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University of Texas Medical Branch, Galveston, Texas 77555-1062
Address all correspondence and requests for reprints to: Yuan-Lin Dong, M.D., Ph.D., Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Medical Research Building, 11.138, Galveston, Texas 77555-1062. E-mail: ydong{at}utmb.edu.
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Abstract |
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Introduction |
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CGRP is a 37-amino acid neuropeptide, resulting from alternative splicing of the primary transcript of the calcitonin gene (4). The peptide is distributed widely in the central and peripheral nervous system and has been shown to have a potent vasodilatory effect on vascular tone (5). The circulating CGRP concentrations were significantly increased in women during pregnancy and fell after delivery (6), suggesting the involvement of CGRP in vascular adaptation in pregnancy. Recently, it has been reported that the magnitude of increases in fetal serum CGRP is parallel to the fetal weight and gestational age (7), indicating that this vasoactive peptide may contribute to the placental development and fetal growth.
CGRP exerts its effect through a 7TM G protein-coupled receptor, calcitonin receptor-like receptor (CRLR). It has been reported that CRLR functions as a receptor for three ligands, CGRP, adrenomedullin, and intermedin, in the presence of its three receptor activity modifying proteins (RAMP1, RAMP2, and RAMP3). Coexpression of CRLR with RAMP1 forms a CGRP receptor, whereas RAMP2 or RAMP3 produces an adrenomedullin receptor, and coexpression of CRLR with either of the three RAMPs mediates intermedin signaling (8). Although, the three ligands are capable of interacting with CRLR, optimal regulation by this G protein-coupled receptor-signaling pathway likely depends on an integrated release of different endocrine/paracrine ligands in a tissue-specific manner. Recently, we have demonstrated the existence of CRLR and RAMP1 in the rat placenta, particularly in the endothelium and smooth muscle cells of the fetoplacental vasculatures (9). The mRNA expression of CRLR and RAMP1 was significantly higher in the rat placenta from gestation d 17 to 22 than during labor. In the human placenta, both CRLR and RAMP1 were expressed in the endothelium and underlying smooth muscle cells in the umbilical, chorionic, and stem villous vessels (10), suggesting that CGRP may play a role in the control of fetoplacental vascular tone. However, the expression of CRLR and RAMP1 and the responsiveness to CGRP in fetoplacental vessels in preeclampsia remains unknown. Therefore, the present studies were designed to examine the alteration of CRLR and RAMP1 in fetoplacental vasculature and the effect of CGRP on vascular tone of various fetoplacental vessels in preeclamptic pregnancies.
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Subjects and Methods |
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The study population consisted of normotensive and preeclamptic pregnant women who were admitted to the Department of Obstetrics and Gynecology in the University of Texas Medical Branch at Galveston, Texas, between April 1999 and March 2001. Patients were excluded from participating in the study if they had multifetal pregnancy, diabetes, fetal anomaly, or clinical evidence of maternal or fetal infection. Obstetricians specializing in high-risk pregnancies made the diagnosis of preeclampsia using the following criteria: blood pressure more than 140/90 mm Hg, plus proteinuria more than 0.3 g/24 h, and/or nondependent edema with no history of hypertension before pregnancy and resolution of hypertension and proteinuria in the postpartum period. This study was approved by the University’s Institutional Review Board and was conducted according to the Declaration of Helsinki Principles. Informed consent was obtained from all participants.
The demographic data of the patients are shown in Table 1
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Placentas were obtained after vaginal delivery or caesarean section immediately after removal of the placentas from the patients. Umbilical arteries and veins were carefully isolated in the 5-cm cord segment proximal to its insertion into the placenta. Chorionic arteries and veins were taken from a secondary branch 5-cm distal to the insertion of the umbilical vessels into the chorionic plate. The vascular tissues were flash frozen in liquid nitrogen, placed in Bouin’s solution, or kept in Krebs solution for additional investigation.
