Expression of Endothelial NO Synthase, Inducible NO Synthase, and Estrogen Receptors Alpha and Beta in Placental Tissue of Normal, Preeclamptic, and Intrauterine Growth-restricted Pregnancies
Ludwig-Maximilians-University Munich, 1st Department of Obstetrics and Gynaecology, Maistrasse 11, 80337 Munich, Germany
Correspondence to: PD Dr. rer. nat. Udo Jeschke, Ludwig-Maximilians-University Munich, 1st Department of Obstetrics and Gynaecology, Maistrasse 11, 80337 Munich, Germany. E-mail: udo.jeschke{at}med.uni-muenchen.de
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Summary |
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Key Words: nitric oxide synthases estrogen receptor alpha/beta intrauterine growth restriction preeclampsia
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Introduction |
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Failure of this process has been associated with complications of pregnancy such as preeclampsia (PE), intrauterine growth restriction (IUGR), and, in severe cases, second-trimester miscarriage (Pijnenborg et al. 1991).
IUGR is diagnosed either by intrauterine growth assessment (sonography) showing an estimated weight below the 5th centile for gestational age or postnatal showing a birth weight below the third centile (Chatelain 2000). Small fetuses resulting from IUGR are at higher risk for poor perinatal and long-term outcome (Baschat and Hecher 2004
).
Preeclampsia is associated with significant maternal and perinatal morbidity in those patients who suffer early onset of PE (Myatt and Miodovnik 1999). Patients with chronic hypertension, pregestational diabetes, or multifetal gestation are at risk for developing preeclampsia as are nulliparous women, but factors defining the risk for multiparous women are yet to be defined. Although factors such as callicrein-creatinine, coagulation, and vascular function tests and oxidant stress parameters as well as placental peptide hormones have been identified as potential markers for patients at risk for PE prospective and longitudinal studies are mandatory to verify the data (Myatt and Miodovnik 1999
).
Nitric oxide (NO) as a potent vasodilator is thought to contribute to the phenomenon of decreasing vascular resistance in uterine circulation, but still its role in normal pregnancy and pregnancies complicated by PE or IUGR is controversial and remains to be clarified (Nasiell et al. 1998). Izumi and coworkers report on vasorelaxation in the human umbilical artery: their data suggest that NO synthase (NOS) is stimulated in endothelial cells and the derived NO activates guanylate cyclase to produce cyclic guanosine monophosphate in umbilical smooth muscle cells (Izumi et al. 1995
). The NO systems also seems to be mainly involved in regulation of uterine quiescence during pregnancy and initiation of labor: Buhimshi et al. demonstrated an NO-cyclic guanosine monophosphate reaction pathway in the human uterus (Buhimschi et al. 1995
).
The question if impaired trophoblast invasion is related to different expression of endothelial NOS (eNOS) and inducible NOS (iNOS) and results in lower NO output or if the remaining elevated impedance at the uterine and spiral arteries causes elevated compensatory NO production is topic of ongoing discussions. Purcell and colleagues showed changing concentrations of iNOS in rat placentas during the course of pregnancy with a decrease after day 16 to day 22 before labor and during delivery (Purcell et al. 1997). The reported data suggest a paracrine role for nitric oxide in regulation of uterine contractility, blood flow and immunosuppression, which are all requested for pregnancy maintenance (Purcell et al. 1997
).
Myatt et al. examined placental villous tissue from normal, PE, and IUGR pregnancies by investigating the expression of eNOS (Eis et al. 1995; Myatt et al. 1997
). They found increased eNOS expression in IUGR, which they interpreted as a possible adaptive response to increased resistance and poor perfusion in these pathological pregnancies (Eis et al. 1995
; Myatt et al. 1997
). Nasiell investigated expression of endothelial constitutive NOS (Nasiell et al. 1998
). Total nuclei acids were prepared and a hybridization technique was used for mRNA analysis. The mRNA expression was significantly higher in pathological groups (IUGR, IUGR+PE, and PE) compared with normal placentas, which might reflect a compensatory mechanism in the disturbed uterine circulation seen in PE or IUGR (Nasiell et al. 1998
). Within the three pathologic groups investigated, no significant differences in elevated endothelial constitutive NOS expression in IUGR, IUGR + PE, and PE alone was found. Based on these findings, elevated NO concentrations in venous umbilical blood in placentas of IUGR have also been reported, suggesting a compensatory response to improve placental circulation (Lyall et al. 1996
; Macara et al. 1996
). Additionally, elevated NO concentrations could also play a role in limiting platelet adhesion aggregation (Lyall et al. 1996
). In contrast, Beinder and coworkers found that NOS activity from patients with preeclampsia was significantly lower in the uterine placental bed (Beinder et al. 1999
).
