Expression of Liver X Receptors in Pregnancies Complicated by Preeclampsia

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Patient selection
  • 2.2. Tissue samples
  • 2.3. RNA isolation from tissue and cDNA synthesis
  • 2.4. mRNA gene analyses by quantitative reverse transcriptase polymerase chain reaction
  • 2.5. Western blots: protein analysis of LXRα and LXRβ
  • 2.6. Measurement of lipids and the fatty acid profile in placental tissues
    • 2.6.1. Lipid class analysis
    • 2.6.2. Fatty acid analysis
    • 2.6.3. Gas chromatographic analysis
  • 2.7. Statistical analysis
• 3. Results
  • 3.1. Clinical characteristics
  • 3.2. mRNA expression of transcription factors in placenta, decidua and adipose tissue in healthy subjects
  • 3.3. Expression of genes in placenta, decidua and adipose tissue in pregnancies complicated by preeclampsia
  • 3.4. Placental protein levels of LXRα and LXRβ in pregnancies complicated by preeclampsia
  • 3.5. Placenta concentrations of lipid and the fatty acid profile in pregnancies complicated by preeclampsia
  • 3.6. Correlation between LXR expression and concentrations of lipids
• 4. Discussion
• Acknowledgements
• References
• Copyright

Abstract 

Preeclampsia is a pregnancy-specific disorder associated with hyperlipidemia. Liver X receptor (LXR) α and LXRβ are key regulators of lipid homeostasis. In the current study, we investigated expression of LXRα, LXRβ and their target genes in human term placenta, decidua and subcutaneous adipose tissue from pregnancies complicated by preeclampsia. Furthermore, we analyzed the protein levels of LXRα and LXRβ in placenta. We also analyzed lipid concentrations in term placental tissue. Gene expression of LXRα, LXRβ and fatty acid transporter CD36 was significantly decreased in placental tissues, while increased expression was observed for LXRα in adipose tissue, from pregnancies complicated by preeclampsia. The placental protein level of LXRβ was reduced, and there was a positive correlation between placental LXRβ mRNA expression and placental free fatty acids in preeclampsia. Our results suggest a possible role for LXRβ as a transcriptional regulator in preeclampsia.

Keywords: Liver X Receptor, Preeclampsia, CD36, Lipids, Decidua, Adipose tissue, Polyunsaturated fatty acid

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1. Introduction 

Preeclampsia is a pregnancy-specific disorder affecting 3–10% of all pregnancies and a significant cause of maternal and neonatal morbidity and mortality. It is clinically defined by hypertension and proteinuria developing after week 20 of gestation. The pathogenesis of preeclampsia is still not fully understood and is most likely multifactorial. However, a key role for the placenta in the etiology of the disease is widely acknowledged (reviewed in [1]).

Hyperlipidemia of pregnancy develops in every pregnant woman, but is significantly increased in women with preeclampsia relative to healthy pregnancies, also prior to clinical onset of the disease [2]. The lipid abnormalities of preeclampsia include hypertriglyceridemia, increased circulating free fatty acids (FFA), increased concentration of small low density lipoproteins and the presence of oxidized low density lipoproteins in maternal circulation [3], [4], [5], that could add to the endothelial dysfunction observed in preeclampsia. However, the regulation of lipid metabolism in preeclamptic placentas has not been studied extensively.

Liver X receptors (LXR) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. Two isoforms are known; LXRα and LXRβ, and both are activated by oxidized cholesterol derivatives, oxysterols [6]. They form obligate heterodimers with the nuclear receptors retinoid X receptors (RXRs). The LXRs have been identified as key regulators of lipid metabolism through the transcriptional regulation of genes involved in de novo fatty acid metabolism, triacylglycerol (TAG) synthesis and cholesterol homeostasis (reviewed in [7], [8]). We previously found that LXR increased the synthesis of fatty acids and inhibited secretion of human chorionic gonadotropin in human placental BeWo cells [9]. A role for the LXRs in placentation and trophoblast invasion has also recently been described [10], as well as in regulation of placental cholesterol transport [11], [12]. These findings suggest that the LXRs may be important in human placentation and feto-placental lipid transport and metabolism.

