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Endocrinology Vol. 147, No. 4 1675-1684
Copyright © 2006 by The Endocrine Society

Expression and Oxygen Regulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Human Placenta during Early Pregnancy

Pascale Hoffmann, Jean-Jacques Feige and Nadia Alfaidy

Institut National de la Santé et de la Recherche Médicale (INSERM) (P.H., J.-J.F., N.A.), Equipe Mixte INSERM 01-05, 38054 Grenoble, France; Département Réponse et Dynamique Cellulaires (P.H., J.-J.F., N.A.), Laboratoire d’Angiogenèse, Commissariat à l’Energie Atomique, 38054 Grenoble, France; Université Joseph Fourier (P.H., J.-J.F., N.A.), 38100 Grenoble, France; and Service de Gynécologie, Obstétrique, et Médecine de la Reproduction (P.H.), Centre Hospitalier Régional Universitaire de Grenoble, 38700 La Tronche, France

Address all correspondence and requests for reprints to: Dr. Nadia Alfaidy, Institut National de la Santé et de la Recherche Médicale EMI 01-05, Département réponse et dynamique cellulaires/Laboratoire d’Angiogenèse, Commissariat à l’Energie Atomique-Grenoble, 17, Rue des Martyrs, 38054 Grenoble, France. E-mail: alfaidybenharouga{at}dsvsud.cea.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiogenesis is a key process of dynamic tissue remodeling occurring during placentation. Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be deregulated in preeclampsia. Recently a new angiogenic factor, endocrine gland-derived VEGF (EG-VEGF), also known as prokineticin 1 (PK1), has been identified, and its expression was shown to be restricted to endocrine glands, including the placenta. In this study we investigated the pattern of expression of EG-VEGF, its related factor Bv8/PK2, and their common receptors, PKR1 and PKR2, in human placenta during the first trimester of pregnancy. We also examined EG-VEGF and PKR1 regulation by oxygen tension in isolated trophoblast cells (TCs). Our results show that EG-VEGF, but not Bv8/PK2, is expressed in human placenta. EG-VEGF is mainly localized to the syncytiotrophoblast layer with the highest expression detected between the 8th and 10th wk of gestation. EG-VEGF expression within placental villi is different from that of VEGF, which is mainly localized in the cytotrophoblast and extravillous trophoblast cells. In TCs, PKR1 mRNA is about 80 times more abundant than PKR2 mRNA. Both EG-VEGF and PKR1 mRNAs appear to be regulated by hypoxia. These findings suggest that EG-VEGF has a direct effect on TCs via its receptor PKR1 and is likely to play an important role in human placentation. The expression pattern of EG-VEGF, its regulation by oxygen tension, and its complementary localization to that of VEGF suggest that this new factor might also be deregulated in preeclampsia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING GESTATION, an adequate and efficient supply of nutrients and oxygen is vital for proper development of the fetus. These fetomaternal exchanges rely on adequate vascularization of both the maternal decidua and the fetus-derived placental villi (1). In the human maternal decidua, vascular remodeling of the intramyometrial portion of the spiral arterioles occurs between the 10th and 12th week of gestation. This transformation is achieved by specialized placental cells, the cytotrophoblasts. During placentation, cytotrophoblasts that are present in anchoring villi generate multilayered columns of highly invasive extravillous trophoblasts that colonize the interstitium of the maternal decidua, the inner third of the myometrium, and the uterine blood vessels. This invasion results in the formation of the low-resistance vascular system that is essential for fetal growth (1). This developmental period (10–12 wk of gestation) is characterized by an important physiological switch in oxygen tension during the opening of the intervillous space. Before the ninth week gestation, placental oxygen tension is low (~20 mm Hg), and after 10–12 wk gestation, it increases to approximately 55 mm Hg (2). At this time, the cytotrophoblasts turn from a proliferative to an invasive phenotype (3). Failure of this transition is associated with clinical complications of pregnancy, including preeclampsia, the most common cause of retarded fetal development (4).

