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Effects of magnesium sulphate on placental expression of endothelin 1 and its receptors in preeclampsia
Clinical Biochemistry 40 (2007) 976 – 980
Effects of magnesium sulphate on placental expression of endothelin 1 and its receptors in preeclampsia Ana Carolina Ariza, Xóchitl Ponce, María Elena González-González, Fernando Larrea, Ali Halhali ⁎ Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Tlalpan, 14000, México D.F., México Received 11 July 2006; received in revised form 16 April 2007; accepted 30 April 2007 Available online 5 June 2007
Abstract Objectives: To investigate the effects of magnesium sulphate (MgSO4) on placental expression of endothelin 1 (ET-1) and its receptors in preeclampsia (PE). Design and methods: Placentas were obtained from 10 normotensive (NT group) and 18 moderate preeclamptic (PE group) women. Among the PE group, 10 patients were treated with 0.9% NaCl solution (PES) and 8 women received MgSO4 (PEMgSO4). Placental mRNAs of ET-1, ET-1A receptor (ET-1AR) and ET-1B receptor (ET-1BR) were evaluated by Northern blot and quantified using densitometry. Results: Placental ET-1BR expression was lower (P b 0.05) in the PES group without significant changes in the mRNAs of ET-1 and ET-1AR when compared with the NT group. MgSO4 treatment was associated with decreased ET-1 and increased ET-1BR (P b 0.05) expression, without significant changes in ET-1AR. Conclusions: The results of the present study showed that moderate PE is associated with low placental expression of ET-1BR, and MgSO4 treatment resulted in placental expression changes of the ET-1/receptors system. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Magnesium sulphate; Endothelin 1; Placenta; Preeclampsia
Introduction Preeclampsia (PE) affects 6–8% of pregnant women and it is characterized by the simultaneous presence of hypertension and proteinuria [1]. In addition, it is well known that PE is associated with decreased uteroplacental blood flux [2]. Although the aetiology of PE still remains unknown, it is generally thought that PE originates in the placenta [3]. The clinical syndrome of PE arises from secondary systemic circulatory disturbances that can be due to generalized endothelial dysfunction and/or results from an imbalance in the production of vasoactive factors [4]. In this regard, it is known that endothelin 1 (ET-1), a potent vasoconstrictor, can lead to paradoxical effects depending on its interaction with two different receptors, thus vasoconstriction results from interaction with the ET-1A receptor (ET-1AR) in the smooth muscle and vasodilatation through binding to ET-1B receptor (ET-1BR) in endothelium [5,6]. ⁎ Corresponding author. Fax: +52 55 56 55 98 59. E-mail address: [email protected] (A. Halhali).
Expression studies of ET-1 in placental tissues from PE women are controversial. Whereas ET-1 expression has been shown to be higher in cultures obtained from trophoblast [7], it shows no changes in placental homogenates [8]. In addition, expression studies of ET-1 receptors in placental tissues from PE women have shown an increase in ET-1AR without changes in ET-1BR [8]. Preeclamptic placental tissue, which is under hypoxic conditions, due to impaired organ perfusion, is also under oxidative stress, since increased free radicals and lipid peroxidation has been documented in PE [9,10]. Furthermore, in a recent study, it has been shown that addition of ET-1 to both placental tissue and cell cultures increased the production of malondialdehyde (MDA), a marker of lipid peroxidation, suggesting a link between ET-1 and oxidative stress [11]. Eclampsia is a complication of PE characterized by the onset of generalized seizures, which can be prevented by treatment with magnesium sulphate (MgSO4) [12,13]. In addition to its anticonvulsive effects, MgSO4 decreases blood pressure [14,15] through a still unknown mechanism. MgSO4 abolishes vaso-
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.04.021
A.C. Ariza et al. / Clinical Biochemistry 40 (2007) 976–980