The LXRs have been extensively studied in rodents in vivo, while clinical data in humans are limited. Due to their regulatory role in lipid metabolism and the dyslipidemia associated with preeclampsia, we hypothesized that the LXRs and some of their target genes involved in lipid metabolism are dysregulated in preeclamptic placenta, decidua and adipose tissue. In order to explore the role of LXR in preeclampsia, we investigated the mRNA and protein expression in placenta, decidua and subcutaneous adipose tissue of LXRα and LXRβ, their target genes, and other transcription factors involved in regulation of lipid metabolism. We found a statistically significant lower placental mRNA expression of LXRα and LXRβ, as well as lower LXRβ protein levels and lower concentrations of placental FFAs in preeclampsia compared to controls, and a correlation between placental mRNA LXRβ expression and placental FFAs in the preeclamptic group. Based on these findings we speculate that placental LXRβ may have a role in regulating FFA levels in the preeclamptic placenta.

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2. Materials and methods 

2.1. Patient selection 

Samples were obtained from an ongoing biobank collection of patient samples from complicated and uncomplicated pregnancies at Oslo University Hospital, Ulleval. Women with singleton pregnancy undergoing cesarean section (n = 62) were included in this study; including 33 women with uncomplicated pregnancy (controls) and 29 with preeclamptic pregnancy. No women with chronic hypertension or renal disease were included. All patients were fasted for a minimum of 6 h; none were in active labor, had ruptured membranes or clinical signs of infection. Preeclampsia was defined as blood pressure augmentation after 20 weeks’ gestation to >140/90 on ≥2 occasions 6 h apart in a previously normotensive woman, combined with proteinuria. Proteinuria was defined as protein dip stick ≥1+ on ≥2 midstream urine samples 6 h apart or a 24-h urine excretion of ≥0.3 g protein, in the absence of urinary infection. Severe preeclampsia was defined by the American College of Obstetricians and Gynecologists criteria (ACOG) [13], including women with blood pressure of 160 mmHg systolic or higher. The newborn birth weight percentiles were calculated according to national birth registry data [14] or an ultrasound based weight percentile [15]. The study protocol was approved by the Regional Committee of Medical Research Ethics in Eastern Norway, and informed written consent was obtained from each patient.

2.2. Tissue samples 

All tissue samples were obtained during cesarean section. Subcutaneous adipose tissue biopsies were sampled adjacent to the lower abdominal incision. Placenta biopsies from a macroscopically normal looking, centrally located cotyledon were collected, omitting the decidual layer. Decidual tissue was collected through vacuum suctioning of the uterine wall underlying the placenta (corresponding to the superficial layer of the placental bed, including minimal myometrial tissue), as described previously [16]. All tissues were snap-frozen in liquid nitrogen and stored at −80 °C.

2.3. RNA isolation from tissue and cDNA synthesis 

The tissues were pulverized in liquid nitrogen and ∼15 mg of tissue was homogenized in 800 μl of RNA lysis buffer using an Ultra-Thurrax homogenizer for 30 s. Total RNA was extracted from placental and decidual tissues using ABI6100 (Applied Biosystems, Foster City, CA, USA) and adipose tissue using RNeasy Lipid Tissue Mini kit (Qiagen, Venlo, Netherland) according to the manufacturers’ instructions. The quality and quantity of the RNA was determined using spectrophotometer (NanoDrop 1000, NanoDrop Technologies, Boston, MA, USA) and capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA) according to manufacturer’s protocol, and was found to be sufficient for the gene expression analysis with 260/280 and 260/230 ratios above 2 and RNA integrity numbers above 7. cDNA was synthesized (20 μl) from extracted total RNA (400 ng) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions.

2.4. mRNA gene analyses by quantitative reverse transcriptase polymerase chain reaction 

Quantitative real-time PCR (qRT-PCR) was performed using custom-made 384-well microfluid cards (TaqMan Low Density Array) and Gene Expression Master Mix (both from Applied Biosystems). The RT-PCR was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Data acquisition and analysis were done according to the manufacturer’s instructions, using the ΔΔCt or comparative Ct method where the Ct value of the sample for each assay is compared to a control value for the same assay [17]. Briefly, each Ct value obtained for each assay was normalized to the endogenous control (YWHAZ, TBP or an average of GAPDH, TBP and 18S) Ct value for the same sample. The delta Ct values of the samples were then compared to a calibrator which was e.g. the delta Ct values of the healthy controls.

