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Pregnancy |
Departments of Obstetrics and Gynecology3
Biochemistry,4 New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cortisol, glucocorticoid receptor, placenta, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transforming growth factor-ß (TGFß) comprises a cytokine family of three closely related peptides (TGFß1, ß2, ß3) that function through membrane receptors (types I and II) [11]. Although the human placenta expresses TGFß, the cellular distribution of the three peptides and their potential function remains the subject of debate [12–14]. TGFß type I and II receptors were also observed to be expressed by human placenta [15–17]. Early studies showed that TGFß treatment stimulated the synthesis of ECM proteins, including FNs, collagens, and their cell-surface integrin receptors in several cell types [18–20]. Oncofetal FN (FFN) is a uniquely glycosylated form of FN that is expressed in fetal tissue, at uterine-placental and chorionic decidual interfaces, and in cancer cell lines [21, 22]. It is of note that TGFß enhanced the expression of FFN in cytotrophoblasts isolated from human term placentas [23, 24]. In light of the relative dearth of information concerning the regulation of ECM protein levels in PMCs, the purpose of the present study was to elucidate the interaction between TGFß and GC in the modulation of ECM protein expression in these cells. We have used FFN as a model ECM protein for our study based on previous reports by our group and others demonstrating its expression in PMCs [4, 25]. Our results indicate that GCs specifically augment the effects of TGFß on specific ECM proteins synthesized by PMCs. These results are important to our understanding of the regulation of the ECM in the human placenta and its potential impact on villous architecture in uncomplicated pregnancies and those associated with villous fibrosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue culture media and dexamethasone (DEX) were obtained from Sigma (St. Louis, MO). Bovine sera were obtained from Gemini Bio- Products (Calabasas, CA). Laboratory plasticware was obtained from Falcon, Becton-Dickinson Labware (Lincoln Park, NJ). ITS+, a mixture containing insulin, transferrin, and selenium, was purchased from Collaborative Research-Becton Dickinson (Bedford, MA). 32P-dCTP was from New England Nuclear (Boston, MA). Plasmids containing cDNAs to fibronectin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from the American Type Culture Collection (Rockville, MD). Other reagents used in tissue culture, ELISA, and Northern blotting procedures were from previously described sources [25, 26].
Cell Culture
Human placental tissue (n = 6) was obtained from uncomplicated pregnancies with normally grown, singleton fetus delivered by Cesarean section at term. A focus of the current study was the examination of GC effects on ECM protein expression. Therefore, tissues were not taken from women in labor because labor is associated with a marked rise in levels of GC in maternal and fetal sera and amniotic fluid [27, 28]. In addition, placentas were also not obtained from pregnancies characterized by toxemia, IUGR, preeclampsia, diabetes, abruptio placentae, or other pathologies. The experimental protocols and informed consent from patients was approved by the Institutional Review Board Committee at New York University School of Medicine.
Placental mesenchymal cells (PMCs) were isolated based on our previous method [8, 25], which is a modification of procedures originally described by Fant and Nanu [29]. Five to 10 g of villous tissue were washed with saline and digested for 45 min in a 1:1 mixture of phenol red-free Ham F12-Dulbecco Modified Eagle medium (basal medium) containing 0.1% collagenase and 0.01% DNase. Dispersed cells were filtered through a 160-µm stainless-steel sieve and centrifuged (500 x g, 5 min). Cells were then resuspended in basal medium supplemented with 10% charcoal-stripped calf serum and ITS+ (i.e., SCS medium) and seeded in two T-75 culture flasks. ITS+ supplementation yields a final concentration of insulin of 6.25 µg/ml; transferrin, 6.25 µg/ml; selenious acid, 6.25 ng/ ml; bovine serum albumin, 1.25 mg/ml; and linoleic acid, 5.35 g/ml. For experiments, PMCs between passage 3 and 10 were plated in SCS medium at a density of 5 x 104 cells/well in a 24-well dish for ELISA studies and 1 x 106 cells per 25 cm2 flask for Northern blot and real-time polymerase chain reaction (PCR) analysis. After 3 days, at approximately 75% confluency, the cells were washed twice with PBS and serum-free medium (SCS medium without serum) was added with and without the indicated concentration of DEX and TGFß. We noted that cells remained firmly attached to the substratum for at least 96 h in serum-free medium with and without 100 nM DEX and 1 ng/ml TGFß. In two independent experiments, cell viability as judged by trypan blue exclusion following trypsinization, was 
90% for cells maintained for 48 or 96 h in serum-free medium with and without DEX and TGFß. In addition, no effect of treatment on cell number was noted. After the specified time in culture, conditioned media were saved for ELISA, and protein and RNA were extracted from adherent cells.