constriction induced by ET-1 and peroxides in placental vasculature [16,17]. Furthermore, our group has demonstrated that MgSO4 is associated with changes in maternal serum levels of MDA and ET-1 in PE [15]. However, it is not known if MgSO4 treatment modifies the expression of placental ET-1 system. Therefore, the aim of this study was to analyze ET-1 gene expression, including ET-1AR and ET-1BR, in placentas obtained from normotensive and MgSO4-treated preeclamptic women. Subjects and methods Placental samples Placentas were collected from patients in accordance with the guidelines of the Declaration of Helsinki, and the study protocol was approved by the Human Ethics Committee of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. All subjects signed a written informed consent. The study was performed cross-sectionally at delivery and included 28 patients: 10 normotensive (NT group) women and 18 moderate preeclamptic (PE group) women. Among the PE group, 10 patients were treated with 0.9% NaCl solution (PES group) and 8 women received MgSO4 (PEMgSO4 group). Magnesium sulphate treatment consisted of a loading dose of 4 g administered intravenously over a period of 30 min followed by a maintenance dose of 1 g/h. The NT group was chosen as control of the PES group, and the former as control of the PEMgSO4 group. The diagnosis of moderate PE was based on the simultaneous presence of hypertension (systolic blood pressure ≥ 140 mm Hg and b160 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg and b110 mm Hg) and proteinuria (≥ 30 mg/dL) in at least two readings [1]. Only those women giving birth to a single newborn with Apgar scores of 7–10 were included in the study. Subjects with preexisting hypertension or previous PE, or liver, renal, heart or any other endocrine disorders, including those under nutritional supplements, diuretics, and hormonal treatments, were excluded from the study. Placentas were collected immediately after delivery. Placental tissue was removed only from the central part of the cotyledons and washed repeatedly in 0.9% NaCl to eliminate blood excess and frozen at −75 °C until assayed. Northern blot The cDNA probes were obtained from human normal placenta tissue by RT-PCR using the Invitrogen SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). The primers used for cDNA probes were for ET-1: sense 5′TTC CGT ATG GAC TTG GAA GC3′ antisense 5′ AAG CCA GTG AAG ATG GTT GG3′ [7]; for ET-1AR: sense 5′TGG CCT TTT GAT CAC AAT GAC TTT3′ antisense 5′TTT GAT GTG GCA TTG AGC ATA CAG GTT3′ [18]; and for ET1BR: sense 5′ACT GGC CAT TTG GAG CTG AGA TGT3′ antisense 5′CTG CAT GCC ACT TTT CTT TCT CAA3′ [18]. The PCR products were electroeluted and labeled with [α-32P] dCTP (110 TBq/mmol) using the Amersham-Biosciences
977
Rediprime II DNA labeling Kit (Amersham-Biosciences, Buckingshamshire, UK). Total RNA from placental tissues was isolated by guanidinium isothiocyanate and the CsCl gradient centrifugation method [19]. For Northern analysis, 20 μg of RNA was size fractionated in 1.2% formaldehydeagarose gels. After electrophoresis, RNA was transferred onto a nylon membrane filter (Millipore-N+ , Bedford, MA) and crosslinked using a UV crosslinker lamp (Amersham, Buckingshamshire, UK). Membranes were prehybridized with rapidhyb buffer (Amersham-Biosciences, Buckingshamshire, UK) at 65 °C for 30 min, and then hybridized with the cDNA probes (53.3 MBq/L) for 2.5 h at 65 °C, and washed once with 2× SSC/ 0.1% SDS at room temperature for 20 min and then twice for 15 min with 0.1× SSC/0.1% SDS at 65 °C. Finally, membranes were exposed to Kodak X-OMAT AR film (Kodak, Rochester, NY) for 18–24 h at − 75 °C with an intensifying screen. Loading was normalized against 28S RNA, and bands were quantified using an image analyzer (Eagle Eye system, Stratagene, USA) by densitometry. Statistical analysis All data are presented as mean ± standard deviation. For clinical characteristics, statistical analysis was performed using analysis of variance (ANOVA) and significant differences among groups were determined by Fisher's protected lastsquare difference test. Unpaired Student's t-test was performed for placental expression comparisons. A value of P b 0.05 was considered significant. Results Clinical characteristics Table 1 summarizes the clinical characteristics of mothers and their newborns belonging to the NT, PES and PEMgSO4 groups. Maternal and gestational ages were similar among groups. As expected, basal diastolic and systolic blood pressures were significantly higher (P b 0.05) in the PE groups when compared to those in the NT group. As shown in this table, moderate PE was not associated with reduced newborn and placental weights.
