Activating protein-1 family of transcription factors in the human placenta complicated by preeclampsia with and without fetal growth restriction

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Patients
    • 2.1.1. Control pregnancies
    • 2.1.2. Preeclamptic cases
  • 2.2. Tissues
  • 2.3. Total RNA extraction and semiquantitative RT-PCR
  • 2.4. Immunohistochemistry
  • 2.5. Statistical analysis
• 3. Results
  • 3.1. Study population
  • 3.2. Semiquantitative RT-PCR
    • 3.2.1. Normal tissues
    • 3.2.2. Pathological tissues
  • 3.3. Immunohistochemistry
    • 3.3.1. Normal tissues
    • 3.3.2. Pathological tissues
• 4. Discussion
• Acknowledgements
• References
• Copyright

Abstract 

Preeclampsia (PE) is a serious disorder of human pregnancy, it is often associated with fetal growth restriction (FGR) which is a failure of the fetus to reach its own growth potential.

Activator protein-1 (AP-1) is a family of transcription factors inducible in response to a variety of extracellular stimuli and functions. AP-1 plays a complex role in the regulation of different fundamental cellular processes, including cell proliferation, survival, death and transformation.

We investigate the expression pattern of AP-1 transcription factors in normal placentas during gestation and in placentas from PE without and with FGR using semiquantitative RT-PCR and immunohistochemistry techniques.

The most interesting data concern the alterations of protein expression patterns of c-fos, Jun D and c-jun in normal gestation as well as in PE and PE-FGR pathologies. In addition, alterations but not significant changes are detected in mRNA expressions for these transcription factors.

We strongly suggest that c-fos is implicated in regulating invasiveness mechanism of extravillous trophoblast in normal gestation as well as in PE placentas. In addition, we suggest that the opposite modulation of Jun D and c-jun in PE and PE-FGR supports the recent hypothesis that PE and PE-FGR could be considered two pathologies with different origin (maternal and placental) each of which has a different molecular pattern of expression.

Keywords: Preeclampsia, FGR, Placenta, Jun, Fos, AP-1

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1. Introduction 

Preeclampsia (PE), a complex and heterogeneous disorder of human pregnancy, is presently a leading cause of fetal and maternal morbidity and mortality worldwide, affecting ∼7% of pregnancies [1]. Clinical diagnosis and symptoms are based on sudden onset of hypertension accompanied by proteinuria and edema. Recent studies on PE show a marked proliferation of villous cytotrophoblastic cells, syncytiotrophoblastic focal necrosis and generally an ischemic placenta with high-resistance vascular bed [2], [3], [4], [5]. PE is often associated with fetal growth restriction (FGR). Failure to develop a functional placenta results in poor pregnancy outcome, ranging from FGR to fetal or perinatal death [2], [3], [4]. Recently, Redman and Sargent [6] proposed a classification of PE in two broad categories, implying different etiologies: “placental” PE, where the main feature is a maldevelopment of the placenta and “maternal” PE. In the latter group maternal metabolic and vascular modifications of pregnancy act as a challenge in a pre-existing low grade systemic inflammation.

Activator protein-1 (AP-1) is a family of transcription factors inducible in response to a variety of extracellular stimuli and functions. AP-1 family plays a complex role in the regulation of different fundamental cellular processes, including cell proliferation, survival, death and transformation [7]. It is a dimeric transcription factor composed of the products of Jun and Fos proto-oncogenes [8]. The Jun proteins (c-jun, Jun B and Jun D) can form homodimers or heterodimers with member of the Fos and ATF protein families. The Fos proteins (c-fos, Fos B, Fra-1 and Fra-2) cannot associate with each other or with ATF proteins, but form stable heterodimers with any of the Jun proteins [8], [9]. Recently, Bamberger et al. [10] showed that AP-1 transcription factors are expressed in normal human placenta where they could be implicated in regulating proliferation and differentiation of the cytotrophoblast. The purpose of this study is to investigate the expression of the AP-1 transcription factors family in placentas from PE with FGR (placental PE) and without FGR (maternal PE) in order to clarify the possible implication of these transcription factors in the pathogenesis of the diseases. For this purpose, each of the seven transcription factors was localized in placental tissues in order to determine the cell types involved in normal gestation as well as in PE pathology (fetal and maternal PE) using immunohistochemistry. In addition, RT-PCR was employed to investigate if mRNA expression was considerably modified during normal and pathological gestations.