Cytotrophoblasts (CTs) were isolated from approximately 45 g of human villous tissue at term following trypsin digestion and centrifugation on Percoll gradients. We have previously employed this procedure [24, 25, 30], originally described by Kliman et al. [31] and modified by Douglas and King [32], to obtain CTs with purities of 95%. Initially, tissue fragments were chopped, washed with saline, and digestions with DNase and trypsin were carried out [30]. The digestate was poured through cheesecloth and two wire-mesh sieves with 0.0038 and 0.0021 inch openings and were centrifuged (500 x g, 5 min). Pellets were resuspended for centrifugation on a continuous Percoll gradient [30]. CTs, sedimenting as a ring of cells at a density of approximately 1.05 g/ml, were washed and then treated with immunomagnetic microspheres conjugated to mouse anti- human CD-45 MAb to remove immune cells [30]. CTs were then resuspended in SCS medium before plating. The yield was approximately 3 x 108 CTs/45 g of villous tissue. For experiments, CTs were plated in SCS medium at a density of 5 x 105 cells/well in a 24-well dish, and after 24 h, the cells were washed twice with PBS and serum-free medium was added with and without 100 nM DEX and 1 ng/ml TGFß. After 48 h, FFN content in culture media was determined by ELISA and normalized to cell protein. Results and statistical analysis are presented as a mean ± SEM from six culture wells obtained in a single experiment representing three identically conducted ones.
PMCs and CTs were maintained at 37°C in SCS medium in a humidified atmosphere of 5% CO2/95% air.
Dose Response Studies
PMCs were plated in 24-well dishes and were maintained for 48 h in serum-free medium with and without 0.01–100 nM DEX and 0.001–1 ng/ ml TGFß. These concentration ranges were chosen based on the observations that DEX and TGFß altered ECM protein synthesis with EC50s of approximately 2 nM and 0.05 ng/ml, respectively [23, 24, 33]. RU486, a GC antagonist, was used to determine whether DEX effects on FFN levels were mediated through the GC receptor. This compound was a gift from Dr. Indrani Bagchi (University of Illinois, Urbana, IL) and was used at 0.01 or 1 µm (i.e., at a 10-fold molar excess compared with DEX) [34]. Levels of FFN in culture media were determined by ELISA in triplicate wells for each experimental condition and are presented as a mean ± SEM. Figure 2 depicts results obtained in a single experiment representative of three identically conducted independent ones. Results were analyzed by ANOVA covering all treatments.
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Hypoxic Treatment
Hypoxic treatment of cells was carried out as described by Esterman et al. [35]. Cultures of PMCs were incubated in sealed Plexiglas chambers (Belleco Glass Co., Vineland, NJ) containing a gas oxygen analyzer (Hudson RCI, Temecula, CA) and a beaker of water to maintain humidity. Air in the chambers was purged with 5% CO2/95% N2 for 10 min after the oxygen meter read 0–1% O2. The sealed chambers were then placed in a 37°C incubator for the indicated time. Under these conditions, a reading of 1–3% O2 was maintained for at least 48 h [35]. Control (i.e., normoxic) cultures were maintained in a standard humidified incubator of 5% CO2/ 95% air (approximately 20% O2). For experiments, cells were maintained for 48 h under normoxia or hypoxia in serum-free medium with and without 100 nM DEX and 1 ng/ml TGFß. Levels of FFN in culture media were determined by ELISA. Results are presented as a mean ± SEM of triplicate wells for each condition from a single experiment representative of three identically conducted ones.
FFN ELISA
FFN content of culture media from PMCs was measured by an ELISA using FDC-6 monoclonal antibody according to information provided by the manufacturer (Adeza Biomedical, Sunnyvale, CA) as we have described [24, 25]. The concentration of FFN in culture media was determined in triplicate wells and was normalized to cell protein using the DC Protein Assay from Bio-Rad Laboratories (Hercules, CA).
Northern Blotting
Northern blotting was used to assess levels of FN mRNA and was carried out as previously reported [8, 25]. Approximately 20 µg RNA were separated on a 1% agarose gel containing formaldehyde, then RNA was transferred to a nylon membrane overnight. Relative levels of FN mRNA expression were determined following simultaneous hybridization of the blot with 32P-labeled cDNAs to FN and GAPDH. Autoradiographic films were scanned using a Hewlett-Packard ScanJet 3C Scanner (Palo Alta, CA). Signal intensities for FN and GAPDH mRNA were quantitated by densitometric analysis using Digital Science 1D Image Software (Eastman Kodak, Rochester, NY). Levels of FN mRNA were normalized to GAPDH mRNA and values for DEX- and TGFß-treated cells are expressed as a percentage change relative to control. Cumulative results and statistical analysis from five independent experiments are presented.