Table 1 Clinical characteristics of the normotensive pregnant (NT), 0.9% NaCl- and MgSO4-treated preeclamptic (PES and PEMgSO4, respectively) women
Maternal age (years) Gestational age (weeks) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Proteinuria (mg/dL) Newborn birth weight (kg) Placental weight (g)
NT (n = 10)
PES (n = 10)
PEMgSO4 (n = 8)
24.7 ± 5.7 39.2 ± 0.6 116 ± 6
25.3 ± 2.8 39.1 ± 1.3 140 ± 9 a
22.4 ± 3.9 38.8 ± 0.71 144 ± 6 a
93 ± 6 a
98 ± 6 a
≥30 3.31 ± 0.98 613 ± 82
≥30 3.07 ± 0.60 552 ± 51
74 ± 6 Trace 3.22 ± 0.35 587 ± 89
Values are given as the mean ± standard deviation. a P b 0.05 vs. NT.
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Fig. 1. Northern blot hybridization of placental ET-1, ET-1AR and ET-1BR. 28S rRNA was used as a control for loading normalization (panel A). The blots were analyzed by densitometry (panel B). Normotensive (NT, n = 10) and 0.9% NaCl treated preeclamptic (UPE, n = 10) groups are represented in panels A and B. Values are given as the mean ± standard deviation, ⁎P b 0.05.
During normal pregnancy, a higher ET-1BR/ET-1AR placental expression ratio is found, which may primarily account for uteroplacental perfusion and vasodilatation [21,22]. Moreover, in vitro and in vivo studies using ET-1 receptor antagonists have demonstrated that ET-1BR has beneficial effects on blood pressure [23–25]. In the present study, we found that ET-1 mRNA showed no changes in the PES when compared with the NT group, which agrees with previous reports where ET-1 expression was similar in placental homogenates from PE and NT pregnancies [8]. In addition, our results showed no changes in ET-1AR placental expression in the PES group. Interestingly, placental ET-1BR expression was significantly lower in this group, suggesting that high placental perfusion pressure seen in preeclampsia [2] may be due, at least in part, to low ET-1 vasodilatation response. In previous study [8], it has been shown decreased ET-1AR and unchanged ET-1BR placental expression in PE women, which differ from our results. In the present study, we used Northern blot analysis instead of semiquantitative RT-PCR method. Furthermore, Faxen et al. [8] studied preterm and term placentas, whereas in the present work only term placentas were used. These two different conditions may account for this discrepancy. Magnesium sulphate (MgSO4) has been used for a long time as a prophylactic anticonvulsive treatment in PE [12,13]. We and others have demonstrated that MgSO4 is involved in blood pressure regulation [15,26,27]; however, the underlying mech-
ET-1, ET-1AR and ET-1BR placental expression in PE To determine whether placental expression of ET-1 and its receptors is modified in PE, we studied mRNA levels in placentas obtained from NT and PES groups. Fig. 1 shows that ET-1 and ET-1AR expression in the PES was similar to that found in NT, whereas ET-1BR expression was significantly lower (P b 0.05). Effects of MgSO4 on ET-1, ET-1AR and ET-1BR placental expression To determine whether MgSO4 treatment is associated with changes in placental expression of ET-1/receptors system, we studied mRNA levels in placentas from PES and PEMgSO4 groups. As shown in Fig. 2, as compared with the PES group, MgSO4 treatment decreased ET-1 and increased ET-1BR (P b 0.05) placental expression, without significant changes in ET-1AR. Discussion It is well known that ET-1 is a potent vasoactive substance and accumulating evidence suggests that ET-1AR mediates vasoconstriction, whereas ET-1BR contributes to vasodilatation of ET-1 action [20].
Fig. 2. Northern blot hybridization of placental ET-1, ET-1AR and ET-1BR. 28S rRNA was used as a control for loading normalization (panel A). The blots were analyzed by densitometry (panel B). 0.9% NaCl-treated preeclamptic (PES, n = 10) and preeclamptic MgSO4-treated (PEMgSO4, n = 8) groups are represented in panels A and B. Values are given as the mean ± standard deviation, ⁎P b 0.05.
