Placenta
Volume 31, Issue 3 , Pages 222-229, March 2010

Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: Gestational diabetes mellitus, intrauterine growth restriction and preeclampsia

  • S.J. Holdsworth-Carson

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia
    • Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia
    • ,
  • R. Lim

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia
    • Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia
    • ,
  • A. Mitton

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia
    • Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia
    • ,
  • C. Whitehead

      Affiliations

    • Department of Obstetrics and Gynaecology, Monash Medical Centre, Monash University, Clayton, Victoria 3168, Australia
    • ,
  • G.E. Rice

      Affiliations

    • Translational Proteomics, Baker IDI, Prahran, Victoria 3004, Australia
    • ,
  • M. Permezel

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia
    • Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia
    • ,
  • M. Lappas

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia
    • Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia
    • Translational Proteomics, Baker IDI, Prahran, Victoria 3004, Australia
    • Corresponding Author InformationCorresponding author. Department of Obstetrics and Gynaecology, University of Melbourne. Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia.

Accepted 8 December 2009. published online 04 January 2010.

Article Outline

Abstract 

Background

Common complications of pregnancy arise in part from dysfunctional placental development, and include gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Peroxisome proliferator-activated receptors (PPARs), and their partner retinoid X receptor a (RXRα), mediate trophoblast differentiation and thus may offer insight into the pathophysiology of these diseases.

Methods

Human placentae were obtained from women at term with GDM and were compared to uncomplicated term placentae. Placentae from women who delivered preterm with IUGR, PE or co-existing PE and IUGR were compared to matched controls. Quantitative RT-PCR and Western blotting were used to examine mRNA and protein expression of PPARα, PPARδ, PPARγ and RXRα. DNA binding activity of PPAR isoforms were measured in nuclear protein extracts.

Results

GDM was associated with significantly lower placental PPARγ mRNA and protein, PPARα protein and RXRα protein expression, while PPAR DNA binding activity remained unchanged. Placentae from women with PE did not demonstrate any changes in mRNA or protein expression or PPAR DNA binding activity, while IUGR/PE placenta showed significant increases in PPARα protein, PPARγ mRNA and protein and RXRα mRNA and protein expression. Significantly elevated protein expression of PPARα and RXRα were associated with IUGR placentae. IUGR and IUGR/PE placentae had significantly higher PPARγ DNA binding activity compared to controls.

Conclusions

The data presented herein suggest that PPARs may be involved in the pathophysiology of GDM, PE and IUGR.

Keywords: Peroxisome proliferator-activated receptor, Placenta, Gestational diabetes mellitus, Intrauterine growth restriction, Preeclampsia

 

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1. Introduction 

The development of the placenta is essential to pregnancy establishment and maintenance. Inadequate placental differentiation is seen in common clinical complications of pregnancy including gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE) [19], [29]. Up to 8% of all pregnancies are complicated by GDM or PE independently [6], [45], and further, approximately 30% of all PE cases also develop IUGR [14], [57]. GDM, IUGR and PE are each associated with an increased risk of developing the other, and share common risk factors including obesity, hypertension, family history of diabetes, collagen vascular disease or previous episodes of GDM, PE and/or IUGR [3], [5], [25], [41], [57]. Thus commonality exists among pathophysiologies, often demonstrating similar abnormalities including irregular placental development, increased pro-inflammatory cytokine secretion and augmented oxidative stress [19], [23], [31], [33], [36], [44], [51].

GDM is impaired glucose tolerance that is first recognised during pregnancy, and results when the maternal insulin supply cannot meet demand [11]. The impact of a diabetic insult has potential deleterious effects on early placental development, resulting in increased vascular resistance [19], [32], [43]. PE is characterised by maternal endothelial dysfunction and manifests clinically by the presence of hypertension and proteinuria [10]. IUGR implies a failure of a fetus to achieve its' genetic growth potential. In practice, IUGR is defined by a birth weight below the 10th percentile for gestational age [24]. PE and IUGR may develop independently of one another depending on the timing of the insult on trophoblast differentiation [29]. In both conditions, the invasiveness of placental trophoblasts are limited resulting in increased placental vascular resistance and reduced placental perfusion [23], [45].

