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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0849
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 620-626
Copyright © 2005 by The Endocrine Society

Circulating Activators of Peroxisome Proliferator-Activated Receptors Are Reduced in Preeclamptic Pregnancy

Leslie L. Waite, Rachel E. Louie and Robert N. Taylor

Center for Reproductive Sciences, University of California San Francisco (L.L.W., R.N.T.), San Francisco, California 94143; and Division of Biological Sciences, Section of Ecology, Behavior, and Evolution, University of California San Diego (R.E.L.), San Diego, California 92093

Address all correspondence and requests for reprints to: Dr. Leslie Waite, Box 0556, San Francisco, California 94143. E-mail: lwaite{at}itsa.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We previously described activators of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in the serum of pregnant women. We have also characterized this activating component by using a hexane-extracted serum fraction to examine PPAR activator levels in normal and preeclamptic (PE) pregnancies. In this study we report that the pregnancy PPAR activator is present in similar concentrations in serum and plasma. We also found that the activating fractions from pregnancy sera stimulate not only PPAR{gamma}, but also PPAR{alpha}, and are capable of inhibiting the production of inflammatory cytokines, consistent with known PPAR ligands. In experiments comparing extracts from normal and PE patients, we found that extracts from women with severe PE showed a reduced level of PPAR activation compared with extracts from normal pregnant women. This reduction was more pronounced for PPAR{gamma} than PPAR{alpha} activation. Finally, this reduction in circulating PPAR activator was observed weeks and sometimes months before the clinical diagnosis of PE. Based on these results, we conclude that PPAR activation is reduced in preeclamptic pregnancy before the onset of maternal symptoms. We speculate that endogenous regulators of PPAR play a role in maternal metabolism and immune function in normal and pathological pregnancies.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PREECLAMPSIA (PE) IS a syndrome of pregnancy characterized by high blood pressure, edema, and proteinuria (1). Additionally, women with PE have evidence of insulin resistance and dyslipidemia. Biochemically, women with PE have been shown to have elevated levels of markers of oxidative stress (2), vasoconstrictive peptides (e.g. endothelin-1) (3), and inflammatory proteins (e.g. TNF-{alpha} and plasminogen activator inhibitor type 1) (4, 5). PE is believed to originate in defective placental development, which is manifested by poor trophoblastic invasion (6, 7). This defective placentation results in the systemic maternal syndrome that is diagnosed as PE.

The peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that are involved in lipid and glucose homeostasis (8). Additionally, they have been found to be important regulators of the inflammatory response and factors involved in the endothelial cell activation of atherosclerosis (9). PPARs are transcriptional activators that bind as heterodimers with the retinoic X receptor (RXR) to PPAR DNA response elements (PPREs) that are found upstream of PPAR target genes. PPARs are highly conserved across species, with mouse, rat, and human sequences showing high levels of amino acid homology (10).

Studies in mouse knockout models have implicated PPARs in placental vascular development (11, 12). Loss of PPAR{gamma} is lethal on embryonic d 9.5–10.5, and histological analyses show major placental defects in these animals. Investigations by ourselves and others studying human placenta have shown that PPAR{gamma} is expressed in trophoblasts and that activation of PPAR{gamma} can regulate trophoblast differentiation, invasion, and secretion of proinflammatory cytokines such as TNF-{alpha} (13, 14, 15, 16, 17, 18). Furthermore, we have discovered circulating activators of PPARs that are increased in pregnancy (13). These studies strongly support the concept that PPARs, in particular PPAR{gamma}, are crucial for proper placentation in humans and may play a role in the maintenance of normal human pregnancy.

We were among the first to formulate the hypothesis that impaired placental invasion leads to the release of factors into the maternal circulation that result in increased levels of inflammatory cytokines, endothelial cell activation, and the clinical signs of PE (19). Given that PPAR{alpha} and PPAR{gamma} have been identified as antiinflammatory regulators, we studied whether the circulating PPAR activation factor that we observed in normal pregnancy is reduced in PE, and thus whether it might contribute to the pathogenesis of PE.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
All serum and plasma samples were collected under approved protocols of the University of California-San Francisco internal review board and met all federal, state, and local requirements for protection and ethical treatment of human subjects.

Unless otherwise stated, chemicals, media, and reagents were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich Corp. (St. Louis, MO).

Patient selection and criteria for PE

All women were nulliparous. Normal pregnant and PE patients were matched for race and gestational age (GA). Criteria for PE were based on American College of Obstetricians and Gynecologists definitions of hypertension combined with proteinuria. Hypertension was defined as a rise of 30 mm Hg systolic or 15 mm Hg diastolic vs. pre-20-wk GA measurements. In the absence of pre-20-wk GA data, an absolute value greater than or equal to 140 mm Hg systolic or 90 mm Hg diastolic was used. Proteinuria was defined as 600 mg/24 h, or a 1+ dipstick reading on a catheterized sample (~30 mg/dl) or a 2+ dipstick reading on a voided sample (≥100 mg/dl).