A.C. Ariza et al. / Clinical Biochemistry 40 (2007) 976–980
anism is still not well understood. Since it has been shown that MgSO4 treatment is associated with changes in circulating levels of vasoactive blood pressure regulators, we were interested to study the local effect of MgSO4 on placental expression of ET-1/receptors system. Interestingly, the results of the present study showed for the first time that MgSO4 treatment is associated with decreased ET-1 and increased ET1BR placental expression. Low ET-1 expression after MgSO4 treatment could have a protective effect against placental oxidative stress since it has been shown that ET-1 increases placental malondialdehyde levels [11]. In support of this concept it has been found that MgSO4 decreases lipid peroxidation in red blood cells obtained from preeclamptic women [28]. The observation in our previous study of increased circulating levels of ET-1 in preeclampsia after MgSO4 treatment [15] and low placental ET-1 expression found in the present study suggest differential effects of MgSO4 on systemic and local compartments. Regarding ET-1 receptors, MgSO4 treatment was not associated with placental ET-1AR expression. However, this treatment resulted in higher placental ET-1BR expression. Taken together, MgSO4 treatment induced low ET-1 and high ET-1BR placental expression, which may contribute to decrease placental perfusion pressure. Indeed, it has been shown that infusion of MgSO4 to isolated placental cotyledons decreased significantly the vasoconstriction induced by ET-1 and Ang II [16,29]. Thus, these results suggest that the effects of MgSO4 on uteroplacental perfusion and blood pressure regulation are mediated, at least in part, by alterations of ET1/receptor system. In further works it would be interesting to establish if changes in placental expression of the ET-1/ receptors system by MgSO4 induce parallel modifications in their respective proteins level and to study the molecular mechanisms involved in these alterations. In summary, the results of our study showed that placental expression of ET-1BR is low in PE without changes in ET-1 and ET-1AR. In addition, our findings demonstrate that MgSO4 treatment was associated with placental changes in the ET-1/ receptor system.
[2]
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12] [13] [14] [15]
[16]
[17]
Acknowledgments This work was supported in part by a grant from the National Council of Science and Technology, CONACyT 42489, México. We thank the scholarship given by CONACyT to Ariza AC. This work was submitted in partial fulfillment of the requirements for the PhD degree of Ana Carolina Ariza at the Program “Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México.” The authors acknowledge with especial thanks to Hospital General Manuel Gea González and Hospital de GinecoObstetricia Luis Castelazo Ayala (IMSS) México, D.F., for placenta samples donation.
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[22]
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Education Program Working Group on high blood pressure in pregnancy. Am J Obstet Gynecol 2000;183:S1–22. Sankaralingam S, Arenas IA, Lalu MM, Davidge ST. Preeclampsia: current understanding of the molecular basis of vascular dysfunction. Expert Rev Mol Med 2006;8:1–20. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science 2005;308:1592–4. Granger JP, Alexander BT, Llinas MT, Bennett WA, Khalil RA. Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction. Hypertension 2001;38:718–22. Hosoda K, Nakao K, Tamura N, et al. Organization, structure, chromosomal assignment, and expression of the gene encoding the human endothelin-A receptor. J Biol Chem 1992;267:18797–804. Liu S, Premont RT, Kontos CD, Huang J, Rockey DC. Endothelin-1 activates endothelial cell nitric-oxide synthase via heteromeric G-protein bg subunit signaling to protein kinase B/Akt. J Biol Chem 2003;278: 49929–35. Napolitano M, Miceli F, Calce A, et al. Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/ endothelial nitric oxide synthase mRNA isoforms from normal and preeclamptic placentas. J Clin Endocrinol Metab 2000;85:2318–23. Faxén M, Nisell H, Kublickiene KR. Altered gene expression of endothelin-A and endothelin-B receptors, but not endothelin-1, in myometrium and placenta from pregnancies complicated by preeclampsia. Arch Gynecol Obstet 2000;264:143–9. Aydin S, Benian A, Madazli R, Uludag S, Uzun H, Oz H. Plasma malondialdehyde, superoxide dismutase, sE-selectin, fibronectin, endothelin-1 and nitric oxide levels in women with preeclampsia. Eur J Obstet Gynecol Reprod Biol 2004;113:21–5. Gupta S, Agarwal A, Sharma RK. The role of placental oxidative stress and lipid peroxidation in preeclampsia. Obstet Gynecol Surv 2005;60:807–16. Fiore G, Florio P, Micheli L, et al. Endothelin-1 triggers placental oxidative stress pathways: putative role in preeclampsia. J Clin Endocrinol Metab 2005;90:4205–10. Sibai BM. Magnesium sulphate is the ideal anticonvulsant in preeclampsia–eclampsia. Am J Obstet Gynecol 1990;162:1141–5. Witlin AG, Sibai BM. Magnesium sulphate therapy in preeclampsia and eclampsia. Obstet Gynecol 1998;92:883–9. Touyz RM. Role of magnesium in the pathogenesis of hypertension. Mol Aspects Med 2003;24:107–36. Ariza AC, Bobadilla N, Fernández C, Muñoz-Fuentes RM, Larrea F, Halhali A. Effects of magnesium sulphate on lipid peroxidation and blood pressure regulators in preeclampsia. Clin Biochem 2005;38:128–33. Holcberg G, Sapir O, Hallak M, et al. Selective vasodilator effect of magnesium sulphate in human placenta. Am J Reprod Immunol 2004;51: 192–7. Walsh SW, Romney AD, Wang Y, Walsh MD. Magnesium sulphate attenuates peroxide-induced vasoconstriction in the human placenta. Am J Obstet Gynecol 1998;178:7–12. Bauer M, Wilkens H, Langer F, Schneider SO, Lausberg H, Schafers H-J. Selective upregulation of endothelin B receptors gene expression in severe pulmonary hypertension. Circulation 2002;105:1034–6. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989. Goraca A. New views on the role of endothelin. Endocr Regul 2002;36: 161–7. Mondon F, Anouar A, Ferré F. Endothelin receptor subtypes in the microvillous trophoblastic membrane of early gestation and term human placentas. Eur J Endocrinol 1998;139:231–7. Cervar M, Huppertz B, Barth S, et al. Endothelin A and B receptors change their expression levels during development of human placental villi. Placenta 2000;21:536–46. Thaete LG, Neerhof MG, Silver RK. Differential effects of endothelin A and B receptor antagonism on fetal growth in normal and nitric oxidedeficient rats. J Soc Gynecol Investig 2001;8:18–23. Thaete LG, Kushner DM, Dewey ER, Neerhof MG. Endothelin and the regulation of uteroplacental perfusion in nitric oxide synthase inhibitioninduced fetal growth restriction. Placenta 2005;26:242–50.
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[25] Madsen KM. Influence of ET(B) receptor antagonism on pregnancy outcome in rats. J Soc Gynecol Investig 2001;8:239–44. [26] Sagsoz N, Kucukozkan T. The effect of treatment on endothelin-1 concentration and mean arterial pressure in preeclampsia and eclampsia. Hypertens Pregnancy 2003;22:185–91. [27] Halhali A, Wimalawansa SJ, Berentsen V, Avila E, Thota CS, Larrea F. Calcitonin gene- and parathyroid hormone-related peptides in preeclampsia: effects of magnesium sulphate. Obstet Gynecol 2001;97:893–7.
[28] Abad C, Teppa-Garran A, Proverbio T, Pinero S, Proverbio F, Marin R. Effect of magnesium sulphate on the calcium-stimulated adenosine triphosphatase activity and lipid peroxidation of red blood cell membranes from preeclamptic women. Biochem Pharmacol 2005;70:1634–41. [29] Kovac CM, Howard BC, Pierce BT, Hoeldtke NJ, Calhoun BC, Napolitano PG. Fetoplacental vascular tone is modified by magnesium sulphate in the preeclamptic ex vivo human placental cotyledon. Am J Obstet Gynecol 2003;189:839–42.
Effects of magnesium sulphate on placental expression of endothelin 1 and its receptors in preeclampsia Ana Carolina Ariza, Xóchitl Ponce, María Elena González-González, Fernando Larrea, Ali Halhali ⁎ Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Tlalpan, 14000, México D.F., México Received 11 July 2006; received in revised form 16 April 2007; accepted 30 April 2007 Available online 5 June 2007
Abstract Objectives: To investigate the effects of magnesium sulphate (MgSO4) on placental expression of endothelin 1 (ET-1) and its receptors in preeclampsia (PE). Design and methods: Placentas were obtained from 10 normotensive (NT group) and 18 moderate preeclamptic (PE group) women. Among the PE group, 10 patients were treated with 0.9% NaCl solution (PES) and 8 women received MgSO4 (PEMgSO4). Placental mRNAs of ET-1, ET-1A receptor (ET-1AR) and ET-1B receptor (ET-1BR) were evaluated by Northern blot and quantified using densitometry. Results: Placental ET-1BR expression was lower (P b 0.05) in the PES group without significant changes in the mRNAs of ET-1 and ET-1AR when compared with the NT group. MgSO4 treatment was associated with decreased ET-1 and increased ET-1BR (P b 0.05) expression, without significant changes in ET-1AR. Conclusions: The results of the present study showed that moderate PE is associated with low placental expression of ET-1BR, and MgSO4 treatment resulted in placental expression changes of the ET-1/receptors system. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Magnesium sulphate; Endothelin 1; Placenta; Preeclampsia
Introduction Preeclampsia (PE) affects 6–8% of pregnant women and it is characterized by the simultaneous presence of hypertension and proteinuria [1]. In addition, it is well known that PE is associated with decreased uteroplacental blood flux [2]. Although the aetiology of PE still remains unknown, it is generally thought that PE originates in the placenta [3]. The clinical syndrome of PE arises from secondary systemic circulatory disturbances that can be due to generalized endothelial dysfunction and/or results from an imbalance in the production of vasoactive factors [4]. In this regard, it is known that endothelin 1 (ET-1), a potent vasoconstrictor, can lead to paradoxical effects depending on its interaction with two different receptors, thus vasoconstriction results from interaction with the ET-1A receptor (ET-1AR) in the smooth muscle and vasodilatation through binding to ET-1B receptor (ET-1BR) in endothelium [5,6]. ⁎ Corresponding author. Fax: +52 55 56 55 98 59. E-mail address: [email protected] (A. Halhali).
