Arch Gynecol Obstet DOI 10.1007/s00404-014-3473-4
MATERNAL-FETAL MEDICINE
Expression of AP-2c in placentas of patients with preeclampsia and of smokers Hannah A. Schneider • Ulrich Gembruch • Rolf Fimmers • Jo¨rg Schmitz • Annette M. Mu¨ller
Received: 23 December 2013 / Accepted: 12 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Introduction There is evidence of a probable key role of the activator protein-2 c (AP-2c) in placental development. It is still an open question whether AP-2c expression may be influenced by preeclampsia, which is a serious pregnancy complication, or by smoking, which has deleterious effects on trophoblastic development. Material and Methods Thus, the expression of AP-2c was studied in trophoblastic epithelium and endothelium of placentas from patients with preeclampsia (n = 43) and smokers (n = 45) as well as placentas of healthy pregnant women (control group, n = 26) between gestational ages 23 and 43 weeks. To allow differential expression in primary, secondary and tertiary villi, AP-2c expression (arbitrary units) was determined immunohistologically. Results In preeclamptic placentas trophoblastic as well as endothelial cells AP-2c expression was significantly higher compared to that in control placentas. Endothelial AP-2c expression in placentas from smokers was similar to that of H. A. Schneider A. M. Mu¨ller (&) Department of Pediatric Pathology, University Clinic Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany e-mail: [email protected] H. A. Schneider Alexianer Hospital, 52064 Aachen, Germany U. Gembruch Department of Gynecology and Obstetrics, University Clinic Bonn, Bonn, Germany R. Fimmers Institue of Medicinal Biometrics, Informatics and Epidemiology, University Clinic Bonn, Bonn, Germany J. Schmitz Institute of Pathology, Grevenbroich, Germany
healthy women while trophoblastic AP-2c expression in smokers’ placenta was insignificantly higher compared to that of control placentas. In all three groups expression rates of AP-2c did not differ between primary, secondary and tertiary villi. Conclusion A correlation between increased trophoblastic and endothelial AP-2c expression in patients with preeclampsia and reduced trophoblastic invasion and migration in preeclampsia has to be discussed. Furthermore, increased AP-2c expression may play a protective role in preeclampsia, protecting from raised blood pressure. The tendency of an enhanced trophoblastic AP-2c expression in smokers may indicate a compensatory response to the disturbed balance between proliferation and differentiation of villi induced by smoking. Keywords Activator protein 2- c AP-2c Preeclampsia Smoker Placenta
Introduction The transcription factor activator protein-2 c (AP-2c) belongs to the family of DNA binding proteins which in humans consists of the five proteins: AP-2a, AP-2b, AP2c, AP-2d, AP-2e. DNA binding results in transcriptional stimulation or inhibition [1–3]. The AP-2 family plays a key role during embryonic development [4] where it induces cell type-specific stimulation of proliferation and suppression of terminal differentiation [3]. AP-2c is essential for the formation of tropho-ectodermal cells following implantation [5]. Heterozygote mice with a mutation in one AP-2c allele are viable and fertile but have a reduced body size. In mice with a complete loss of AP-2c, intrauterine growth is
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retarded and embryos die between days 7 and 9 [6]. An immunohistochemical analysis revealed in these embryos a lost proliferation of normally AP-2c expressing trophoectodermal cells. Hence, embryonal death is caused by insufficient placental nutrition of the developing embryo. Moreover, in blastocyte cultures AP-2c is involved in the regulation of the adenosine deaminase gene. This gene is expressed at the materno-fetal interface and is important in purine metabolism. Summing up, AP-2c steers genetic programs controlling proliferation and differentiation of extra-embryonic tropho-ectodermal cells [6]. Furthermore, AP-2c is enriched in the trophoblast lineage where its gene expression is regulated by the human transcription factors Sp1 and Sp2 [7]. Like AP-a, AP-2c stimulates promotor activities for human placental lactogen and human chorion gonadotropin in human cytotrophoblasts. Whereas mRNA of AP-a increases, mRNA of AP2c decreases during differentiation [8]. Trophoblast invasion in primary extravillous trophoblasts in the presence of epidermal growth factor (EGF) was reduced when AP-2c was inhibited by small interfering RNA (siRNA). This indicates a functional role of AP-2c for human trophoblast invasion in placentas of the first trimester [9]. Preeclampsia is a serious complication during pregnancy and a major cause of maternal mortality (15–20 % in developed countries) and morbidities (acute and long term), perinatal deaths, preterm birth, and intrauterine growth restriction [10]. Although its etiology has not yet been completely elucidated, it is believed to be an immune maladaptation at the implantation site, which impairs the invasion of extravillous trophoblast [11]. Recently, elevated expressions of AP-2-a and AP-2c were found in preeclamptic placentas in comparison to age-matched control placentas [11]. Smoking induces deleterious effects on the placenta and here the trophoblast resulting in placenta insufficiency, fetal growth retardation and various malformations [12]. Women smoking actively or passively carry a higher risk of spontaneous abortion, preterm birth, lower birth weight or premature rupture of membrane [13]. It is well known that smoking disturbs the balance between trophoblast proliferation and differentiation [14]. The numbers of trophoblast columns and proliferating trophoblast cells are diminished whereas the number of cell isles is increased [15]. Already in the first trimester trophoblast basal membrane is thickened, the collagen content increased and vascularization reduced. In addition to these morphological changes, metabolic and enzymatic alterations of the placenta are associated with smoking [16]. Besides, benzopyren, a component of cigarette smoke, acts as anti-proliferative agent on trophoblast stem cells [17]. Interestingly, despite its many adverse effects on fetal growth and development, smoking is also
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known to consistently reduce the risk of preeclampsia and gestational hypertension [18]. Due to the numerous effects of smoking on trophoblast development and the important role of AP-2c in placenta development, we wondered whether the expression of this activator protein may be influenced in trophoblastic epithelial and in endothelial cells in placentas of smokers and likewise its expression in placentas from patients with preeclampsia, paying interest on differences in trophoblastic and endothelial AP-2c expression. Using immunolocalization allowed the differentiation of AP-2c expression in trophoblast and endothelium of primary, secondary and tertiary villi.