Given the above abnormalities in early placental development and the occurrence of GDM, IUGR and PE, examination of mediators of trophoblast differentiation may offer insight into better understanding these conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that work in co-operation with retinoid X receptor a (RXRα). PPARs were first identified as integral regulators of placental development in mouse studies where embryos of PPAR-null mutants demonstrated lethality as a consequence of failed trophoblast differentiation [1], [2], [59]. In humans, a role for PPARγ and PPARδ in trophoblast invasion, implantation and decidualisation has been established [21], [38], [52]. Physiologically, PPARs play a part in lipid homeostasis and inflammation [7], [17], [20], [34], [53]. As PE, IUGR and GDM placentae demonstrate elevated levels of pro-inflammatory cytokines, soluble phospholipase A2 and prostaglandins, reactive oxygen species and fatty acids [13], [19], [23], [31], [32], [33], [36], [40], [44], [51], [55], [59], it is conceivable that PPARs may play a role in the pathophysiology of these diseases.

Thus far however, data is largely lacking regarding a role for PPARs in abnormal human placental pathologies. At term, basal protein expression of PPARγ is lower in human placentas from GDM women compared to uncomplicated controls [31]. Similarly, serum from women with severe PE exhibit weaker PPARγ activation compared to normal pregnant women [56]. To date, examination of placentae from IUGR-affected pregnancies has not identified a change in placental PPARs [22], [46]. The objective of this study was to examine the gene and protein expression of PPAR isoforms and RXRα and the activity of PPARs in GDM, PE and IUGR. Identifying differences in PPAR/RXR in association with placental pathologies may assist in improved clinical intervention and/or management of commonplace complications of pregnancy.

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2. Materials and methods 

2.1. Reagents 

Mouse monoclonal β-actin antibody (A5316) and TRI reagent were supplied by Sigma (St. Louis, Missouri, USA). SuperScript VILO cDNA Synthesis Kit was purchased from Invitrogen (Carlsbad, California, USA). Predesigned bioinformatically validated QuantiTect primer assays for 18s QT00199367, β-actin QT00095431, PPARα QT00017451, PPARδ QT00078064, PPARγ QT00029841 and RXRα QT00005726 were purchased from Qiagen (Germantown, Maryland, USA). SensiMixPlus SYBR & Fluorescein was purchased from Quantace (Alexandria, NSW, Australia). Coomassie Plus Protein Assay reagent was from Pierce (Rockford, Illinois, USA). The following antibodies and reagents were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, California, USA), Western blotting luminol reagent, rabbit polyclonal PPARα sc-9000, rabbit polyclonal PPARβ(δ) sc-7197, rabbit polyclonal PPARγ sc-7196, rabbit polyclonal RXRα sc-553, goat anti-rabbit IgG-HRP sc-2004 and goat anti-mouse IgG-HRP sc-2005. PPARα, δ, γ, Complete Transcription Factor Assay Kit was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

2.2. Patient participation and tissue collection 

Human placental tissues were collected at the Mercy Hospital for Woman (Heidelberg, Victoria, Australia) with the approval of The Research Ethics Committee of Mercy Health and Aged Care. All placentae were collected prior to labour onset, at Caesarean section (CS). Gestational age, recorded as completed weeks of gestation, was calculated from the date of last menstrual period and/or from ultrasound. Placentae obtained from preterm gestations were from women who delivered before 37 weeks' completed gestation. Tissues obtained from term pregnancies were from women who delivered between 37 and 42 weeks of gestation. All placentas collected from preterm gestations were swabbed for microbiological culture investigations and histopathological examination. Patients with chorioamnionitis were excluded from analyses.