Hemochorial placentation is also dependent on the establishment and maintenance of a competent fetoplacental vascular network formed by branching (first and second trimesters) and nonbranching (third trimester) angiogenesis. In human placenta, branching angiogenesis is important for both the development of the villous vasculature and the formation of terminal villi. Consequently, careful coordination of development, proliferation, and differentiation of both trophoblasts and endothelial cells is required during the early stages of placental development. Angiogenic growth factors are considered to be the main mediators of these processes. Mouse models have demonstrated the importance of two families of ligands, namely vascular endothelial growth factors (VEGFs) and angiopoietins and their tyrosine kinase receptors in fetal and placental angiogenesis (4, 5, 6, 7). The discrete phenotypes of the null animals suggest distinct roles for individual families with VEGF family members being important for the initial stages of vasculogenesis and angiogenesis and angiopoietins being involved in the latter stages of fetal blood vessel maturation (8). Besides its mitogenic action on endothelial cells, VEGF has also been reported to stimulate the proliferation of the choriocarcinoma cell line BeWo (9) and extravillous trophoblasts (10). Low VEGF levels or increased production of VEGF antagonists such as the soluble form of its receptor-1 (VEGF-R1/flt-1), have been proposed as possible mediators of preeclampsia (11, 12, 13, 14). Although VEGF and angiopoietins are selectively acting on any type of endothelial cell, they are ubiquitously expressed.

The existence of organ-specific angiogenic factors has been postulated for many years (15, 16, 17, 18) but only recently received confirmation when such a factor, named endocrine gland-derived vascular endothelial growth factor (EG-VEGF), was characterized and sequenced (19). This new factor was found to be expressed in testis, adrenal gland, ovary, and placenta (19). In addition, its angiogenic action appeared to be restricted to endothelial cells derived from endocrine tissues (19). In endothelial cells isolated from steroidogenic tissues, EG-VEGF was shown to promote proliferation, survival, and chemotaxis (19, 20). Very interestingly, in vivo delivery of adenoviruses encoding EG-VEGF resulted in endocrine tissue-specific angiogenesis (21). EG-VEGF is a member of a class of proteins that also includes Bv8, a frog peptide purified from the skin secretion of the yellow-bellied toad, Bombina variegata (19), also known as prokineticin (PK)-2. Human EG-VEGF and Bv8 proteins present 83% identity, exert the same functions (21), and share the same G protein-coupled receptors (PKRs), termed PKR1 and PKR2 (22)

EG-VEGF expression in the placenta was briefly described in the initial report by LeCouter et al. (19). Since then, no further characterization of its cellular localization and expression during pregnancy has been reported. The specificity of EG-VEGF expression in endocrine glands and its similarity of action with VEGF suggested to us that this new factor might be important in human placentation. The aims of this study were therefore to examine the expression of EG-VEGF, Bv8, PKR1, and PKR2 in human placentas during the first trimester of pregnancy, determine their sites of expression within the chorionic villi, and examine the effect of oxygen tension on their expression levels in isolated human trophoblast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection
First-trimester human placentas from 4 to 12 wk of gestation, corresponding to 6–14 wk amenorrhea were obtained from elective terminations of pregnancies. The sampling was as follows [4 wk of gestation (n = 3); 5 wk of gestation (n = 3); 6 wk of gestation (n = 3); 7 wk of gestation (n = 4), 8 wk of gestation (n = 4), 9 wk of gestation (n = 4), 10 wk of gestation (n = 4), 11 wk of gestation (n = 3), and 12 wk of gestation (n = 3)]. Shortly after collection, tissue was snap frozen in dry ice and stored at –80 C (for protein and RNA extraction), fixed in paraformaldehyde at room temperature (for immunohistochemistry), or placed in ice-cold Hanks’ balanced salt solution (Ca2+-, Mg2+-HBSS) and transported to the laboratory for in vitro primary culture. A total of eight placentas from 7 to 10 wk of gestation were used for primary culture. Collection and processing of human placentas was approved by the University Hospital Ethics Committee, and informed consent was obtained from each patient. Mouse testis was also collected and used as positive control in this study. Animal surgery was conducted following both institutional and European Community guidelines for the use of experimental animals.