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2. Materials and methods 

2.1. Patients 

We studied 7 first trimester, 7 second trimester and 15 third trimester placentas from normal pregnancies in order to determine the expression trend of AP-1 factors during gestation (Table 1). In addition, we studied 20 pregnancies complicated by preeclampsia (PE), 10 with and 10 without FGR. All recruited women had a singleton pregnancy and were of Caucasian race. The study was approved by Piedmont Region and Hospital Ethics Committee and informed consent was obtained from each woman.

Table 1. Clinical characteristics of normal (A) and pathologic pregnancies (B).

(A)	1st trim median (25th–75th) or N (%)	2nd trim median (25th–75th) or N (%)	3rd trim SpD median (25th–75th) or N (%)	3rd trim CSD median (25th–75th) or N (%)	p value
N	7	7	7	8
Maternal age at delivery (years)	28 (19–32)	30 (21–35)	35 (32–36)	32.5 (31–35)	n.s.
Pre-pregnancy BMI (kg/m2)	n.a.	20.4 (18.4–22.8)	19.3 (18.8–24.2)	20.4 (19.4–23.5)	n.s.
Gestational weight gain (kg)	n.a.	4* (0–5)	11* (9–13)	13 (11–14)	*0.001
Nulliparae	3 (42.9)	3 (42.9)	2* (28.6)	7* (87.5)	*0.035
Gestational age at delivery (weeks)	10*° (9–12)	16*^ (15–16)	39°^ (38–39)	38.5 (37–39)	*°^0.001
Neonatal birth weight (g)	n.a.	125* (65–140)	3220* (3100–3280)	3275 (2942.5–3540)	*0.001
Neonatal weight Z-score	n.a.	n.a.	−0.19 (−0.65 to 0.26)	−0.05 (−0.32 to 0.94)	n.s.
Placental weight (g)	n.a.	107.5* (70–125)	560* (500–560)	550 (507.5–590)	*0.006
Placental weight/neonatal weight ratio	n.a.	0.89* (0.62–0.96)	0.16* (0.16–0.21)	0.17 (0.16–0.18)	*0.006
Blood pressure (mmHg)
systolic	n.a.	110 (100–120)	110 (100–120)	110 (100–110)	n.s.
diastolic		70 (60–80)	70 (70–75)	70 (65–70)	n.s.
Family risk factors	n.a.	3 (42.9)	3 (42.9)	5 (62.5)	n.s.
Smoking mothers	0 (0)	3 (42.9)	0 (0)	0 (0)	n.s.
Patients receiving
corticosteroids	0 (0)	0 (0)	1 (14.3)	1 (12.5)	n.s.
antihypertensives	0 (0)	0 (0)	0 (0)	0 (0)	n.s.
antibiotics	0 (0)	2 (28.6)	2 (28.6)	2 (25.0)	n.s.