Women were diagnosed with GDM when a 75 g oral glucose tolerance test (OGTT) revealed either a fasting venous plasma glucose level of 5.5 mmol/L glucose and/or at 2 h plasma glucose level >8.0 mmol/L glucose (in accordance with the Australian Diabetes in Pregnancy Society (ADIPS)). Insulin was commenced in those women in whom blood glucose monitoring revealed more than two high values in a week as defined by a fasting glucose higher than 5.5 mmol/L or 2 h postprandial blood glucose >6.5 mmol/L [26], [42]. Insulin-controlled GDM placentae were collected from term women, with normal 12 week pregnancy BMI (body mass index) (20–25 kg/m2) who delivered healthy singleton infants (n = 7). GDM patients were control-matched with uncomplicated term pregnancies with normal glucose tolerance (NGT) (n = 7) (Table 1). The diagnosis of PE was in accordance with the Australasian Society for the Study of Hypertension in Pregnancy (ASSHP) classification; hypertension (systolic BP = 140 mmHg or diastolic BP = 90 mmHg) arising for the first time after 20 weeks gestation plus one or more of the following clinical diagnoses; proteinuria (0.3 g/day), renal insufficiency (serum/plasma creatinine = 0.09 mmol/L (Pr-Cr ratio)), liver disease, neurological problems (requiring prophylaxis (magnesium sulphate)), haematological disturbances or IUGR [10]. Generalized oedema is usually associated with PE [10], and was therefore included as a symptom. Babies were classified as IUGR when birth weight was below the 10th percentile for gestational age and matched for gender in accordance with an Australian population [24]. PE and IUGR participants were then categorized as PE alone (n = 7), IUGR alone (n = 7) or as co-existing IUGR/PE (n = 10). All IUGR, PE and IUGR/PE cases where from singleton preterm gestations and were matched to control patients who also delivered preterm (n = 8) (Table 2). Indications for preterm delivery (in the absence of IUGR or PE) were prolonged pre-labour rupture of the fetal membranes (PROM), placenta praevia, antepartum haemorrhage (APH) or Rhesus isoimmunisation.

Table 1. Clinical characteristics of GDM and control women.
Clinical featuresNGT n = 7GDM n = 7
Maternal age (mean ± SEM)32.4 ± 1.734.4 ± 1.5
Maternal BMI (mean ± SEM)20.6 ± 0.623.0 ± 0.4a
Gravida (mean ± SEM)2.4 ± 0.43.1 ± 0.4
Parity (mean ± SEM)2.3 ± 0.32.1 ± 0.3
OGTT fasting4.5 ± 0.14.7 ± 0.2
1 h5.1 ± 0.810.3 ± 0.4a
2 h4.9 ± 0.58.6 ± 0.5a
Weeks gestation (mean ± SEM)38.7 ± 0.338.5 ± 0.2
Birth weight (g) (mean ± SEM)3395.0 ± 135.43348.6 ± 154.5
5 min Apgar (mean ± SEM)9.4 ± 0.29.4 ± 0.2
Gender female (%)43%43%
Male (%)57%57%
Mode of deliviery CS (%)100%100%
Labour Onset (No Labour) (%)100%100%

aSignificantly different to NGT.

