Objective
Plasma adrenomedullin (AM) concentrations are increased in fetal and maternal circulation in response to exogenous glucocorticoids administration. The role of corticosteroids and progesterone in regulating AM synthesis and secretion was investigated in amnion and chorion trophoblast cells of the fetal membranes and in placental trophoblast cells.
Study Design
Cells were treated with betamethasone, hydrocortisone, and progesterone. Changes in AM output were measured with radioimmunoassay. Protein expression was evaluated with Western blot and immunohistochemistry.
Results
Betamethasone stimulated AM secretion and protein expression in placental trophoblast cells and in amnion cells of the fetal membranes. Hydrocortisone and progesterone did not induce any effect either on secretion or protein expression in placenta and fetal membranes cells.
Conclusion
Glucocorticoids regulate AM secretion and expression by human placenta thereby promoting increased AM concentration in maternal and fetal circulation in circumstances characterized by increased cortisol levels.
Adrenomedullin (AM), a multifunctional peptide that belongs to the calcitonin gene-related peptide family, is expressed in a variety of tissues including the placenta. Plasma concentrations of AM are much higher in pregnant than in nonpregnant women and it has been shown that human placenta and fetal membranes contribute significantly to circulating AM. There is evidence that AM is implicated in several functions, including the regulation of the hypothalamus-pituitary-adrenal axis. Furthermore, hormonal factors such as steroid hormones participate in the regulation of AM production in vascular tissue. In cultured vascular smooth muscle cells dexamethasone and progesterone increase AM concentration in a dose-dependent manner, in contrast estradiol does not induce any effect on AM output.
Maternal and fetal glucocorticoid concentrations increase during human pregnancy, raising the possibility that positive feedback of glucocorticoid on placental AM output might be important. Recently we reported that glucocorticoids given to pregnant women in the third trimester of pregnancy increase plasma AM levels in maternal and fetal circulation, and that this increase may derive from the placenta.
To gain insight into the cellular mechanisms of regulation of AM expression in the human placenta, in the present study we investigated the effects of corticosteroids (betamethasone and hydrocortisone) and progesterone on AM production in trophoblast cells and amnion and chorion cells isolated from human term placentas.
Materials and Methods
Placental trophoblast along with amnion and chorion trophoblast cells were isolated from placenta and fetal membranes obtained from uncomplicated normal term pregnancies, with elective cesarean section in the absence of labor. Patient consent and ethical approval were obtained in accordance with the guidelines of the Research Ethics Committees of the University of Rome. A modification of Kliman’s protocol was used. Within 10 minutes from delivery, placentas (n = 7) were immediately collected in Hanks phosphate saline buffer (Gibco Laboratories Inc, New York, NY). The chorion was peeled off the amnion. The tissue was digested 3 times in a buffer containing 0.125% trypsin; Sigma, St Louis, MO), 0.02% deoxyribonuclease I (Sigma), and 0.2% collagenase (Roche Diagnostics Corp, Indianapolis, IN) in phenol red free Dulbecco modified Eagle medium (DMEM), in a shaking water bath at 36°C. To reduce the possible contamination with the adherent decidua and fibroblast cells, cell suspensions obtained from the first digest were discarded. Cell suspension was filtered with a 200-μm metal strainer and loaded onto a continuous Percoll (Sigma) gradient (5-7% in 5% steps of 3 mL each). After centrifuging for 20 minutes at 1200 g , cytotrophoblasts were collected between density markers of 1.049 and 1.062 g/mL. Cells were washed in DMEM and resuspended in DMEM containing 10% heat inactivated fetal bovine serum (Sigma) and 1% antibiotic-antimycotic solution (10,000 U/mL penicillin G, 10 mg/mL streptomycin sulfate, 25 mg/mL amphotericin B in 0.9% sodium chloride; Sigma). Cells were plated in 24-well (16-mm) plates at a density of 0.5 × 10 6 cells/dish, for medium collection and protein extraction. For immunohistochemistry cells were plated on glass coverslides, in 8-well plates at a density of 0.15 × 10 6 cells/well and cultured at 37°C in 5% carbon dioxide and 95% air atmosphere.
Time response to betamethasone (Laboratorio Farmacologico Milanese srl, Caronno Pertusella, VA, Italy), hydrocortisone (Gruppo Lepetit spa, Lainate, MI, Italy), and progesterone (AMSA srl, Rome, Italy) was performed with the concentration of 10 –7 M in 100% ethanol. Final ethanol concentrations in the culture dishes were 0.1% (vol/vol). Ethanol 0.1% had no effect on AM output when compared to the DMEM control (data not shown). Treatments of placenta trophoblast cells were added to culture medium at time 0 (when cells were plated) or >48 hours, for 48 hours. In this case, cells were washed twice with DMEM and serum starved overnight, for approximately 15 hours, in the absence of antibiotics. Culture medium was then replaced with fresh DMEM (fetal bovine serum and antibiotic free) containing treatments. The effect of treatment at each time point was compared to a vehicle control sample (0.1% ethanol in DMEM). Control samples were maintained in the same conditions as the treated ones.