(B)	3rd trim CSD median (25th–75th) or N (%)	PE median (25th–75th) or N (%)	PE-FGR median (25th–75th) or N (%)	p value
N	8	10	10
Maternal age at delivery (years)	32.5 (31–35)	35 (31–37)	33.5 (28–37)	n.s.
Pre-pregnancy BMI (kg/m2)	20.4 (19.4–23.5)	22.0 (21.6–24.6)	23.7 (21.1–27.3)	n.s.
Gestational weight gain (kg)	13 (11–14)	14.3 (8.5–18)	11.3 (6.5–15)	n.s.
Nulliparae	7 (87.5)	5 (50)	8 (80)	n.s.
Gestational age at delivery (weeks)	39*° (37–39)	31* (29–33)	30° (29–31)	*°<0.001
Neonatal weight (g)	3275*° (2942.5–3540)	1425*^ (1150–1575)	995°^ (960–1260)	*°<0.001; ^0.022
Neonatal weight Z-score	−0.05*° (−0.32 to 0.94)	−1.19*^ (−1.22 to −0.75)	−2.05°^ (−2.20 to −1.61)	*^0.001; °<0.001
Placental weight (g)	550° (507.5–590)	392.5^ (250–570)	210°^ (150–250)	°<0.001; ^0.004
Placental weight/neonatal weight ratio	0.17 (0.16–0.18)*°	0.27 (0.21–0.37)*	0.21 (0.19–0.24)°	*<0.001; °0.008
Blood pressure (mmHg)
systolic	110*° (100–120)	158.5* (150–160)	145° (140–155)	*°<0.001
diastolic	70*° (70–75)	92.5* (90–100)	95° (90–100)	*°<0.001
Proteinuria	*°	*^	°^	*°<0.001; ^0.033
<1 g/24 h	0 (0)	0 (0)	5 (50)
<5 g/24 h	0 (0)	7 (70)	3 (30)
>5 g/24 h	0 (0)	3 (30)	2 (20)
Family risk factors	5 (62.5)	6 (60)	6 (60)	n.s.
Smoking mothers	0 (0)°	2 (20)	5 (50)°	°0.036
Presence of severe symptoms	n.a.	6 (60)	6 (60)	n.s.
Patients receiving
corticosteroids	1 (12.5)*°	9 (90)*	10 (100)°	*<0.001; °0.003
antihypertensives	0 (0)*°	10 (100)*	10 (100)*	*°<0.001
antibiotics	2 (25.0)	2 (20)	0 (0)	n.s.

BMI: body mass index; 1st trim: first trimester; 2nd trim: second trimester; 3rd trim: third trimester; n.a.: not available; SpD: spontaneous delivery; CSD: caesarean delivery; n.s.: not significant. *, ° and ^ symbols indicate the pair of compared values.

Term controls were normotensive pregnancies with normal fetal growth and normal uterine and umbilical Doppler flow velocimetry. Eight of them were delivered by caesarean section (CSD) and 7 had a spontaneous vaginal delivery (SpD). First and second trimester placentas were obtained from healthy patients undergoing termination of pregnancy for psychological or social reasons.

First, second and third trimester SpD and CSD placentas were analysed to evaluate the trend of AP-1 transcription factors expression in placental tissue during pregnancy.

Pathological pregnancies were included in our study according to the following criteria: 1) diagnosis of PE defined by appearance of hypertension (systolic pressure ≥ 140 mmHg or diastolic pressure ≥ 90 mmHg) accompanied by proteinuria (≥300 mg/24 h) after twenty weeks of gestational age in previously normotensive patients [11]; 2) pathologic Uterine Doppler Flow velocity waveforms, defined as the presence of bilateral notching [12].

Furthermore, pregnancies complicated by PE were divided in two subgroups:

The diagnosis of FGR was made according to the following criteria: ultrasound measurement of the fetal abdominal circumference below the 10th centile [13] or fetal growth rate below 5 mm/week at serial measurements, and/or birth weight below the 10th centile according to our birth weight references [14] with abnormal Doppler flow velocity waveforms of the uterine [12] and/or of the umbilical arteries [15].

Patients were managed according to our guidelines for the treatment of severe PE.

AP-1 expression in pathologic cases (all delivered with CSD) was compared with the expression in third trimester CSD controls. Obviously all parameters of third trimester controls (CSD and SpD) were compared.

PE cases and IIIrd trimester pregnancies were dated by an ultrasound scanning before 20 weeks of gestational age.