Real-time PCR
For each experiment, Tri Reagent (Sigma) was used to extract RNA from duplicate flasks maintained for 48 h without (control) or with both 100 nM DEX and 1 ng/ml TGFß. Before use in real-time PCR, 2.5 µg of total RNA were reverse transcribed in a 20-µl reaction containing 50 ng of random hexamers, 0.5 mM of each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 40 U RNase inhibitor, and 50 U SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Control reactions were set up lacking reverse transcriptase to assess the level of contaminating genomic DNA. All reactions were carried out at 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. The RNA template was removed from the cDNA:RNA hybrid by incubation with 2 U RNase H at 37°C for 20 min. Amplification was performed on an iCycler iQ real-time PCR detection system (Bio-Rad). Primers for amplification of cDNAs were designed using Beacon Designer software (Bio-Rad) from Genbank cDNA sequences, and the uniqueness of primers was established using BLAST search analysis. Real-time PCR information is summarized in Table 1. Primers were purchased from Invitrogen. The following primers were used: GAPDH; FN; laminin; collagens I, III, and IV. Aliquots containing 25 ng of cDNA were amplified in a total volume of 25 µl containing 12.5 µl of a 2x iQ SYBR Green Supermix and 0.5 µM of each primer. All samples were run in triplicate. The reactions were heated at 95°C for 3 min to activate Taq polymerase followed by 40 cycles of 95°C for 10 sec, 56°C for 30 sec, and 72°C for 30 sec. Melting curves of the products were obtained after cycling by stepwise increase of temperature from 55 to 95°C. PCR products were electrophoresed on agarose gels to confirm the presence of a single product of the expected size. For the relative quantification of gene expression, the comparative threshold cycle (CT) method was employed as described in User Bulletin #2 for ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). CT represents the PCR cycle at which an increase in reporter fluorescence above a background signal can first be detected (10 times the standard deviation of the baseline). First, GAPDH CT values were subtracted from the gene of interest CT values to derive a 
CT value. The relative expression of the gene of interest was then evaluated using the expression 2–CT, where the value for CT was obtained by subtracting the CT of the calibrator (i.e., the lowest control value within each experiment) from each CT in that experiment. Cumulative results are presented as mean ± SEM of the fold change relative to the calibrator in five independent experiments.
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Statistics
Results are expressed as a mean ± SEM. Data were analyzed by ANOVA or Student t-test using SigmaStat software from Jandel Scientific (San Rafael, CA). P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PMCs were incubated for 48 or 96 h in serum-free medium with and without 100 nM DEX and 1 ng/ml TGFß. In four independent experiments, we observed that treatment of PMCs with DEX and TGFß alone did not significantly affect FFN content in culture media (Fig. 1). However, the combination of DEX and TGF stimulated a fourfold increase (P < 0.05) in FFN levels. We observed that DEX and TGFß effects on FFN expression in PMCs were dose-dependent between 0.01 and 100 nM DEX and 0.001 and 1 ng/ml TGFß (Fig. 2). We noted that FFN levels in cells treated with 100 nM DEX and 1 ng/ml TGFß were greater than all other treatment groups (P < 0.05, denoted by a single asterisk). In addition, FFN expression in cells treated with 10 nM DEX and 1 ng/ml TGFß or 100 nM DEX and 0.1 ng/ml TGFß were significantly higher than control (P < 0.05, denoted by a double asterisk). It is of note that FFN levels in cells treated with DEX and TGF and the GC antagonist RU486 were not significantly different from controls, suggesting that enhancement of FFN expression in TGFß-treated cells by DEX was mediated through the GC receptor.
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Northern Blot Analysis
PMCs were maintained in serum-free medium with and without 100 nM DEX and 1 ng/ml TGFß for 48 h and Northern blotting was carried out (Fig. 3). We observed that DEX increased FN mRNA 36% ± 26%, but the increase was not significant. However, FN mRNA expression increased 259% ± 46% (P < 0.01) by TGFß and 317% ± 61% (P < 0.01) by DEX and TGFß.