For immunocytochemistry, cells were treated with betamethasone, hydrocortisone, progesterone, or vehicle, for 48 hours at time 0 or 48 hours.
Chorion and amnion cells of fetal membranes were serum starved overnight and treated with betamethasone, hydrocortisone, or progesterone 10 –7 mol/L, for 2, 8, or 24 hours. The effect of treatment at each time point was compared to a vehicle control sample (0.1% ethanol).
For immunohistochemistry, cells were treated with betamethasone, hydrocortisone, or progesterone (10 –7 mol/L) or vehicle, for 2, 8, or 24 hours.
AM concentration in cultured medium was measured without extraction and purification as reported, using a specific radioimmunoassay (Phoenix Pharmaceuticals Inc, Mountain View, CA) with rabbit polyclonal antibody raised against human AM 1-52. The antibody cross-reacts 100% with human AM and no cross-reactivity was reported with rat AM, amylin, calcitonin gene-related peptide, endothelin-1, or α-atrial natriuretic peptide. The intraassay and interassay coefficients of variance were 4.8% and 10.4%, respectively. The sensitivity of the assay was 2 pg/mL.
Cells were lysed with radioimmunoprecipitation assay buffer containing protease inhibitors, complete mini EDTA-free (Roche Diagnostics Corp). Protein concentration was measured using the Bradford protein assay (BioRad, Hercules, CA). Proteins (30 μg) were loaded onto a 10% sodium dodecylsulfate gel, separated with electrophoresis, and transferred onto nitrocellulose membrane. Blots were incubated overnight at 4°C with 5% milk/phosphate-buffered saline (PBS)-Tween (wt/vol), and then for 1 hour (room temperature) with a primary polyclonal rabbit antibody against human AM (1:500). Membranes were then hybridized for 1 hour at room temperature with a secondary antirabbit (1:3000) antibody (Amersham Biosciences Europe Gmbh, Cologno Monzese, Italy) conjugated with horseradish peroxidase. Blots were incubated for 1 minute with Western Lightning Chemiluminescence reagent Plus (Perkin Elmer, Boston, MA) and then exposed on film. For β-actin, membranes were stripped with a glycine buffer (pH = 4.1) and incubated with a primary monoclonal antibody against β-actin (1:10000) (Amersham Biosciences) followed by the antimouse antibody (1:10000) (Amersham Biosciences).
Cells cultured on coverslips were washed with PBS and fixed with 4% paraformaldehyde/PBS (vol/vol) as described. Coverslips were then stored in 95% ethanol at 4°C, until used for immunocytochemistry. Before staining, cells were rehydrated in 50% and 70% ethanol and then in PBS (2 times 5 minutes each). Nonspecific binding was blocked with 10% fetal bovine serum in PBS. The cells were stained using the avidin-biotin peroxidase technique (Dako Italia S.p.A., Milan, Italy), and polyclonal antibody raised in rabbits against purified human AM 1-52 (Peninsula Laboratories Inc, Belmont, CA) at a dilution of 1:600. Cells were incubated overnight with AM antibody. After washing with PBS a secondary antibody was applied. For negative controls primary antibodies were omitted. Vectashield mounting medium (Vector Laboratories Inc, Burlingame, CA) was used to mount coverslips. The cells were examined by light microscopy. The number of positive cells was quantified using a quantitative system (field = 0.175 mm 2 at 250× magnification). Randomly selected fields were counted by 3 different examiners (E.M., A.S., K.P.) and the proportion of stained cells was expressed as a percentage of the total cells (stained and unstained). Intensity of staining was scored on a scale of 1-4 (1 = weak, 2 = moderate, 3 = strong, 4 = very strong). Examiners were blinded as to treatment.
Kruskal-Wallis analysis of variance followed by Dunn multiple comparison test was used to analyze the effects of treatment on media concentrations. When the data passed the tests for equal variance and normal distribution, a 1-way analysis of variance followed by Tukey test was applied. Data are expressed as mean ± SE, relative to their controls. To compare proportions between treatments and controls Fisher’s exact test was used, whereas to determine whether there was a difference in the intensity of the staining contingency table analysis of the score was performed. Statistical analysis was performed with Sigma Stat (Jandel Scientific Software, San Rafael, CA). Limit of significance was fixed as P < .05.
Results
Trophoblast cells and chorion and amnion cells in culture secrete AM. AM output increased over the time in unstimulated chorion cells, with concentrations 6-fold higher at 24 hours, whereas basal output of AM did not change significantly at different time points, either in trophoblast or amnion cells. Treatment with betamethasone markedly up-regulated AM output by placental trophoblast cells at 48 and 96 hours over control ( Figure 1 , A). AM concentrations in medium collected at 48 hours were 2 times higher than in control, being 860 ± 90 pg/10 6 cells and 413 ± 60 pg/10 6 cells, respectively.
