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Endocrinology, doi:10.1210/en.2003-1747
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Endocrinology Vol. 145, No. 10 4583-4591
Copyright © 2004 by The Endocrine Society

Lipids from Oxidized Low-Density Lipoprotein Modulate Human Trophoblast Invasion: Involvement of Nuclear Liver X Receptors

Laëtitia Pavan, Axelle Hermouet, Vassilis Tsatsaris, Patrice Thérond, Tatsuya Sawamura, Danièle Evain-Brion and Thierry Fournier

Institut National de la Santé et de la Recherche Médicale, Unité 427, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes (L.P., A.H., V.T., D.E.-B., T.F.); Maternité Port-Royal, Hôpital Cochin, Université René Descartes (V.T.); Laboratoire de Biochimie Clinique et Métabolique, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes (P.T.), 75006 Paris, France; and Department of Bioscience, National Cardiovascular Center Research Institute (T.S.), Suita, Osaka 565-8565, Japan

Address all correspondence and requests for reprints to: Dr. Thierry Fournier, Institut National de la Santé et de la Recherche Médicale, Unité 427, Faculté de Pharmacie, 75006 Paris, France. E-mail: thierry.fournier{at}univ-paris5.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human embryonic implantation involves major invasion of the uterine wall and remodeling of the uterine arteries by extravillous cytotrophoblast cells (EVCT). Abnormalities in these early steps of placental development lead to poor placentation and fetal growth defects and are frequently associated with preeclampsia, a major complication of human pregnancy. We recently showed that oxidized low-density lipoproteins (oxLDLs) are present in situ in EVCT and inhibit cell invasion in a concentration-dependent manner. The aim of the present study was to better understand the mechanisms by which oxLDL modulate trophoblast invasion. We therefore investigated the presence of oxLDL receptors in our cell culture model of human invasive primary EVCT. We found using immunocytochemistry and immunoblotting that the lectin-like oxLDL receptor-1 was the scavenger receptor mainly expressed in EVCT and was probably involved in oxLDL uptake. We next examined the effect of low-density lipoprotein oxidative state on trophoblast invasion in vitro using EVCT cultured on Matrigel-coated Transwell. We demonstrated that only oxLDL containing a high proportion of oxysterols and phosphatidylcholine hydroperoxide derivatives that provide ligands for liver X receptor (LXR) and peroxisomal proliferator-activated receptor {gamma} (PPAR{gamma}), respectively, reduced trophoblast invasion. We next investigated the presence and the role of these nuclear receptors and found that in addition to PPAR{gamma}, human invasive trophoblasts express LXRß, and activation of these nuclear receptors by specific synthetic or natural ligands inhibited trophoblast invasion. Finally, using a PPAR{gamma} antagonist, we suggest that LXRß, rather than PPAR{gamma}, is involved in oxLDL-mediated inhibition of human trophoblast invasion in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMPLANTATION OF THE human conceptus involves invasion of the uterine epithelium and the underlying stroma by trophoblastic cells, which undergo a complex process involving proliferation, migration, and differentiation. Human placentation is unusual due to the high degree of trophoblast invasion during the first trimester (1), unparalleled in other mammals. The trophoblast, known as an extravillous cytotrophoblast (EVCT), invades the uterine wall and the associated spiral arteries, where it replaces the endothelial lining and most of the musculoelastic tissue of the vessel wall. This arteriole remodeling generates the low resistance vessels that supply the fetus with maternal blood for its growth (2).

Unlike tumor invasion, human trophoblastic invasion is precisely regulated. It is temporally restricted to early pregnancy and is spatially confined to the endometrium, the first third of the myometrium and the associated uterine arteries (3, 4). Defective invasion of the uterine spiral arteries is directly involved in preeclampsia, a major and frequent complication of human pregnancy with serious fetal and maternal consequences (for review, see Ref.5). One of the characteristic pathological lesions seen in the uteroplacental bed of preeclampsia patients is a necrotizing arteriopathy, consisting of fibrinoid necrosis, accumulation of foam cells or lipid-laden macrophages in the decidua, fibroblast proliferation, and a perivascular infiltrate. This lesion, termed acute atherosis, resembles endothelial lesions occurring during atherosclerosis, where oxLDLs [oxidized low-density lipoproteins (LDLs)] play an important role (6).