The following data were collected: maternal age at delivery, smoking habits, body mass index (BMI = kg/m²), gestational weight gain, gestational age at birth, mode of delivery, neonatal sex and weight at birth, parity, placental weight, utero and feto-placental Doppler ultrasound velocimetry indexes, blood pressure, urinary protein, exposure to pharmaceuticals (such as antihypertensives, corticosteroids, antibiotics, aspirin) and family history.

2.2. Tissues 

Immediately after delivery and gross examination of the placentas, three zones were identified: the central one (near the umbilical cord insertion), the peripheral one (the most distal from the umbilical cord) and the intermediate one (between the others). Two placental tissue samples from each zone were then taken:

2.3. Total RNA extraction and semiquantitative RT-PCR 

Frozen placental tissues (20–25 mg) were homogenized in a 1 ml of TRI-Reagent (Sigma, St. Louis, MO, USA) and total RNA was extracted according to the procedure described by the manufacturer. Briefly, the samples were disrupted in lysis buffer, separated into a phenol–chloroform phase and precipitated. Pellets were dissolved into RNAse-free water. Total RNA yield and purity were assessed by spectrophotometry at A260 and A280 nm.

First-strand cDNA was synthesized using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas Life Science, Lithuania), following supplier’s protocols. Briefly, 3 μg of total RNA was diluted in 5-strength reaction buffer containing 200 U of RVHminus reverse transcriptase, 20 U of RNase inhibitor, deoxy-NTPs (dNTPs: 1 mM each of dGTP, dATP, dTTP, and dCTP), and 0.2 μg of random hexamer primer in 20 μl of volume. The mixture was incubated at 25 °C for 10 min, 42 °C for 60 min and 70 °C for 10 min. The blank for each RT reaction was prepared omitting the reverse transcriptase.

The expression level of c-jun, Jun B, Jun D, c-fos, Fos B, Fra-1 and Fra-2 mRNA were evaluated by semiquantitative RT-PCR of total RNA of normal and preeclamptic placental tissues. The human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as housekeeping gene to normalise the data. Briefly, 2 μl of RT reaction product was added to a mixture containing 10-strength reaction buffer (with [NH₄]₂SO₄), MgCl₂ (for final concentration see Table 2), dNTP mixture (final concentration 0.25 mM), 1.0 U Taq DNA polymerase (Fermentas Life Science, Lithuania) and specific primer pairs (final concentration 0.4 mM) listed in Table 2 (Sigma-Genosys, Milan, Italy) in a volume of 25 μl.

After an initial denaturation of DNA (4 min at 94 °C), amplifications were carried out for 1 min at 94 °C, 1 min at the appropriate annealing temperature (AT) (see Table 2), and 1 min at 72 °C for 35 cycles followed by a final 10 min at 72 °C. The appropriate AT values were selected to obtain: a) clearly visible amplification products on an agarose gel, b) amplification in the exponential range and c) a constant intensity of G3PDH bands. For each reaction, a blank was prepared using 2 μl of the corresponding RT blank. Ten μl of each PCR solution was fractionated by electrophoresis in a 1.8% agarose gel containing ethidium bromide. PCR products were visualized by UV illumination and acquired by a digital camera with a specific filter. The intensities of the respective G3PDH and AP-1 bands were quantified by Image J software analysis (version 1.9). Results were expressed as relative intensity (RR), that is the ratio between the different AP-1 values divided by those of the housekeeping gene G3PDH, and were the mean of three independent experiments.

Table 2. Primers used in this study.

2.4. Immunohistochemistry 

Immunohistochemistry was performed as previously described [16]. Briefly, deparaffined parallel sections were incubated overnight at 4 °C with one of the primary antibodies listed in Table 3 and the peroxidase ABC method (Vector Laboratories, Burlingame, CA) was performed for 1 h at RT and 3′,3′ diaminobenzidine hydrochloride (Sigma, St Louis, MO, U.S.A.) was used as chromogen.

