Expression and localization of ATP binding cassette transporter A1 (ABCA1) in first trimester and term human placenta
Article Outline
- Abstract
- 1. Introduction
- 2. Materials and methods
- 3. Results
- 4. Discussion
- Funding
- Acknowledgement
- References
- Copyright
Abstract
ATP binding cassette transporter A1 (ABCA1) is a membrane transporter which performs cellular efflux of cholesterol and phospholipid. ABCA1's cholesterol transporting role in human placenta appears to be crucial for normal fetal development. Despite the critical importance of cholesterol in fetal development, expression of ABCA1 in the human placenta throughout gestation and its specific cellular localization have not been known yet. We therefore investigated ABCA1 expression in human placenta at first trimester and term by western blot and quantitative real-time PCR (qRT-PCR) analysis. Furthermore, its localization was investigated by immunohistochemistry and confocal microscopy. Expression of ABCA1 did not differ significantly between first trimester and term placenta at both protein and mRNA levels. Immunohistochemical data demonstrated that ABCA1 was widely localized in the villous and extravillous cytotrophoblast as well as in some stromal and endothelial cells. Confocal microscopy imaging data showed that ABCA1 was localized largely at the basolateral and to some extent at the apical side of first trimester villous cytotrophoblast cell membranes. Placental expression of ABCA1 throughout the gestation and its specific cellular localization indicate that this transporter may play an important role in materno-fetal cholesterol transfer.
Keywords: Placenta, Pregnancy, ABCA1, Expression, Localization
1. Introduction
Placenta is the vital organ liable as a protective barrier and a site for nutrient and waste exchange between mother and the fetus. Placental transport and metabolism of cholesterol and lipid are critical for the fetal development and its survival. Cholesterol is the integral part of cell membranes, precursor of steroid hormones such as progesterone and metabolic mediators such as oxysterol [1]. Cholesterol is essential for both activation and propagation of Hedgehog signaling. Sonic Hedgehog (SHH) is responsible for patterning and development of the central nervous system [2], [3], [4]. There are two routes by which cholesterol is available to the fetus, the de novo synthesis and exogenous source. The individual lacking de novo cholesterol synthesis may develop lethal congenital birth defects [5], [6].
Convincing evidence shows that maternal cholesterol is a source of fetal cholesterol [7], [8]. In vivo studies in murine and in vitro assay using the choriocarcinoma cell line, BeWo have demonstrated that cholesterol is transported across the trophoblast cells [9], [10]. The uptake and utilization of cholesterol by trophoblast through very low density lipoprotein (VLDL) receptor, low density lipoprotein receptor-related protein (LRP), LDL receptor, and Scavenger Receptor B I (SR-BI) have been reported [11], [12]. Placenta is composed of different cell types, including trophoblasts, endothelial cells, fibroblasts, as well as blood cells in the intervillous space and fetal vessels. But the actual barrier between maternal and fetal circulation is made up of trophoblast cells. In order to acquire maternal cholesterol by the fetus cholesterol must be taken through the apical side of the placental trophoblast cells and exit through the basolateral side of the trophoblast layer to enter into the fetal circulation. Experimental evidence suggests that trophoblasts efflux cholesterol from cells like any other polarized cells [1]. So far three different mechanisms for cholesterol efflux have been proposed namely aqueous diffusion and protein independent pathway based on concentration gradient, SR-BI mediated efflux and ATP binding cassette transporter A1 (ABCA1) mediated efflux [13], [14]. Apparently all the processes may occur in placenta through the basolateral membrane of trophoblast layer as placenta possesses SR-BI [11] and ABCA1 [15]. ABCA1 is one of the efflux transporters highly expressed in human placenta [15]. It performs cholesterol and phospholipids efflux to lipid poor Apolipoprotein A-I (ApoA-I), precursor of high density lipoprotein. Moreover, along with cholesterol homeostasis, ABCA1 is also involved with phosphatidylserine (PS) translocation [16], apoptosis [17] and immune responses [18], [19]. Apoptosis and PS translocation processes are extremely important for placental development [20], [21] and immune function is important to orchestrate the peri-implantation placenta development environment. ABCA1 deficiency is associated with Tangier disease and familial low density lipoprotein deficiency in humans [22], [23]. Moreover, dysfunction of ABCA1 in mice resulted in severe placental malformation with structural abnormalities, intrauterine growth retardation and increased neonatal death [24]. Reduced expression of placental ABCA1 was observed in women with antiphospholipid syndrome [25] and ABCA1 was reported as a potential target for in utero therapy of Smith–Lemli–Opitz syndrome [26]. Very recently, Stefulj et al. [27] demonstrated the presence of ABCA1 in endothelial cells of term placenta and its involvement in cholesterol transfer towards fetal circulation. However, no information is available on the expression of ABCA1 in human placenta throughout the gestation. In the present paper, we report the expression of ABCA1 and its cellular localization in first trimester and term human placenta.