Materials and methods Tissue samples Histological slices for immunohistochemical staining were prepared from paraffin-embedded placentas (of gestational weeks 23–43). The samples were taken from a control group (healthy pregnant women without pregnancy-related complications) and time-adequate placental development (n = 26), from patients with preeclampsia (n = 43) and from smokers (n = 45). All samples were processed and stained simultaneously. For immunohistochemical staining the primary antibody Ap2c (1:500, microwave) from Santa Cruz Biotechnology (Heidelberg, Germany) was used. Further reagents were citrate buffer (pH 6.0), protease no. 6911, hematoxylin Mayer (Sigma-Aldrich, St. Gallen, Switzerland), antibody diluent S2022, buffer kit K5006, detection kit K5003, horseradish peroxidase S2023 (all from Dako, Hamburg, Germany), and Aquatex (Merck, Darmstadt, Germany). Tissue processing Placental tissue was fixed in formaldehyde, embedded in paraffin according to well-established protocols. Serial sections were obtained (Mikrotom, Rotationsmikrotom HM 66 355; MICROM Laborgera¨te GmbH), collected on coated slides (SuperFrostÒ Plus; Menzel), dewaxed, dehydrated in ascending concentrations of ethanol, and placed in citrate buffer. To unmask antigens, an antigen retrieval procedure was performed by treating the samples in a microwave oven at 600 W. After cooling at room temperature, the sections were washed in phosphate-buffered saline and blocked with normal human serum. After 30 min incubation with blocking solution, sections were incubated in a moist chamber with the appropriate antibody against AP-2c. Before washing avidin was added, which binds selectively to biotin. After another wash step, color
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was developed (Vectastatin Elite ABC Kit S 74 standard; PK 6100 Firma Vector). The resulting signal was developed with diaminobenzidine (DAB). For computer analysis from all placentas 12 details representing the different villi types were photographed (magnification 209). Photographs were taken from all specimens using a microscope with integrated camera (Carl Zeiss Axioskop, Leica DM 3000), using for all photos the same magnification (209), and saved as BMP (24 bits per pixel). Immunohistochemical expression intensity of AP-2c was determined using computer-assisted image analysis as this method allows to register unbiased even minimal variations in expression intensity in a standardized fashion. The image analysis (Delphi 6.0 based, developed by J.S) allows (following white background balance) an optical densitometry of the protein resp. and antibody expression (=brown staining). Besides the unbiased analysis this program allows the detection and analysis even of very weak staining results resp protein expressions which would escape the detection resp analysis of intensity by the human eye. As stained regions of interest (trophoblast, endothelium) in primary, secondary and tertiary villi were marked semi-automatically even weak protein staining could be taken into account. A mean grey value per region was used to scale the staining intensity. The grey values of the three channels red, green and blue (RGB) were weighted so that the brown shade of the immune-histochemical staining color equaled maximum contrast while blue (=staining of the nuclei) was widely suppressed. Staining intensity was indicated by arbitrary units, defined as ratio of staining intensity to computer-determined reference value. For all preparations the mean values of two runs each of endothelial and trophoblast epithelial staining intensity measurements were calculated. Statistical analysis Statistical analysis was performed using IBM SPSS statistics program (version 19). AP-2c expression in trophoblast epithelium and endothelial cells in the different patient groups was described as median and 25 and 75 % quartiles, as mean values, standard deviation and 95 % upper and lower limits of the means. Expression was determined separately for primary, secondary and tertiary villi. AP-2y values were compared between patient groups with analysis of variance followed by pairwise comparison with Tukeys method (Fig. 1). Differences between primary, secondary and tertiary villi were evaluated by multivariate analysis of variance. For all statistical analyses level of significance was p \ 0.05.