Table 2. Clinical characteristics of IUGR, IUGR/PE, PE and control women.
Clinical FeaturesIUGR n = 7IUGR/PE n = 10PE n = 7Control n = 8
Maternal age (mean ± SEM)30.1 ± 2.829.7 ± 1.832.6 ± 1.830.9 ± 1.3
Maternal BMI (mean ± SEM)27.4 ± 2.631.3 ± 2.633.0 ± 3.826.3 ± 1.8
Gravida (mean ± SEM)3.1 ± 0.7c1.4 ± 0.2a1.9 ± 0.33.0 ± 0.3
Parity (mean ± SEM)1.9 ± 0.51.2 ± 0.11.4 ± 0.22.1 ± 0.3
Weeks gestation (mean ± SEM)34.3 ± 0.731.7 ± 0.931.7 ± 0.933.4 ± 1.1
Birth weight (g) (mean ± SEM)1615.0 ± 144.01321.8 ± 139.3a1775.1 ± 235.52051.0 ± 204.3
5 min Apgar (mean ± SEM)9.1 ± 0.1b9.1 ± 0.3b8.1 ± 0.39.0 ± 0.2
Gender Female (%)57%60%57%37.5%
Male (%)43%40%43%62.5%
Mode of deliviery CS (%)100%100%100%100%
Labour onset (No Labour) (%)100%100%100%100%
Blood pressure elevated (%)N/A100%100%N/A
Proteinuria elevated >0.3g/day (%)N/A100%100%N/A
Pr-Cr ratio elevated >0.09 mmol/L (%)N/A60%43%N/A
Oedema (%)N/A70%100%N/A
Magnesium sulphate administration (%)N/A90%71%N/A

Pr-Cr Ratio, protein-creatinine ratio.

aSignificantly different to control.

bSignificantly different to PE.

cSignificantly different to IUGR/PE.

Placentae were prepared within 10–15 min of delivery. Tissues was dissected and repeatedly rinsed until free from blood in chilled (4 °C) PBS. Placental lobules were always taken from the central region of the placenta and the basal plate and chorionic surface were removed with the villous tissue obtained from the middle cross section. Dissected tissue was immediately snap frozen in liquid nitrogen and stored at −80 °C until ready for RNA or protein extraction.

2.3. RNA extraction and qRT-PCR 

Total RNA was extracted from 100 mg of frozen placenta using TRI reagent as per manufacturers' instructions. RNA absorbance ratios (A260/A280) were between 1.8 and 2.0. RNA (1 μg) was converted to cDNA using SuperScript VILO cDNA Synthesis Kit (Invitrogen). qRT-PCR was performed by the two-step method as described previously [28]. Each reaction plate was run with an internal β-actin control. 18s ribosomal RNA primers were used for data normalisation. The RT-PCR was performed using an iQ5 Multicolour Real-Time PCR Detection system (iCycler) (Bio-Rad Laboratries, Hercules, California, USA). iQ Standard Edition software (Version 2.0.148.60623) (Bio-Rad) was used for CT measurements and melt curve analysis. Relative mean fold change expression ratios were calculated using the 2−ΔΔCT method [39].

2.4. Nuclear protein extraction and Western blotting 

Nuclear proteins from 200 mg of frozen placenta for Western blotting analysis were extracted as described previously [35]. Protein lysates were prepared in the presence of protease inhibitors (10 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM AEBSF, 1 mM Na3VO4 and 1 mM NaF). Protein concentrations were determined using the Coomassie Plus Protein Assay (Pierce).

Fifty μg of protein were resolved in 10% SDS-PAGE gels or 7.5% pre-cast Criterion Tris–HCl gels (Bio-Rad Laboratories, Hercules, California, USA) at 200 V for 1 h and transferred onto PVDF membrane (Millipore Corporation, Billerica, Massachusetts, USA) at 105 mAmps for 1 h. The membrane was blocked with 5% (w/v) skim milk in TBST for 1 h at room temperature. Primary antibody incubations occurred for between 2 h at room temperature and 48 h at 4 °C, depending on antigen. PPARα and PPARδ primary antibody dilutions were 1:150, while PPARγ and RXRα primary antibody dilutions were 1:500 in 5% (w/v) skim milk in TBST. Horseradish peroxidase-conjugated secondary antibody diluted between 1:2500 and 1:5000 was incubated for 30 min at room temperature. Western blot luminal was used to detect the chemiluminescent signal. Western blots were normalised using β-actin [8]. Densitometry values were measured using Quantity One software (Version 4.6.5) (Bio-Rad Laboratories, Hercules, California, USA). Molecular weights were determined using kaleidoscope prestained standards (Bio-Rad Laboratories, Hercules, California, USA).