For severe PE, proteinuria was defined as greater than 5 g in 24 h. In addition, severe preeclamptics met one of the following additional criteria: an absolute blood pressure reading of more than 160 mm Hg systolic or 110 mm Hg diastolic, platelet count less than 140,000/ml, or liver function tests (aspartate aminotransferase or alanine aminotransferase) of 1.5 times the upper normal range. Blood hemoglobin and hematocrit measurements were used to evaluate the degree of hemoconcentration. In all cases, abnormal measurements returned to normal levels postpartum.

Plasmids

Galactosidase-4 (GAL4)-human (h) PPAR fusion constructs were a gift from Dr. Thomas Scanlan (University of California-San Francisco). PPAR{gamma}, PPAR{alpha}, GAL4-hRXR{alpha}, and PPRE3 constructs were provided by Dr. Ronald Evans (The Salk Institute, La Jolla, CA). All plasmids were described previously (20, 21). Briefly, the PPAR genes were inserted downstream of a cytomegalovirus promoter that allows constitutive expression. The PPRE3 plasmid [PPRE3-thymidine kinase (TK)-luciferase (luc)] contains three consensus PPRE sequences upstream of a minimal TK promoter and firefly luc gene. GAL4 fusion receptors contain the ligand binding domains of the human PPAR or RXR genes inserted downstream of the GAL4 DNA binding domain and nuclear localization sequence. GAL4 UAS plasmid contains the GAL4 response element upstream of the firefly luc gene. pRL-TK (Promega Corp., Madison, WI) is an internal control vector containing Renilla luc under control of a constitutively expressed thymidine kinase promoter.

Cell culture of choriocarcinoma cells

JEG-3 cells (American Type Culture Collection, Manassas, VA) were cultured in MEM/Eagle’s medium with Earle’s salts plus 10% fetal bovine serum (Omega Engineering, Stamford, CT) and antibiotics. Three days before treatment, cells were passaged using trypsin and transferred to medium containing 2% dextran-charcoal-stripped serum (Omega Scientific, Inc., Tarzana, CA) instead of fetal bovine serum to allow quiescence. One day before treatment, cells were plated into 12-well plates at a density of 3 x 105 cells/well, again in dextran-charcoal-stripped serum medium and allowed to attach overnight. These cells were then treated as described in the text and figures.

TNF-{alpha} production in U937 cells

Human U937 cells (American Type Culture Collection) were cultured according to the manufacturer’s directions. Cells were plated at 2 x 105 cells/well in 24-well plates and induced to undergo differentiation with 10 nM phorbol dibutyrate (PDB). Some wells were also treated with increasing concentrations of either pregnancy serum extracts or 15-deoxy-{Delta}12,14-prostaglandin J2 (PGJ2) as described in the text and figures. Approximately 24 h after treatment, conditioned media were collected from the wells, and TNF-{alpha} levels were measured using a Quantikine TNF-{alpha} ELISA kit (R&D Systems, Minneapolis, MN).

Cell transfection

Superfect (Qiagen, Valencia, CA) was used to transfect cells according to the manufacturer’s instructions. Optimal concentrations of DNA per well were determined empirically for each vector (Waite, L. L., unpublished observations). The vector concentrations used in these experiments were 1.8 µg PPRE, 45 ng pRL-TK, and 2 µg PPAR{gamma} or PPAR{alpha}. For GAL4 fusion experiments, final DNA levels per well were 160 ng GAL4-X (X = RXR{alpha}, PPAR{alpha}, PPARß, or PPAR{gamma}) and 480 ng GAL4-UAS. Cells were transfected for 2 h before treatment.

Hexane extraction of serum

Serum and plasma were extracted as previously described (22). Briefly, acidified serum or plasma was extracted twice with hexane, and the combined organic fractions were dried under nitrogen, then resuspended in ethanol equal to 0.1 times the original sample volume. This was added to serum-free medium and used to treat cells. In some experiments hexane fractions were additionally extracted with ice-cold methanol, and the methanol fraction was dried under nitrogen. No difference in activity was observed between hexane vs. methanol fractions (data not shown). In the text, the percent serum or plasma extract refers to the extract equivalent to a given volume of serum or plasma. For example, if 100 µl serum were extracted, resuspended in 10 µl ethanol and added to a final volume of 1 ml medium, this would be referred to in the text as 10% extract.