Isolation of RNA and RT-PCR
Total RNA was isolated from the vascular tissues using TRIzol Reagent (Life Technologies Products, Inc., Grand Island, NY) (11). The homogenate was centrifuged at 12,000 g at 4 C for 30 min. The top phase was removed and mixed with an equal volume of cold isopropanol to precipitate RNA. After centrifugation at 12,000 g at 4 C, the pellets were washed with 75% ethanol and lightly dried. Isolated RNA was treated with deoxyribonuclease DNase I, according to the manufacturer’s protocol (Ambion, Austin, TX). The quality and quantity of RNA were assessed on denaturing agarose gel stained with ethidium bromide. The bands of 28s and 18s rRNA were sharp, and the ratio of 28s to 18s rRNA was 2:1.
The first-strand cDNA was synthesized by reverse transcription as prescribed previously (11). Briefly, 2 µg RNA was added to the reaction mixture containing 2.5 mM MgCl2; 50 mM KCl; 10 mM Tris-HCl; 0.5 mM deoxy-GTP, deoxy-ATP, deoxythymidine triphosphate, and deoxy-CTP; 5 µl ribonuclease inhibitor; 10 U murine leukemia virus reverse transcriptase; 0.05 µg random (hexamer) primer; and diethylpyrocarbonate water to 20 µl of volume. For reverse transcription, samples were placed into a thermal cycler Progene (Techne Inc., Princeton, NJ) for one cycle at 25 C for 10 min, 42 C for 40 min, and 94 C for 2 min. The PCR was initiated by the specific primer set for CRLR and RAMP1. Primer sets were: CRLR, 5'-TGCTCTGTGAAGGCATTTAC-3' and 5'-CAGAATTGCTTGAACCTCTC-3'; and RAMP1, 5'-GAGACGCTGTGGTGTGACTG-3' and 5'-TCGGCTACTCTGGACTCCTG-3'. Primer sequences for CRLR and RAMP1 were derived from published sequences (12, 13). The PCRs were carried out at the following conditions: an initial denaturation step at 95 C for 7 min, followed by 35 cycles of 30 sec at 95 C, 1 min at 60 C, and 30 sec at 72 C. Reactions were terminated by a 7-min elongation step at 72 C. The PCR products were loaded on 1.8% agarose gel containing 0.5 µg/ml ethidium bromide and run in 0.5% Tris-borate buffer at 100 V for 2 h. Gels were placed on a UV light box, imaged, and then analyzed with Sigma gel system (Sigma, St. Louis, MO). Expressions of CRLR and RAMP1 were normalized to 18s (Ambion). The identity of the amplified sequences has been verified by sequencing the gel-extracted PCR product.
Generation and characterization of the polyclonal antibodies to the CRLR and RAMP1
Polyclonal antibodies were raised against the peptides corresponding to the intracellular C-terminal domain of CRLR and RAMP1 and affinity purified (11). Specificity of the affinity-purified antisera was checked by blocking the antibody with the corresponding antigen and using the blocked antibody for Western blotting, in which total blockage of the respective band was observed, and linearity of the antibodies was examined by loading increasing concentration of the antigen. Western blot showed a linear increase in the intensity of the bands (14).
Immunofluorescent staining
Human fetoplacental vascular tissues and villi were rinsed thoroughly in cold PBS (0.1 M, pH 7.4) and fixed in Bouin’s fixative (11). After routine tissue processing procedure of dehydration in ascending grades of ethanol, cleaning in xylene, and infiltration with paraffin, the tissue was embedded in paraffin. Sections (5-µm thick) were rinsed with 3% normal goat serum and Triton X-100 for 30 min at room temperature, then incubated with avidin-biotin blocking buffer to reduce nonspecific staining. The primary polyclonal antibody for CRLR and RAMP1 in 1% normal goat serum was applied to the slides and incubated overnight in the cold room (4 C). Preimmune serum from the rabbits has been used as a negative control antibody. After washing with PBS, the slides were incubated with fluorescence conjugated secondary antibody Alexa Fluor 594 (Molecular Probes, Eugene, OR) at room temperature for 4 h. The slides were then rinsed with PBS, mounted using 4',6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA), covered with coverslips, and viewed under an Olympus microscope with image-ProPlus software (Olympus Optical Co. LTD., Tokyo, Japan).