2. Materials and methods
2.1. Tissue sampling and preservation
First trimester placental tissues were collected after elective termination of pregnancy at 7–12 weeks of gestation (n = 23). Term placental tissues were collected from uncomplicated deliveries via caesarean section (n = 25). Placental tissues were collected with the informed consent of patient and approval of the Regional Committee of Medical and Health Research Ethics in Siena (Siena, 2004) and in accordance with the guidelines of Helsinki declaration. First trimester placental tissues were subdivided into 3–6 portions; some of these were immediately snap frozen in liquid Nitrogen and stored at −80 °C for protein extraction and RNA isolation. The others were fixed in 10% buffered formalin, embedded in paraffin and stored at room temperature until slide preparation. Term placenta full-thickness biopsies were excised in duplicate from the central periumbilical region. One of these was processed for preparation of paraffin blocks and the other was centrally subdivided into 4–5 portions and snap frozen in liquid Nitrogen and stored at −80 °C for protein extraction and RNA isolation or fast frozen in liquid nitrogen chilled isopentane for cryosection.
2.2. Tissue sectioning
Both first trimester and term paraffin-embedded placental tissues were cut at 5 μm thickness and mounted on SuperFrost Plus slides (Menzel, Braunschweig, Germany). Tissue sections were stained in haematoxylin of Mayer (Merck, Darmstadt, Germany) to evaluate the morphology and integrity of the samples. Only samples showing good tissue integrity were selected for immunohistochemistry and confocal microscopy. All samples for cryosectioning were cut at 7 μm thickness at −20 °C cryostat (Leica Microsystems Nussloch GmbH, Heidelberger, Germany) and mounted on SuperFrost plus slides and stored at −80 °C until further experimentation.
2.3. Western blot analysis
Western blot was performed on both first trimester (n = 19) and term placental tissues (n = 11) extracts. Membrane proteins from frozen placental tissues were extracted by ProteoExtract Native Membrane Protein Extraction Kit (M-PEK) (Calbiochem, Darmstadt, Germany) according to the manufacturer's instructions. Extracted membrane protein concentration was determined by quick start Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein sample (25 μg) was subjected to SDS-PAGE electrophoresis on 7% Tris–acetate gel (Invitrogen, Carlsbad, CA, USA) at 120 V for 2 h. To preserve the integrity of ABCA1, samples were not heated prior to loading on 7% Tris–acetate gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Hybond-C, Amersham International, Little Chalfont, UK) by electroblotting at 30 V for 1 h. The western blot was performed using XCellII blot module (Invitrogen). The membrane blot was blocked in 7% non-fat dry milk in phosphate buffered saline (PBS) with 0.1% Tween20 (PBST) for 1 h. The blot was then incubated with mouse anti-human monoclonal ABCA1 antibody (1: 3000) (Abcam, Cambridge, UK) in 7% non-fat dry milk in PBST overnight at 4 °C. Following incubation, the blot was washed three times with PBST, each for 10 min. Then, the membrane was incubated with goat anti-mouse secondary antibody (1:3000) (Bio-Rad Laboratories) diluted in 7% milk in PBST for 1 h at room temperature. The membrane was washed three times with PBST and labelled with peroxidase (Bio-Rad Laboratories) at room temperature for 5 min. The chemiluminescence was visualized by the West Pico chemiluminescent substrate (Pierce, Rockford, IL). Equal loading of the protein was assessed in accordance with Moore and Viselli [28] by staining the membrane with 10% (v/v) Ponceau S solution (2% Ponceau S in 30% trichloroacetic acid/30% sulfosalicylic acid) (Sigma–Aldrich Co., MO). The bands were quantified using the Bio-Rad's Quantity One 1-D analysis software (Bio-Rad Laboratories).
