVA was used, followed by Dunnett’s test, to compare each treatment dose.
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Results |
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Using RT-PCR and specific primers, common for all the different CRH-R2 subtypes, a specific DNA fragment of 126 bp was amplified from human normal placenta cDNA (Fig. 1A). In addition, expression of UCN II and UCN III mRNA was evaluated by RT-PCR using cDNA from human placenta and term myometrium (positive control). Results showed that both UCN II and UCN III mRNA are expressed in term human placenta (Fig. 1A). The nucleic acid sequence of the fragments confirmed the identity of all of the PCR products. FISH was used to localize the cellular distribution of these peptides; results showed that the mRNA for both peptides is expressed around the syncytiotrophoblast with very little expression within the structure of the villi (Fig. 1B). Similar experiments were carried out to localize the cellular distribution of the CRH-R2 mRNA by using specific oligonucleotide probes. Results showed intense cytoplasmic staining of the CRH-R2 mRNA detectable in syncytiotrophoblast of chorionic villi and around placenta microvasculature (Fig. 1B). No specific staining was detected in both tissues when a sense oligonucleotide probe was used. Immunofluorescent analysis using specific antibodies indicated that both peptides are also expressed at protein level. Syncytiotrophoblast from secondary and tertiary villi are the primary cell type where an intense signal for both receptors was detected. A less intense signal was also noted around the blood vessels within trophoblast villi (Fig. 1C).
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). Similar experiments, using an eNOS-specific antibody, showed that eNOS expression is widespread within the cytoplasm of the chorionic villi and that there is colocalization of CRH-R with eNOS expression (Fig. 2). No specific staining was detectable in tissues where the primary antibody had not been added. The DNA-specific dye DAPI was used in these experiments for the identification of individual cells in both tissues.
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The effect of CRH and UCN on placental NOS isoforms (eNOS and iNOS) and sGC expression was assessed using an in vitro human placental explant system. Initially we measured mRNA expression in cultured placental explants and in snap-frozen biopsies from the same placenta using quantitative RT-PCR. Results revealed that there is no apparent change in the mRNA expression of these proteins even after 1 d in culture (data not shown) confirming the suitability and the functional integrity of the in vitro system.
Treatment of normal placental explants with CRH or UCN induced a 2- to 2.5-fold increase in eNOS mRNA, but not in iNOS or sGC (Fig. 3, A and B). This effect was dose dependent, with maximal activation at 100 nM, and was apparent 30 min after treatment, with a maximum effect at 2.5 h, returning to basal levels at 4 h (data not shown). CRH or UCN had no effect on either iNOS or sGC at higher concentrations or after prolonged treatment. In contrast, treatment of PE placental explants with CRH or UCN failed to induce eNOS mRNA expression levels (Fig. 3, A and B). To assess whether CRH-induced up-regulation of normal placental eNOS, mRNA was associated with increased eNOS protein expression, placental explants were treated with CRH (100 nM) for 2 h, and the eNOS protein expression was determined by immunoblotting analysis. Results showed that CRH treatment significantly increased (by 60–90%; P < 0.001) expression of a placental protein with a molecular mass of 140 kDa recognized by a specific eNOS protein (Fig. 4), thus confirming that induction of eNOS mRNA levels was translated into increased protein expression. In contrast, CRH treatment had no effect on iNOS or sGC expression (Fig. 4), in agreement with our quantitative RT-PCR data. Similar results were obtained with UCN (data not shown).
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We further assessed the interactions between CRH and the NO pathway by treating normal placental explants with CRH or CRH-like peptides and determining the cGMP response. CRH and UCN caused a significant (P < 0.001) increase in cGMP production (90 ± 5% above basal), with no apparent difference in the potency of the two peptides. The cGMP response was rapid (within 5 min) and dose dependent, with a threshold of 1 nM and a maximum response at 100 nM (Fig. 5A).
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), suggesting that the cGMP response primarily requires placental NOS and sGC activation.
To evaluate the relative contribution of the type 2 CRH-R in mediating NOS activation and cGMP production, we tested the ability of the specific CRH-R2 agonist, UCN II, to stimulate placental cGMP production. Treatment of placental explants from normal-term pregnancies with UCN II caused a modest increase in cGMP production (42% above basal) (Fig. 5B). This effect was rapid and dose dependent, reaching maximal activation at 100 nM, and was abolished when placental explants were pretreated with either L-NAME or ODQ (data not shown).