The following TaqMan gene expression assays were employed: LXRα (Hs00172885_m1), LXRβ (Hs00173195_m1), fatty acid synthase (FAS) (Hs00188012_m1), fatty acid elongase 5 (Elovl5) (Hs01094711_m1), CD36 (Hs00169627_m1), lipoprotein lipase (LPL) (Hs00173425_m1), low density lipoprotein receptor (LDLR) (Hs01092525_m1), apolipoprotein E (ApoE) (Hs00171168_m1), ATP-binding cassette (ABC), sub-family A, member 1 (ABCA1) (Hs00194045_m1), ABC, sub-family G, member 1 (ABCG1) (Hs01555189_m1), leptin (Hs00174877_m1). Four genes, all commonly used as endogenous controls; 18S (Hs99999901-s1), glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1), TATA box binding protein (TBP) (Hs99999910_m1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) (Hs00237047_m1) were included in the expression analysis and the expression values of each investigated gene in all tissue samples were normalized against the most stable endogenous control in each tissue, which was YWHAZ for placenta and decidua tissue and TBP for adipose tissue. For the comparison of LXRα and LXRβ between the tissues (Fig. 1), the average of three endogenous controls (GAPDH, TBP and 18S) was used, to minimize the differential expression of the endogenous controls between the tissues. We also thoroughly tested the RNA and cDNA material to assure that there was no inhibition of the qRT-PCR reaction (data not shown).

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• Fig. 1 

  Gene expression of LXRα and LXRβ in placenta, decidua and adipose tissue in healthy subjects. Gene expression was analyzed on total RNA extracted from controls using qRT-PCR normalized to the average of GAPDH, TBP and 18S. The results are presented as mean fold change ± standard error of the mean (SEM) relative to controls.

2.5. Western blots: protein analysis of LXRα and LXRβ 

A ∼5 mg piece of homogenized placental tissue was added to a tube containing 1 spoon of glass beads and 300 μl of lysis buffer on ice (1% Nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS and 15 μl/ml of proteinase inhibitor cocktail). The tissue was homogenized using a Precellys24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) at 5000 rpm for 2 × 20 s. Protein concentration was quantified using the Bio-Rad colorimetric assay system with BSA as protein standard (Bio-Rad Laboratories, Hercules, CA, USA). 50 μg of total proteins were separated on SDS polyacrylamide gels (Bio-Rad Laboratories, Inc.), and LXRα and LXRβ levels were determined by blotting with mouse monoclonal anti-human LXR antibodies (R&D Systems, Perseus Proteomics, Inc., Tokyo, Japan: PP-K8607-00 (LXRα) or PP-K8917-00 (LXRβ), 1: 500). Levels of β-actin were determined using mouse monoclonal anti-β-actin antibody (Sigma–Aldrich Inc.: A5441, 1: 10000). Secondary anti-mouse IgG antibody (Abcam plc, Cambridge, UK: ab6728, 1: 10000).

2.6. Measurement of lipids and the fatty acid profile in placental tissues 

Homogenized placenta tissue (100–200 mg), that was initially crushed under liquid nitrogen, was removed from −80 °C storage and transferred to a frozen vial with a frozen spatula. Immediately 1.5 ml methanol was added and the vial was vortex-mixed to avoid metabolism of free fatty acids. Lipids were then extracted by the Folch method, evaporated to dryness by vacuum centrifugation and the lipids dissolved in 200 μl hexane. The hexane containing the lipids was aliquoted into two vials, one for polar lipid analysis and one for apolar lipids analysis. Lipid classes were separated by normal phase HPLC and detected with Evaporative Light Scattering Detector. Apolar lipids were separated with 0.5% acetic acid in heptane delivered at 2.0 ml/min on a Merck Purospher Si, 100 × 4.6 mm, while polar lipids were analyzed in separate method using the same column but with a mobile phase consisting of 0.5% acetic acid and 5% methyl tert butyl ether in heptane.