Fig. 1 Immune-histochemical staining with the antibody against AP2c in a placenta from a smoker: the cytoplasm and nuclei of a villous trophoblast epithelium and endothelium show––according to digital estimation—an intermediate AP-2c expression
Results Trophoblast epithelium The trophoblastic AP-2c expression intensity in control placentas ranged from 3 to 387. The median AP-2c expression intensity in these controls was 89, while it was 186 in preeclampsia (p = 0.002) (Fig. 2). In placentas from smokers median AP-2c expression was 136. This increase in comparison to control placentas did not reach statistical significance (p = 0.312) (Fig. 2). The AP-2c expression did not vary between primary, secondary and tertiary villi (median values of expression rates 90, 39 and 89, respectively) in placentas from control patients. In placentas of preeclampsia patients, the AP-2c expression in tertiary villi (median 266) was insignificantly higher compared to the undifferentiated analysis (median 186). Within the smokers group AP-2c expression was nearly identical in all villous subtypes (Fig. 2). Endothelial cells The endothelial AP-2c expression intensity in control placentas ranged from 3 to 142. The median endothelial AP-2c expression intensity in preeclamptic placentas (115) was almost twice as high as in control placentas (55) (p = 0.011) while median endothelial AP-2c expression of smokers’ placentas (72) was nearly the same as that of control placentas (p = 0.793) (Fig. 3). Like in trophoblast epithelium the endothelial AP-2c expression did not vary between primary, secondary and tertiary villi (median values of expression rates were 49, 59 and 46, respectively) in placentas from control patients. In placentas of
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Discussion In comparison to placentas from healthy women, AP-2c expression was increased in endothelium and trophoblast epithelium of preeclamptic placentas. The enhanced
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Fig. 3 Boxplots of AP-2c expression in endothelia of placentas from control group (CTRL), patients with preeclampsia (Preecl) and smokers (Smok). Graphs show median values, upper whiskers as 75 % ? 1.5 interquartile range (IQR) and lower whiskers as 25% quartiles - 1.5 IQR for all villi (undifferentiated), primary (prim), secondary (sec) and tertiary (tert) villi. AP-2c expression in preeclampsia was increased versus control group (p\0.05). There was no change versus controls in smokers. AP-2c expression did not vary between primary, secondary and tertiary villi
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Fig. 2 AP-2c expression in trophoblast epithelium of placentas from the control group (CTRL), patients with preeclampsia (Preecl) and smokers (Smok) with median values, upper whiskers as 75 % ? 1.5 interquartile range (IQR) and lower whiskers as 25 % quartiles - 1.5 IQR for all villi (undifferentiated), primary (prim), secondary (sec) and tertiary (tert) villi: AP-2c expression in preeclampsia was increased versus control group (p \ 0.05). There was a nonsignificant (n.s.) alteration versus controls in smokers. AP-2c expression did not vary between primary, secondary and tertiary villi
expression in trophoblasts as well as in endothelial cells could be demonstrated for all types of villi. Higher AP-2a and AP-2c expression in preeclamptic placentas in comparison to time-matched controls was also reported by Kotani et al. [11]. These authors studied preeclamptic placentas of similar gestation age (pregnancy weeks 33.3 ± 2.7; mean ± SEM of n = 12) as in our study. However, they did not differentiate between endothelium and trophoblast epithelium. According to them AP-2c causes a reduction of the migratory and invasive properties of the trophoblastic cells, which is a prerequisite for vascular remodeling in the developing placenta. Furthermore, Kotani et al. [11] discuss tumor necrosis factor (TNF)-a as
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a potential inducer of increased AP-2 expression in preeclampsia, as stimulation by TNF-a led to higher trophoblast expression of AP-2a and AP-2c in comparison to unstimulated HTR-8/SVneo cells. An elevated TNF-a
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concentration in serum in preeclampsia and a higher concentration in preeclamptic decidua than in serum has been described [19–21]. Another functional aspect of AP-2 during pregnancy and especially in preeclampsia was evaluated by Nomura et al.