Immunohistochemistry
Placental tissues collected at 4–12 wk of gestation were fixed for 24 h at 4 C in 4% (vol/vol) paraformaldehyde, embedded in paraffin, and cut into 5-µm sections as described previously (23). To control tissue integrity and select the most representative sections, every 10th section was stained with hematoxylin and eosin. Adjacent sections were stained using specific antibodies and the avidin-biotin immunoperoxidase detection method. Endogenous peroxidase activity was quenched by pretreatment with 3% (vol/vol) hydrogen peroxide in methanol for 30 min. Tissue sections were then washed in PBS and incubated with normal goat serum (10%) that served as a blocking agent for nonspecific binding. Immunoreactive EG-VEGF or Bv8 was detected using rabbit polyclonal antibodies raised against the following peptides: human EG-VEGF peptide 84–96, LLCSRFPDGRYRC; and a mixture of Bv8-derived peptides 26–40, DDAVITGACDKDSQC, and 94–108, CLRTSFNRFICLAQK. The immunization was performed for our laboratory by Covalab (Lyon, France). For immunohistochemical detection, anti-EG-VEGF and anti-Bv8 antibodies were incubated with the tissue sections for 18 h at 4 C and used at final concentrations of 0.33 and 0.72 µg/ml, respectively. The tissue sections were subsequently washed three times with PBS and incubated with biotinylated goat antirabbit IgG (1:250 dilution in blocking solution; Sigma Aldrich, Saint-Quentin Fallavier, France) for 1 h at 4 C. After three PBS washes, the slides were incubated with an avidin-biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 1 h. After a final PBS wash, the immunoreactive proteins were visualized after the addition of 3,3'diaminobenzidine (Dako, Trappes, France) for 2 min and then counterstained with heamatoxylin. Control sections were treated with anti-EG-VEGF and anti-Bv8 antibodies that had been preabsorbed overnight at 4 C with the appropriate antigen peptides or preimmune serum.

Western blotting analysis
Frozen placental samples were homogenized on ice for 1 min in radioimmunoprecipitation assay lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin] as previously described (24). The homogenates were centrifuged (15,000 x g at 4 C) for 15 min, and the supernatants were collected. Proteins were also extracted from cultured trophoblast cells using the same procedure. Protein concentrations were determined using the Bradford assay. Because of the weakness of the signal in the preliminary experiments, a specific protocol was set up to detect EG-VEGF protein. One hundred micrograms placental protein and 70 µg protein extracted from trophoblast cells were electrophoretically separated on 0.1% sodium dodecyl sulfate-17% polyacrylamide gels and electrically transferred onto 0.25-µm polyvinylidene difluoride membranes (Millipore, Bedford, MA). Appropriate transfer was confirmed by protein staining with Ponceau S (Sigma-Aldrich). The blots were washed with PBS-Tween 20 0.1% (PBS-T) and incubated overnight in blocking solution (5% skimmed milk in PBS-T). Subsequently membranes were immunoblotted with a rabbit antibody against EG-VEGF (0.48 µg/ml) (Covalab) for 2 h. Blots were then rinsed three times with PBS-T and incubated with biotinylated goat antirabbit IgG (450 ng/ml, 1:2000, dilution in blocking solution; Dako) for 30 min. After three PBS-T washes, the membrane was incubated with a peroxidase-conjugated extravidin (1:2000 dilution in blocking solution; Sigma Aldrich) for 30 min. Blots were washed six times with PBS-T, and the antibody-antigen complex was detected using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). The membranes were then exposed to radiographic films (Kodak Scientific Imaging Products, Rochester, NY).

The intensities of immunoreactive bands were measured by scanning the photographic film and analyzing the image on a desktop computer using Scion Image software (version 4.0.2; Scion Corp., Frederick, MD). The mean pixel density for each band was analyzed to obtain relative OD units for EG-VEGF protein. To standardize for sample loading, the blots were stripped in [6.25 mM Tris-HCl (pH 6.6), 10 mM ß-mercaptoethanol, 2% sodium dodecyl sulfate] and reprobed with an anti-Gß antibody (Transduction Laboratories, Lexington, KY) as an internal control for protein loading. The Gß antibody detects a 37-kDa protein whose abundance does not vary during gestation. The sensitivity of the EG-VEGF antibody was also tested by Western blotting using recombinant human EG-VEGF (Tebu-Bio, Le Perray en Yvelines, France).