Based on the hypothesis that heat exposure disrupts placental structure and reduces placental eNOS protein expression, Galan et al. described reduced eNOS protein content in the hyperthermic group (Galan et al. 1999,2001
). Myatt et al. showed that eNOS is expressed by the syncytiotrophoblast (Myatt et al. 1993
). Trophoblast differentiation is associated with expression of estrogen receptors (ER)
and ERß (Bukovsky et al. 2003a
,b
). Interestingly, the ER
expression has been associated with a inhibition of angiogenesis in cancer cells (Ali et al. 2000
), but if this also is the case in human pregnancy remains unknown (Ali et al. 2000
). We therefore combined the immunohistochemical detection of eNOS and iNOS with investigations on ER
and ERß expression in normal, PE, and IUGR placentas.
The aims of this study were (a) clarifying the relation between expression of iNOS/eNOS and (b) assessing the expression of ER and ERß in normal, preeclamptic and growth restricted human placental tissue by immunohistochemistry and Western blot experiments.
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Materials and Methods |
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Immunohistochemistry
Immunohistochemistry on paraffin sections (7 µm) of the different specimens was done by incubating the slides in methanol/H2O2 (30 min) to inhibit endogenous peroxidase activity, followed by washing in PBS (5 min) and treating with goat serum (20 min, 22C) to reduce nonspecific background staining. Incubation with the primary antibody (Table 1) was done overnight at 4C. Sections were then thoroughly incubated with the biotinylated secondary anti-mouse or anti-rabbit antibody (1 hr, 22C) and avidin-biotinylated peroxidase (45 min, RT). Between each step, sections were washed with PBS (pH 7.4). Peroxidase staining reaction was done with diaminobenzidine/H2O2 (1 mg/ml; 5 min) and stopped in tap water (10 min). Sections were counterstained in hemalum (1 min) and then cover-slipped. In controls, the primary antibody was replaced with preimmune mouse serum with positive and negative controls being included. From each section, five digital images were obtained with a 3CCD color camera (JVC; Victor Company of Japan, Japan) and a Leitz (Wetzlar, Germany) microscope.
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Immunohistochemical Evaluation and Statistical Analysis
The intensity and distribution patterns of the staining reaction was evaluated by two blinded, independent observers, including a gynecological pathologist (PH), using a semiquantitative score (graded as 0 = no, 1 = weak, 2 = moderate, and 3 = strong staining) and without knowing the pathological evaluation, the diagnosis, or the standard performed hematoxylin reaction of each specimen. The SPSS/PC software package, version 6.01 (SPSS; Munich, Germany), was used for collection, processing, and statistical analysis of all data. Statistical analysis was performed using the nonparametrical Wilcoxon's signed rank tests for comparison of the means. The Spearman rho coefficient was used to assess any significant correlations between the analyzed substances within the distinct groups. p<0.05 values were considered statistically significant.
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Results |
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Results of immunochemical detection (Western blots) of ER in villous trophoblast cell lysates are shown in Figure 8A. The ER
monoclonal antibody generates a main protein band in the 67-kDa molecular mass range. Normal villous trophoblast tissue (Lane 1), preeclamptic trophoblast tissue (Lane 2), and IUGR tissue (Lane 3) showed almost the same staining intensity.