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[22]. They studied gene regulation and physiological function of oxytocinase, an enzyme predominantly expressed in trophoblasts and syncytio-trophoblasts. Oxytocinase physiologically degrades uterotonic oxytocin but also contributes to hydrolysis of various peptides like vasopressin and angiotensin III. Thus, this enzyme plays a role in blood pressure control during pregnancy. The activity of oxytocinase increases in the course of a normal pregnancy but is diminished in patients with severe preeclampsia. As a consequence the degradation of oxytocin but also of vasopressin and angiotensin III is impaired leading to a disturbance in the homeostasis of vasoconstrictor peptides. Elevated concentrations of vasopressin and angiotensin III are suspected to contribute to development of hypertension which is a clinical hallmark of preeclampsia. An elevated AP-2 c expression in preeclamptic placentas as found in our study and also reported by Kotani et al. [11] is of importance in the context of an increased promoter activity of oxytocinase in trophoblast cells by AP-2. Hence, an increased AP-2 expression during preeclampsia would counteract the reduced oxytocinase activity. Thus, it may compensate for the overweight of vasoconstrictory peptides and constitute a protective mechanism of the placenta. The deleterious effects of smoking on placenta and trophoblast with increased risk for spontaneous miscarriage, premature birth, low birth weight, preterm placenta detachment and death births after active and passive smoking of pregnant women are well known [12, 13]. Smoking during pregnancy disturbs the balance between trophoblastic proliferation and differentiation as trophoblast columns and proliferating trophoblasts are reduced, whereas the number of isle cells is increased [15]. In addition, metabolic and enzymatic alterations of the placenta are associated with smoking, as e.g. changes in endothelial nitric oxide synthase, decreased flow of uterine blood to the placenta, increased calcification in term placentas, alterations in progesterone production, estrogen metabolism, amino acid transport and activity of drugmetabolizing enzymes. Nicotine, as only one toxic substance of the cigarette contents, suppresses amino acid uptake in placenta villi and inhibits trophoblast invasion. Interestingly, AP-2c expression in trophoblastic epithelium of smokers was increased versus control patients by 60 %. Although this alteration did not reach statistical significance, it indicates a marked tendency towards elevated AP-2c in all types of chorionic villi of smokers. Thus far, no other studies on the influence of smoking on placenta AP-2 expression have yet been reported. The transcription factor AP-2c induces effects on the placenta which are opposite in many aspects to those triggered by smoking. AP-2 is essential for placenta development as it steers proliferation and differentiation of tropho-
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ectodermal cells [6] and stimulates cell-specific proliferation and suppresses terminal differentiation [3, 4]. Smoking disturbs the balance between villous proliferation and differentiation which may cause placenta insufficiency [12]. Various components of cigarette smoke, especially benzpyren and its derivatives [17] are discussed causing inhibition of placenta proliferation [15]. While opposite effects of AP-2 and smoking on proliferation and differentiation of placenta preclude a causal relation of the observed increase in AP-2c expression to the deleterious effect of smoking on placenta morphology and function, on the contrary, the increased AP-2c expression in placentas of smokers has to be discussed as a reaction to prevent or to diminish the negative effects of cigarette smoke on placenta development. As with preeclampsia, smoking too, leads to placental hypoxia [15, 23–26]. The increased expression of AP-2c in patients with preeclampsia and in smokers can be interpreted as reaction to placental hypoxia as AP-2c-induced trophoblast proliferation may thus counteract diminished oxygen supply. Increased serum concentrations of serum TNF-a are found in smokers and during preeclampsia [20, 21, 27, 28]. As the inflammatory marker TNF-a can induce AP-2a as well as AP-2c expression in the trophoblastic cell line HTR-8/SVneo [11], TNF-a should be discussed as an activator candidate for the observed stimulated-AP-2c expression in placentas of smokers. Conflict of interest of interests.
The authors declare that they have no conflict
References 1. Williams T, Tjian R (1991) Characterization of a dimerization motif in AP-2 and its function in heterologous DNA-binding proteins. Science 251:1067–1071 2. Wankhade S, Yu Y, Weinberg J, Tainsky MA, Kannan P (2000) Characterization of the activation domains of AP-2 family transcription factors. J Biol Chem 275(38):29701–29708 3. Eckert D, Buhl S, Weber S et al (2005) The AP-2 family of transcription factors. Genome Biol 6:246 4. Hilger-Eversheim K, Moser M, Schorle H, Buettner R (2000) Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control. Gene 260:1–12 5. Auman HJ, Nottoli T, Lakiza O et al (2002) Transcription factor AP-2 gamma is essential in the extraembryonic lineages for early postimplantation development. Development 129:2733–2747 6. Werling U, Schorle H (2002) Transcription factor gene AP-2c essential for early murine development. Mol Cell Biol 22(9): 3149–3156 7. Li M, Kellems RE (2003) Sp1 and Sp3 are important regulators of AP-2c gene transcription. Biol Repro 69(4):1220–1230 8. Richardson BD, Cheng Y-H, Langland RA, Handwerger S (2001) Differential expression of AP-2c and AP-2a during human trophoblast differentiation. Life Sci 69(18):2157–2165 9. Biadasiewicz K, Sonderegger S, Haslinger P et al (2011) Transcription Factor AP-2c promotes EGF-dependent invasion of human trophoblast. Endocrinology 152:1458–1469