Approximately 40 mg placenta tissue powder, that was initially crushed under liquid nitrogen, was removed from −80 °C storage, transferred to a frozen vial with a frozen spatula and immediately 0.9 ml HCl Methanol was added and the vial was vortex-mixed. Transmethylation was performed in an ultrasound bath held at 70 °C for 30 min, then for additional 120 min at 80 °C without ultrasound. After cooling and neutralization by KOH, fatty acid methyl esters were extracted with 500 μl hexane.

Analyses were performed using a 6890N GC with a split/splitless injector, a 7683B automatic liquid sampler, and flame ionization detection (Agilent Technologies, Palo Alto, CA, USA). Separation was performed with a Supelco SP2380 (30 m × 0.25 mm i.d. × 0.25 μm film thickness) GC column.

Temperature program, initial: 90 °C with 0.5 min hold, ramp 50 °C/min to 150 °C, 10 °C/min to 225 °C, 120 °C/min to 245 °C with hold 3 min. Carrier gas was H₂ with a flow of 2.2 ml/min. Fatty acid analysis was performed by auto injection of 0.5 μl of each sample at a split ratio of 0.1:1, constant flow mode, injector temperature 250 °C. The flame ionization detector temperature was 270 °C. The sampling frequency was 10 Hz. The run time for a single sample was 12.37 min. Theoretical response factors were used.

2.7. Statistical analysis 

Statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS-PC), version 16.0. For gene expression data and lipid concentration data, the results were normally distributed and the significance was calculated using Student’s t-test and correlations using linear regression. For the patient characteristics, differences in continuous variables between the control and preeclamptic group were tested by non-parametric Mann–Whitney tests. A probability level of <0.05 was considered statistically significant.

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3. Results 

3.1. Clinical characteristics 

Clinical characteristics of the 29 preeclamptic and 33 control pregnant women included in the study on placenta are shown in Table 1. Not all samples were available or used for all analyses (decidua: 30 controls and 27 preeclampsia; adipose tissue: 29 controls and 29 preeclampsia; placenta tissue lipid analysis: 12 controls and 12 preeclampsia, western blot analysis: 6 controls and 6 preeclampsia). In the patient groups used for decidual and placental lipid analyses, there were no significant differences in diastolic blood pressure <week 20 between the preeclamptic group and the control group. In the placental lipid analysis group, there was also no difference in body mass index (BMI) prior to pregnancy or at delivery between preeclamptic and controls. The remaining clinical variations between the patient groups were not significantly different from the results presented in Table 1. 20 of the preeclamptic patients had severe preeclampsia according to ACOG criteria [13]. In the preeclampsia group, 20 patients delivered prematurely, before week 37, and 17 of these delivered prior to week 34. In the uncomplicated pregnancy group, there were no premature deliveries. Further, three of the preeclamptic patients had evidence of HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome [13]. There was a significantly higher median BMI (weight (kg)/height (meter)²) in the preeclamptic group compared to controls, while birth weight, neonatal weight percentile and gestational age at delivery were significantly lower in the preeclamptic group as compared to controls.

Table 1. Clinical characteristics of the control and preeclamptic (PE) patient groups included in the placental gene expression analysis (n = 63). Values shown are median (and minimum-maximum). The P-values are given for PE compared to control. P-values * <0.05 and ** <0.01. n.s. = not statistically significant.

3.2. mRNA expression of transcription factors in placenta, decidua and adipose tissue in healthy subjects 

We have previously shown expression of the LXRs in placental trophoblast cells [9]. To further investigate the expression of LXR during gestation, mRNA expression levels of LXRα and LXRβ were analyzed in placenta, decidua and adipose tissue from healthy controls using an average of GAPDH, TBP and 18S as endogenous controls (Fig. 1). LXRα and LXRβ were similarly expressed in the gestational tissues, with an approximately equal expression in placenta and decidua. In adipose tissue, LXRβ was similarly expressed, whereas higher expression of LXRα was observed compared to placenta and decidua.