Results of immunochemical detection (Western blots) of ERß in villous trophoblast cell lysates are shown in Figure 8B. The ERß monoclonal antibody generates a main protein band in the 57-kDa molecular mass range. Normal villous trophoblast tissue (Lane 1) and PE trophoblast tissue (Lane 2) showed almost the same staining intensity, whereas in IUGR tissue (Lane 3), ERß expression is reduced.
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Discussion |
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There are several reports on NO and its fetoplacental synthases (NOS) distribution in normal and preeclamptic placentas. PE is characterized by hypertension, edema, and proteinuria and affects 510% of all pregnancies (Bartl and Muller-Tyl 1985
). PE is associated with IUGR and impaired uterine blood flow (Schonfelder et al. 2004
; Takagi et al. 2004
; Torry et al. 2004
). IUGR, on the other hand, is not necessarily associated with PE symptoms such as hypertension and proteinuria. In PE, fetoplacental NOS activity and NO concentrations in the umbilical circulation are altered. Some studies have described a decreased or unchanged placental NOS activity in preeclampsia (Lee et al. 1997
; Nasiell et al. 1998
; Faxen et al. 2001
; Schonfelder et al. 2004
). Elevated nitrite/nitrate concentrations in umbilical vein blood from PE patients compared with control patients have also been demonstrated (Lyall et al. 1995
; Norris et al. 1999
), suggesting an increase of NO production in the fetoplacental unit in PE. NO in the fetoplacental circulation is derived from eNOS activity, found predominantly in the syncytiotrophoblast (Lyall et al. 1995
; Norris et al. 1999
; Ayuk et al. 2002
). Ayuk and coworkers described investigations on L-arginine as precursor for NO synthesis. They found no differences of the transport systems of L-arginine in relation to NO production in normal-term pregnancies or those complicated by IUGR or PE (Ayuk et al. 2002
; Speake et al. 2003
). Their findings lead to speculate that decreased or lowered NO concentration is not the result of a reduced substrate concentration in PE or IUGR. In this study, we identified only moderate elevated eNOS expression in the syncytiotrophoblast of PE placentas, although differences were not significantly different from normal controls. Differences in eNOS expression were significant between IUGR placentas and normal controls and IUGR placentas and PE placentas. Both iNOS and eNOS expression is reduced in IUGR placentas, especially in the extravillous trophoblast (Figures 1F and Figure 2F). In addition, Witlin et al. demonstrated that L-NAMEtreated rats show increased decidual necrosis and deficient fetal vessel development. Application of adrenomedullin did not ameliorate hypertension or growth restriction (Witlin et al. 2003
).
Bukovsky and coworkers recently demonstrated both in vivo and in vitro that ER+/ERß trophoblast cells differentiate into ER
+/ERß+ in the first trimester of pregnancy, changing into ER
/ERß+ in mature states. They demonstrated a dynamic and maturity-dependent expression of ER
and ERß (Bukovsky et al. 2003a
,b
). In the present study, we identified a raised expression of ER
/ERß in PE placentas but a reduced ERß expression in IUGR placentas compared with normal controls, but without statistical significance. ER
is known to play an important role in the proliferation and so does ERß in the maturation of estrogen-dependent cells (Bukovsky et al. 2003a
,b
). Bukovsky et al. also stated that significantly enhanced expression of ERß in differentiating trophoblast cells and stimulation of trophoblast differentiation by estrogens, indicates a unique role of the ERß hormone-binding domain in the regulation of placental function (Bukovsky et al. 2003a
,b
). Because the trophoblast is a major source of placental hormones, ERß expression by trophoblast cells may be involved in stimulation of placental hormonal production by estrogens.
In summary, we demonstrated reduced ERß, iNOS, and eNOS expression in trophoblast cells in placentas of growth-restricted pregnancies. Regarding these two findings, one may speculate that ER trophoblast differentiation in pathological pregnancies is altered. Whether reduced iNOS/eNOS expression results in altered differentiation of ER to ERß or if an unchanged status of ER expression results in reduced expression of iNOS/eNOS merits further investigation. The significant association between ER
and NOS in normal pregnancies suggest an important role in the establishment of the fetoplacental unit and the physiological ongoing of pregnancy. This is underlined by the lack of association between IUGR and PE placentas and might suggest that during pathogenesis of IUGR and PE, the normal relation between ER and NOS is disrupted, although additional data are still needed.