Isolation and treatment of trophoblasts
Placental cytotrophoblasts were isolated from first-trimester human placentas (7–10 wk of gestation, n = 14) and cultured using a combination of two techniques described by Kliman et al. (25) and Tarrade et al. (26). Briefly, the tissue was thoroughly washed in 50 ml cold sterile Ca2+-Mg2+-HBSS until the supernatant was nearly free of blood. Areas rich in chorionic villi were selected and were minced into small pieces between scalpels. Tissue was incubated in HBSS containing 0.125% trypsin (Sigma), 4.2 mM MgSO4, 25 mM HEPES, and 50,000 U/ml DNase IV (Sigma) for 35 min at 37 C without agitation to remove extravillous trophoblast cells. After tissue sedimentation, the supernatant was discarded and the chorionic villi were submitted again to sequential 30 min trypsin (0.25%) and DNase (0.2 mg/ml) digestions, as previously described (25, 27). The dispersed placental cells were filtered through 100-µm nylon gauze and loaded onto a discontinuous Percoll gradient (5–70% in 5% steps of 3 ml each) and then centrifuged at 1200 x g for 20 min at room temperature to separate the different cell types. Cytotrophoblast cells that sedimented between the density markers of 1.049 and 1.062 g/ml were collected and washed with DMEM. Isolated cells were then incubated with anti-CD9 antibodies and subjected to negative immunomagnetic separation using MiniMacs columns (Miltenyi Biotech, Paris, France). This allowed the elimination of CD9-positive placental mesenchymal cells, placental macrophages, and blood monocytes. Briefly, 10 µl of anti-CD9 antibody (Biosource International, Camarillo, CA) were added to 107 freshly isolated cells and the mixture was incubated for 40 min on ice with gentle agitation. Twenty microliters of goat antirat IgG microbeads (Miltenyi Biotech) were mixed with the cell suspension, and mixture was kept at 4 C for 15 min under gentle rotation.

The unabsorbed cell fraction containing trophoblasts was collected, washed three times, and seeded at a density of 106 cells/ml in DMEM supplemented with 2 mM glutamine, 10% fetal bovine serum, 25 mM HEPES, 100 IU penicillin, and 10 µg/ml streptomycin. Approximately 4–5 x 106 cells were obtained per gram of chorionic villi. The dispersed trophoblasts were cultured for 24 h at 37 C in 5% CO2-95% air to allow attachment. The cells were then divided into two groups: half were incubated under standard tissue culture conditions (95% air-5% CO2) and half were incubated under hypoxic conditions (3% O2/92% N2/5% CO2) for 24 h. Trophoblast cells were also treated with hypoxia-mimicking drugs such as deferoxamine (DFO, 200 µM) and cobalt chloride (CoCl2, 200 µM) for 24 h. In some experiments trophoblast cells were treated by 50 µg/ml of 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), a potent RNA polymerase inhibitor, or 10 µg/ml cycloheximide, a translation inhibitor. Purity of the cell preparation was assessed at the end of the experiment by immunofluorescent staining for cytokeratin, an epithelial cell lineage marker (Dako), or vimentin, a mesenchymal cell lineage marker (Dako). After 48 h of culture, both mononucleated cytotrophoblasts and aggregates of placental trophoblasts forming syncytial clumps were observed; 95 ± 5% of the cultured cells were cytokeratin positive and vimentin negative, suggesting the presence of a majority of cytrophoblasts and a minority of fibroblasts or decidual cells. Cell viability, assessed by Trypan blue exclusion, was more than 95% before and after incubation.

RNA isolation and RT-PCR analysis
Total RNA was extracted from placental tissue and isolated trophoblasts after 48 h of cell culture. The extraction was done in 4 M guanidium isothiocyanate buffer using a rapid RNA isolation system (RNAgents; Promega, Charbonnieres, France). The integrity of extracted RNA was analyzed using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). Reverse transcription was performed on 1 µg total RNA with Superscript II-RNaseH reverse transcriptase (Invitrogen, Cergy Pontoise, France) under conditions recommended by the manufacturer.