3.3. Expression of genes in placenta, decidua and adipose tissue in pregnancies complicated by preeclampsia 

We and others have previously shown the involvement of LXRs in lipid metabolism and uptake in placental cells [9], [12]. To further look at the LXRs during gestation, we investigated the expression of the LXRs in placenta, decidua and adipose tissue. Placenta and decidua were chosen because of their key roles in healthy and preeclamptic pregnancies. Adipose tissue was chosen because of the important role of LXRs in this tissue and the potential importance of adipose tissue for the changes in maternal serum lipids during healthy and preeclamptic pregnancies [8], [18]. The results from the LXR expression analysis according to patient groups are shown in Fig. 2. We found a statistically significant lower mean level of expression of LXRα and LXRβ in the placentas of the preeclamptic group compared to those of the control group (Fig. 2a), while no change in expression was observed in decidua (Fig. 2b). Interestingly, we found a statistically significant higher mean level of expression of LXRα in adipose tissue of the preeclamptic group compared with those of the control group (Fig. 2c). Since the LXRs regulate transcription of genes involved in lipid metabolism [8], we further investigated the expression of some of these target genes in placenta, decidua and adipose tissue. These genes are coding for proteins involved in transport of fatty acids and cholesterol, and genes coding for fatty acid metabolizing enzymes and proteins involved in lipoprotein particle metabolism. The results from the expression analysis of known LXR target genes are shown in Table 2. There was a significantly lower expression of both CD36/FAT and ApoE, and a significantly higher expression of LDLR in preeclamptic placentas as compared with the controls (Table 2). We included placental leptin expression as a positive control because it is upregulated in preeclampsia in both serum and placental tissue [19] and we found a 12-times higher expression in preeclamptic placentas compared to control placentas (P = .0001). In decidua, we found a statistically significant lower mean level of expression of ABCG1 in the preeclamptic group compared to the control group, and in adipose tissue we found a statistically significant higher mean level of expression of ABCA1 (Table 2). There were no differences in the expression of any of the other genes that were analyzed in either placenta, decidua or adipose tissue (Table 2).

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• Fig. 2 

  Gene expression of LXRα and LXRβ in preeclampsia in placenta, decidua and fat compared to controls. The expression of LXRα and LXRβ were analyzed in total RNA extracted from preeclamptic (PE) and control tissues, using qRT-PCR normalized to YWHAZ for placenta and decidua and TBP for adipose tissue. A) Placenta B) Decidua C) Adipose tissue. The results are presented as mean fold change ± SEM relative to controls. P-values * <0.05 and ** <0.01. D) Placental protein levels of LXRα and LXRβ in preeclampsia compared to controls. 50 μg total proteins isolated from six patients in the control group (C1-C6) and six patients in the preeclampsia group (PE1-PE6) were subjected to SDS-PAGE and blotted using anti-LXRα, anti-LXRβ or anti-β-actin antibodies as indicated. The control lane (C) contains exogenously expressed human LXRα/LXRβ in a human hepatoma cell line and is used for positive control. Arrow points at LXRα, LXRβ or β-actin proteins.

Table 2. Gene expression of potential LXR target genes in preeclampsia in placenta, decidua and fat compared to controls. The gene expression was analyzed in total RNA extracted from preeclamptic (PE) and control tissues, using qRT-PCR normalized to YWHAZ for placenta and decidua and TBP for adipose tissue. The results are presented as mean fold change ± SEM relative to controls. P-values * <0.05 and ** <0.01.