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Footnotes |
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Literature Cited |
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Ali SH, O'Donnell AL, Balu D, Pohl MB, Seyler MJ, Mohamed S, Mousa S, et al. (2000) Estrogen receptor-alpha in the inhibition of cancer growth and angiogenesis. Cancer Res 60:70947098
Ayuk PT, Theophanous D, D'Souza SW, Sibley CP, Glazier JD (2002) L-arginine transport by the microvillous plasma membrane of the syncytiotrophoblast from human placenta in relation to nitric oxide production: effects of gestation, preeclampsia, and intrauterine growth restriction. J Clin Endocrinol Metab 87:747751
Bartl W, Muller-Tyl E (1985) Placental morphology and clinical correlations in pregnancies complicated by hypertension. Biol Res Pregnancy Perinatol 6:173176[Medline]
Baschat AA, Hecher K (2004) Fetal growth restriction due to placental disease. Semin Perinatol 28:6780[CrossRef][Medline]
Beinder E, Mohaupt MG, Schlembach D, Fischer T, Sterzel RB, Lang N, Baylis C (1999) Nitric oxide synthase activity and Doppler parameters in the fetoplacental and uteroplacental circulation in preeclampsia. Hypertens Pregnancy 18:115127[Medline]
Brosens I, Robertson WB, Dixon HG (1967), The physiological response of the vessels of the placental bed to normal pregnancy. J Pathol Bacteriol 93:569579[Medline]
Buhimschi I, Yallampalli C, Dong YL, Garfield RE (1995) Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol 172:15771584[CrossRef][Medline]
Bukovsky A, Caudle MR, Cekanova M, Fernando RI, Wimalasena J, Foster JS, Henley DC, et al. (2003a) Placental expression of estrogen receptor beta and its hormone binding variantcomparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells. Reprod Biol Endocrinol 1:36[CrossRef][Medline]
Bukovsky A, Cekanova M, Caudle MR, Wimalasena J, Foster JS, Henley DC, Elder RF (2003b) Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol. Reprod Biol Endocrinol 1:13[CrossRef][Medline]
Chatelain P (2000) Children born with intra-uterine growth retardation (IUGR) or small for gestational age (SGA): long term growth and metabolic consequences. Endocr Regul 34:3336[Medline]
Di Paolo S, Volpe P, Grandaliano G, Stallone G, Schena A, Greco P, Resta L, et al. (2003) Increased placental expression of tissue factor is associated with abnormal uterine and umbilical Doppler waveforms in severe preeclampsia with fetal growth restriction. J Nephrol 16:650657[Medline]
Eis AL, Brockman DE, Pollock JS, Myatt L (1995) Immunohistochemical localization of endothelial nitric oxide synthase in human villous and extravillous trophoblast populations and expression during syncytiotrophoblast formation in vitro. Placenta 16:113126[CrossRef][Medline]
Faxen M, Nisell H, Kublickiene KR (2001) Altered mRNA expression of ecNOS and iNOS in myometrium and placenta from women with preeclampsia. Arch Gynecol Obstet 265:4550[CrossRef][Medline]
Galan HL, Hussey MJ, Barbera A, Ferrazzi E, Chung M, Hobbins JC, Battaglia FC (1999) Relationship of fetal growth to duration of heat stress in an ovine model of placental insufficiency. Am J Obstet Gynecol 180:12781282[Medline]
Galan HL, Regnault TR, Le Cras TD, Tyson RW, Anthony RV, Wilkening RB, Abman SH (2001) Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model of fetal growth restriction. J Appl Physiol 90:24202426
Izumi H, Makino Y, Shirakawa K, Garfield RE (1995) Role of nitric oxide on vasorelaxation in human umbilical artery. Am J Obstet Gynecol 172:14771484[CrossRef][Medline]
Lee CN, Chang SW, Cho NH, Cho SH (1997) Nitrous oxide synthase expression in placenta of preeclampsia. J Korean Med Sci 12:532538[Medline]