Blood lipid concentrations are generally elevated during pregnancy, particularly in women with preeclampsia (7). Increases in plasma triglyceride concentrations have been reported (8). LDLs, which are involved in the plasma transport of triglycerides, are smaller, denser, and more susceptible to oxidation during pregnancy (9, 10). In addition, lipid peroxide concentrations in maternal blood increase during pregnancy (11). Lipid peroxides are secreted by the human placenta (12), and oxLDL are metabolized by human trophoblasts (13). We hypothesized that the lipids constituting oxidatively modified LDL particles in blood and/or the placental bed might participate in the modulation of trophoblast invasion during the early steps of placental development. We recently showed by immunohistochemistry that oxLDL are present in cytotrophoblasts of villous and extravillous origin in sections of first trimester human placenta, and preliminary studies showed that oxLDL inhibit cell invasion in a concentration-dependent manner (14). The aim of the present study was to better understand the mechanisms by which oxLDL modulate trophoblast invasion. Therefore, we used our cell culture model of human invasive primary extravillous cytotrophoblasts to investigate the presence of oxLDL receptors in these cells and the influence of LDL oxidation state on trophoblast invasion in vitro. This led us to investigate the effect on cell invasion of oxysterol (7-ketocholesterol) and phosphatidylcholine or cholesteryl ester hydroperoxide derivatives, which are putative ligands for liver X receptor (LXR) and peroxisomal proliferator-activated receptor {gamma} (PPAR{gamma}), respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues
Placental tissues from patients who voluntarily and legally chose to terminate pregnancy during the first trimester (7–12 wk) were obtained from Broussais Hospital (Paris, France). All patients gave informed consent. The tissue was washed in Ca2+- and Mg2+-free Hanks’ balanced salt solution supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. Chorionic villi were dissected, rinsed, and minced for cell isolation.

Isolation and purification of trophoblasts differentiating into EVCT
Extravillous cytotrophoblasts were isolated from chorionic villi by trypsin-deoxyribonuclease digestion and discontinuous Percoll gradient fractionation as recently described (15, 16). Purified EVCT were cultured in Ham’s F-12/DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin on Matrigel-coated (5 mg/ml; Collaborative Biomedical Products, Le Pont de Claix, France), 35-mm Falcon culture dishes or Matrigel-coated Transwell for invasion assays.

Cytotoxicity assay
Lactate dehydrogenase (LDH) release into the cell supernatant was measured to assess cell integrity. Culture medium was collected for the extracellular LDH assay, and the corresponding cells were lysed in 1% Triton X-100 in PBS for determination of intracellular LDH activity. LDH activity was measured spectrophotometrically at 340 nm by monitoring the NADH-dependent conversion of pyruvate to lactate. LDH release was expressed as a percentage of total cellular activity (intracellular plus extracellular).

Isolation and copper oxidation of LDLs
LDLs (1.019 < density < 1.063) were isolated by sequential ultracentrifugation of serum samples from normolipidemic donors as previously described (17). EDTA (0.40 g/liter) was added to the serum to prevent spontaneous lipid peroxidation. LDL purity was checked by agarose gel electrophoresis and by determining its chemical composition. The total protein concentration was measured by the Lowry technique using BSA as a standard. After adjusting the LDL concentration to 1.5 g/liter (expressed as the total LDL concentration), lipoprotein preparations were dialyzed against 100 vol 10 mM sodium phosphate buffer, pH 7, containing 150 mM sodium chloride for 18 h at 4 C in the dark. After dialysis, oxidation was initiated at 37 C by the addition of 0.03, 0.3, or 5 µM cupric chloride (oxLDL) type -C, -B, or -A, respectively) for a period corresponding to the termination phase (Tmax) or to half the termination phase (1/2P). Oxidation was stopped by adding an EDTA solution (20 µM final concentration) and cooling on ice.

Measurement of molecular species of cholesteryl ester (CE) and phosphatidylcholine hydroperoxides (PC)
Lipids were extracted from aliquots of LDL solutions using methanol/hexane (4:10, vol/vol) as previously described (17). Briefly, the hexane (upper phase containing CE) and the methanol/water (lower phase containing PC) layers were separated by centrifugation at 1500 x g for 5 min and dried under a nitrogen stream. The dried PC residue was dissolved in methanol, loaded on the HPLC system, and subjected to phosphatidylcholine molecular species separation as previously described (18) with a 250 x 4.6-mm C18 Kromasil column with 10 mM 6% ammonium acetate (pH 5)/94% methanol as the mobile phase. The dried CE residue was dissolved in methanol containing 1% hexane. CE separation was performed with a 150 x 4.60-mm C18 Spherisorb column and methanol as a mobile phase. Molecular species of PC and CE were detected at 205 nm, and the eluate was then mixed with the chemiluminescence reagent, prepared as described by Yamamoto et al. (19) with slight modifications previously validated by Thérond et al. (18, 20).