Statistics

Data are presented as the mean ± SEM of combined replicate experiments and as the mean ± SD for representative single experiments performed in triplicate. Due to the small sample sizes, Mann-Whitney U tests were used to compare results between normal and PE groups. For hemoglobin and hematocrit analyses comparing the four patient groups (early-onset severe PE, late-onset severe PE, mild PE, and normal pregnant), the Kruskal-Wallis test was used. For simultaneous evaluation of patient diagnosis and extract concentration in PGJ2-stimulated JEG-3 cells, a two-way repeated measures ANOVA was used. In all statistical analyses, P = 0.05 was set as a threshold for statistically significant differences.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
PPAR{gamma} activators partition to the organic phase of extracted serum and plasma

Previously, we established that serum obtained from normal pregnant women contained activators of PPAR{gamma} (13). Additionally, we showed that albumin acts as an inhibitor of naturally occurring PPAR activators in our in vitro system (23). Therefore, to optimize our ability to detect activation and to eliminate potentially confounding factors due to differences in albumin isoforms (24), we purified the activating component(s) of serum away from albumin and other potential protein modifiers by organic extraction as described in Subjects and Methods. These extracts were used to treat JEG-3 cells that were transfected as described in Subjects and Methods. JEG-3 cells are a choriocarcinoma cell line that we and others have successfully used as a transfection competent model for placental cells. Serum extracts exhibit a relative activity level 2- to 4-fold higher than the original whole serum for PPAR{gamma} activation, suggesting the successful removal of inhibitory components (e.g. albumin) and the partial purification of activating component(s) (Fig. 1Go).



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  		FIG. 1. Activation of PPAR{gamma} by organic extracts of serum and plasma from normal pregnancy. Quiesced JEG-3 cells were transfected and treated for 24 h with 10% serum or the equivalent amount of extracted serum or plasma from the same patient. Lysed cells were analyzed for luc activity relative to controls. Values represent the average of triplicate wells ± SD.

 
We also determined that the pregnancy activator was present in plasma as well as serum. Serum contains cellular components released during clotting ex vivo that do not reflect normal circulating factors. To verify that the activator we observed in serum was indeed a circulating factor and not a byproduct of coagulation, we extracted a matched set of serum and plasma collected from the same normal pregnant woman obtained at a single blood draw. Heparin was found to inhibit activation of PPAR{gamma} in our cell model (Waite, L. L., unpublished observations), preventing our ability to use heparinized plasma in our studies. However, as shown Fig. 1Go, we found that extracted EDTA plasma and serum contained a similar level of activator(s), confirming that the activating principle in pregnancy serum is a circulating factor.

Serum extracts activate PPAR{gamma} and PPAR{alpha}, but not PPARß or their heterodimeric partner RXR{alpha}

Transcription of PPAR-regulated genes also can occur when the heterodimeric partner of the PPARs, RXR{alpha}, is activated. Because we do not know the identity of the PPAR-activating component(s) in the blood of pregnant women, it is possible that the PPRE activation we observed is due to activation of the endogenous RXR{alpha}. Also, because many of the known, naturally occurring ligands for PPARs activate more than one member of the PPAR family, we were curious as to whether our activating component might stimulate other related nuclear receptors. We therefore used a series of GAL4-nuclear receptor fusion constructs to determine which individual receptors were activated by the hexane extract. In this assay, the DNA binding domain of the yeast GAL4 gene was fused in-frame to the ligand binding domain of PPAR{alpha}, PPARß, PPAR{gamma}, or RXR{alpha}. When activated by ligand binding, the receptor fusion protein activates transcription via a GAL4 response element (GAL4 UAS) engineered upstream of the firefly luc gene. We used extracts from normal pregnant women’s sera to treat JEG-3 cells transfected with the GAL4 UAS plasmid and one of the four GAL4 fusion vectors. As a positive control for each experiment, some cells also were treated with a ligand specific to each receptor [WY,14643 or 8(S)HETE for PPAR{alpha}, PGA1 for PPARß, rosiglitazone for PPAR{gamma}, and 9-cis-retinoic acid for RXR{alpha}]. As shown in Fig. 2Go, the extracts were able to activate PPAR{alpha} and PPAR{gamma}, but neither PPARß nor RXR{alpha}. In all cases, positive controls showed strong activation of their respective receptors (data reported in figure legend as fold activation).



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  		FIG. 2. Signaling by GAL4 fusion receptors in JEG-3 cells treated with normal pregnant serum extracts. Cells were transfected with cDNA for GAL4-PPAR or GAL4-RXR fusion receptors as described in the text. Triplicate wells were treated with extracts from sera as indicated. Signaling was normalized to similarly transfected controls. Values represent the average of triplicate wells ± SD. The fold activation of positive controls is as follows: Wy14,643 (PPAR{alpha}), 6.3; PGA1 (PPARß), 7.8; rosiglitazone (PPAR{gamma}), 33.9; and 9-cis-retinoic acid (RXR{alpha}), 170.4.