Table 3. Antibodies used in this study.

mAb, monoclonal antibody; pAb, polyclonal.

Sections were counterstained in Mayer’s haematoxylin, dehydrated and mounted with Eukitt solution (Kindler GmbH and Co., Freiburg, Germany). Pretreatments by microwaved (3 times for 5 min) were made for AP-1 proteins and pretreatments with 0.1% trypsin (Sigma Chemical Co, St Louis, MO, USA) in Tris–HCl for 5–10 min at 37 °C were made for vimentin (used to identify decidual cells), cytokeratin and cytokeratin 7 (used to identify extravillous cytotrophoblastic cells). Negative controls were performed by omitting the first antibody or the secondary antibody. Further negative control was performed using non-immune rabbit or murine serum. Histological and immunohistochemical evaluations were performed independently by two morphologists (D.M. and M.C.). Nuclei were scored as positive when a brown color was present. Percentages were determined by counting positive nuclei of at least 100 nuclei in multiple microscopic fields for each structure (syncytium, villous cytotrophoblastic cells, villous stroma, vessels, extravillous cytotrophoblastic cells). Counting was performed on at least three different sections from each placenta. In summary we counted 300 nuclei for each structure present in each placenta using an image analysis software (Lucia, v.4.6.).

2.5. Statistical analysis 

Patient age, BMI, weight gain, gestational age, birth weight, blood pressure readings, urinary protein levels, AP-1 gene expression levels and AP-1 immunohistochemical scores were reported as median values (lower-upper quartile). Medians among groups were analysed by non-parametric Kruskal–Wallis test, followed by Mann–Whitney U test for pair-wise comparison Categorical and nominal values (parity, smoking habit, exposure to pharmaceuticals and risk factors for PE) were analysed by the chi-squared test (χ²). Fisher’s exact test was used for small sample sizes. All tests were 2-sided, a value of p ≤ 0.05 was considered significant. Statistical evaluation was performed with SPSS 17 for Windows (SPSS, Chicago, IL).

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3. Results 

3.1. Study population 

The characteristics of the study groups are reported in Table 1A. Obviously groups delivering in different trimesters significantly differ for gestational weight gain, gestational age at delivery, neonatal birth weight, placental weight and placental/neonatal weights ratio. Third trimester Spontaneus Delivery (3rd trim SpD) and third trimester Caesarean Delivery (3rd trim CSD) differ only for parity: nulliparas are more frequent in 3rd trim CSD (p = 0.035) (Table 1A).

Furthermore, data and placentas were obtained from 20 preeclamptic pregnancies (10 with FGR and 10 without). All cases were early-onset PE (symptoms appearance <34 weeks’ gestational age) and delivered by caesarean section. Twelve pregnancies (6 PE and 6 PE-FGR) presented with severe symptoms (blood pressure ≥160/110 mmHg or proteinuria ≥3 g/24 h) [17]. Accordingly with inclusion criteria, all PE cases had abnormal uterine artery Doppler flow velocimetry (DV), while only PE-FGR group had abnormal umbilical artery DV. The two subgroups of pregnancies complicated by PE differed for neonatal weight, placental weight and proteinuria (Table 1B).

Third trimester controls differed from all PE (with or without FGR) for pre-pregnancy BMI, gestational age at delivery, neonatal and placental weights, placental weight/neonatal weight ratio, numbers of smoking mothers, percentage of patients receiving corticosteroids or antihypertensives, blood pressure and urinary protein values (Table 1B).

3.2. Semiquantitative RT-PCR 

G3PDH showed constant mRNA expression across gestation assessed by semiquantitative RT-PCR.

During normal pregnancy c-jun, Jun B and Jun D gene expressions tended to increase, but the increase resulted significant only for c-jun. On the contrary, c-fos and Fos B mRNA levels seemed to decrease throughout gestation, while Fra 1 and Fra 2 showed a maximum gene expression in the second trimester of gestation, but only for Fra 2 there was a significant difference (Table 4). The comparison between III trimester SpD and CSD showed no significant differences for AP-1 gene expression (Table 4).