Arch Gynecol Obstet 10. Sibai B, Dekker G, Kupferminc M (2005) Pre-eclampsia. Lancet 365:785–799 11. Kotani T, Iwase A, Ino K et al (2009) Activator protein-2 impairs the invasion of a human extravillous trophoblast cell line. Endocrinol 150:4276–4285 12. Einarson A, Riordan S (2009) Smoking in pregnancy and lactation: a review of risks and cessation strategies. Eur J Clin Pharmacol 65:325–330 13. Rodriguez-Thompson D (2012) Smoking and pregnancy. Up To DateÒ 19.3 Jan 2012 14. Genbacev O, Bass KE, Joslin RJ, Fisher SJ (1995) Maternal smoking inhibits early human cytotrophoblast differentiation. Reprod Toxicol 9:245–255 15. Zdravkovic T, Genbacev O, McMaster MT, Fisher SJ (2005) The adverse effects of maternal smoking on the human placenta: a review. Placenta 26(Suppl A):S81–S86 16. Janiaux E, Burton GJ (2007) Morphological and biological effects of maternal exposure to tobacco smoke on the feto-placental unit. Early Hum Dev 83(11):699–706 17. Rappolee DA, Awonuga AO, Puscheck EE (2010) Benzopyrene and experimental stressors cause compensatory differentiation in placental trophoblast stem cells. Syst Biol Repro Med 56:168–183 18. Karumanchi SA, Levine RJ (2010) How does smoking reduce the risk of preeclampsia? Hypertension 55(5):1100–1101 19. Lockwood CJ, Yen CF, Basar M, Kayisli UA, Martel M, Buhimschi M, Buhimschi C, Huang SJ, Krikun G, Schatz F (2008) Preeclampsia-related inflammatory cytokines regulate
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interleukin-6 expression in human decidual cells. Am J Pathol 172:1571–1579 Xie C, Yao MZ, Liu JB, Xiong LK (2011) A meta-analysis of tumor necrosis factor alpha, interleukin-6, and interleukin-10 in preeclampsia. Cytokine 56(3):550–559 Zhou P, Luo X, Qi HB et al (2012) The expression of pentraxin 3 and tumor necrosis factor-alpha is increased in preeclamptic placental tissue and maternal serum. Inflamm Res 61(9):1005–1012 Nomura S, Ito T, Yamamoto E et al (2005) Gene regulation and physiological function of placental leucine aminopeptidase/oxytocinase during pregnancy. Biochim Biophys Acta 1751(1):19–25 Collet M, Beillard C (2005) Consequences of smoking on fetal development and risk of intra-uterine growth retardation or in utero fetal death. J Gynecol Obstet Biol Reprod 34(1):3S135–3S145 Shea AK, Steiner M (2008) Cigarette smoking during pregnancy. Nicotine Tob Res 10(2):267–278 Lorquet S, Pequeux C, Munaut C, Foidart JM (2010) Aetiology and physiopathology of preeclampsia and related forms. Acta Clin Belg 65(4):237–241 George EM, Granger JP (2011) Mechanisms and potential therapies for preeclampsia. Curr Hypertens Rep 13(4):269–275 Petrescu F, Voican SC, Silosi S (2010) Tumor necrosis factor-a serum levels in healthy smokers and nonsmokers. Int J Chron Obstruct Pul Dis 5:217–222 Nunes SO, Vargas HO, Brum J et al (2011) A comparison of inflammatory markers in depressed and nondepressed smokers. Nicotine Tob Res 14(5):540–546
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MATERNAL-FETAL MEDICINE
Expression of AP-2c in placentas of patients with preeclampsia and of smokers Hannah A. Schneider • Ulrich Gembruch • Rolf Fimmers • Jo¨rg Schmitz • Annette M. Mu¨ller
Received: 23 December 2013 / Accepted: 12 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Introduction There is evidence of a probable key role of the activator protein-2 c (AP-2c) in placental development. It is still an open question whether AP-2c expression may be influenced by preeclampsia, which is a serious pregnancy complication, or by smoking, which has deleterious effects on trophoblastic development. Material and Methods Thus, the expression of AP-2c was studied in trophoblastic epithelium and endothelium of placentas from patients with preeclampsia (n = 43) and smokers (n = 45) as well as placentas of healthy pregnant women (control group, n = 26) between gestational ages 23 and 43 weeks. To allow differential expression in primary, secondary and tertiary villi, AP-2c expression (arbitrary units) was determined immunohistologically. Results In preeclamptic placentas trophoblastic as well as endothelial cells AP-2c expression was significantly higher compared to that in control placentas. Endothelial AP-2c expression in placentas from smokers was similar to that of H. A. Schneider A. M. Mu¨ller (&) Department of Pediatric Pathology, University Clinic Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany e-mail: [email protected] H. A. Schneider Alexianer Hospital, 52064 Aachen, Germany U. Gembruch Department of Gynecology and Obstetrics, University Clinic Bonn, Bonn, Germany R. Fimmers Institue of Medicinal Biometrics, Informatics and Epidemiology, University Clinic Bonn, Bonn, Germany J. Schmitz Institute of Pathology, Grevenbroich, Germany