 
Pregnancy PPAR activator inhibits production of TNF-{alpha} in PDB-differentiated U937 cells

Activation of PPAR{gamma} has been shown to reduce the production of inflammatory cytokines in vascular cells and monocytes. Many of these cytokines are known to be increased in PE, suggesting a loss of PPAR activity in this syndrome. To determine whether the pregnancy PPAR activator might play a role in the regulation of inflammatory cytokines in pregnancy, we differentiated U937 cells, a model human cell line for monocyte/macrophage function, with PDB for 24 h in the presence or absence of the pregnancy activator. These cells previously have been shown to express endogenous PPAR{gamma} and to up-regulate the production of TNF-{alpha} in response to phorbol esters (25, 26). Furthermore, it has been shown that TNF-{alpha} production in these cells is inhibited by known PPAR activators (26). As shown in Fig. 3Go, production and secretion of TNF-{alpha} by U937 cells were reduced in a dose-responsive manner by the PPAR pregnancy activator and by PGJ2, a natural PPAR{gamma} agonist used as a positive control to confirm the antiinflammatory effects of PPAR{gamma} activation in this system. The findings confirm that the pregnancy PPAR activator can act as a negative regulator of the inflammatory response in monocytic cells.



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  		FIG. 3. TNF-{alpha} production in differentiated U937 cells treated with pregnancy PPAR activator. Conditioned medium was collected from cells treated with 10 nM PDB in the presence or absence of increasing amounts of plasma extract from normal pregnancy or PGJ2, then analyzed for TNF-{alpha} by ELISA.

 
PPAR{gamma} activators are reduced in preeclamptic pregnancy

Given that the circulating PPAR activator in pregnancy is capable of mitigating cellular inflammation, and because PE is known to reflect an environment of immune activation, we investigated the hypothesis that the pregnancy PPAR activator was attenuated in PE compared with normal pregnancy. We used serum extracts from 14 PE and 14 race- and GA-matched normal, nulliparous pregnant women; five subjects experienced severe early-onset PE (≤32 wk gestation), five women had severe late-onset PE (after 32 wk gestation), and four had mild PE. Extracts from each patient’s serum were used to treat JEG-3 cells that had been transfected with plasmids containing cDNA for PPAR{gamma} and the PPRE as well as a control vector (pRL-TK). As shown in Fig. 4Go, serum extracts from women with severe early-onset PE showed a reduction in activation of PPAR{gamma} of more than 60% compared with that in serum extracts of normal pregnant women (P = 0.02). Similarly, severe late-onset PE showed a significant, albeit lesser (55%), decrease in PPAR{gamma} activation relative to matched normal pregnant controls (P = 0.05). We failed to observe a decrease in PPAR{gamma} activation in mild PE (P = 0.42; Fig. 4Go). However, the latter blood samples were collected at or very near term, and we noted that this result reflected an overall GA-dependent reduction in activator levels in the normal controls rather than an increase in the level of activator in mild PE (Fig. 5Go). The mean GAs of each of the three groups were 30.4, 35.4, and 37.8 wk, respectively. These results have been confirmed using samples from different patients (data not shown).



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  		FIG. 4. PPAR{gamma} activation by serum extracts from normal pregnant (NL) women and preeclamptics. Quiesced JEG-3 cells were transfected and treated for 24 h with 10% serum extracts as described in the text. Lysed cells were analyzed for luc activity relative to cells treated with serum-free medium. Values represent the average of five triplicate samples ± SEM. *, Differences between NL and PE treatments were assessed by Mann-Whitney analysis (P = 0.02, P = 0.05, and P = 0.39 for early-onset severe, late-onset severe, and mild preeclamptics, respectively).

 


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  		FIG. 5. Comparison of PPAR{gamma} signaling in normal and PE patients at various GAs. Quiesced JEG-3 cells were transfected and treated for 24 h with 10% serum extracts as described in the text. Lysed cells were analyzed for luc activity relative to transfected untreated cells. Values represent the averaged results combined from several experiments ± SEM. *, Differences between normal pregnant women (NL) and mild PE extracts were statistically significant by Mann-Whitney analysis for 21–26 (P = 0.003) and 36–40 (P = 0.007) wk GA. Numbers in parentheses indicate the number of NL and PE samples, respectively, used for analysis at the given GA.

 
To assess levels of hemoconcentration in our patients, we checked hemoglobin and hematocrit values for all four groups of women (early-onset severe PE, late-onset severe PE, mild PE, and normal pregnant). No significant differences were observed in these values among the groups (P = 0.49 for hemoglobin; P = 0.46 for hematocrit; determined by Kruskal-Wallis analysis).