Expression studies of ET-1 in placental tissues from PE women are controversial. Whereas ET-1 expression has been shown to be higher in cultures obtained from trophoblast [7], it shows no changes in placental homogenates [8]. In addition, expression studies of ET-1 receptors in placental tissues from PE women have shown an increase in ET-1AR without changes in ET-1BR [8]. Preeclamptic placental tissue, which is under hypoxic conditions, due to impaired organ perfusion, is also under oxidative stress, since increased free radicals and lipid peroxidation has been documented in PE [9,10]. Furthermore, in a recent study, it has been shown that addition of ET-1 to both placental tissue and cell cultures increased the production of malondialdehyde (MDA), a marker of lipid peroxidation, suggesting a link between ET-1 and oxidative stress [11]. Eclampsia is a complication of PE characterized by the onset of generalized seizures, which can be prevented by treatment with magnesium sulphate (MgSO4) [12,13]. In addition to its anticonvulsive effects, MgSO4 decreases blood pressure [14,15] through a still unknown mechanism. MgSO4 abolishes vaso-
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.04.021
A.C. Ariza et al. / Clinical Biochemistry 40 (2007) 976–980
constriction induced by ET-1 and peroxides in placental vasculature [16,17]. Furthermore, our group has demonstrated that MgSO4 is associated with changes in maternal serum levels of MDA and ET-1 in PE [15]. However, it is not known if MgSO4 treatment modifies the expression of placental ET-1 system. Therefore, the aim of this study was to analyze ET-1 gene expression, including ET-1AR and ET-1BR, in placentas obtained from normotensive and MgSO4-treated preeclamptic women. Subjects and methods Placental samples Placentas were collected from patients in accordance with the guidelines of the Declaration of Helsinki, and the study protocol was approved by the Human Ethics Committee of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. All subjects signed a written informed consent. The study was performed cross-sectionally at delivery and included 28 patients: 10 normotensive (NT group) women and 18 moderate preeclamptic (PE group) women. Among the PE group, 10 patients were treated with 0.9% NaCl solution (PES group) and 8 women received MgSO4 (PEMgSO4 group). Magnesium sulphate treatment consisted of a loading dose of 4 g administered intravenously over a period of 30 min followed by a maintenance dose of 1 g/h. The NT group was chosen as control of the PES group, and the former as control of the PEMgSO4 group. The diagnosis of moderate PE was based on the simultaneous presence of hypertension (systolic blood pressure ≥ 140 mm Hg and b160 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg and b110 mm Hg) and proteinuria (≥ 30 mg/dL) in at least two readings [1]. Only those women giving birth to a single newborn with Apgar scores of 7–10 were included in the study. Subjects with preexisting hypertension or previous PE, or liver, renal, heart or any other endocrine disorders, including those under nutritional supplements, diuretics, and hormonal treatments, were excluded from the study. Placentas were collected immediately after delivery. Placental tissue was removed only from the central part of the cotyledons and washed repeatedly in 0.9% NaCl to eliminate blood excess and frozen at −75 °C until assayed. Northern blot The cDNA probes were obtained from human normal placenta tissue by RT-PCR using the