Table 4. AP-1 mRNA levels in normal tissues.

*, ° and ^ symbols indicate the pair of compared values.

The AP-1 gene expression variation observed in PE pregnancies (with and without FGR) is compared to 3rd trim CSD values. Interestingly, we found an abnormal decrease of Fos B and Fra 2 mRNA levels in PE and PE-FGR samples, respectively. In addition, c-fos mRNA levels decrease in PE and PE-FGR placentas. The differences between 3rd trim CSD and pathological placentas as well as between PE and PE-FGR placentas were not significant.

3.3. Immunohistochemistry 

C-jun showed a staining pattern similar to p-c-jun but c-jun was localized in the nuclei and cytoplasm of the cells. In particular, p-c-jun antibody showed nuclear staining and a minor number of positive cells (not significant) than those observed using c-jun antibody.

The immunostaining for p-c-jun showed a decrease in the chorionic villi while the extravillous trophoblast showed an unchanged positive staining pattern during gestation (see for details Table 5, Table 6, Fig. 1a–c). Fetal vessels showed a positive staining for p-c-jun in the first half of gestation. Jun B was localized in the trophoblast (syncytium and villous trophoblast) as well as in the extravillous trophoblast. Jun B immunostaining was significantly increased at the end of gestation (Table 5, Table 6).

Table 5. Immunohistochemical staining of normal tissues.

++: 100–75% positive cells; +: 75–50 positive cells; ±: 50–25% positive cells; −: < 25–0%.

Table 6. Percentage of AP-1 immunopositive cells in normal tissues.

*, ° and ^ symbols indicate the pair of compared values.

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• Fig. 1 

  Immunohistochemical analysis of the expression pattern of AP-1 family proteins in normal human placenta. a, d, g, l, o, p, q, r: 9th week of gestation. b, e, j, m: 16th week of gestation. c, f, i, n, s: 39th week of gestation. Negative control (39th week of gestation): t. p-c-jun is predominantly expressed in the extravillous cytotrophoblastic cells (a, b, c). Jun D expression decreases in the villous trophoblast from first trimester to third trimester of gestation (d, e, f). Jun D is expressed in extravillous cytotrophoblastic cells (g, h, i). c-fos is positive in the extravillous trophoblast (l, n) and in the villous trophoblast as well as in the fetal vessels (m). Fos B shows an evident immunostaining in extravillous cytotrophoblastic cells of cell columns as well as in the villous trophoblast (o). Villous cytotrophoblastic cells are positive for Fra-1 while the syncytiotrophoblast is mainly negative (p). Fra-1 is expressed in extravillous cytotrophoblastic cells of cell column (q). Fra-2 shows immunopositive reaction in the fetal vessel walls (r, s).

Jun D showed an evident immunostaining in the first trimester while it showed a weak or negative immunoreaction of the chorionic villi in the second and third trimester of gestation (Fig. 1d–f). The fetal vessel walls were immunolabeled for Jun D in the first and second trimester while they were negative in the third trimester of gestation. The extravillous trophoblast was positive for Jun D during gestation (Table 5, Table 6, Fig. 1g–i).

c-fos immunoreactivity decreased throughout gestation in the chorionic villi as well as in the extravillous cytotrophoblast (Table 5, Table 6, Fig. 1l–n).

Fos B expression was mainly unchanged in the chorionic villi as well as in the extravillous trophoblast during gestation (Table 5, Table 6, Fig. 1o).

Fra-1 was expressed in the villous cytotrophoblast during gestation while it was detected in the syncytium starting from second trimester of gestation. The fetal vessel walls as well as the extravillous trophoblastic cells were mainly positive during gestation (Table 5, Table 6, Fig. 1p,q).