Lyall F, Greer IA, Young A, Myatt L (1996) Nitric oxide concentrations are increased in the feto-placental circulation in intrauterine growth restriction. Placenta 17:165168[Medline]
Lyall F, Young A, Greer IA (1995) Nitric oxide concentrations are increased in the fetoplacental circulation in preeclampsia. Am J Obstet Gynecol 173:714718[CrossRef][Medline]
Macara L, Kingdom JC, Kaufmann P, Kohnen G, Hair J, More IA, Lyall F, et al. (1996) Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 17:3748[Medline]
Myatt L, Brockman DE, Eis AL, Pollock JS (1993) Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta 14:487495[Medline]
Myatt L, Eis AL, Brockman DE, Greer IA, Lyall F (1997) Endothelial nitric oxide synthase in placental villous tissue from normal, pre-eclamptic and intrauterine growth restricted pregnancies. Hum Reprod 12:167172[CrossRef]
Myatt L, Miodovnik M (1999) Prediction of preeclampsia. Semin Perinatol 23:4557[Medline]
Nasiell J, Nisell H, Blanck A, Lunell NO, Faxen M (1998) Placental expression of endothelial constitutive nitric oxide synthase mRNA in pregnancy complicated by preeclampsia. Acta Obstet Gynecol Scand 77:492496[CrossRef][Medline]
Norris LA, Higgins JR, Darling MR, Walshe JJ, Bonnar J (1999) Nitric oxide in the uteroplacental, fetoplacental, and peripheral circulations in preeclampsia. Obstet Gynecol 93:958963
Papageorghiou AT, Yu CK, Bindra R, Pandis G, Nicolaides KH (2001) Multicenter screening for pre-eclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks of gestation. Ultrasound Obstet Gynecol 18:441449[CrossRef][Medline]
Papageorghiou AT, Yu CK, Cicero S, Bower S, Nicolaides KH (2002) Second-trimester uterine artery Doppler screening in unselected populations: a review. J Matern Fetal Neonatal Med 12:7888[Medline]
Pijnenborg R, Anthony J, Davey DA, Rees A, Tiltman A, Vercruysse L, van Assche A (1991) Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol 98:648655[Medline]
Pijnenborg R, Bland JM, Robertson WB, Brosens I (1983) Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy. Placenta 4:397413[Medline]
Purcell TL, Buhimschi IA, Given R, Chwalisz K, Garfield RE (1997) Inducible nitric oxide synthase is present in the rat placenta at the fetal-maternal interface and decreases prior to labour. Mol Hum Reprod 3:485491[Abstract]
Schonfelder G, Fuhr N, Hadzidiakos D, John M, Hopp H, Paul M (2004) Preeclampsia is associated with loss of neuronal nitric oxide synthase expression in vascular smooth muscle cells of the human umbilical cord. Histopathology 44:116128[CrossRef][Medline]
Speake PF, Glazier JD, Ayuk PT, Reade M, Sibley CP, D'Souza SW (2003) L-Arginine transport across the basal plasma membrane of the syncytiotrophoblast of the human placenta from normal and preeclamptic pregnancies. J Clin Endocrinol Metab 88:42874292
Takagi Y, Nikaido T, Toki T, Kita N, Kanai M, Ashida T, Ohira S, et al. (2004) Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction. Virchows Arch 444:4955[CrossRef][Medline]
Torry DS, Hinrichs M, Torry RJ (2004) Determinants of placental vascularity. Am J Reprod Immunol 51:257268[CrossRef][Medline]
Witlin AG, Gangula PR, Wimalawansa SJ, Grafe M, Grady JJ, Yallampalli C (2003) Adrenomedullin requires an intact nitric oxide system to function as an endogenous vasodilator in rat gestation. Hypertens Pregnancy 22:924[CrossRef][Medline]
Yagel S, Anteby EY, Shen O, Cohen SM, Friedman Z, Achiron R (1999) Placental blood flow measured by simultaneous multigate spectral Doppler imaging in pregnancies complicated by placental vascular abnormalities. Ultrasound Obstet Gynecol 14:262266[CrossRef][Medline]
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