Oxysterol measurement
Oxysterol concentrations were measured using a modified version of the procedure described by Brown et al. (21). Briefly, total lipids from the LDL preparations (1.5 g/liter total LDL) were extracted using chloroform/methanol (2:1, vol/vol). 5{alpha}-Cholestane (Sigma-Aldrich Corp., St. Louis, MO) was used as an internal standard. The chloroform layer was dried under a nitrogen stream and saponified for 10 min at 70 C by adding 0.5 M methanolic KOH. Total cholesterol was then extracted using hexane, and the hexane layer was dried under a nitrogen stream. Trimethylsilyl ether derivatives of the oxysterols were prepared by adding Fluka II Silylating Mixture (Supelco, Inc., Bellefonte, PA) for 15 min at 70 C. The trimethylsilyl ether derivatives of oxysterols were separated by gas chromatography (GC-14 A capillary gas chromatograph; Shimadzu Scientific Instruments, Columbia, MD) using a fused carbon-silica column (inside diameter, 30 m x 0.25 mm) coated with (5% phenyl) methylpolysiloxane (film thickness, 1 µm; DB-SMS, Supelco, Inc) and detection by flame ionization (17).

Western blot immunoassay of carbonylation and carbonylated fragmentation of apolipoprotein B (apo B)
Oxidative modification of apo B was determined by quantification of apo B carbonyl groups with 2,4-dinitrophenyl hydroxylase and immunoblot assay as previously described (17). Reagents were from the OXYBLOT Oxidized Protein Detection Kit from Oncor (Quantum Appligene, Illkirch, Bas-Rhin, France). Briefly, native LDL, oxLDL solutions (1.5 g/liter), or irradiated ovalbumin as an external standard was treated with dinitrophenyl hydroxylase (DNPH) derivatization solution and separately analyzed by 4–10% exponential gradient SDS-PAGE as described by Fairbanks et al. (22). Separated proteins were electroblotted onto nitrocellulose membrane. Dinitrophenyl apo B bands and external standard were visualized by chemiluminescence with SuperSignal West Pico Chemiluminescent Substrate from Pierce Chemical Co. (PerBio Science France, Bezons, France) and used to expose radiographic film.

Invasion assays
Transwell inserts (6.5 mm; Costar, Cambridge, MA) containing polycarbonate filters with 8-µm pores coated with Matrigel (10 µl 5 mg/ml Matrigel) were used as previously described (16). The cells (25 x 104 cells/insert) were treated with 50 µg/ml native or oxLDLs, 3 µM 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE, Cayman Chemical, Ann Arbor, MI], 1 µg/ml 7-ketocholesterol (Sigma-Chemie, Saint Quentin Fallavier, France), 1 µM rosiglitazone (BRL 49653, Cayman Chemical), or 5 µM T0901317 (Cayman Chemical); all reagents were dissolved in ethanol. At the concentration used, these compounds did not affect cell integrity (as assayed by LDH measurement), nuclei condensation, or fragmentation [as tested by 4',6-diamido-2-phenylindole hydrochloride (DAPI) staining]. To abolish the activity of PPAR{gamma}/retinoid X receptor (RXR) heterodimers, we used the PPAR{gamma} antagonist bisphenol A diglycidyl ether (BADGE; 50 µM; Fluka, St. Quentin Fallavier, France). After 48 h of culture, cells were immunostained using cytokeratin 7 antibody (1:200; OV-TL 12/30, Dako, Trappes, France) and fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Filters were dissected and mounted in mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA), and the number of cells crossing the membrane was quantified on a BX60 epifluorescence microscope (Olympus Corp., New Hyde Park, NY). For each independent culture, the invasion assay was run in triplicate, and the number of invasive cells that had crossed the porous membrane were counted in fields (10 fields corresponding to ~300–600 invasive cells) and normalized to the corresponding control values.

Immunodetection of scavenger receptors and LXRß
Cells were cultured for 48 h on Matrigel-coated plates. They were then fixed for 20 min in 4% paraformaldehyde [CD36, lectin-like oxLDL receptor-1 (LOX1), LXRß] or for 5 min in ice-cold methanol [scavenger receptor expressed by endothelial cells (SREC)]. After preincubation with 7% donkey serum, ascites containing mouse anti-LOX-1 monoclonal antibody (23) (1:200 dilution), monoclonal CD36 (1:40; Neomarkers, Fremont, CA), polyclonal SREC (G14; 1:40; TEBU, Le Perray-en-Yvelines, France), or monoclonal LXRß (K8917; 1:100; PPMX, Tokyo, Japan) was applied for 2 h at room temperature. Bound antibodies were revealed after 1-h incubation with a 1:100 dilution of a fluorescein-conjugated donkey antimouse antibody (CD36, LOX-1), a 1:200 dilution of a biotinylated donkey IgG antigoat antibody (SREC; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or a 1:200 dilution of a biotinylated goat IgG antimouse antibody (LXRß; Amersham Pharmacia Biotech, Les Ulis, France). This step was followed by a 1-h incubation with streptavidin-fluorescein diluted 1:200 (Interchim, Montluçon, France). In all cases, cells were extensively washed in PBS containing 0.1% Tween 20 between steps. Finally, a drop of fluorescent DAPI mounting medium (Vector Laboratories, Inc.) was added to slides, coverslips were put in place, and slides were examined under an epifluorescent microscope. To ensure the specificity of the immunological reaction, a negative control was performed by replacing the primary antibody with nonimmune serum. Human umbilical vascular endothelial cells (HUVEC) treated with 50 ng/ml phorbol myristate acetate (PMA) for 4 h (24) and U937 cells treated with 10 ng/ml PMA for 5 d were used as positive controls for SREC and CD36 immunodetections, respectively.