2.5. PPAR transcription factor DNA binding activity assay 

Transcription factor DNA binding activity was measured using the commercially available PPARα, δ, γ, Complete Transcription Factor Assay Kit following the manufacturers' instructions (Cayman Chemical) as described previously [27], [28]. Forty μg of nuclear protein was loaded per well per PPAR isoform. Transcription factor binding activity to the PPAR response element (PPRE) was measured at 450 nm (minus the blank).

2.6. Statistical analysis 

Statistical analyses were performed using a commercially available statistical software package, Statgraphics Plus (Version 3.1) (Statistical Graphics Corp., Rockville, Maryland, USA). For analysis of GDM data, patient clinical information, qRT-PCR and Western blot densitometry were analysed by two-sample comparison and Students t-test to compare the means. For analysis of IUGR and PE data, patient clinical information, qRT-PCR, Western blot densitometry and PPAR DNA binding activity were analysed by multiple comparison (ANOVA) using LSD correction. In addition, all data for PPAR DNA binding activity were analysed by two-sample comparison and Students t-test to compare the means. Data are expressed as mean ± standard error the mean (SEM). Statistical difference was indicated by a p value of less than 0.05.

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3. Results 

3.1. PPAR and RXRα in GDM women compared to control pregnant women 

3.1.1. Clinical characteristics of the patients 

Table 1 presents the clinical characteristics for the patients included in the evaluation of GDM women (n = 7) compared to NGT women (n = 7). No significant difference was identified between maternal age, gravida, parity, weeks gestation, birth weight or 5 min Apgar score. GDM women had a significantly greater BMI at their first antenatal visit (12 weeks gestation), however both NGT and GDM values were within the normal range for BMI. While fasting OGTT levels were not significantly different between groups, both 1 h and 2 h OGTT readings were significantly greater for GDM women.

3.1.2. PPAR and RXRα mRNA and protein expression 

qRT-PCR and Western blotting were used to determine PPARα, δ and γ, and RXRα mRNA and nuclear protein expression between term NGT and GDM placenta (Fig. 1). Placental PPARα, PPARδ and RXRα mRNA expression were unaltered when comparing GDM patients to normal patients (Fig. 1a, b and d). Placental PPARγ mRNA and protein expression was significantly lower in the GDM group compared to normal women (Fig. 1c and g). Additionally, PPARα and RXRα protein expression was also significantly reduced in GDM placenta compared to NGT women (Fig. 1e and h). PPARδ protein expression remained unaltered (Fig. 1f).

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  • Fig. 1 

    qRT-PCR and Western blotting were performed on NGT and GDM term placenta. mRNA expression of a) PPARα, b) PPARδ, c) PPARγ and d) RXRα. Protein expression for e) PPARα, f) PPARδ, g) PPARγ and h) RXRα. Representative Western blot images are also shown for NGT and GDM expression of e) PPARα, f) PPARδ, g) PPARγ and h) RXRα, all normalised for β-actin. All data were compared by two-sample comparison (Students t-test) with P values < 0.05 denoted with *. Expression of mRNA is displayed as the mean fold change ratio ± SEM and expression of nuclear protein is displayed as the mean optical density (OD/cm2).

3.1.3. PPAR DNA binding activity 

To demonstrate if there are differences in PPAR transcription factor activity between normal and GDM women, DNA binding activity was measured from nuclear protein extracts. For each PPAR isoform, no difference in DNA binding activity was detected between GDM and normal women (data not shown).