Fra-2 was mostly localized in the fetal vessel walls (Table 5, Table 6, Fig. 1r,s). The extravillous trophoblast showed a positive immunostaining only in the second trimester of gestation (Table 5, Table 6).

PE placental tissues showed only a very weak immunostaining in the extravillous cytotrophoblast of the basal plate for p-c-jun (Fig. 2a). In addition, p-c-jun was clearly detected in the basal plate and it was weakly expressed in the syncytium in PE-FGR placentas (Table 7, Table 8, Fig. 2b). Jun B staining pattern was unchanged in PE with and without FGR and in control placental tissues (Table 7, Table 8). Jun D immunostaining was mainly expressed in the extravillous trophoblast and it was mainly negative in the PE-FGR placentas (Table 7, Table 8 Fig. 2c,d). c-fos was expressed only in the extravillous cytotrophoblast into the basal plate in PE (Table 7, Table 8, Fig. 2e) as well as in PE-FGR placental tissues but the latter showed a stronger immunostaining (Table 7, Table 8, Fig. 2f). Immunostaining for Fos B showed a similar staining pattern in PE and PE-FGR placental samples (Table 7, Table 8). Fra-1 was expressed in the chorionic villi, in the extravillous trophoblast as well as in the fetal vessel walls in all pathologic tissues (Table 7, Table 8). Fra-2 was mainly expressed in the fetal vessel walls, particularly in the large vessels. Small fetal vessels were negative for Fra-2 in PE (Table 7, Table 8, Fig. 2g) and PE-FGR (Table 7, Table 8, Fig. 2h).

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• Fig. 2 

  Immunohistochemical analysis of the expression pattern of AP-1 family proteins in PE and PE-FGR placentas. a, c, e, g: PE; b, d, f, h: PE-FGR. i: negative control (PE) p-c-jun shows immunopositivity of extravillous cytotrophoblastic cells (a, b). p-c-jun shows few positive cells into the basal plate (*) in PE placentas (a) while PE-FGR placentas show many cells positive for p-c-jun (b). Jun D is moderately positive in PE (c) while it is negative in PE-FGR (d). c-fos is weakly expressed into the basal plate of PE placentas (e) and it is strongly positive in PE-FGR (f). Fra-2 is positive in the large fetal vessels in PE (g) as well as in PE-FGR (h) placentas.

Table 7. Immunohistochemical staining of pathological tissues.

++: 100–75% positive cells; +: 75–50 positive cells; ±: 50–25% positive cells; −: <25–0%.

Table 8. Percentage of AP-1 immunopositive cells in pathological tissues.

*, ° and ^ symbols indicate the pair of compared values.

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4. Discussion 

This is the first study that analyzes the protein expression pattern of AP-1 transcription factors in PE with and without FGR and that quantifies mRNA in normal and PE placental tissues. Bamberger et al. [10] showed that these transcription factors were specifically expressed in the intermediate (extravillous) trophoblast in normal human placenta. These authors suggested that AP-1 proteins could be implicated in regulating proliferation, and/or differentiation during placental development [10]. Our results concerning normal placental tissues generally confirm previous findings on extravillous trophoblast [10]. In addition, we have observed a protein expression pattern of c-fos different to that described from Bamberger et al. [10] during normal pregnancy. c-fos was mainly localized in the extravillous cytotrophoblst showing a reduction of expression from first to third trimester of gestation. Additionally, we have observed a reduction in c-fos mRNA total levels during gestation. Interestingly, c-fos is usually considered to be associated with the progression of tumors [18], [19], [20]. However in some cases [21], [22] it has been hypothesized to have a tumor suppressor activity. Regarding normal placenta, our data show that c-fos correlates positively with the grade of invasiveness. This is true in pregnancy progression from first to third trimester as well as in the case of PE without FGR, suggesting for c-fos a similar behavior in PE and normal tissues. The present study also shows a high protein expression of c-fos in PE with FGR while the c-fos mRNA levels result unchanged in the two pathological conditions. These c-fos mRNA data confirm previous findings reported by Faxén et al. [23]. We demonstrate that c-fos is localized in numerous extravillous cytotrophoblastic cells in PE-FGR, while PE shows a reduced number of extravillous cytotrophoblastic cells positive for c-fos. Although the etiology of preeclampsia remains unclear, it is accepted that preeclampsia is associated with a generalized impairment of trophoblast invasion [2], [3]. Interestingly, a recent study of Ahenkorah et al. [24] suggests that the trophoblast in preeclamptic placentas might be cytoskeletally weaker and therefore tends to be deported in greater quantity. In fact both interstitial and endovascular trophoblast invasion are reduced. Our data support the hypothesis that c-fos may be involved in different mechanisms responsible of the pathogenesis of PE with FGR (placental PE) and PE without FGR (maternal PE), thus confirming the different pathogenesis of the two diseases [6], [16].