Immunoblot analysis of LOX-1 and SREC expressions
EVCT primary cells were untreated or treated with Amax oxLDL (50 µg/ml) for 48 h. HUVEC treated with PMA served as a positive control for SREC (24) and LOX-1 (25) expressions. Cell pellets were sonicated in Laemmli or RIPA buffer (1% Nonidet P-40, sodium deoxycholate 0.5%, SDS 0.1% in PBS 1x) for LOX-1 or SREC immunoblot analysis, respectively. After centrifugation (15 min at 12,000 x g), the extracts were heated at 100 C for 3 min and subjected to SDS-PAGE on a 7.5% gel. One hundred micrograms of total proteins from primary EVCT or HUVEC cell lysates were loaded on the minigel, and separated proteins were electrotransferred onto nitrocellulose membranes using a liquid-blotting apparatus (Bio-Rad Laboratories, Hercules, CA). Transfer was performed in 25 mM Tris, 192 mM glycine, 20% ethanol (vol/vol), and 0.1% sodium dodecyl sulfate (wt/vol) at pH 8.3 for 1 h. The membranes were then washed in 10 mM Tris, 150 mM NaCl, and 0.05% Tween and incubated with LOX-1 (1:1,000) or SREC (1:100) antibody, followed by a 1-h incubation with peroxidase-conjugated antimouse or antigoat secondary antibody, respectively, and were developed using an enhanced chemiluminescence kit (Pierce Chemical Co., Rockford, IL). To correct for differences in protein loading, the membranes were washed and reprobed with a 1:1,000 dilution of polyclonal antibody against human actin (Sigma-Aldrich Corp.).

Statistical analysis
Results represent the mean ± SEM of at least three different cultures obtained from individual placental villi, each run in triplicate. Data were analyzed using ANOVA for multiple comparisons (StatView F4.5 software; SAS Institute Inc., Cary, NC). Results were considered significantly different at P < 0.05 by Mann-Whitney test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of scavenger receptors by invasive EVCT
We have recently localized oxLDL in situ in trophoblasts of villous and extravillous origin (14). In the present study we investigated the presence of the cell surface receptors associated with oxLDL uptake by EVCT in primary EVCT cultured on Matrigel-coated Transwells for 48 h. Figure 1Go depicts LOX-1, CD36, and SREC immunodetections (left panels) and SREC and LOX-1 immunoblots (right panels). The CD36 antibody used for immunocytochemistry was not suitable for immunoblotting. PMA-treated U937 and HUVEC cells were used as positive controls for CD36 and SREC expressions, respectively. Our results show that CD36 was not found in primary EVCT. SREC appears to be weakly immunolabeled compared with background (nonspecific IgG), but immunoblot analysis clearly shows that SREC was not present in EVCT cell lysates. In contrast, LOX-1 was strongly immunolabeled in EVCT, and LOX-1 expression was confirmed by immunoblotting (lanes 1 and 2). In lysates prepared from primary EVCT, the antibody revealed one single band of approximately 70 kDa (lanes 1 and 2). This band was weakly expressed by the positive control, HUVEC, which also expressed two major bands of 54 and 60 kDa (lane 3). To confirm that the bands obtained after immunoblotting of the EVCT cell lysates with LOX-1 antibody corresponded to LOX-1 proteins, the same membrane was stripped and incubated with LOX-1 antibody previously preabsorbed with HUVEC cell lysates known to express LOX-1 proteins. The bands corresponding to LOX-1 were eliminated, demonstrating that the signal was specific (lanes 4 and 5). Similarly, preabsorption of LOX-1 antibody with EVCT cell lysates eliminated the band obtained with HUVEC (lane 6), confirming that EVCT contain LOX-1 proteins. The same membrane was then reprobed with actin antibody to confirm that the proteins were still present. The bands corresponding to LOX-1 and actin were scanned, and after normalization to actin, we found a 1.3-fold increase in LOX-1 from Amax oxLDL-treated EVCT (lane 2) vs. control cells (lane 1).