3.2. PPAR and RXRα in IUGR, IUGR/PE and PE women compared to preterm controls 

3.2.1. Clinical characteristics of the patients 

Table 2 presents the clinical characteristics for the women included in the evaluation of IUGR (n = 7), IUGR/PE (n = 10) and PE (n = 7) patients compared to matched controls (n = 8). There was no statistical difference between pathological and control groups when comparing maternal age, maternal BMI, parity or gestational age at birth. IUGR/PE women had significantly lower gravida compared to the control and the IUGR groups. The five-minute Apgar score for the PE group was also significantly reduced compared to the IUGR and IUGR/PE groups. IUGR patients had a mean birth weight less than the control group, and while this did not reach statistical significance (perhaps due to the small number of cases), all IUGR patients fell below the 10th percentile for gestational age (matched for gender). The IUGR/PE group mean birth weight was however significantly less than the control group.

3.2.2. PPAR and RXRα mRNA and protein expression 

qRT-PCR and Western blotting were used to determine PPARα, δ and γ, and RXRα mRNA and protein expression differences between placenta from IUGR, IUGR/PE, PE and matched controls (Fig. 2). Placental PPARα protein, but not PPARα mRNA, was significantly increased in the IUGR and IUGR/PE group compared to matched controls (Fig. 2a and e). The IUGR/PE group also had significantly elevated PPARα protein compared to the PE group (Fig. 2e). Placental expression of PPARδ mRNA and protein did not change significantly compared to placenta from control women (Fig. 2b and f). PPARγ mRNA and protein were significantly higher in the IUGR/PE group; with PPARγ mRNA expression higher in comparison to the IUGR group and PPARγ protein expression elevated compared to the control group (Fig. 2c and g). Placental RXRα mRNA expression was significantly increased in the IUGR/PE group compared to the control and IUGR group (Fig. 2d). RXRα protein expression was increased in the IUGR and IUGR/PE groups compared to the control group (Fig. 2h).

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  • Fig. 2 

    qRT-PCR and Western blotting were performed on IUGR, IUGR/PE, PE and control placenta. mRNA expression of a) PPARα, b) PPARδ, c) PPARγ and d) RXRα. Protein expression (including representative Western blot images) of e) PPARα, f) PPARδ, g) PPARγ and h) RXRα. Expression of mRNA is displayed as the mean fold change ratio ± SEM and expression of protein is displayed as the mean optical density (OD/cm2). All data were compared by multiple comparison (ANOVA) using LSD correction. For mRNA expression, significant differences versus control * (p < 0.05), and versus IUGR † (p < 0.05). For protein expression, significant differences versus control * (p < 0.05), versus PE † (p < 0.05).

3.2.3. PPAR DNA binding activity 

To demonstrate if there are differences in placental PPAR transcription factor DNA binding activity from women with IUGR, PE and IUGR/PE compared to matched control women, PPRE-binding activity was measured from nuclear protein extracts. For the PPARα and d isoforms, no difference in DNA binding activity was detected (data not shown). In contrast, PPARγ DNA binding activity was significantly higher in the IUGR placenta compared to preterm control placentas (Students t-test) and also in the IUGR/PE group versus controls (Students t-test and ANOVA) (Fig. 3). PE placental PPARγ DNA binding activity did not reach significance compared to the control group.

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  • Fig. 3 

    PPARγ DNA binding activity from preterm control (n = 7), IUGR (n = 7), IUGR/PE (n = 7) and PE (n = 7) placenta. DNA binding activity was compared by ANOVA and two-sample comparison (Students t-test). DNA binding activity is displayed as the mean optical density (at 450 nm) minus the blank ± SEM with * denoting a significant difference compared to control (Students t-test, p < 0.05).

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4. Discussion 

The aim of this investigation was to study the gene and protein expression of PPAR isoforms and RXRα and PPAR DNA binding activity in placentas from GDM, IUGR and PE patients. We found that GDM was associated with a downregulation in placental PPARα, PPARγ and RXRα expression without a change in PPAR DNA binding activity. Women with PE were unchanged from matched controls, while PE co-existing with IUGR (IUGR/PE) demonstrated significant increases in PPARα, PPARγ and RXRα expression and PPARγ DNA binding activity. IUGR alone was also found to have enhanced PPARα and RXRα expression and PPARγ DNA binding activity. Overall, the data presented herein suggest that PPARs may be involved in the pathophysiology of GDM, PE and IUGR.