Invitrogen SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). The primers used for cDNA probes were for ET-1: sense 5′TTC CGT ATG GAC TTG GAA GC3′ antisense 5′ AAG CCA GTG AAG ATG GTT GG3′ [7]; for ET-1AR: sense 5′TGG CCT TTT GAT CAC AAT GAC TTT3′ antisense 5′TTT GAT GTG GCA TTG AGC ATA CAG GTT3′ [18]; and for ET1BR: sense 5′ACT GGC CAT TTG GAG CTG AGA TGT3′ antisense 5′CTG CAT GCC ACT TTT CTT TCT CAA3′ [18]. The PCR products were electroeluted and labeled with [α-32P] dCTP (110 TBq/mmol) using the Amersham-Biosciences
977
Rediprime II DNA labeling Kit (Amersham-Biosciences, Buckingshamshire, UK). Total RNA from placental tissues was isolated by guanidinium isothiocyanate and the CsCl gradient centrifugation method [19]. For Northern analysis, 20 μg of RNA was size fractionated in 1.2% formaldehydeagarose gels. After electrophoresis, RNA was transferred onto a nylon membrane filter (Millipore-N+ , Bedford, MA) and crosslinked using a UV crosslinker lamp (Amersham, Buckingshamshire, UK). Membranes were prehybridized with rapidhyb buffer (Amersham-Biosciences, Buckingshamshire, UK) at 65 °C for 30 min, and then hybridized with the cDNA probes (53.3 MBq/L) for 2.5 h at 65 °C, and washed once with 2× SSC/ 0.1% SDS at room temperature for 20 min and then twice for 15 min with 0.1× SSC/0.1% SDS at 65 °C. Finally, membranes were exposed to Kodak X-OMAT AR film (Kodak, Rochester, NY) for 18–24 h at − 75 °C with an intensifying screen. Loading was normalized against 28S RNA, and bands were quantified using an image analyzer (Eagle Eye system, Stratagene, USA) by densitometry. Statistical analysis All data are presented as mean ± standard deviation. For clinical characteristics, statistical analysis was performed using analysis of variance (ANOVA) and significant differences among groups were determined by Fisher's protected lastsquare difference test. Unpaired Student's t-test was performed for placental expression comparisons. A value of P b 0.05 was considered significant. Results Clinical characteristics Table 1 summarizes the clinical characteristics of mothers and their newborns belonging to the NT, PES and PEMgSO4 groups. Maternal and gestational ages were similar among groups. As expected, basal diastolic and systolic blood pressures were significantly higher (P b 0.05) in the PE groups when compared to those in the NT group. As shown in this table, moderate PE was not associated with reduced newborn and placental weights.
Table 1 Clinical characteristics of the normotensive pregnant (NT), 0.9% NaCl- and MgSO4-treated preeclamptic (PES and PEMgSO4, respectively) women
Maternal age (years) Gestational age (weeks) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Proteinuria (mg/dL) Newborn birth weight (kg) Placental weight (g)
NT (n = 10)
PES (n = 10)
PEMgSO4 (n = 8)
24.7 ± 5.7 39.2 ± 0.6 116 ± 6
25.3 ± 2.8 39.1 ± 1.3 140 ± 9 a
22.4 ± 3.9 38.8 ± 0.71 144 ± 6 a
93 ± 6 a
98 ± 6 a
≥30 3.31 ± 0.98 613 ± 82
≥30 3.07 ± 0.60 552 ± 51
74 ± 6 Trace 3.22 ± 0.35 587 ± 89
Values are given as the mean ± standard deviation. a P b 0.05 vs. NT.
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Fig. 1. Northern blot hybridization of placental ET-1, ET-1AR and ET-1BR. 28S rRNA was used as a control for loading normalization (panel A). The blots were analyzed by densitometry (panel B). Normotensive (NT, n = 10) and 0.9% NaCl treated preeclamptic (UPE, n = 10) groups are represented in panels A and B. Values are given as the mean ± standard deviation, ⁎P b 0.05.