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  		FIG. 1. Immunocytochemistry and immunoblots of scavenger receptors. Primary EVCT were cultured on Matrigel for 48 h. PMA-treated U937 cells were used as positive controls for CD36, and HUVEC were used for LOX-1 or SREC detections. Immunoblot analysis for SREC and LOX-1 (right panels) were performed to validate results obtained by immunocytochemistry (left panels). For SREC, the weak signal detected in EVCT was not detected by immunoblot analysis compared with PMA-treated HUVEC (single band of 86 kDa). For LOX-1 immunoblot analysis, EVCT were incubated for 48 h in the absence (lanes 1 and 4) or the presence of 50 µg/ml Amax oxLDL (lanes 2 and 5). EVCT were strongly immunostained for LOX-1, and immunoblot analysis showed a unique 70-kDa protein (lanes 1 and 2). A similar, but weak, band was found in HUVEC in addition to two major bands corresponding to 54- and 60-kDa proteins (lane 3). To confirm the specificity of the signal, the same blot was stripped and reprobed after saturation of LOX-1 antibody with HUVEC (lanes 4 and 5) or EVCT (lane 6) lysates. Preabsorption of LOX-1 antibody with HUVEC lysates abrogated LOX-1 immunodetection in EVCT (lanes 4 and 5), and preabsorption of LOX-1 antibody with EVCT lysates abrogated LOX-1 immunodetection in HUVEC (lane 6). Bands were scanned, and LOX-1 values were normalized to actin values. Amax ox-LDL treatment (lane 2) induced a 1.3-fold increase in LOX-1 contents compared with untreated EVCT (lane 1).

 
Effect of LDL oxidation state on trophoblast invasion
We next investigated the effect of the level of oxLDL oxidation on trophoblast invasion. LDL was oxidized with increasing concentrations of cupric chloride (C < B < A) for a period corresponding to the termination phase (max) or to half the termination phase (1/2P). As EVCT are able to invade an extracellular matrix in vitro, we used Matrigel-coated Transwells to analyze cell invasion (Fig. 2AGo). The invading trophoblasts crossing the porous membrane were visualized and quantified after immunostaining with cytokeratin-7 antibodies and counterstaining with DAPI (Fig. 2BGo). We used this model to investigate the effect of the LDL oxidation state on the invasive properties of EVCT. LDLs (native or oxLDL) were used at the same concentration (50 µg/ml) and incubated with EVCT for 48 h. Figure 2CGo demonstrates that only Amax oxLDL significantly inhibited EVCT invasion by about 50% (P < 0.01), whereas native LDLs or less oxLDLs (A1/2P, Bmax, or Cmax) did not significantly alter cell invasion.



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  		FIG. 2. Effect of oxLDL oxidation state on trophoblast invasion. Scheme of a Matrigel-coated Transwell used for invasion assay (A). EVCT were cultured on Matrigel-coated Transwells for 48 h with 50 µg/ml native LDL or various types of oxLDL (Cmax, Bmax, A1/2P, or Amax). EVCT were immunostained with anticytokeratin 7 antibody, and nuclei were counterstained with DAPI. Fluorescein isothiocyanate-immunostained invasive cells that crossed the porous membrane are visible at the inferior side of the membrane (B). The number of cells crossing the 8-µm diameter pores of the membrane was quantified and expressed as an invasion index relative to the control (C). Results represent the mean ± SEM of three independent cultures obtained from individual placentas run in triplicate. **, P < 0.01, treated vs. controls.

 
We next checked that the inhibitory effect of 50 µg/ml Amax oxLDL on cell invasion was not due to cell toxicity. LDH activity was measured in cell supernatants after 48 h of culture of EVCT in the absence or presence of 50 µg/ml Amax oxLDL, the concentration used in the invasion assay. LDH activity in cell supernatants remained under the detection limit of the assay (10 U/liter; controls, 4.75 ± 2.75; 50 µg/ml Amax oxLDL, 6.25 ± 2.5). LDH release was expressed as a percentage of total cellular activity, and the results were not significantly different (P = 0.39, by Mann-Whitney test; controls, 5.2% ± 2.4; Amax oxLDL, 6.8% ± 1.8).

Furthermore, no sign of apoptosis (condensed and fragmented nuclei) was observed after staining with DAPI in control or treated EVCT during quantification of invasive cells.