2.4. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was isolated from snap frozen placental tissues. Total RNA was extracted using TRIzol reagent (Invitrogen) followed by chloroform extraction and isopropanol precipitation, and quantified spectrophotometrically. The integrity of isolated RNA was checked by ethidium bromide staining in formaldehyde containing 1% agarose gel. Only RNA samples demonstrating 28S to 18S rRNA ratio of 2:1 or more (as determined by densitometry analysis) were included in study (first trimester n = 6 and term placental tissues n = 5). cDNA was prepared from 1 μg of total RNA using high fidelity reverse transcription (RT) kit (Applied Biosystems, Foster City, CA). All RNA samples were reverse transcribed in a single reaction cycle. The cDNA obtained was amplified by real-time quantitative Polymerase Chain Reaction (PCR) using a pre-developed ABCA1 specific primer and probe mix and TaqMan probe based detection system (Applied Biosystems). Step one fast real-time PCR instrument (Applied Biosystem) was used for cDNA amplification and gene expression analysis. In all placental samples 18S rRNA was used as endogenous control. For detection of 18S rRNA in the placental RNA samples, a pre-developed primer and probe mix was purchased from Applied Biosystems. The corresponding real-time PCR efficiency was calculated according to Livak and Schmittgen [29]. Investigated transcripts showed high real-time PCR efficiency rates; for 18S, 1.93; and ABCA1, 1.95 both in the first trimester and term placental tissues. Quantitative real-time for both ABCA1 and 18S rRNA was performed for every sample at the same assay. The threshold cycle (CT) values of each samples was recorded. Relative gene expression was determined by 2−ΔΔCT method [29]. The first trimester placental tissue was considered as control and relative expression in term placental tissue was determined in comparison with first trimester placenta. The data were normalized using the ratio of the target cDNA (ABCA1) concentration to that of the control cDNA (18 S rRNA).
2.5. Immunohistochemistry
Tissue sections were prepared from first trimester (n = 15) and term (n = 10) placenta. Formalin-fixed, paraffin-embedded tissues were selected for immunohistochemistry on the basis of tissue integrity visualized by Haematoxylin staining. Slides were heated at 60 °C for 10 min, then dewaxed, rehydrated and washed in Tris-buffered saline (TBS) (20 mM Tris–HCl and 150 mM NaCl, pH 7.6). Then the slides were subjected to antigen retrieval in sodium citrate buffer (10 mM pH 6.0) in a microwave oven at 750 W for 15 min (three times for 5 min each). Afterwards, the slides were cooled for 20 min at room temperature and rinsed three times in TBS, 5 min each. The slides were preincubated with 10% normal rabbit serum (DakoCytomation, Glostrup, Denmark) to block the non-specific binding of the antibodies. After blocking, anti-human mouse monoclonal ABCA1 antibody (1: 200 Abcam) was applied and incubated overnight at 4 °C followed by three washes in TBS, 5 min each. Then the slides were incubated with biotinylated rabbit anti-mouse antibody (1: 200) (DakoCytomation). The reaction was revealed using streptavidin–biotin complex (DakoCytomation). The sections were also counterstained with Mayer's haematoxylin, then mounted with an aqueous mounting medium (Merck) and examined under a light microscope. Negative controls were performed for each staining by substituting the anti-ABCA1 antibody with TBS. All antibodies and serum were diluted in TBS.