PPARs have long been linked to the diabetic phenotype [50], [58]. Adipose tissue from obese GDM women have shown reduced PPARγ expression compared to uncomplicated pregnant obese patients [13] and term human placentas have demonstrated lower basal protein expression of PPARγ in GDM patients [31]. Our results support these findings, with lower mRNA and protein expression of PPARγ in term GDM placentae. Further, we have also shown that placental PPARα and RXRα protein expression are reduced in association with GDM. The decline in expression of PPARs was not reflected in PPAR DNA binding activity, which was unchanged in GDM placenta. In the absence of active PPRE-binding, it is possible that PPARs may function via transrepression mechanisms, regulating neighbouring transcription factors and their target genes [7]. For example, PPAR is capable of transrepressing the pro-inflammatory nuclear factor-κB (NF-κB) [16], a transcription factor that we have shown to be important for the manifestation of GDM [15], [37]. An environment deficient in PPAR:RXR may therefore encourage the development of GDM by alleviating transrepression of NF-κB and allowing gene transcription of GDM-mediators, including pro-inflammatory cytokines, thus inflicting the disease phenotype.

Like in placenta from GDM pregnancies, there is limited information showing a relationship between PPARs and PE and IUGR. Available data show that serum from severe PE patients exhibit weaker PPARγ activity compared to normal pregnant women, and it is suggested that in serum, a reduction in PPAR activation reflects the nature of PE with an overall loss of anti-inflammatory control [56]. In contrast, our study found that PE had no significant effect on PPAR transcription factor activity, however localised to placental tissue possibly indicating tissue-specific differences in PPAR activity. However, the methods and specimens used in our investigation differ to Waite and colleagues (2005) who examined firefly luc gene transcription assays in JEG-3 cells treated with maternal serum extracts. Our results demonstrated no difference in mRNA or protein expression of PPARs or RXRα in human placental tissues in association with PE. Similarly, Rodie and colleagues have previously reported that in the placenta, the expression of PPARγ, PPARδ and RXRα protein were unchanged between PE women and control patients [46]. On the other hand we found that when PE co-exists with IUGR, PPAR expression and activity is altered compared to control placentae, with elevated PPARγ and RXRα mRNA, elevated PPARα, PPARγ and RXRα protein and greater PPARγ activity. The concurrent presence of the two diseases more often represents heightened severity and earlier onset [29], thus it is conceivable to see changes with IUGR/PE and not in PE alone.

Alternatively, the differences seen in the IUGR/PE group may purely be a consequence of IUGR. In support of this, our findings demonstrate an increase in PPARα and RXRα protein expression and PPARγ transcriptional activity in placental samples in association with IUGR. PPARγ activity was elevated in the absence of an increase in PPARγ protein in IUGR placentae, indicating that PPAR DNA binding activity can occur independent of changes in abundance. Many additional factors mediate receptor activity, including ligand accessibility, co-activator/co-repressor availability and post-transcriptional modifications of receptors [7]. For example, patients with cardiovascular disease with associated triglyceride accumulation have unchanged PPARα transcript levels, but increased PPARα activity [48]. The accumulation of fatty acids provide natural ligands for PPAR, thus the elevation of fatty acids may result in an increase in PPAR activity and not PPAR expression [48].

Our findings are in contrast to previous studies where the expression of PPARγ, PPARδ and RXRα mRNA and protein were found to be unchanged in placental tissues between IUGR placentas and control patients [22], [46]. Such discrepancies may be explained by differences in patient selection criteria. For example, the study by Dy and colleagues (2008) specifically examined only term cases from vaginal deliveries, while Rodie et al., did not exclude for labour status (or delivery mode) and included placentae from the broad range of gestational ages across the third trimester. Our previous work emphasises the importance of separating women according to labour status and gestational age, where temporal changes in PPAR expression are apparent dependent on labour onset (not in labour compared to during or post-labour) [28]. Further, within the third trimester, preterm placentas also demonstrate differences in PPAR expression compared to complete gestation [27], thus our more stringent patient selection criteria restricts the possibility of such variation.