healthy women while trophoblastic AP-2c expression in smokers’ placenta was insignificantly higher compared to that of control placentas. In all three groups expression rates of AP-2c did not differ between primary, secondary and tertiary villi. Conclusion A correlation between increased trophoblastic and endothelial AP-2c expression in patients with preeclampsia and reduced trophoblastic invasion and migration in preeclampsia has to be discussed. Furthermore, increased AP-2c expression may play a protective role in preeclampsia, protecting from raised blood pressure. The tendency of an enhanced trophoblastic AP-2c expression in smokers may indicate a compensatory response to the disturbed balance between proliferation and differentiation of villi induced by smoking. Keywords Activator protein 2- c AP-2c Preeclampsia Smoker Placenta
Introduction The transcription factor activator protein-2 c (AP-2c) belongs to the family of DNA binding proteins which in humans consists of the five proteins: AP-2a, AP-2b, AP2c, AP-2d, AP-2e. DNA binding results in transcriptional stimulation or inhibition [1–3]. The AP-2 family plays a key role during embryonic development [4] where it induces cell type-specific stimulation of proliferation and suppression of terminal differentiation [3]. AP-2c is essential for the formation of tropho-ectodermal cells following implantation [5]. Heterozygote mice with a mutation in one AP-2c allele are viable and fertile but have a reduced body size. In mice with a complete loss of AP-2c, intrauterine growth is
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retarded and embryos die between days 7 and 9 [6]. An immunohistochemical analysis revealed in these embryos a lost proliferation of normally AP-2c expressing trophoectodermal cells. Hence, embryonal death is caused by insufficient placental nutrition of the developing embryo. Moreover, in blastocyte cultures AP-2c is involved in the regulation of the adenosine deaminase gene. This gene is expressed at the materno-fetal interface and is important in purine metabolism. Summing up, AP-2c steers genetic programs controlling proliferation and differentiation of extra-embryonic tropho-ectodermal cells [6]. Furthermore, AP-2c is enriched in the trophoblast lineage where its gene expression is regulated by the human transcription factors Sp1 and Sp2 [7]. Like AP-a, AP-2c stimulates promotor activities for human placental lactogen and human chorion gonadotropin in human cytotrophoblasts. Whereas mRNA of AP-a increases, mRNA of AP2c decreases during differentiation [8]. Trophoblast invasion in primary extravillous trophoblasts in the presence of epidermal growth factor (EGF) was reduced when AP-2c was inhibited by small interfering RNA (siRNA). This indicates a functional role of AP-2c for human trophoblast invasion in placentas of the first trimester [9]. Preeclampsia is a serious complication during pregnancy and a major cause of maternal mortality (15–20 % in developed countries) and morbidities (acute and long term), perinatal deaths, preterm birth, and intrauterine growth restriction [10]. Although its etiology has not yet been completely elucidated, it is believed to be an immune maladaptation at the implantation site, which impairs the invasion of extravillous trophoblast [11]. Recently, elevated expressions of AP-2-a and AP-2c were found in preeclamptic placentas in comparison to age-matched control placentas [11]. Smoking induces deleterious effects on the placenta and here the trophoblast resulting in placenta insufficiency, fetal growth retardation and various malformations [12]. Women smoking actively or passively carry a higher risk of spontaneous abortion, preterm birth, lower birth weight or premature rupture of membrane [13]. It is well known that smoking disturbs the balance between trophoblast proliferation and differentiation [14]. The numbers of trophoblast columns and proliferating trophoblast cells are diminished whereas the number of cell isles is increased [15]. Already in the first trimester trophoblast basal membrane is thickened, the collagen content increased and vascularization reduced. In addition to these morphological changes, metabolic and enzymatic alterations of the placenta are associated with smoking [16]. Besides, benzopyren, a component of cigarette smoke, acts as anti-proliferative agent on trophoblast stem cells [17]. Interestingly, despite its many adverse effects on fetal growth and development, smoking is also
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known to consistently reduce the risk of preeclampsia and gestational hypertension [18]. Due to the numerous effects of smoking on trophoblast development and the important role of AP-2c in placenta development, we wondered whether the expression of this activator protein may be influenced in trophoblastic epithelial and in endothelial cells in placentas of smokers and likewise its expression in placentas from patients with preeclampsia, paying interest on differences in trophoblastic and endothelial AP-2c expression. Using immunolocalization allowed the differentiation of AP-2c expression in trophoblast and endothelium of primary, secondary and tertiary villi.