2.6. Immunofluorescence and confocal scanning laser microscopy
The localization of ABCA1 was investigated by confocal laser scanning immuofluorescence microscopy on paraffin-embedded first trimester (n = 12) and term placental tissues (n = 8). Mouse anti-human monoclonal ABCA1 antibody (1: 200) (Abcam) was used in immunofluorescence staining. The sections were processed as immunohistochemistry until reaching the step of blocking. For blocking, the sections were preincubated with 5% goat serum (Sigma–Aldrich Co.) for 1 h at room temperature. After blocking, mouse anti-human ABCA1 monoclonal antibody (1: 200) (Abcam) was applied to the sections and incubated overnight at 4 °C. Afterwards the sections were washed with 0.2% BSA (Bovine Serum Albumin) in phosphate buffered saline (PBS) for 3 times, 10 min each. Then the sections were incubated with Cy2 conjugated affinipure goat anti-mouse antibody (Jackson ImmunoResearch, PA) as described by the manufacturer followed by washing with 0.2% BSA in PBS for 3 times, 10 min each. The nuclei were counterstained with propidium iodide (PI) by incubating the sections with PI (100 ng/mL) for 10 min followed by wash with 0.2% BSA in PBS. Then the slides were mounted with mowiol containing 0.025% DABCO (1,4-Diaza Bicyclo 2.2.2 octane). After mounting, the slides were dried at 37 °C for 1 h and preserved at 4 °C until watched under a confocal microscope.
For double and triple labelling, cryostat sectioned samples were used. Double labelling and triple labelling were performed on term placenta. The slides were fixed with 3% paraformaldehyde and washed with 0.2% BSA and blocked with 5% goat serum for 1 h at room temperature. The incubation with anti-ABCA1 primary antibody was performed as described above. Both for double and triple color labelling, mouse anti-human cytokeratin 7 antibody (Sigma–Aldrich Co.) was used by means of the Zenon Mouse IgG labelling kit (Molecular Probes, Leiden, The Netherlands) according the manufacturer's instructions. Shortly, after overnight incubation with anti-ABCA1 and subsequent washing steps, the sections were incubated with Cy3 conjugated affinipure goat anti-mouse antibody (Jackson ImmunoResearch) as described by the manufacturer. Subsequently, after the washing steps, the sections were again incubated 3 h with Zenon-labelled anti-cytokeratin 7 antibody. Then the sections were washed and mounted. For triple labelling, the nuclei were additionally stained with To-PRO 3 (Invitrogen). Specimens were analyzed with an LSM-510 meta confocal microscope equipped with 63× objective (Zeiss, Jena, Germany).
2.7. Statistical analysis
All data were shown as mean ± SEM. The western blot and qRT-PCR data were statistically analyzed by the Student t-tests using GraphPad Prism Version 4.0 (GraphPad Software, Inc., San Diego, CA). The qRT-PCR data were also analyzed by the gene expression analysis software REST-384 Version 2 [30]. Differences were considered significant if p < 0.05.
3. Results
Western blotting analysis using the extracted membrane proteins of placental tissues showed the expression of ABCA1 in human placenta at first trimester and term of gestation. A band at 254 kDa corresponding to the molecular mass of the ABCA1 protein was detected in all the samples analyzed (Fig. 1A). Densitometric analysis performed with Bio-Rad's Quantity One 1-D analysis software showed no significant difference of ABCA1 protein expression level between the first trimester and term placental tissue (Fig. 1B).