Radio-labeled CGRP binding assay
Membranes were prepared from chorionic artery, and placental villi and radio-labeled CGRP binding assay was performed (15). Membrane preparations (50 µg of protein) were incubated with 10–11 M of 125I-CGRP in a total volume of 300-µl assay buffer with 0.5% heat-inactivated BSA for 150 min at 4 C. After incubation, 600 µl of assay buffer was added to each tube and centrifuged at 12,000 g for 5 min at 4 C. The bound radioactivity remaining in the pellets was counted in a gamma counter. Specific binding was calculated by subtracting the labeled CGRP bound in the presence of 0.5 µM unlabeled CGRP from the total amount of labeled CGRP bound, and the receptor density in the placental tissues was expressed as CGRP femtomoles bound per milligram of protein.
Isometric force measurement
Arteries were isolated from the umbilical cords (dissected from Warton’s jelly) and chorionic plates. These vessels were placed in cold Krebs solution (sodium chloride 119 mM, potassium chloride 4.7 mM, magnesium sulfate 1.2 mM, potassium phosphate 1.2 mM, calcium chloride 2.5 mM, sodium bicarbonate 25 mM, dextrose 11.1 mM, and sodium EDTA 0.034 mM) and processed as described previously (5, 11). Briefly, the vessels were cut into 4-mm rings, mounted onto stainless steel wire stirrups (200 µm), and placed in 5-ml organ baths containing Krebs solution maintained at 37 C. A gas mixture of 5% carbon dioxide, 21% oxygen, and 74% nitrogen was constantly bubbled through the organ bath solution. The isometric force generated by the vessels was monitored with Harvard isometric transducers (Harvard Apparatus, South Natick, MA) and analyzed with DATAQ system (DATAQ Instruments, Akron, OH). The passive tension was gradually increased to the optimal levels of 2 g during an equilibration period of 2 h. Each vessel ring was contracted repeatedly with potassium chloride (60 mmol/liter) until a stable contraction was obtained. After washing out the potassium chloride, ED70 of serotonin (5-HT) was determined for each vessel, and then the 5-HT at ED70 was used as a precontracted dose for the CGRP dose response. Varying concentrations of CGRP (10–10 to 10–6 M) were applied to the chamber in a cumulative manner. The relaxation responses to CGRP were calculated as a percentage of 5-HT-induced initial tension of the vessel. In these experiments, the logEC50, the CGRP concentration at which the initial tension was reduced 50%, was also calculated using nonlinear regression curve system (Prism GraphPad Software, Inc., San Diego, CA).
Statistical analysis
In the present study, six patients were recruited in each group. mRNA expression for CRLR and RAMP1 was determined in all umbilical vessels, chorionic vessels, and villous tissues from each placenta. The in vitro isometric force measurement was carried out in all of the umbilical and chorionic arteries, but only three placentas were examined from each group for CGRP receptor immunofluorescent staining with anti-CRLR and -RAMP1 antibodies. Data are presented as mean ± SEM. Relaxation to CGRP was expressed as a percentage of the initial precontraction to 5-HT. Raw data for individual concentration response curves were also compared by two-way repeated measurement ANOVA. The Bonferroni/Dunn post hoc test was used for determining significant differences between factors. Student’s unpaired t test was used for statistical comparison for logEC50 values. P values less than 0.05 were considered statistically significant.