Materials and methods Tissue samples Histological slices for immunohistochemical staining were prepared from paraffin-embedded placentas (of gestational weeks 23–43). The samples were taken from a control group (healthy pregnant women without pregnancy-related complications) and time-adequate placental development (n = 26), from patients with preeclampsia (n = 43) and from smokers (n = 45). All samples were processed and stained simultaneously. For immunohistochemical staining the primary antibody Ap2c (1:500, microwave) from Santa Cruz Biotechnology (Heidelberg, Germany) was used. Further reagents were citrate buffer (pH 6.0), protease no. 6911, hematoxylin Mayer (Sigma-Aldrich, St. Gallen, Switzerland), antibody diluent S2022, buffer kit K5006, detection kit K5003, horseradish peroxidase S2023 (all from Dako, Hamburg, Germany), and Aquatex (Merck, Darmstadt, Germany). Tissue processing Placental tissue was fixed in formaldehyde, embedded in paraffin according to well-established protocols. Serial sections were obtained (Mikrotom, Rotationsmikrotom HM 66 355; MICROM Laborgera¨te GmbH), collected on coated slides (SuperFrostÒ Plus; Menzel), dewaxed, dehydrated in ascending concentrations of ethanol, and placed in citrate buffer. To unmask antigens, an antigen retrieval procedure was performed by treating the samples in a microwave oven at 600 W. After cooling at room temperature, the sections were washed in phosphate-buffered saline and blocked with normal human serum. After 30 min incubation with blocking solution, sections were incubated in a moist chamber with the appropriate antibody against AP-2c. Before washing avidin was added, which binds selectively to biotin. After another wash step, color
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was developed (Vectastatin Elite ABC Kit S 74 standard; PK 6100 Firma Vector). The resulting signal was developed with diaminobenzidine (DAB). For computer analysis from all placentas 12 details representing the different villi types were photographed (magnification 209). Photographs were taken from all specimens using a microscope with integrated camera (Carl Zeiss Axioskop, Leica DM 3000), using for all photos the same magnification (209), and saved as BMP (24 bits per pixel). Immunohistochemical expression intensity of AP-2c was determined using computer-assisted image analysis as this method allows to register unbiased even minimal variations in expression intensity in a standardized fashion. The image analysis (Delphi 6.0 based, developed by J.S) allows (following white background balance) an optical densitometry of the protein resp. and antibody expression (=brown staining). Besides the unbiased analysis this program allows the detection and analysis even of very weak staining results resp protein expressions which would escape the detection resp analysis of intensity by the human eye. As stained regions of interest (trophoblast, endothelium) in primary, secondary and tertiary villi were marked semi-automatically even weak protein staining could be taken into account. A mean grey value per region was used to scale the staining intensity. The grey values of the three channels red, green and blue (RGB) were weighted so that the brown shade of the immune-histochemical staining color equaled maximum contrast while blue (=staining of the nuclei) was widely suppressed. Staining intensity was indicated by arbitrary units, defined as ratio of staining intensity to computer-determined reference value. For all preparations the mean values of two runs each of endothelial and trophoblast epithelial staining intensity measurements were calculated. Statistical analysis Statistical analysis was performed using IBM SPSS statistics program (version 19). AP-2c expression in trophoblast epithelium and endothelial cells in the different patient groups was described as median and 25 and 75 % quartiles, as mean values, standard deviation and 95 % upper and lower limits of the means. Expression was determined separately for primary, secondary and tertiary villi. AP-2y values were compared between patient groups with analysis of variance followed by pairwise comparison with Tukeys method (Fig. 1). Differences between primary, secondary and tertiary villi were evaluated by multivariate analysis of variance. For all statistical analyses level of significance was p \ 0.05.
Fig. 1 Immune-histochemical staining with the antibody against AP2c in a placenta from a smoker: the cytoplasm and nuclei of a villous trophoblast epithelium and endothelium show––according to digital estimation—an intermediate AP-2c expression
Results Trophoblast epithelium The trophoblastic AP-2c expression intensity in control placentas ranged from 3 to 387. The median AP-2c expression intensity in these controls was 89, while it was 186 in preeclampsia (p = 0.002) (Fig. 2). In placentas from smokers median AP-2c expression was 136. This increase in comparison to control placentas did not reach statistical significance (p = 0.312) (Fig. 2). The AP-2c expression did not vary between primary, secondary and tertiary villi (median values of expression rates 90, 39 and 89, respectively) in placentas from control patients. In placentas of preeclampsia patients, the AP-2c expression in tertiary villi (median 266) was insignificantly higher compared to the undifferentiated analysis (median 186). Within the smokers group AP-2c expression was nearly identical in all villous subtypes (Fig. 2). Endothelial cells The endothelial AP-2c expression intensity in control placentas ranged from 3 to 142. The median endothelial AP-2c expression intensity in preeclamptic placentas (115) was almost twice as high as in control placentas (55) (p = 0.011) while median endothelial AP-2c expression of smokers’ placentas (72) was nearly the same as that of control placentas (p = 0.793) (Fig. 3). Like in trophoblast epithelium the endothelial AP-2c expression did not vary between primary, secondary and tertiary villi (median values of expression rates were 49, 59 and 46, respectively) in placentas from control patients. In placentas of
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Discussion In comparison to placentas from healthy women, AP-2c expression was increased in endothelium and trophoblast epithelium of preeclamptic placentas. The enhanced
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Fig. 3 Boxplots of AP-2c expression in endothelia of placentas from control group (CTRL), patients with preeclampsia (Preecl) and smokers (Smok). Graphs show median values, upper whiskers as 75 % ? 1.5 interquartile range (IQR) and lower whiskers as 25% quartiles - 1.5 IQR for all villi (undifferentiated), primary (prim), secondary (sec) and tertiary (tert) villi. AP-2c expression in preeclampsia was increased versus control group (p\0.05). There was no change versus controls in smokers. AP-2c expression did not vary between primary, secondary and tertiary villi
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Fig. 2 AP-2c expression in trophoblast epithelium of placentas from the control group (CTRL), patients with preeclampsia (Preecl) and smokers (Smok) with median values, upper whiskers as 75 % ? 1.5 interquartile range (IQR) and lower whiskers as 25 % quartiles - 1.5 IQR for all villi (undifferentiated), primary (prim), secondary (sec) and tertiary (tert) villi: AP-2c expression in preeclampsia was increased versus control group (p \ 0.05). There was a nonsignificant (n.s.) alteration versus controls in smokers. AP-2c expression did not vary between primary, secondary and tertiary villi
expression in trophoblasts as well as in endothelial cells could be demonstrated for all types of villi. Higher AP-2a and AP-2c expression in preeclamptic placentas in comparison to time-matched controls was also reported by Kotani et al. [11]. These authors studied preeclamptic placentas of similar gestation age (pregnancy weeks 33.3 ± 2.7; mean ± SEM of n = 12) as in our study. However, they did not differentiate between endothelium and trophoblast epithelium. According to them AP-2c causes a reduction of the migratory and invasive properties of the trophoblastic cells, which is a prerequisite for vascular remodeling in the developing placenta. Furthermore, Kotani et al. [11] discuss tumor necrosis factor (TNF)-a as
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a potential inducer of increased AP-2 expression in preeclampsia, as stimulation by TNF-a led to higher trophoblast expression of AP-2a and AP-2c in comparison to unstimulated HTR-8/SVneo cells. An elevated TNF-a
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concentration in serum in preeclampsia and a higher concentration in preeclamptic decidua than in serum has been described [19–21]. Another functional aspect of AP-2 during pregnancy and especially in preeclampsia was evaluated by Nomura et al.