PPAR activation is reduced weeks before clinical onset of PE

If dysregulation of PPAR activation plays any causal role in the maternal syndrome of PE, reduced serum activators should appear before the onset of clinical symptoms of PE. We performed a prospective, cross-sectional study of pregnant women across all GAs. Serum extracts from PE patients (21 samples from 11 women) and normal pregnant controls (18 samples from seven women) were used in our cell model. As shown in Fig. 5Go, significantly reduced levels of PPAR activators were seen as early as 21–26 wk, a full 10–15 wk before presentation of symptoms in these mild preeclamptics. As in the results of Fig. 4Go, the level of PPAR activators decreased as samples were collected closer to delivery, and activation was indistinguishable between PE and controls by 48 h postpartum.

PPAR activator in both normal and PE women shows similar receptor activation profiles

The reduction that we found in PPAR activation by sera of PE patients could be explained in several ways. First, women with PE could be making different compounds or metabolizing the activating components for PPARs. Second, women with PE might be synthesizing similar levels of activating compounds, but also be making an additional inhibitory compound(s) that impairs PPAR activation. Finally, women with PE could be making the same compounds, but simply have a lower steady state level of the PPAR activator than normal pregnant women. To determine whether the activating components in normal and PE pregnancy might be different compounds, we examined the abilities of PE extracts to activate the three PPAR receptors and the RXR receptor, as we had done previously with normal serum using GAL4 fusion constructs. As observed with normal serum extracts, the activating component from PE patients stimulated PPAR{alpha} and PPAR{gamma}, but not PPARß or RXR{alpha} (Fig. 6AGo). Stimulation of PPAR{alpha} and PPAR{gamma} was reduced in PE, consistent with our results using the intact PPAR{gamma} receptor. Thus, it appears that the activating fraction from PE patients, although present in lower concentrations, has biochemical characteristics similar to those of extracts from normal pregnant women’s serum.



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  		FIG. 6. A, Comparison of the PPAR stimulation profile of serum extracts from normal pregnant (NL) and PE women. Quiesced JEG-3 cells were transfected with a GAL4 UAS-luc DNA construct plus a fusion receptor containing the GAL4 DNA binding domain and the ligand binding domain from one of the PPAR receptors or RXR-{alpha} as indicated above and in the text. After treatment with serum extract for 24 h, cells were lysed and analyzed for luc activity. Bars represent average values of triplicate wells ± SD. Values for treatment with positive controls were: 8(S)HETE (PPAR{alpha}), 6.8; PGA1 (PPARß), 372.5; rosiglitazone (PPAR{gamma}), 13.4; and 9-cis-retinoic acid (RXR{alpha}), 378.8. B, PPAR{alpha} signaling in JEG-3 cells treated with serum extracts from NL and early-onset severe PE women. Quiesced JEG-3 cells were transfected with PPAR{alpha} and treated for 24 h with 10% serum extracts as described in the text. Lysed cells were analyzed for luc activity relative to transfected untreated cells. Values represent the mean ± SD for triplicate wells, except for average values, which represent the mean ± SEM. Differences between NL and PE treatments were not statistically significant by Mann-Whitney analysis (P = 0.07).

 
PPAR{alpha} activation by serum extracts is not significantly reduced in PE pregnancy

The results of the GAL4 fusion assays (Figs. 2Go and 6AGo) suggested that the pregnancy PPAR activator could exert effects via PPAR{alpha} as well as PPAR{gamma}. We therefore examined activation of the full-length PPAR{alpha} receptor by normal and PE serum extracts. We repeated the experiment described in Fig. 4Go, using severe early-onset PE extracts and full-length PPAR{alpha} instead of PPAR{gamma}. Although a consistent trend of reduced PPAR activation was seen in these experiments, it did not reach statistical significance (Fig. 6BGo; P = 0.07). Additionally, the overall stimulation of the PPAR{alpha} receptor was much lower than what we observed using PPAR{gamma} (2- vs. 6.5-fold), suggesting that the pregnancy PPAR activator is a more effective agonist of PPAR{gamma}.

Reduction of PPAR{gamma} activation in PE extracts is due to lower levels of activating factors

We next examined whether PPAR{gamma} activation by extracts from pooled normal and PE sera was dose dependent. As shown in Fig. 7Go, increasing concentrations of either type of serum extract resulted in increasing levels of PPAR activation. As expected, extracts from the pooled PE samples showed lower levels of activation than extracts from normal pregnant women at all tested concentrations. Thus, both types of serum contain PPAR{gamma}-activating components.