Fig. 1
Western blot and qRT-PCR analysis of ABCA1 in human placenta tissues. (A) Top, representative western blot analysis of ABCA1 protein at first trimester (lanes 1–6) and term human placenta (lanes 7–8) using mouse anti-human ABCA1 monoclonal antibody. Bottom, Membrane stained with ponceau S to assess total loaded protein in each lane. (B) The histogram represents densitometric measurement of western blot bands of first trimester (n = 19) and term (n = 11) placental tissues. Each bar represents the mean ± SEM at each gestational age range. (C) Quantification of ABCA1 mRNA in human placenta during the first trimester (n = 6) and term (n = 5) of gestation using qRT-PCR. Bars represent mean ± SEM ABCA1/18S rRNA ratio at each gestational age range.
mRNA expression profile of ABCA1 in the placenta during gestation was investigated by qRT-PCR using the RNA isolated both from first trimester and term placental tissues. Higher expression of ABCA1 mRNA was detected in term placental tissues in comparison with first trimester but the difference was not statistically significant (Fig. 1C).
Immunohistochemistry studies using anti-human ABCA1 monoclonal antibodies showed the expression of ABCA1 in both first trimester and term placental tissues (Fig. 2). The staining demonstrated that ABCA1 was widely localized in the villous cytotrophoblast layer and in the extravillous cytotrophoblast cells of first trimester chorionic villi (Fig. 2A and B). ABCA1 was also localized in some stromal and endothelial cells of the villous core (Fig. 2A and B). A similar tissue distribution for ABCA1 was found in term placental tissues (Fig. 2C and D). Here more distinct staining was observed in endothelial cells of the chorionic villi, while less staining was observed in the epithelial layer of the placenta possibly due to the lower number of the cytotrophoblast cells in term placenta (Fig. 2C and D). No staining was observed in the syncytiotrophoblast in both first trimester and term placental tissues (Fig. 2A–D). No positive staining was observed when the antibody was substituted with TBS (negative control) (Fig. 2E)
Fig. 2
Representative images of immunohistochemical staining using mouse anti-human ABCA1 monoclonal antibody. First trimester (A,B) and term (C,D) human placenta. Positive staining is shown in red. In first trimester placenta, ABCA1 was widely expressed in the villous (arrow heads) and extravillous (asterisk) cytotrophoblast (A,B). Staining was also observed in some stromal cells (triangles) of the chorionic villi (A,B). In term placenta, ABCA1 staining was widely distributed in the stromal (triangles) and endothelial cells (thin arrows) of the villous core and in the few cytotrophoblast cells present in epithelial layer (C,D). No positive staining was observed in the syncytiotrophoblast (arrows) of first trimester and term tissues (A–D). Negative control performed in absence of ABCA1 antibody, showed no staining in any cell type (insert, E). Scale bar = 50 μm for image A–D.
The studies using confocal microscopy imaging demonstrated ABCA1's cell-specific localization in first trimester and term placenta (Fig. 3). ABCA1 was principally localized at the basolateral (Fig. 3A and C) and partly at the apical side (Fig. 3B) of the villous cytotrophoblast cell membrane in first trimester tissues. Cell membrane staining was also observed in the extravillous cytotrophoblast cells of first trimester tissues (Fig. 3D), while, more diffuse staining was observed in the cytotrophoblast cells of term placenta tissues (Fig. 3E). A punctuate staining was observed in the syncytial brush border of few villi, only in some sections (Fig. 3A). As in immunohistochemistry, staining in endothelial cells of chorionic villi was clearly evident in both first trimester and term tissues (Fig. 3C,E and 4C). Since it was difficult to distinguish between syncytiotrophoblast and cytotrophoblast cells in the term placental tissues, we did double and triple labelling of the term placental tissue sections using anti-ABCA1 and cytotrophoblast-specific anti-cytokeratin 7 antibodies along with To-Pro 3 (far-red) as nuclear stain in triple labelling. These staining confirmed a diffuse localization of ABCA1 in the few villous cytotrophoblast cells in term placenta (Fig. 4A–C).