Composition of LDL after various degree of oxidation
To better investigate the molecular species involved in Amax oxLDL-mediated inhibition of EVCT invasion, we analyzed the lipid and protein (apo B) composition of oxLDL depending on the length of the exposure period and the concentration of cupric chloride used (26). At the termination phase (Tmax) of oxidation (Fig. 3AGo), the oxidation products of oxLDL type A were composed of a higher proportion of oxysterols (250 mol/mol LDL) and phosphatidylcholine hydroperoxides (PCOOH; 35 mol/mol LDL) than other oxLDL preparations; Cmax (oxysterols, 4 mol/mol LDL; PCOOH, 8 mol/mol LDL), Bmax (oxysterols, 35 mol/mol LDL; PCOOH, 15 mol/mol LDL), and A1/2P (oxysterols, 17 mol/mol LDL; PCOOH, 4 mol/mol LDL). oxLDL Cmax, Bmax, and A1/2P were predominantly composed of CE hydroperoxides (CEOOH). Furthermore, oxLDL type A at Tmax (oxLDL Amax) was the most oxLDL preparation, as confirmed by Western blot analysis of the oxidative modification of apo B, i.e. carbonylation and fragmentation of apo B (Fig. 3BGo).



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  		FIG. 3. Lipid and protein compositions of oxLDL depending on level of oxidation. A, Concentrations of oxysterols ({blacksquare}), CEOOH ({cjs2108}), and PCOOH ({square}) in oxLDL (1.5 g/liter total LDL) at the Tmax of oxidation with 0.03 µM (type C) and 0.3 µM (type B) copper and at the half-time of the propagation phase (T1/2P) and the termination phase (Tmax) of oxidation with 5 µM copper (type A). Oxysterols, CEOOH, and PCOOH were isolated after extracting lipids from oxLDL, measured by gas chromatography for oxysterols, separated by HPLC, and measured by chemiluminescence for CEOOH and PCOOH. B, Western blot analysis of apo B from copper oxLDL, at T1/2P and Tmax for LDL oxidized with 5 µM (type A) copper and at Tmax for LDL oxidized with 0.3 µM (type B) or 0.03 µM (type C) copper. Carbonylated apo B, carbonylated apo B fragments, and irradiated ovalbumin were derivatized with DNPH and were detected by an immunoassay with antidinitrophenyl antibodies.

 
PPAR{gamma} and LXR ligands inhibit EVCT invasion
We demonstrated that only Amax oxLDLs containing a high proportion of oxysterols and phosphatidylcholine hydroperoxides decreased trophoblast invasion. Oxysterols, on the one hand, and PCOOH or CEOOH, on the other hand, provide potential ligands of two nuclear receptors, LXR and PPAR{gamma}, respectively. They form heterodimers exclusively with RXR. We previously demonstrated that PPAR{gamma} and RXR{alpha} are present in primary EVCT and that PPAR{gamma}/RXR{alpha} heterodimers control trophoblast invasion (16). We therefore investigated whether activation of LXR might alter the invasive properties of EVCT. First, we immunodetected LXRß in the nuclei of primary EVCT (Fig. 4AGo, left panel). We next examined the effect of LXR ligands on trophoblast invasion using the above-mentioned in vitro system. Figure 4AGo (right panel) shows that 7-ketocholesterol (the main oxysterol in oxLDL) (26) and the synthetic LXR agonist T0901317 inhibited cell invasion by about 75% and 55%, respectively. The role of PPAR{gamma} in the oxLDL-mediated decrease in trophoblast invasion was investigated, as shown in Fig. 4BGo. Amax oxLDL inhibited significantly trophoblast invasion by about 30%, whereas 15(S)-HETE (derived from PCOOH or CEOOH) and the synthetic PPAR{gamma} agonist rosiglitazone (BRL 49653) induced a significant 50% inhibition. Finally, to determine whether Amax oxLDL inhibited trophoblast invasion by activating PPAR{gamma}/RXR heterodimers, we incubated cells with oxLDL alone or in combination with the PPAR{gamma} antagonist (BADGE). The results clearly showed that the PPAR{gamma} antagonist did not abrogate the effect of Amax oxLDL. Indeed, preincubation of EVCT with BADGE and concomitant incubation with oxLDL led to significant 40% and 50% inhibitions compared with controls or BADGE alone, respectively.