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  		FIG. 7. PPAR{gamma} signaling in JEG-3 cells treated with increasing concentrations of serum extracts from normal pregnant (NL) and PE women. Wells were treated with the indicated concentrations of pooled serum extracts from NL and PE subjects compared with control cells. Values represent the mean ± SD for triplicate determinations.

 
The lower level of PPAR{gamma} activation observed in PE extracts could be due to lower levels of activator(s), higher levels of inhibitor(s), or a combination of the two. To determine whether PE sera also contained inhibitory factors that prevented full activation of PPAR{gamma}, we mixed increasing amounts of PE serum extract with a fixed amount of normal pregnant serum extract to determine how this affected PPAR{gamma} stimulation. We chose 6% normal pregnant serum extract, because this concentration approximated the 50% effective concentration for PPRE stimulation. We deduced that if the PPAR activator in normal pregnancy serum was also present in PE serum, but simply in lower amounts, then mixing the two extracts together would result in an increase in PPAR activation. If, however, PE serum also contains inhibitory factors, then mixing the two extracts together should inhibit the ability of normal serum extracts to stimulate PPAR{gamma}. As shown in Fig. 8Go, the addition of increasing levels of PE serum extract to 6% normal pregnant serum extract resulted in augmented PPAR{gamma} activation. This suggests that the lower PPAR{gamma} activation seen in PE extracts is predominantly due to a reduction of activating factors, rather than the presence of an inhibitory compound(s).



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  		FIG. 8. PPRE activation in cells treated with normal serum extracts and increasing amounts of PE serum extract. JEG-3 cells were transfected as described in the text, and triplicate wells were treated with 6% pooled normal pregnant serum extract and the indicated amount of pooled PE serum extract. After 24 h of treatment, cells were lysed and analyzed for luc activity. Values represent the mean ± SD for each triplicate determination.

 
We wanted to confirm this result using a naturally occurring known PPAR{gamma} ligand. We therefore repeated the experiment in Fig. 8Go using 50 nM PGJ2 as our baseline PPAR{gamma} stimulation. This concentration of PGJ2 activates PPAR{gamma} at a level similar to 10% normal serum extract. When increasing concentrations (2.5–15%) of serum extract from either normal pregnant or PE women were added to these PGJ2-stimulated cells, both types of serum extract showed a significant increase in PPAR{gamma} stimulation (1.8-fold at 15% extract; P < 0.001. by two-way repeated measures ANOVA; data not shown). This effect did not differ between the two groups of subjects (P = 0.48). These data support our interpretation that both normal pregnant and PE extracts contain PPAR activators, and that PE extracts do not contain additional inhibitory components.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present studies we define organic extracts of serum and plasma that contain a circulating activator of PPARs that we previously described (13). This activating fraction is present throughout gestation in normal pregnancy, but drops significantly in women destined to develop PE weeks before the onset of symptoms. We show in this study that although the activating fraction is capable of stimulating the activity of both PPAR{alpha} and PPAR{gamma}, the loss of activity in PE is only statistically significant for PPAR{gamma}. Finally, we demonstrate that the activating fraction is capable of inhibiting production of the proinflammatory cytokine TNF-{alpha}, suggesting a role in the regulation of inflammation during pregnancy.

It is well known that hemoconcentration can occur in severe PE (27). Our measurements of hemoglobin and hematocrit levels of the patients in our studies suggest that hemoconcentration is not occurring in these women and is therefore unlikely to play a role in the effects we observed. However, even in cases where hemoconcentration might occur, it would be expected to result in increased circulating PPAR activators in severe PE, not decreased levels as we observed in our study. Therefore, the drop in activator levels appears to reflect a substantial inhibition in synthesis or an accelerated catabolism of PPAR-activating components. This loss of PPAR activator levels could create a feed-forward loop of increasing proinflammatory cytokine production that further reduces PPAR activity (28).

It is interesting that clinically mild and severe forms of PE had similar levels of PPAR signaling. This result emphasizes the fact that the similarity of PPAR activation in mild preeclamptics is due less to a rise in PPAR activator levels in this group and more to a decrease in PPAR activation in normal pregnancy as GA advances. The data indicate that PPAR activation decreases naturally toward term. Such a reduction might reflect an overall shift in humoral signals that trigger the onset of labor. Because this fall in PPAR activation occurs prematurely in women with PE, one result might be conflicting maternal-fetal signals that balance the maintenance of pregnancy vs. delivery. This conflict could be part of the resulting maternal syndrome of PE. This idea is consistent with recent work from Dunn-Albanese et al. (29). They found that the expression of PPAR{gamma} protein in fetal membranes decreases with labor and suggest that a drop in PPAR{gamma} activity might facilitate parturition. It is possible that the reduced PPAR{gamma} protein levels they observed are related to the loss of circulating activator we describe in this report.