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Analysis of DEX and TGFß Effects on ECM Protein Expression in PMCs by Real-time PCR
Real-time PCR analysis was used to determine whether combined treatment of PMCs with DEX and TGFß coordinately affected the levels of expression of several ECM protein genes. For experiments, expression of FN, collagen (Col) I, II, III, and IV were analyzed and normalized to GAPDH levels (methodologic information is shown in Table 1). We observed that the combined treatment promoted a statistically significant two- to fourfold enhancement of FN (P < 0.001), Col IV (P < 0.01) and Col I (P < 0.05) expression, whereas no effect on laminin or Col III levels was noted (Fig. 4). This indicated that GC and TGFß treatment selectively enhanced the expression of specific ECM protein genes in PMCs.
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Effect of Hypoxic Treatment on FFN Levels in PMCs
Because hypoxia has been demonstrated to enhance ECM protein levels in several cell types [36–38], in the current study, we wished to determine whether hypoxic treatment altered levels of FFN in PMCs treated with and without DEX and TGFß. As shown in Figure 5, hypoxic treatment for 48 h significantly increased FFN levels in controls by 41% (P < 0.05), whereas no effect of hypoxia was noted in cells treated with DEX and TGFß alone or TGFß plus DEX. Under both normoxic and hypoxic conditions, the combined treatment with DEX and TGFß increased FFN expression to a level that was statistically different from the three other treatment groups (P < 0.01, denoted by double asterisk).
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Regulation of FFN Expression in Cytotrophoblasts (CTs) by DEX and TGFß
To test whether the pattern of FFN regulation in human placenta showed cell-type-specific differences, we examined the effects of DEX and TGFß treatment on FFN expression in CTs isolated from human term placentas. CTs were maintained for 48 h in serum-free medium with and without 100 nM DEX and 1 ng/ml TGFß, and levels of FFN in the culture medium were determined by ELISA (Fig. 6). We observed that TGFß treatment alone significantly increased FFN expression in CTs 182% compared with control (P < 0.01), a similar trend to that noted with PMCs. However, in sharp contrast with the results obtained with PMCs, we observed that DEX treatment significantly reduced levels of FFN in CTs maintained without and with TGFß by 34% (P < 0.01) and 41% (P < 0.01), respectively. These results suggest that GCs regulate FFN expression in placenta in a cell-type-specific manner.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results suggest that the combined treatment of PMCs with GC and TGFß most likely enhanced ECM protein expression by a transcriptional mechanism because effects on ECM mRNAs noted in Northern blot and real-time PCR analyses were consistent with changes in protein noted by ELISA. It is possible that GC enhanced TGFß action in PMCs by altering the expression of TGFß receptors; both types I and II have been localized to the human placenta [15–17]. Alternatively, GC treatment could affect downstream signaling of TGFß by phosphorylated Smads (Sma and MAD gene homologues in Caenorhabditis elegans and Drosophila melanogaster) that bind to TGFß-responsive 5'-upstream sequences [11]. Song and colleagues have demonstrated a direct, albeit inhibitory, interaction between glucocorticoid receptor (GR) and Smad3/4 binding to TGFß-responsive sequences in the plasminogen activator inhibitor-1 gene [39]. It remains to be determined whether GR and Smads interact to regulate ECM protein expression.
It is of note in the present study that in the absence of DEX, TGFß effects on FN mRNA expression detected by Northern blotting (approximately 260% increase vs. control) were greater than its effects on levels of FFN protein determined by ELISA (approximately 65% stimulation vs. control). These differences may suggest that not all FN molecules released by PMCs contain the oncofetal epitope. This idea is supported by a previous study in which immunofluorescence revealed more staining for FN compared with FFN in the ECM of cultured placental fibroblasts [4]. It is possible that, in addition to its stimulatory effects on FN transcription, TGFß may also inhibit protein glycosylation and/or stability, resulting in lower levels of immunodetectable FFN.
Significantly, real-time PCR analysis revealed that DEX and TGFß treatment enhanced expression of FN, Col IV, and Col I to a lesser extent, whereas levels of laminin and Col III were unaffected by treatment. These results suggest that GC and TGFß effects on ECM protein synthesis in PMCs were specific. The effects of DEX and TGFß treatment on FN and Col IV levels are of particular interest in that these proteins were demonstrated by immunofluorescence to be expressed at high levels in the villous stroma of both first-trimester and term placentas [2–4].
In the current study, we compared FFN levels in PMCs maintained for 48 h under normoxic (20% O2) and hypoxic (1–3% O2) conditions. We observed that hypoxic treatment moderately increased (approximately 40%) FFN levels in PMCs not treated with DEX and TGFß. These results are consistent with an earlier report demonstrating an increase in FFN levels of approximately 50% and 100% in placental fibroblasts maintained for 5 days under oxygen tensions of 3.4% and 0.4%, respectively [4]. The smaller effect of hypoxic treatment observed in our study may reflect the shorter period of exposure to hypoxia. It is noteworthy that, in the present study, hypoxic treatment did not augment FFN levels in cells treated with TGFß. This may indicate that hypoxia enhances FFN expression in PMCs through an autocrine mechanism involving induction of TGFß. The observation that hypoxic treatment stimulated TGFß expression in several cell types [40–42] supports this hypothesis. Thus, it is likely that GC, TGFß, and hypoxia are all important regulators of ECM protein synthesis in PMCs.