The capacity of CRH and CRH-like peptides to induce cGMP production in placental explants from PE pregnancies was also evaluated. CRH- and UCN-induced cGMP response was significantly reduced in PE placental explants, compared with normal (70 ± 6% reduction in maximal cGMP response) (Fig. 5B), and just reached statistical significance above basal levels (25 ± 10% above basal). Interestingly, when PE placental explants were treated with UCN II, there was no apparent activation of cGMP (Fig. 5B).
To investigate this phenomenon further, we assessed the relative levels of mRNA expression of molecules involved in the cGMP signaling, namely eNOS, iNOS, sGC, and mGC, in both normal and PE placentas using quantitative RT-PCR. We also determined the mRNA level of CRH-R2 in both normal and PE placentas. Results showed no difference in the mRNA content of iNOS and sGC and a slight increase, just failing to reach significance, in eNOS in PE placentas when compared with normal (Fig. 6A). Interestingly, a significant down-regulation (50 ± 20%) of CRH-R2 and a significant increase (120 ± 30%) of mGC mRNA were found in PE placentas (Fig. 6A).
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To compare the relative CRH-R2 protein expression in placentas obtained from normal and PE pregnancies, SDS-PAGE was carried out, followed by immunoblotting analysis of proteins using an antibody raised against a peptide mapping near the C terminus of the human CRH-R2 protein. A band with apparent molecular mass of 55 kDa was detected in preparations from all the different samples (Fig. 6B). The specificity of the response was confirmed by preincubation of the CRH-R2 antibody with the blocking peptide. Using densitometric analysis, it was shown that there was a significant decrease (55%; P < 0.01) in the expression of immunocomplexes in the samples corresponding to preeclamptic patients (Fig. 6B). To ensure homogeneous protein content, the same placental membranes were subjected to immunoblotting using anti-ß-actin antibody, which showed no apparent difference in expression between the different placental homogenates (Fig. 6B).
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Discussion |
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Previous studies have identified both types of CRH-R in human placenta (17, 18). In our study, CRH-R staining appears almost exclusively to be membrane-distributed, mainly in syncytiotrophoblast and some around vessels. Most importantly, the CRH-Rs appear to colocalize with eNOS protein, signifying the potential for paracrine or autocrine regulation of eNOS expression and activation by CRH and CRH-like peptides within the chorionic villi. Moreover, we previously showed that the human placenta expresses two CRH-R1 subtypes, namely CRH-R1 and CRH-R1c, a receptor splice variant which has a 40-amino-acid deletion at the N terminus and unknown function (19). The expression of CRH-R2ß subtype has also been demonstrated in human placentas (18). The current study confirms this finding by using RT-PCR and FISH with specific oligonucleotide primers that can amplify a DNA fragment common for all CRH-R2 subtypes. We have previously been unable to detect any CRH-R2 subtypes in placental tissue preparations (17); therefore, this data suggest that the R2ß is probably the specific CRH-R2 receptor isoform present in human placenta. In line with our previous findings (17), FISH studies using antisense RNA probes specific for CRH-R2 showed that significant levels of hybridization were evident in syncytiotrophoblast cells within the chorionic villi.