3.4. Placental protein levels of LXRα and LXRβ in pregnancies complicated by preeclampsia 

To dissect the biological implications of LXR in placenta, we next investigated the protein levels of LXRα and LXRβ in both healthy controls and pregnancies complicated by preeclampsia. Total proteins (50 μg) from 6 patients in each group were analyzed for LXRα and LXRβ expression using specific LXR antibodies (Fig. 2d). To validate specificity, overexpressed FLAG-tagged LXR proteins produced in a liver hepatoma cell line (Huh7) were included as positive controls of LXRα and LXRβ. They were loaded as size controls together with the placenta samples, and were detected both with the LXR antibodies and the antibody recognizing the FLAG-tag (“C” in Fig. 2d and data not shown). Both LXRα and LXRβ were detected in the control group. The protein level of LXRβ was clearly down-regulated in the preeclamptic group compared to the control group, indicating an important role for LXRβ in this tissue. In the case of LXRα, no difference in the expression level was seen between the controls and the preeclamptic group. Comparing mean LXRα and LXRβ mRNA levels for the whole patient groups to the mean LXRα and LXRβ mRNA levels for the selected patient groups used for protein analyses (data not shown) did not reveal any selection bias for the group used for protein analysis, indicating that this was a representative selection of the total study group.

3.5. Placenta concentrations of lipid and the fatty acid profile in pregnancies complicated by preeclampsia 

Because of the identified dysregulation of the LXR genes and genes involved in lipid metabolism in preeclamptic placenta tissue (Fig. 2 and Table 2), we were interested in analysing the placental lipid classes and total fatty acid profile in preeclamptic compared to control pregnancies. The results from the analysis of placental lipid classes are shown in Table 3a. A statistically significant lower mean concentration of FFAs was observed in placenta tissues from the preeclampsia group compared to the control group. No statistically significant difference in concentration was observed for any of the other classes of placental lipids between the patient groups.

Table 3. Concentration of different classes of lipids and PUFAs in placenta and maternal serum lipids from preeclamptic (PE) compared to control pregnancies. The results are shown as mean and 95% confidence interval concentrations. A) Placental lipids (mg/g placental wet weight) B) Placental PUFAs (esterified and non-esterified together, g/100 g fatty acid methyl ester (FAME)). The P-values are given for PE compared to controls. P-values * <0.05. n.s. = not statistically significant.

We further analyzed the total fatty acid profile (esterified and non-esterified fatty acids together) in placenta, which included all saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). We found a small but significantly higher concentration of total n-6 PUFAs (the total of linoleic acid, 18:2n-6 (LA), dihomo gamma linolenic acid, 20:3n-6 (DGLA) and arachidonic acid, 20:4n-6) (ARA) in placenta tissues from pregnancies complicated by preeclampsia compared to controls (Table 3b). No statistically significant difference in concentration was observed for any of the other fatty acids or groups of fatty acids in preeclamptic placentas compared to controls (data not shown).

3.6. Correlation between LXR expression and concentrations of lipids 

There was a significant positive correlation between LXRβ expression in preeclamptic placentas and FFA concentration in preeclamptic placentas (Fig. 3). The same, but statistically non-significant trend, was observed between placental concentrations of FFA and placental expression of LXRα and CD36 in preeclampsia (data not shown). There was no correlation between placental expression of LXRα, LXRβ or CD36 and placental FFA concentration in the control group (data not shown).

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• Fig. 3 

  Correlation between expression of LXRβ and concentrations of FFA. Linear regression between placental LXRβ expression and placental concentrations of FFA. Preeclamptic group alone, r² = 0.237, P-value < 0.05.

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4. Discussion 

There is an increasing interest regarding the role of LXRα and LXRβ in placental biology [9], [12], [20], [21], [22], [23], however, their regulation in placenta in pathological pregnancy has to the best of our knowledge not previously been addressed. In this paper, we show a statistically significant lower mRNA expression of LXRα and LXRβ in preeclamptic placentas as compared to controls. Furthermore, we are able to detect both LXRα and LXRβ proteins expression in healthy controls, and we found the protein level of LXRβ to be down-regulated in the preeclamptic placentas compared to controls. However, we did not find any regulation of LXRα protein expression in preeclamptic placentas as compared to controls. The observed discrepancies between the transcript and protein level could suggest mRNA processing or post-translational modifications of LXRα in placenta dissimilar to that of LXRβ. LXRα has previously been reported to be differentially expressed on mRNA and protein levels in macrophages [24], and recently the amount of LXRα protein was shown to be highly regulated by LXRα agonists, preventing the protein from degradation by ubiquitination [25].