Our results are consistent with other clinical evidence that loss of antiinflammatory regulation by PPAR{gamma} may be an initiating factor in the maternal syndrome of PE. It has been proposed that pregnancy is an inflammatory state, and that PE is a hyperinflammatory state (30). It is reasonable to expect that careful regulation of inflammatory cytokines would be critical to maintain normal pregnancy and prevent PE. PGF2{alpha}, which is stimulated by factors in the plasma of women with PE (31), inhibits PPAR{gamma} (32) The inflammatory cytokines TNF-{alpha} and IL-1, which are in a negative feedback loop with PPARs that operates via the nuclear factor-{kappa}B pathway, are also elevated in PE (33, 34). Reduced levels of PPAR activators could result in increases in these compounds; conversely, elevated levels of these compounds would be predicted to result in a decrease in PPAR activation in PE pregnancy relative to that in normal pregnancy. The loss of PPAR activation in PE that we demonstrate here could account for the widespread increase in inflammatory cytokines and endothelial cell activation that have been described as the maternal response component of PE (33). Additionally, the fact that this reduction in PPAR activators occurs months before the onset of clinical disease suggests that it may be an early step in the evolution of PE. The circulating pregnancy PPAR activator may thus be a good candidate for an early diagnostic marker of PE and potentially could represent a targeted therapeutic. We are currently undertaking studies to purify and determine the identity of this activator.


   Footnotes
 
This work was supported by NIH Grant HL-73469.

First Published Online November 23, 2004

Abbreviations: GA, Gestational age; GAL, galactosidase; h, human; luc, luciferase; PDB, phorbol dibutyrate; PE, preeclampsia; PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; RXR, retinoic X receptor; TK, thymidine kinase.

Received May 5, 2004.