During normal pregnancy, a higher ET-1BR/ET-1AR placental expression ratio is found, which may primarily account for uteroplacental perfusion and vasodilatation [21,22]. Moreover, in vitro and in vivo studies using ET-1 receptor antagonists have demonstrated that ET-1BR has beneficial effects on blood pressure [23–25]. In the present study, we found that ET-1 mRNA showed no changes in the PES when compared with the NT group, which agrees with previous reports where ET-1 expression was similar in placental homogenates from PE and NT pregnancies [8]. In addition, our results showed no changes in ET-1AR placental expression in the PES group. Interestingly, placental ET-1BR expression was significantly lower in this group, suggesting that high placental perfusion pressure seen in preeclampsia [2] may be due, at least in part, to low ET-1 vasodilatation response. In previous study [8], it has been shown decreased ET-1AR and unchanged ET-1BR placental expression in PE women, which differ from our results. In the present study, we used Northern blot analysis instead of semiquantitative RT-PCR method. Furthermore, Faxen et al. [8] studied preterm and term placentas, whereas in the present work only term placentas were used. These two different conditions may account for this discrepancy. Magnesium sulphate (MgSO4) has been used for a long time as a prophylactic anticonvulsive treatment in PE [12,13]. We and others have demonstrated that MgSO4 is involved in blood pressure regulation [15,26,27]; however, the underlying mech-
ET-1, ET-1AR and ET-1BR placental expression in PE To determine whether placental expression of ET-1 and its receptors is modified in PE, we studied mRNA levels in placentas obtained from NT and PES groups. Fig. 1 shows that ET-1 and ET-1AR expression in the PES was similar to that found in NT, whereas ET-1BR expression was significantly lower (P b 0.05). Effects of MgSO4 on ET-1, ET-1AR and ET-1BR placental expression To determine whether MgSO4 treatment is associated with changes in placental expression of ET-1/receptors system, we studied mRNA levels in placentas from PES and PEMgSO4 groups. As shown in Fig. 2, as compared with the PES group, MgSO4 treatment decreased ET-1 and increased ET-1BR (P b 0.05) placental expression, without significant changes in ET-1AR. Discussion It is well known that ET-1 is a potent vasoactive substance and accumulating evidence suggests that ET-1AR mediates vasoconstriction, whereas ET-1BR contributes to vasodilatation of ET-1 action [20].
Fig. 2. Northern blot hybridization of placental ET-1, ET-1AR and ET-1BR. 28S rRNA was used as a control for loading normalization (panel A). The blots were analyzed by densitometry (panel B). 0.9% NaCl-treated preeclamptic (PES, n = 10) and preeclamptic MgSO4-treated (PEMgSO4, n = 8) groups are represented in panels A and B. Values are given as the mean ± standard deviation, ⁎P b 0.05.
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anism is still not well understood. Since it has been shown that MgSO4 treatment is associated with changes in circulating levels of vasoactive blood pressure regulators, we were interested to study the local effect of MgSO4 on placental expression of ET-1/receptors system. Interestingly, the results of the present study showed for the first time that MgSO4 treatment is associated with decreased ET-1 and increased ET1BR placental expression. Low ET-1 expression after MgSO4 treatment could have a protective effect against placental oxidative stress since it has been shown that ET-1 increases placental malondialdehyde levels [11]. In support of this concept it has been found that MgSO4 decreases lipid peroxidation in red blood cells obtained from preeclamptic women [28]. The observation in our previous study of increased circulating levels of ET-1 in preeclampsia after MgSO4 treatment [15] and low placental ET-1 expression found in the present study suggest differential effects of MgSO4 on systemic and local compartments. Regarding ET-1 receptors, MgSO4 treatment was not associated with placental ET-1AR expression. However, this treatment resulted in higher placental ET-1BR expression. Taken together, MgSO4 treatment induced low ET-1 and high ET-1BR placental expression, which may contribute to decrease placental perfusion pressure. Indeed, it has been shown that infusion of MgSO4 to isolated placental cotyledons decreased significantly the vasoconstriction induced by ET-1 and Ang II [16,29]. Thus, these results suggest that the effects of MgSO4 on uteroplacental perfusion and blood pressure regulation are mediated, at least in part, by alterations of ET1/receptor system. In further works it would be interesting to establish if changes in placental expression of the ET-1/ receptors system by MgSO4 induce parallel modifications in their respective proteins level and to study the molecular mechanisms involved in these alterations. In summary, the results of our study showed that placental expression of ET-1BR is low in PE without changes in ET-1 and ET-1AR. In addition, our findings demonstrate that MgSO4 treatment was associated with placental changes in the ET-1/ receptor system.
[2]
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12] [13] [14] [15]
[16]
[17]
Acknowledgments This work was supported in part by a grant from the National Council of Science and Technology, CONACyT 42489, México. We thank the scholarship given by CONACyT to Ariza AC. This work was submitted in partial fulfillment of the requirements for the PhD degree of Ana Carolina Ariza at the Program “Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México.” The authors acknowledge with especial thanks to Hospital General Manuel Gea González and Hospital de GinecoObstetricia Luis Castelazo Ayala (IMSS) México, D.F., for placenta samples donation.
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