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  		FIG. 4. The oxLDL-derived putative ligands for LXR and PPAR{gamma} control trophoblast invasion. A, Left panel, LXRß was immunodetected on primary EVCT after 48 h of culture on Matrigel compared with cells incubated with nonimmune antibody; nuclei were counterstained with DAPI. Right panel, EVCT were cultured on Matrigel-coated Transwells for 48 h with LXR natural (1 µg/ml 7-ketocholesterol) or synthetic (5 µM T0901317) ligands. Invasion was quantified as described in Fig. 2Go. Values represent the mean ± SEM of three independent cultures obtained from individual placentas run in triplicate. *, P < 0.05, treated vs. controls. B, EVCT were cultured on Matrigel-coated Transwells for 48 h with PPAR{gamma} natural [3 µM 15(S)-HETE] or synthetic (1 µM BRL 49653) ligands and with Amax oxLDL (50 µg/ml) alone or in combination with 50 µM PPAR{gamma} antagonist (BADGE). Invasion was quantified as described in Fig. 2Go. Values represent the mean ± SEM of three independent cultures obtained from individual placentas and run in triplicate. *, P < 0.05, treated vs. controls; $, P < 0.05, treated vs. BADGE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human pregnancy is characterized by a major invasion of the uterine wall by trophoblastic cells of extravillous origin and major alterations in lipid metabolism, with LDL becoming more susceptible to oxidation (2, 9, 27). The mechanisms involved in the generation of oxLDL in vivo are not well defined, but several methods have been used to oxidize LDL in vitro, including copper oxidation (28). Metal ion-oxLDLs have altered chemical and physical properties, including a diverse array of oxidized sterols and fatty acids, and degradation fragments of apo B-100 (29). The lipid and protein compositions of oxLDL depend strictly on their level of oxidation. Therefore, to investigate the effect of oxLDL on trophoblast invasion, we tested LDLs that had been oxidized for various amount of time and with various concentrations of cupric chloride. We found that only highly oxidized LDLs (Amax oxLDL), which are predominantly composed of oxysterols, CEOOH, and PCOOH, significantly inhibited trophoblast invasion. This effect was not due to oxLDL-induced cytotoxicity as assayed by measurement of LDH activity in cell supernatants. Furthermore, it was not due to inhibition of cell growth, because primary extravillous cytotrophoblasts cultured on Matrigel do not proliferate (30). Finally, it was not a result of inhibition of cell adhesion, because EVCT were allowed to adhere for 2 h before incubation with oxLDL.

The cellular uptake of oxLDL involves the expression of membrane receptors belonging to the scavenger receptor family. We showed that invasive extravillous cytotrophoblasts strongly express the scavenger receptor LOX-1, whereas expression of other scavenger receptors involved in oxLDL uptake (SREC and CD36) was not detected. Immunoblot analysis showed a discrepancy between EVCT and HUVEC-LOX-1 molecular weights. This difference might result from variable N-glycosylation in the posttranslational processing, as suggested by others (31, 32). LOX-1 is an endothelial cell receptor that belongs to the lectin-like family of oxLDL receptors and has been detected in total human placental extracts (23). LOX-1 recognizes the protein moiety of oxLDL, whereas lipid constituents of oxLDL do not appear to be necessary for oxLDL binding to LOX-1 (33). Three other scavenger receptors have been detected in total human placental extracts: SREC, an endothelial cell scavenger receptor (34); CL-P1, mainly present in endothelial cells, but not in monocyte-macrophage cells (35); and SR-B1 (36), the major function of which is cholesterol transport, in particular, uptake of high density lipoprotein cholesterol esters within cells of the adrenal gland, ovary, and liver. Fixation, internalization, and degradation of acetylated LDLs or oxLDL has been clearly demonstrated in chorionic villi by cytotrophoblasts of villous origin (syncytiotrophoblasts) (13, 37). In agreement with our results, LOX-1 has recently been shown to be expressed in a trophoblastic cell line, the choriocarcinoma JAR, in which it mediates 40–50% of oxLDL uptake (38). Up-regulation of scavenger receptors by oxLDL has been described in macrophages, where oxLDL stimulate expression of CD36 (39). In the present study we showed that Amax oxLDL increased LOX-1 protein content by 30% in EVCT, which is consistent with a recent study performed on human coronary artery endothelial cells (40).

The role of oxLDL on cell migration or invasiveness has not been described in detail. However, one study reported that in vitro human oxLDL inhibit the migration of bovine aortic endothelial cells in a concentration- and oxidation-dependent manner (41). This effect on migration has been attributed to the lipid content of oxLDL. Furthermore, highly oxidized LDL was shown to attract monocytes and to inhibit macrophage migration in vitro (42).