Enhancement of FFN levels in TGF-ß-treated PMCs by DEX is surprising in light of the antagonistic interaction of GC and TGFß previously observed in several cell types [24, 43, 44] and in cytotrophoblasts in the present study. The well-known detrimental effect of GCs on wound healing is suggested to be due to suppression of TGFß-mediated matrix protein production at the site of a wound [45]. Our results also demonstrated cell-type-specific regulation of ECM protein synthesis in human placenta by GC and TGFß. We specifically noted that GC enhanced TGFß action in PMCs but inhibited TGFß effects in CTs. This finding supports our previous study in which GC treatment enhanced and suppressed FFN levels in amnion fibroblasts and epithelial cells, respectively. This same pattern of response to GC was also noted in epithelial cells and fibroblasts isolated from baboon amnion [8].
Our findings predict that pregnancies associated with excessive placental exposure to GC would result in overexpression of ECM proteins by PMCs. Placentas from pregnancies with intrauterine growth restriction (IUGR) showed excessive villous fibrosis and ECM production by PMCs [9, 10, 46]. IUGR pregnancies typically have elevated umbilical cord Dopplers or absent diastolic flow, which is indicative of increased placental vascular resistance [47, 48]. The enhanced ECM protein production by PMCs that we have demonstrated in our study and the villous fibrosis seen on histological examination [10, 46] may contribute to the clinical observation of increased placental vascular resistance. These changes in the villous stroma have been suggested to reduce the flow of nutrients from mother to fetus in pregnancies with IUGR [9, 10, 46].
The level of GC in fetal sera was found to be higher in pregnancies with IUGR compared with gestational-age- matched controls [49, 50]. The source of elevated periplacental GC in these pregnancies has been attributed to reduced placental levels of 11-hydroxysteroid dehydrogenase-2 (the enzyme that irreversibly converts cortisol to the receptor inactive cortisone) [51, 52]. In addition, a direct correlation has been noted between the number of doses of antenatal GC given to women at risk for preterm delivery and the severity of villous fibrosis [53]. Based on the results of the current study, we suggest that aberrant placental ECM protein synthesis in pregnancies with IUGR may be in part due to GC-mediated enhancement of TGFß effects in PMCs. Our results suggest that GC and TGFß effects on ECM protein synthesis play a key role in modulating placental architecture.
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FOOTNOTES |
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2 Correspondence and current address: Seth Guller, Yale University School of Medicine, Department of OB/GYN, 333 Cedar Street - 339 FMB, P.O. Box 208063, New Haven, CT 06520-8063. FAX: 203 785 4713; seth.guller{at}yale.edu
Received: 12 August 2003.
First decision: 8 September 2003.
Accepted: 2 December 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5ß1. Biol Reprod 1999 60:828-838This article has been cited by other articles:
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  A. E. Michael and A. T. Papageorghiou Potential significance of physiological and pharmacological glucocorticoids in early pregnancy Hum. Reprod. Update, September 1, 2008; 14(5): 497 - 517. [Abstract] [Full Text] [PDF]
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 |
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 X. Zhang, A. F. Clark, and T. Yorio Regulation of Glucocorticoid Responsiveness in Glaucomatous Trabecular Meshwork Cells by Glucocorticoid Receptor-{beta} Invest. Ophthalmol. Vis. Sci., December 1, 2005; 46(12): 4607 - 4616. [Abstract] [Full Text] [PDF]
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M.-J. Lee, Z. Wang, H. Yee, Y. Ma, N. Swenson, L. Yang, S. S. Kadner, R. N. Baergen, S. K. Logan, M. J. Garabedian, et al. Expression and Regulation of Glucocorticoid Receptor in Human Placental Villous Fibroblasts Endocrinology, November 1, 2005; 146(11): 4619 - 4626. [Abstract] [Full Text] [PDF]
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 C.-P. Chen, Y.-C. Yang, T.-H. Su, C.-Y. Chen, and J. D. Aplin Hypoxia and Transforming Growth Factor-{beta}1 Act Independently to Increase Extracellular Matrix Production by Placental Fibroblasts J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1083 - 1090. [Abstract] [Full Text] [PDF]
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