The placental CRH/CRH-R system has been associated with the pathological mechanisms leading to a poorly perfused fetoplacental unit, abnormal vascular resistance and PE. Elevated CRH levels are found in pregnancies complicated with PE (8), and our previous studies have shown decreased CRH-R1 expression in preeclamptic placentas (9). In this study, we demonstrate for the first time a dramatic reduction in the expression of CRH-R2 mRNA and protein levels in placentas associated with PE, raising the possibility that a common PE-related mechanism is involved in the down-regulation of both types of CRH-R. The signaling pathways regulating CRH-R expression in the placenta or other peripheral tissues have not yet been identified; however, it is likely that these are tissue specific. Studies have demonstrated that long-term CRH stimulation can down-regulate its own CRH-R1 receptor in the pituitary by decreasing mRNA levels (20), whereas CRH can positively regulate the promoter activity of CRH-R1 when transfected in human myometrial cells, leading to increased mRNA expression (21), Most importantly, the decrease of CRH-R levels in preeclamptic placentas appears to have significant functional consequences, such as the dampening of the effect of CRH and CRH-like peptides on eNOS mRNA expression and NOS activation and cGMP production. This appears to be specific for the CRH-R because we failed to detect any alterations in the mRNA expression of other molecules involved in placental NO/cGMP signaling pathways, such as sGC, iNOS, and eNOS, in agreement with previous published data (22, 23). Given that NOS-inhibition can induce a PE-like syndrome (10), the placental CRH-R down-regulation and associated inability of CRH and CRH-like peptides to stimulate the NO/cGMP pathway might contribute to the abnormalities of pathways regulating placental NOS expression and/or activity and the pathogenesis of PE. Interestingly these studies demonstrated elevated mGC mRNA levels in preeclamptic placentas. This finding in association with the previously observed increase in plasma atrial natriuretic peptide concentrations found in PE (24) raises the possibility that hyperactivity of the atrial natriuretic peptide/mGC pathway might contribute to abnormal vascular resistance and PE. Although the CRH effects on placental cGMP stimulation appear to be dependent exclusively on the NOS/sGC system, the presence of a functional interaction between CRH/CRH-R and placental mGC is also possible.
In conclusion, in normal placenta CRH and CRH-like peptides can stimulate eNOS mRNA expression and NOS activity and cGMP production, pathways that potentially mediate the vasodilatory action of CRH and UCN. However, in PE, down-regulation of both CRH-R1 and -R2 leads to diminished regulation of the NO/cGMP pathway, which might result in a disturbance of the balance controlling vascular tone toward vasoconstriction.
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Footnotes |
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First Published Online March 22, 2005
Abbreviations: CRH-R, CRH receptor; DAPI, 4,6-diamido-2-phenylindole; eNOS, endothelial NO synthase; FISH, fluorescent in situ hybridization; iNOS, inducible NO synthase; L-NAME, L-NG-nitro-L-arginine methyl ester; mGC, membrane-bound guanylyl cyclase; NO, nitric oxide; NOS, NO synthase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PE, preeclampsia; sGC, soluble guanylyl cyclase; UCN, urocortin.
Received November 10, 2004.
Accepted March 11, 2005.
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in human preeclamptic and growth restricted placentas: inverse relationship to CRH circulating levels. J Clin Endocrinol Metab 88:363–370 (CRH-1) and the CRH-C variant receptor. J Clin Endocrinol Metab 83:1376–1379This article has been cited by other articles:
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  A. Imperatore, Wei Li, F. Petraglia, and J. R. G. Challis Urocortin 2 Stimulates Estradiol Secretion From Cultured Human Placental Cells: An Effect Mediated by the Type 2 Corticotrophin-releasing Hormone (CRH) Receptor Reproductive Sciences, June 1, 2009; 16(6): 551 - 558. [Abstract] [PDF]
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 E. W. Hillhouse and D. K. Grammatopoulos The Molecular Mechanisms Underlying the Regulation of the Biological Activity of Corticotropin-Releasing Hormone Receptors: Implications for Physiology and Pathophysiology Endocr. Rev., May 1, 2006; 27(3): 260 - 286. [Abstract] [Full Text] [PDF]
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A.-M. Bamberger, V. Minas, S. N. Kalantaridou, J. Radde, H. Sadeghian, T. Loning, I. Charalampopoulos, J. Brummer, C. Wagener, C. M. Bamberger, et al. Corticotropin-Releasing Hormone Modulates Human Trophoblast Invasion through Carcinoembryonic Antigen-Related Cell Adhesion Molecule-1 Regulation Am. J. Pathol., January 1, 2006; 168(1): 141 - 150. [Abstract] [Full Text] [PDF]
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, P < 0.05 compared with normal placentas. Inset, Representative real-time RT-PCR measurements demonstrating significant increase in eNOS mRNA levels in normal placental explants upon treatment with or without CRH (100 nM). These differences in mRNA expression can be seen from the large difference in amplification efficiency as it is demonstrated by the delay in amplification (intercept cycle) of the control sample. NS, No supplement.