Accepted November 14, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Roberts J, Redman C 1993 Pre-eclampsia: more than pregnancy-induced hypertension. Lancet 341:1447–1451[CrossRef][Medline]
  2. Hubel CA 1999 Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med 222:222–235[Abstract/Free Full Text]
  3. Taylor RN, Varma M, Teng NNH, Roberts JM 1990 Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J Clin Endocrinol Metab 71:1675–1677[Abstract]
  4. Chappell L, Seed P, Briley A, Kelly F, Hunt B, Charnock-Jones D, Mallet A, Poston L 2002 A longitudinal study of biochemical variables in women at risk of preeclampsia. Am J Obstet Gynecol 187:127–136[CrossRef][Medline]
  5. Conrad KP, Miles TM, Benyo DF 1998 Circulating levels of immunoreactive cytokines in women with preeclampsia. Am J Reprod Immunol 40:102–111
  6. Meekins JW, Pijnenborg R, Hanssens M, McFadyen IR, van Asshe A 1994 A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies. Br J Obstet Gynaecol 101:669–674[Medline]
  7. Brosens IA, Robertson WB, Dixon HG 1972 The role of the spiral arteries in the pathogenesis of preeclampsia. Obstet Gynecol Ann 1:177–191[Medline]
  8. Evans R, Barish G, Wang Y-X 2004 PPARs and the complex journey to obesity. Nat Med 10:1–7[CrossRef]
  9. Duval C, Chinetti G, Trottein F, Fruchart J, Staels B 2002 The role of PPARs in atherosclerosis. Trends Mol Med 8:422–430[CrossRef][Medline]
  10. Kersten S, Desvergne B, Wahli W 2000 Roles of PPARs in health and disease. Nature 405:421–424[CrossRef][Medline]
  11. Barak Y, Nelson M, Ong E, Jones Y, Ruiz-Lozano P, Chien K, Koder A, Evans R 1999 PPAR{gamma} is required for placental, cardiac, and adipose tissue development. Mol Cell 4:585–595[CrossRef][Medline]
  12. Barak Y, Liao D, He W, Ong E, Nelson M, Olefsky J, Boland R, Evans R 2002 Effects of peroxisome proliferator-activated receptor {delta} on placentation, adiposity, and colorectal cancer. Proc Natl Acad Sci USA 99:303–308[Abstract/Free Full Text]
  13. Waite LL, Person EC, Zhou Y, Lim KH, Scanlan TS, Taylor RN 2000 Placental peroxisome proliferator-activated receptor-{gamma} is up-regulated by pregnancy serum. J Clin Endocrinol Metab 85:3808–3814[Abstract/Free Full Text]
  14. Schaiff WT, Carlson MG, Smith SD, Levy R, Nelson DM, Sadovsky Y 2000 Peroxisome proliferator-activated receptor-{gamma} modulates differentiation of human trophoblast in a ligand-specific manner. J Clin Endocrinol Metab 85:3874–3881[Abstract/Free Full Text]
  15. Schild R, Schaiff W, Carlson M, Cronbach E, Nelson D, Sadovsky Y 2002 The activity of PPAR {gamma} in primary human trophoblasts is enhanced by oxidized lipids. J Clin Endocrinol Metab 87:1105–1110[Abstract/Free Full Text]
  16. Capparuccia L, Marzioni D, Giordano A, Fazioli F, De Nictolis M, Busso N, Todros T, Castellucci M 2002 PPAR{gamma} expression in normal human placenta, hydatidiform mole and choriocarcinoma. Mol Hum Reprod 8:574–579[Abstract/Free Full Text]
  17. Tarrade A, Schoonjans K, Pavan L, Auwerx J, Rochette-Egly C, Evain-Brion D, Fournier T 2001 PPAR{gamma}/RXR{alpha} heterodimers control human trophoblast invasion. J Clin Endocrinol Metab 86:5017–5024[Abstract/Free Full Text]
  18. Lappas M, Permezel M, Georgiou H, Rice G 2002 Regulation of proinflammatory cytokines in human gestational tissues by peroxisome proliferator-activated receptor {gamma}: effect of 15-deoxy-{Delta}12,14-PGJ2 and troglitazone. J Clin Endocrinol Metab 87:4667–4672[Abstract/Free Full Text]
  19. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK 1989 Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 161:1200–1204[Medline]
  20. Forman B, Tontonoz P, Chen J, Brun R, Spiegelman B, Evans R 1995 15-deoxy-{Delta}12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR {gamma}. Cell 83:803–812[CrossRef][Medline]
  21. Kliewer S, Umesono K, Noonan D, Heyman R, Evans R 1992 Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358:771–774[CrossRef][Medline]
  22. de Urquiza A, Liu S, Sjoberg M, Zetterstrom R, Griffiths W, Sjovall J, Perlmann T 2000 Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain. Science 290:2140–2144[Abstract/Free Full Text]
  23. Person EC, Waite LL, Taylor RN, Scanlan TS 2001 Albumin regulates induction of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) by15-deoxy-{Delta}12–14-prostaglandin J2 in vitro and may be an important regulator of PPAR{gamma} function in vivo. Endocrinology 142:551–556[Abstract/Free Full Text]
  24. Vigne J-L, Murai J, Arbogast B, Jia W, Fisher S, Taylor R 1997 Elevated nonesterified fatty acid concentrations in severe preeclampsia shift the isoelectric characteristics of plasma albumin. J Clin Endocrinol Metab 82:3786–3792[Abstract/Free Full Text]
  25. Hornung D, Waite L, Ricke E, Bentzien F, Wallwiener D, Taylor R 2001 Nuclear peroxisome proliferator-activated receptors {alpha} and {gamma} have opposing effects on monocyte chemotaxis in endometriosis. J Clin Endocrinol Metab 86:3108–3114[Abstract/Free Full Text]
  26. Jiang C, Ting A, Seed B 1998 PPAR-{gamma} agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82–86[CrossRef][Medline]
  27. Lindheimer MD, Cunningham FG, Roberts JM (ed.) 1999 Chesley’s hypertensive disorders of pregnancy. Stamford, CT: Appleton, Lange
  28. Chinetti G, Fruchart JC, Staels B 2000 Peroxisome proliferator-activated receptors (PPARs): nuclear receptors at the crossroads between lipid metabolism and inflammation. Inflamm Res 49:497–505[CrossRef][Medline]
  29. Dunn-Albanese L, Ackerman WI, Xie Y, Iams J, Kniss D 2004 Reciprocal expression of peroxisome proliferator-activated receptor {gamma} and cyclooxygenase-2 in human term parturition. Am J Obstet Gynecol 190:809–816[CrossRef][Medline]
  30. Redman C, Sargent I 2003 Pre-eclampsia, the placenta and the maternal systemic inflammatory response: a review. Placenta 24:S21–S27
  31. de Groot C, Murai J, Vigne J-L, Taylor R 1998 Eicosanoid secretion by human endothelial cells exposed to normal pregnancy and preeclampsia plasma in vitro. Prostaglandins Leukotriene Essent Fatty Acids 58:91–97[CrossRef][Medline]
  32. Reginato MJ, Krakow SL, Bailey ST, Lazar MA 1998 Prostaglandins promote and block adipogenesis through opposing effects on peroxisome proliferator-activated receptor {gamma}. J Biol Chem 273:1855–1858[Abstract/Free Full Text]
  33. Waite LL, Atwood AK, Taylor RN 2002 Preeclampsia, an implantation disorder. Rev Endocr Metab Disord 3:151–158[CrossRef][Medline]
  34. Daynes R, Jones D 2002 Emerging roles of PPARs in inflammation and immunity. Nat Rev Immunol 2:748–759[CrossRef][Medline]




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