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Results |
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As shown in Table 1
, there were no significant differences between normotensive and preeclamptic pregnancies in the maternal age, gestational age, gravidity, parity, and the rate of cesarean section. There were three patients who underwent C-section in the normotensive group (two repeat C-section and one breech) and three patients who underwent C-section in the preeclamptic group (repeat C-section, failure to progress, and failed induction). However, the infant birth weights were substantially lower in patients with preeclampsia compared with normotensive individuals, indicating that fetoplacental circulation may have been compromised in preeclampsia.
mRNA expression for CGRP receptors by human fetoplacental tissues
RT-PCR was performed to elucidate the existence of CGRP receptor components, CRLR and RAMP1. As shown in Figs. 1 and 2, mRNA encoding both CRLR and RAMP1 was identified in human umbilical artery and vein, chorionic artery and vein, and stem villous tissues, confirming the existence of CGRP receptors in the human fetoplacental unit. After normalizing the expressions of mRNA for CRLR and RAMP1 to that of 18s, no significant differences were observed among all the tissues examined, implying similar levels of expression of CGRP receptor in fetoplacental tissues in normotensive pregnancies. When compared with normotensive pregnancies, the mRNA expressions for both CRLR and RAMP1 are substantially lower in the tissues from preeclamptic patients, indicating reduced CGRP receptors in fetoplacental unit with preeclampsia.
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Using immunofluorescent staining, we found that both CRLR and RAMP1 were present in the umbilical artery and vein (Fig. 3), chorionic artery and vein (Fig. 4), and the vessels in stem villi (Fig. 5). CRLR and RAMP1 proteins are abundantly expressed in the endothelium and smooth muscle layer of the umbilical vessels, chorionic vessels, and stem villous vessels, as well as the trophoblast layer in normal placentas. However, the intensity of CRLR and RAMP1 protein staining in both blood vessels and trophoblasts of placentas from patients with preeclampsia is lower compared with that in normal patients.
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Abundant binding sites for CGRP are present in chorionic artery and villous tissues from normotensive pregnancies (Fig. 6), indicating the presence of CGRP receptor and the specificity of the binding in normal placentas, but these binding sites are markedly reduced in the chorionic artery and villous tissues in the placentas from the preeclamptic patients.
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To compare the vasodilatory effects of CGRP on the fetoplacental circulation between normotensive women and preeclamptic patients, we mounted the rings of umbilical and chorionic plate vessels in organ bath and measured the changes in isometric tension with DATAQ system. 5-HT-induced vascular contraction in umbilical artery was maintained in the absence of CGRP. Addition of CGRP in the organ bath relaxed 5-HT (5 x 10–7 M)-preconstricted umbilical artery (Fig. 7) in a dose-dependent manner, but the relaxation response to CGRP in umbilical arteries was profoundly attenuated in preeclamptic pregnancies. When we analyzed the data obtained from six patients in each group (Fig. 8), we found that even at its highest dose of 1 x 10–6 M, CGRP failed to relax the vessels from patients with preeclampsia, indicating the profound decrease in vasorelaxation to CGRP in preeclamptic pregnancies. Similar to the effects in umbilical arteries, CGRP dose-dependently relaxed 5-HT-preconstructed chorionic artery (Fig. 9), but the relaxation response to CGRP in the chorionic artery was substantially reduced in preeclamptic compared with the normotensive pregnancies (Figs. 9 and 10). The logEC50, the CGRP concentration at which the initial tension was reduced 50%, was calculated using nonlinear regression analysis. As shown in Table 2, the logEC50 values of CGRP in the umbilical artery and chorionic artery from preeclamptic pregnancy were significantly increased compared with the values of normotensive pregnancies (P < 0.01, ANOVA), implying decreased sensitivity to CGRP in umbilical and chorionic arteries from preeclamptic women.