Interestingly, our immunohistochemical pattern indicates that the majority of fetal vessels, small and large vessels, are positive for AP-1 factors in first and second trimester of gestation, whereas only the larger fetal vessels are weakly positive for the majority of AP-1 factors in third trimester of gestation. Particularly, p-c-jun, Jun D, c-fos and Fos B were weakly expressed in first and second trimester of gestation and no immunostaining was observed in third trimester as well as in PE and PE-FGR pathologies. Fra-1 and Fra-2, were expressed in the placental fetal vessel walls during normal gestation as well as in PE and PE-FGR pathologies. Our findings are supported by previous studies showing that AP-1 factors are involved in smooth muscle cell differentiation using in vitro as well as in vivo techniques [7], [25], [26], [27]. In particular, Rusovici and LaVoie [26] showed that blood vessels, i.e. arterioles and arteries were immunopositive for most AP-1 factors in ovarian tissues suggesting a possible role in vessel remodeling. Vasculogenesis or the novo formation of blood vessels occurs in the first trimester while angiogenesis or growth and branching of existing vessels is associated with the second and third trimester expansion of the villous tree and the maturation of placental villi. Consequently, we suggest that Fra-1 and Fra-2 may be mainly related to angiogenesis (growth vessels) and the other transcription factors to vasculogenesis (the novo formation of blood vessels).

It should also be noted that our RT-PCR data showed mRNA expression of the majority of AP-1 factors similar to the expression of respective proteins with the exception of Jun D and c-jun in normal as well as pathological samples. Our data show that during normal gestation, Jun D and c-jun mRNA show increased while Jun D and c-jun proteins decreased. In pathological placentas, Jun D and c-jun mRNAs were similarly expressed. On the contrary, Jun D protein was evidently expressed in PE placentas while it was mainly negative in PE-FGR placental tissues. In addition, c-jun protein was mainly negative in PE and positive in PE-FGR. Because Jun D and c-jun proteins were expressed in the extravillous cytotrophoblst we suggest that these proteins may be critical for regulating placental development and for controlling the terminal differentiation of extravillous cytotrophoblast. The opposite modulation of these two proteins in PE and PE-FGR supports the recent hypothesis [4], [6] that PE and PE-FGR could be considered two pathologies with different origin (maternal and placental) each of which has a different molecular pattern of expression. Future research should be performed to better clarify the pathogenesis of maternal (PE) and placental preeclampsia (PE-FGR).

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Acknowledgements 

We gratefully acknowledge the generous support of Fondazione Cassa di Risparmio di Fabriano e Cupramontana. We are indebted to Dr Rosamaria Fiorini (Department of Biochemistry, Biology and Genetic – Polytechnic University of Marche) for useful discussions. Thanks are due to Rosella Vitali for excellent technical assistance. This study was supported by grants from Polytechnic University of Marche to D.M., M.C., PRIN 2003 to T.T. and M.C. and CIPE to T.T.

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