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[22]. They studied gene regulation and physiological function of oxytocinase, an enzyme predominantly expressed in trophoblasts and syncytio-trophoblasts. Oxytocinase physiologically degrades uterotonic oxytocin but also contributes to hydrolysis of various peptides like vasopressin and angiotensin III. Thus, this enzyme plays a role in blood pressure control during pregnancy. The activity of oxytocinase increases in the course of a normal pregnancy but is diminished in patients with severe preeclampsia. As a consequence the degradation of oxytocin but also of vasopressin and angiotensin III is impaired leading to a disturbance in the homeostasis of vasoconstrictor peptides. Elevated concentrations of vasopressin and angiotensin III are suspected to contribute to development of hypertension which is a clinical hallmark of preeclampsia. An elevated AP-2 c expression in preeclamptic placentas as found in our study and also reported by Kotani et al. [11] is of importance in the context of an increased promoter activity of oxytocinase in trophoblast cells by AP-2. Hence, an increased AP-2 expression during preeclampsia would counteract the reduced oxytocinase activity. Thus, it may compensate for the overweight of vasoconstrictory peptides and constitute a protective mechanism of the placenta. The deleterious effects of smoking on placenta and trophoblast with increased risk for spontaneous miscarriage, premature birth, low birth weight, preterm placenta detachment and death births after active and passive smoking of pregnant women are well known [12, 13]. Smoking during pregnancy disturbs the balance between trophoblastic proliferation and differentiation as trophoblast columns and proliferating trophoblasts are reduced, whereas the number of isle cells is increased [15]. In addition, metabolic and enzymatic alterations of the placenta are associated with smoking, as e.g. changes in endothelial nitric oxide synthase, decreased flow of uterine blood to the placenta, increased calcification in term placentas, alterations in progesterone production, estrogen metabolism, amino acid transport and activity of drugmetabolizing enzymes. Nicotine, as only one toxic substance of the cigarette contents, suppresses amino acid uptake in placenta villi and inhibits trophoblast invasion. Interestingly, AP-2c expression in trophoblastic epithelium of smokers was increased versus control patients by 60 %. Although this alteration did not reach statistical significance, it indicates a marked tendency towards elevated AP-2c in all types of chorionic villi of smokers. Thus far, no other studies on the influence of smoking on placenta AP-2 expression have yet been reported. The transcription factor AP-2c induces effects on the placenta which are opposite in many aspects to those triggered by smoking. AP-2 is essential for placenta development as it steers proliferation and differentiation of tropho-
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ectodermal cells [6] and stimulates cell-specific proliferation and suppresses terminal differentiation [3, 4]. Smoking disturbs the balance between villous proliferation and differentiation which may cause placenta insufficiency [12]. Various components of cigarette smoke, especially benzpyren and its derivatives [17] are discussed causing inhibition of placenta proliferation [15]. While opposite effects of AP-2 and smoking on proliferation and differentiation of placenta preclude a causal relation of the observed increase in AP-2c expression to the deleterious effect of smoking on placenta morphology and function, on the contrary, the increased AP-2c expression in placentas of smokers has to be discussed as a reaction to prevent or to diminish the negative effects of cigarette smoke on placenta development. As with preeclampsia, smoking too, leads to placental hypoxia [15, 23–26]. The increased expression of AP-2c in patients with preeclampsia and in smokers can be interpreted as reaction to placental hypoxia as AP-2c-induced trophoblast proliferation may thus counteract diminished oxygen supply. Increased serum concentrations of serum TNF-a are found in smokers and during preeclampsia [20, 21, 27, 28]. As the inflammatory marker TNF-a can induce AP-2a as well as AP-2c expression in the trophoblastic cell line HTR-8/SVneo [11], TNF-a should be discussed as an activator candidate for the observed stimulated-AP-2c expression in placentas of smokers. Conflict of interest of interests.
The authors declare that they have no conflict
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