Fig. 3
Confocal laser scanning microscopic localization of ABCA1 using mouse anti-human ABCA1 monoclonal antibody in first trimester (A–D) and term (E) human placenta. Positive staining of ABCA1 is shown in green. Red staining represents nuclei (propidium iodide stained). In first trimester placenta, ABCA1 staining was found in the basolateral (windows pointers) and in the apical part (arrow heads) of villous cytotrophoblast cell membrane, rarely observed staining in syncytial brush border (triangle) and in endothelial cells (arrows) of the villous core (A–C). (D) ABCA1 positive staining in the cell membrane of most extravillous cytotrophoblast cells (star frame). In term placenta (E), diffuse ABCA1 positive staining in the cytotrophoblast cells (triangle frame). (F) Negative control in first trimester placenta. Inserts show the higher magnification of the selected part. Scale bar = 20 μm for all the images.
Fig. 4
Confocal laser scanning microscopic localization of ABCA1 and cytokeratin 7 on cryostat sections of term placental tissues. Duel color staining with anti-human ABCA1 and anti-human cytokeratin 7 (a marker for the cytotrophoblast cells) antibodies. Green color indicates the cytokeratin 7 and red color indicates the ABCA1 positive staining. (A) A1 and A2, labelling with cytokeratin 7 and ABCA1 antibodies are green and red, respectively. (B,C) Triple labelling with cytokeratin 7 (green), ABCA1 (red) and TO-PRO 3 (Far-red), a nuclear stain. Cytokeratin 7 and ABCA1 were predominantly observed in cytotrophoblast cells (A–D). Arrows indicate ABCA1 positive endothlial cells. Scale bar = 20 μm for all the images.
4. Discussion
To the best of our knowledge, this is the first evidence demonstrating the expression and specific cellular localization of ABCA1 in human placenta throughout the gestation. We showed that ABCA1 is present in the first trimester and term placenta but with no significant difference both at mRNA and protein levels. ABCA1 was predominately localized at the basolateral and infrequently at apical part of the cytotrophoblast cell layer of first trimester human placenta. It was also expressed in cell membranes of the majority of extravillous cytotrophoblast cells and in some other cell types of the placenta including stromal and endothelial cells of chorionic villi. In term placenta, the signal was observed in the few villous cytotrophoblast cells but more intense staining was observed in endothelial cells of fetal vessels compared with the vessels of first trimester placenta. These findings on ABCA1 expression and localization in human placenta greatly support the role of ABCA1 as an important component of feto-placental transport function. Albrecht et al. [25] reported immunohistochemical localization of ABCA1 in the syncytium in term placenta by using a rabbit polyclonal antibody. On the other hand, we found a positive staining mainly in cytotrophoblast cells both in the first trimester and term placenta. The use of different antibodies may be one of the reasons for getting different results, as in the present experiments monoclonal antibodies were used. Although there was no significant difference of ABCA1 in mRNA and protein levels between first trimester and term placental tissue, relatively higher level of ABCA1 protein was present in first trimester than at term despite the fact that ABCA1 mRNA level was greater in term placental tissue.