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Discussion |
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Fetal growth and well-being depends mainly on uteroplacental and fetoplacental blood flow. In most systemic vascular beds, large arteries and veins contribute little to the total vascular resistance of the circulation and, therefore, are not important to the control of organ blood flow. However, in the human fetoplacental circulation, the umbilical artery and vein are extremely long, and the resistance of the placental microcirculation is extremely low. As reported in the sheep model, almost one half of the total placental vascular resistance resides in the umbilical vessels and their major branches (16). In the human, the umbilical vessels are, on average, four times longer than those in the sheep. Therefore, umbilical vessels and chorionic plate vessels in the human may make even more of a contribution to total fetoplacental vascular resistance. Factors regulating the fetoplacental circulation in both normal and pathological states remain elusive. The lack of innervation in fetoplacental vasculature implies that circulating vasoactive agents may be implicated in this regulation (17). Previous studies identified a number of vasoactive substances that may contribute to the regulation of fetoplacental vascular tone. These include bradykinin, angiotensin, oxytocin, 5-HT, histamine, different types of prostaglandins, and the neuropeptide CGRP. The present study confirmed that CGRP receptors are abundantly expressed in the endothelium and smooth muscle layer of the fetoplacental vasculature. Furthermore, in the in vitro force measurement, CGRP dose-dependently relaxed umbilical and chorionic arteries, indicating that CGRP may play a role in the control of fetoplacental vascular tone and in the fetoplacental vascular adaptive responses in normal pregnancies.
Preeclampsia represents a significant health problem during human pregnancy, and it is often associated with fetal growth restriction (18). This syndrome is clinically characterized by hypertension and proteinuria or edema. Decreased circulatory volume and cardiac output are observed in preeclampsia, along with greater vascular resistance than seen in normal pregnancies. It is believed that increased fetoplacental resistance compromised uteroplacental perfusion, which may lead to reduced fetoplacental uptake of oxygen and other nutrients in preeclampsia (19), but the etiology of the disease is largely unknown. Serum CGRP levels in women with preeclampsia have been measured by several groups (20, 21, 22), and the majority of these studies suggest that CGRP levels declined in preeclamptic patients compared with normotensive pregnancy. However, the present study is the first study to assess the changes in CGRP receptors and the responsiveness to CGRP in fetoplacental vasculature in preeclampsia. The current study demonstrated that CGRP receptors and CGRP binding sites declined in the fetoplacental vessels with preeclampsia and the relaxation responsiveness to CGRP of fetoplacental vessels is compromised in preeclamptic pregnancies. Therefore, it is possible that the decreased expression of CGRP receptors and lower CGRP binding in the fetoplacental vessels may contribute to the compromised CGRP-dependent vascular relaxation, resulting in increased resistance of the fetoplacental circulation occurring in preeclampsia.
The presence of CGRP receptors in trophoblast implies that CGRP may play roles in the regulation of trophoblast function also. During early pregnancy, the interface at the site of the placental bed is composed of trophoblast and placental villous tissues on one side and deciduas and myometrium on the other side. The interactions at this interface are vital for implantation, fetal development, and parturition. Many of the mediators produced at the trophoblast bed are released into the maternal circulation, and they are related to the systemic physiological changes in the vasculature and the intermediary metabolism of the pregnant women (23). The present study shows that both the mRNA and proteins for CRLR and RAMP1 are substantially reduced in the trophoblast cells from preeclamptic patients, suggesting that CGRP may play a role in the control of trophoblast function and reduced CGRP receptor expression in trophoblast cells may contribute to the pathogenesis of preeclampsia. Apparently, studies assessing the effects of CGRP actions on trophoblast functions, such as trophoblast proliferation, differentiation, hormonal production, and nutrient transfer, are required to fully understand the role of CGRP in the pathogenesis of preeclampsia.
Vascular reactivity is altered in preeclamptic patients compared with normal pregnant women. For example, relaxation to lactate was markedly inhibited in both placental arteries and veins of women with severe preeclampsia compared with vessels from uncomplicated term or preterm pregnancies (24). In human omental resistance arteries, the bradykinin-induced relaxation was significantly reduced for the preeclamptic group (25). Furthermore, vasorelaxation to sodium nitroprusside was substantially attenuated in placental arteries in preeclampsia compared with normal pregnancy (26). The present study added to the previous findings and confirmed that preeclampsia may be related to an altered response of the fetal placental vascular system to vasoactive substances.
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Acknowledgments |
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Footnotes |
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First Published Online December 28, 2004
Abbreviations: CGRP, Calcitonin gene-related peptide; CRLR, calcitonin receptor-like receptor; 5-HT, serotonin; RAMP, receptor activity modifying protein.
Received July 27, 2004.
Accepted December 16, 2004.
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