Oxysterol and to a lesser extent PCOOH concentrations were higher in Amax oxLDL than in the other oxLDL types, which were mainly composed of CEOOH. PCOOH and CEOOH contain arachidonic derivatives such as 15(S)HETE and prostaglandins, but also linoleic derivatives such as 9(S)-hydroxyoctadecadienoic acid [9(S)-HODE] and [13(S)-hydroxyoctadecadienoic acid [13(S)-HODE], which are all activators of the nuclear receptor PPAR{gamma} (43). PPAR{gamma} controls the expression of a large array of genes in a ligand-dependent manner. This receptor is essential for the development of adipose tissue, plays a critical role in glucose homeostasis, and inhibits the expression of a number of proinflammatory genes (44). Taking into account the results from our previous work showing that PPAR{gamma}/RXR{alpha} heterodimers control trophobast invasion, we hypothesized that oxLDL compounds might inhibit trophoblast invasion by activating PPAR{gamma}/RXR. Under our experimental conditions, we showed that potential PPAR{gamma} ligands, such as 15(S)HETE (Fig. 4BGo) or 9(S)HODE and 13(S)HODE (data not shown), inhibited trophoblast invasion. Oxidized lipids may therefore provide endogenous agonists of PPAR{gamma} in the human placenta. Accordingly, Schild et al. (45) reported that 15(S)HETE, 9(S)HODE, and 13(S)HODE activated PPAR{gamma} in villous cytotrophoblasts, resulting in enhanced hCG production, a marker of villous cytotrophoblast differentiation. We next determined whether inhibiting PPAR{gamma} reversed the oxLDL-mediated effect and found that the PPAR{gamma} antagonist BADGE failed to reverse Amax oxLDL-induced inhibition of EVCT invasion in vitro.

Oxysterols contain agonists of LXR, another nuclear receptor that regulates genes controlling lipid metabolism and is expressed in liver, kidney, intestine, and spleen (46). We show in the present study that EVCT expressed LXRß and that 7-ketocholesterol, a known activator of LXR that is mainly present in the oxysterol fraction (26), inhibited trophoblast invasion. Thus, oxysterols might also be responsible for the action of oxLDL on cell invasion. The role of LXR was confirmed by using the specific LXR agonist T0901317, demonstrating for the first time the role of this nuclear receptor in the modulation of human trophoblast invasion. Although the role of oxysterols in cell motility has not been extensively investigated, one study performed in humans showed that 7-ketocholesterol and 27-hydroxycholesterol extracted from atherosclerotic lesions inhibited the migration of vascular smooth muscle cells in vitro, whereas free cholesterol and cholesterol ester had no effect (47). Castrillo et al. (48) demonstrated that activation of LXR{alpha} and -ß inhibited basal and cytokine-inducible expression of matrix metalloprotease-9 mRNA in macrophages, an enzymatic endopeptidase that degrade extracellular matrix components during normal and tumoral tissue remodeling, suggesting that LXR regulates cell invasion process.

In conclusion, our results clearly indicate that oxLDL, predominantly composed of oxysterols, CEOOH, and PCOOH, abrogate the invasive properties of human EVCT in vitro, probably through LOX-1-mediated uptake. Oxysterols, CEOOH, and PCOOH provide ligands for PPAR{gamma} and LXR, and we demonstrated that activation of these nuclear receptors inhibit trophoblast invasion. However, the inhibitory effect of oxLDL on trophoblast invasion did not appear to be mediated through activation of PPAR{gamma} alone, and we suggest that LXRß plays a major role in mediating this effect. This study improves our understanding of preeclampsia, a condition in which lipid peroxidation is increased, and trophoblast invasion is defective (7, 49, 50).


   Acknowledgments
 
We thank Dr. Kalus (Hoffmann-La Roche, Inc., Basel, Switzerland) for providing RO26-5405. We also thank the Department of Obstetrics and Gynecology, Broussais Hospitals (Paris, France), for donating placental tissues.


   Footnotes
 
This work was supported by Roche Vitamines (Neuilly sur Seine, France), the Medical Research Foundation, and la Caisse d’Assurance Maladie des Professions Libérales-Province (Paris, France).

Abbreviations: apo B, Apolipoprotein B; BADGE, bisphenol A diglycidyl ether; CE, cholesteryl ester; CEOOH, cholesteryl ester hydroperoxide; DAPI, 4',6-diamido-2-phenylindole hydrochloride; DNPH, dinitrophenyl hydroxylase; EVCT, extravillous cytotrophoblast cell; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; HUVEC, human umbilical vascular endothelial cell; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; LOX1, lectin-like oxidized receptor-1; LXR, liver X receptor; oxLDL, oxidized LDL; 1/2P, half the termination phase; PC, phosphatidylcholine hydroperoxide; PCOOH, phosphatidylcholine hydroperoxide; PMA, phorbol myristate acetate; PPAR, peroxisomal proliferator-activated receptor; RXR, retinoid X receptor; SREC, scavenger receptor expressed by endothelial cells; Tmax, termination phase.

Received December 23, 2003.

Accepted for publication June 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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