ABCA1 is responsible for cellular cholesterol and phospholipids efflux. Localization of ABCA1 in the villous cytotrophoblast cells and more at the basolateral part of the cell membrane is of particular interest and indicates that efflux may be directed towards the fetal circulation from the maternal circulation. Stefulj et al. [27] recently showed that endothelial cells of the fetal vessels successfully deliver cholesterol towards the fetal circulation. The present findings indicate that ABCA1 of the cytotrophoblast may facilitate cholesterol and phospholipid exit from the trophoblast layer to enter into the chorionic villous core. In addition to its lipid export activity, it has been demonstrated that ABCA1 plays an important role in immune responses [18], [19], [31]. Mice lacking ABCA1 have an enhanced inflammatory response to lipopolysaccharide (LPS) [31]. Incubating apoA-I with activated ABCA1-expressing macrophages suppressed production of the inflammatory cytokines interleukin 1β, interleukin-6, and tumor necrosis factor-α [18]. We have recently shown the involvement of ABCA1 in modulating the secretion of the cytokine macrophage migration inhibitory factor (MIF) in human first trimester placenta [32]. These studies raise the possibility that apolipoprotein/ABCA1 interaction or ABCA1 alone have multiple biological effects that are lipid export dependent or independent. Thus the expression of ABCA1 in the villous and extravillous trophoblast may reflect additional functions other than those involved in cholesterol homeostasis. In term placental tissue, ABCA1 was also present in cytotrophoblast cells but diffused in the whole cell without any specific localization. Some staining in the term placental cytotrophoblast cells suggests it to be in intracellular endocytic compartment. ABCA1 is actually a membrane transporter although its expression in the intracellular endocytic compartment has also been reported in different cell types [17], [33]. The functional role of intracellular ABCA1 in trophoblast cells remains to be elucidated. To date, two mechanisms have been suggested to explain ABCA1-mediated cholesterol efflux to apoA-I [34], [35], [36]. One mechanism, apoA-I forms complexes with phospholipid and cholesterol at the cell surface in a process promoted by ABCA1 activity [36]. The other mechanism, apoA-I binds ABCA1 at the cell surface and is subsequently internalized and targeted to late endosomes, where apoA-I picks up lipids. The apolipoprotein–lipid complexes are then resecreted from the cell by exocytosis [36]. Azuma et al. [37] have shown that apoA-I internalizes inside the cell and co-localizes with the cell surface-derived ABCA1 on endosomal compartments contributing to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.
In addition to biosynthetic route, fetus can receive the exogenous cholesterol through or from the yolk sac and/or placenta [38], [39], [40]. The yolk sac begins to regress and becomes non-functional at eight weeks of gestation [41], but the placenta remains functional throughout gestation [42]. In first trimester placenta, there are more cytotrophoblast cells. The turnover of cytotrophoblast cells to syncytiotrophoblast is a continuous process. The quantity of cytotrophoblast cells reduces during the progression of gestation and at near term. At term, cytotrophoblast occupies only 15% to the total volume of the villous trophoblast [43]. In term placenta, ABCA1 is more predominantly and intensely expressed in endothelial cells and proportion of vessels rise from 37% in first trimester to 63% at term [44]. Due to this increase of vessels structure at term, the total ABCA1 expression may remain the same as in first trimester placenta even after reduction of cytotrophoblast cell number at term. Based on our data, we only can speculate that ABCA1 may be significantly involved in materno-fetal cholesterol transfer throughout the gestation period. Although the role of syncytiotrophoblast cells in cholesterol uptake from maternal circulation was reported [13], [45], further functional studies of ABCA1 on cholesterol efflux in trophoblast cells are required to fully understand as to how the up taken cholesterol enters into the fetal circulation.
Cholesterol plays a vital role during embryogenesis and normal embryo development. Our data on ABCA1 expression throughout the gestation and its localization suggest ABCA1 can be involved in cholesterol transport across the trophoblast as well as other biological functions such as immune responses. Lack of expression or function of ABCA1 plays a role in pathological pregnancies such as intrauterine growth restriction (IUGR) in mice [24] and antiphospholipid syndrome in human [25]. Further studies are required to explore the role of ABCA1 in normal and pathological pregnancies. Our study provides foundation for performing future research into physiological and molecular mechanisms involving ABCA1 regulation in human placenta.
Funding
This work was supported by the European Union Sixth Framework, ReProTect (LSHB-CT-2004-503257).
Acknowledgement
We would like to thank Luca Formoso, Molecular Medicine Section, Department of Neuroscience, University of Siena, Siena, Italy, for his technical support.
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PII: S0143-4004(10)00094-9
doi:10.1016/j.placenta.2010.02.015
© 2010 Elsevier Ltd. All rights reserved.