Changes in PPAR:RXR protein expression were not reflective of mRNA, where nuclear protein expression levels differed significantly to that of the matched control while mRNA remained unaltered. Similar inconsistencies regarding PPAR and RXRα mRNA/protein relationships have been observed previously and most likely represent a post-transcriptional event [28], [30]. Discordance between mRNA and protein expression have been reported before in patients with PE, while protein remained unaltered, placental RXRα mRNA expression was higher in relation to PE compared to controls [46].

Similar to our findings, a rodent model of IUGR demonstrated increased expression PPARγ and RXRα in adipose and PPARγ in placenta [18], [60]. Desai and co-workers suggest that an increase in PPAR:RXR expression in adipose may provide offspring with enhanced lipid storage capabilities in preparation for a nutrient-poor environment [18]. Similarly we propose that the increased expression of PPAR:RXR during IUGR (with or without PE) and enhanced PPARγ activation may be an adaptive response to the IUGR placenta, whereby PPARs may provide protection against hypoxia and/or nutrient deficiency caused from insufficient placental development [9], [29], [51]. Further strengthening this, placental trophoblasts in response to PPAR:RXR ligands, have been found to enhance lipid uptake and accumulation by modulating PPAR target genes involved in fatty acid transport [9], [47]. Future studies might entail analysis of PPAR target genes in IUGR-effected placenta to see which genes are altered by elevated PPARγ activity. Alternatively, the augmented PPAR activity may be causative of IUGR as per the findings of Schaiff et al. (2007), who propose that in mice, enhanced PPARγ-specific activation leads to a reduction in wild type placental and fetal size.

In conclusion, this study identifies differing expression profiles of PPAR:RXR in the pathophysiology of placental diseases. The observed decrease in PPAR:RXR expression in GDM may be associated with promotion of the disease phenotype. While an increase in the activity of PPARγ and overall expression of PPAR:RXR in IUGR (with or without PE) may be causative or an adaptive response to the disease phenotype. The upstream mechanisms responsible for these alterations in PPAR expression and transcriptional activation are yet to be fully defined. Studies in non-gestational tissues have identified post-transcriptional modifications which both downregulate and upregulate PPAR function [4], [12] and specific agonists that downregulate PPAR expression (autoregulation) [49], [54]. Therefore further investigations are warranted in human gestational tissues to provide better understanding of the role PPARs play in placental pathologies, which may overall offer resolution to future clinical management.

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Funding 

The work described in this paper was funded in part by NHMRC Project grants (Grant No. 454310 and 526686), Trevor Basil Kilvington Bequest and Diabetes Australia Research Trust.

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Acknowledgements 

Dr. Martha Lappas is the recipient of a National Health and Medical Research Council (NHMRC) RD Wright Fellowship (Grant No. 454777). Professor Greg Rice is an NHMRC Principal Research Fellow. The authors gratefully acknowledge the assistance of Michelle Colomiere (from the Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women) her assistance with GDM placenta specimen collection. We would like to thank Dr Stephen Tong (from the Department of Obstetrics and Gynaecology, Monash Medical Centre and Monash University) for supplying us with three of the PE placental samples. Finally, the authors acknowledge Clinical Research Midwives; Anne Beeston, Astrid Tiefholz, Renee Grant and Gabrielle Fleming and the Obstetrics and Midwifery staff of the Mercy Hospital for Women (Heidelberg, Victoria) for their support and co-operation.

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PII: S0143-4004(09)00399-3

doi:10.1016/j.placenta.2009.12.009

Placenta
Volume 31, Issue 3 , Pages 222-229, March 2010

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