Taken together, the significant altered placental expression of LXRβ, suggests that this nuclear receptor, might play a regulatory role during preeclampsia. Although the observed changes in gene expression are small, weak changes in gene expression, especially of transcription factors, can have major differences in vivo as they can alter the expression of many different genes [26].

We observed a decreased expression of CD36 in preeclamptic placentas compared to controls. CD36 is a direct LXR target gene in liver and a fatty acid transport protein [27]. However, Laivuori et al did not find any difference in expression of CD36 in preeclamptic placentas compared with those of controls [28]. This contrasting data may be due to study differences in mode of delivery, differences in gestational age or other differences between the study populations. CD36 is important for the LXR mediated increase in liver FFA concentrations in mice [27]. We demonstrated a statistically significant decreased concentration of FFA in preeclamptic placentas compared to controls. LXR activation is previously reported to increase FFA levels in pancreatic β cells and liver tissue [27], [29], in our study we observed a positive correlation in placenta between expression of LXRβ and levels of FFA in preeclamptic subjects. Taken together, these data suggest that LXRβ could play a role in the control of preeclamptic placental concentrations of FFA.

We found significantly higher concentrations of total n-6 PUFAs (esterified and non-esterified fatty acids together) in preeclamptic placentas as compared to controls. This could indicate a selective uptake or reduced oxidation of n-6 PUFAs by the placenta in order to meet the fetal demands of long-chain PUFAs. Circulating free fatty acid levels of linoleic acid have been reported to be increased in preeclampsia in one study [30]. Wang et al found lower concentrations of total non-esterified n-6 and n-3 PUFAs mainly due to lower concentrations of ARA and docosahexaenoic acid (DHA) in preeclamptic placentas compared to controls [31]. This discrepancy between our findings could be due to differences in the study populations, such as differences in delivery mode and inclusion of diabetic patients in their study. It could possibly also be due to the difference in measuring non-esterified fatty acids alone or esterified and non-esterified fatty acids together.

The expression of LXRα and LXRβ is influenced by the metabolic changes of fasting and subsequent refeeding [32], [33] and by oxidative stress in different tissues [34]. Strength of our study is that our results were not confounded by the influence of variations in fasting-feeding as all women were fasted for a minimum of 6 h. In addition, elective cesarean section secured no variation in labor duration and thereby possible variations in oxidative stress and stretching of fetal membranes.

Placenta is composed of a number of different cell types. LXRα and LXRβ are reported to be expressed in both trophoblasts, macrophages and endothelial cells, which are also present in the placenta [9], [35], [36]. To explore which placental cells are important for the differences in gene expression and protein levels observed between the preeclamptic and control group, it would be necessary to investigate the protein expression of LXRα and LXRβ in the different placental cell types, e.g. using immunohistochemistry (IHC). However, due to the relative high background detecting LXRs by western blotting, and the less controlled antibody-specificity in IHC (due to lack of size controls), better LXR antibodies need to be developed. These experiments are ongoing in our laboratory.

There is a significant difference in gestational age between our two study groups with the preeclamptic group delivering earlier than the control group. Premature deliveries of uncomplicated pregnancies are ethically unacceptable and not available as a control group. Similarly, premature deliveries are normally due to pathological conditions, such as inflammation/infection or placental abruption and therefore not suitable as controls. Correcting for gestational age is mathematically possible but it is not necessarily biologically correct, as premature delivered women with preeclampsia will generally have a more severe form of the disease than women delivered at term [37]. Still we cannot exclude that differences in gestational length between the study groups could potentially affect our conclusions.

In summary, the present study shows a down-regulation of LXRα and LXRβ mRNA levels and LXRβ protein levels in preeclamptic placentas compared to controls. Further, it suggests a role for placental LXRβ in regulating placental FFA concentrations in third trimester preeclamptic pregnancy.

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Acknowledgements 

The work is supported by research grants from the Medical faculty, University of Oslo, the Regional Health Authority of South-Eastern Norway, Oslo University Hospital, Ulleval and the Norwegian Research Council.

We are grateful for the contribution of Drs Nina K. Harsem and Kristin Brække in recruiting some of the patients included in the study, as well as to the patient inclusion and biobank organization of Lise Levy, all from Oslo University Hospital, Ulleval.

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