Real-time PCR analysis
EG-VEGF, Bv8, PKR1, PKR2, VEGF mRNA, and 18S rRNA expression was quantified by real-time RT-PCR using a Light Cycler apparatus (Roche Diagnostics, Meylan, France). The PCR was performed using the primers shown in Table 1Go and SYBR green PCR core reagents (Light Cycler-FastStart Master SYBR Green I, Roche Diagnostics) according to the manufacturer’s instructions. For negative controls, we used a complete DNA amplification mix in which the target cDNA template was replaced by water. PCR conditions were: step 1, 94 C for 10 min; step 2, 45 cycles consisting of 95 C for 15 sec, temperature indicated in Table 1Go for 5 sec, and 72 C for 10 sec. VEGF primers were designed to amplify the four human VEGF isoforms. The results were normalized to 18S rRNA expression levels. To assess linearity and efficiency of PCR amplification, standard curves for all transcripts were generated by using serial dilutions of cDNA. For standard-curve generation, we used cDNAs from human placenta or untreated trophoblast cells. A melt curve analysis was carried out on the products of amplification reaction to ascertain the melting temperature of the product. The RealQuant analysis software (Roche Diagnostics) was used to quantify levels of expression.


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  		TABLE 1. Primers used for semiquantitative (SQ) and real-time (RT) PCR

 
Semiquantitative PCR
Semiquantitative PCR was used to examine the effect of oxygen tension on EG-VEGF and PKR1 expression in isolated trophoblast cells. Before PCR, quantities of cDNA samples were adjusted to yield equal amplification. Ribosomal 18S was used as the internal reference. Specific oligonucleotide primers for EG-VEGF and PKR1 were designed using MacVector software (Table 1Go). PCRs were performed in a final volume of 25 µl containing appropriate quantities of cDNAs in 1 x PCR buffer, 1.5 mM MgCl2, 200 mM deoxynucleotide triphosphate, and 400 mM each primer, 0.5 U Taq polymerase (Q. Biogene, Illkirch, France). In addition, several control reactions were routinely run in parallel including RT-PCR run in the absence of reverse transcriptase to confirm the absence of genomic DNA contamination and reverse transcription reactions without RNA to check for reagent contamination. The PCR conditions were: step 1, 94 C for 1 min; step 2, 25–35 cycles at hybridization temperature indicated in Table 1Go; and step 3, 72 C for 5 min. To ensure semiquantitative results in the RT-PCR assays, the number of PCR cycles was selected to be in the linear range of amplification. PCR products were visualized after electrophoresis on 1.5 to 2% gels by ethidium bromide staining.

Statistical analysis
All data are expressed as mean ± SE. Statistical comparisons were made using one-way ANOVA test and Student’s t test. Calculations were performed using SigmaStat (Jandel Scientific Software, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EG-VEGF protein expression in human placenta during early pregnancy
Immunohistochemistry.
Immunohistochemistry was used to define the pattern of expression of EG-VEGF and Bv8 proteins in human placenta between 4 and 12 wk of gestation. A total of 18 placentas were used (two different placental samples for each gestational age). Immunoreactive EG-VEGF (Ir-EG-VEGF) was observed from 6 to 10 wk of gestation. No staining was observed before 6 or after 10 wk of gestation. At 6 wk of gestation, expression was limited to the syncytiotrophoblast layer of the chorionic villi (Fig. 1AGo). At advanced gestational ages, the staining for EG-VEGF was more intense in the syncytiotrophoblast layer (7–10 wk) and became apparent in the inner cytotrophoblast layer. No staining was observed in the extravillous trophoblasts forming the anchoring villi (Fig. 1BGo). Ir-EG-VEGF was also found in fetal Hofbauer cells within the placental villous mesenchyme as early as 6 wk of gestation. In contrast to Ir-EG-VEGF, Bv8 immunoreactivity was barely detectable in human placenta from 4 to 12 wk of gestation. A very faint staining was observed in the syncytiotrophoblast layer between 6 and 10 wk. Because our antibody was designed to react with both human and mouse Bv8 protein, we used mouse testis as a positive control for Bv8 immunoreactivity. As expected, Bv8 immunoreactivity was detectable in primary spermatocytes and Leydig cells (Fig. 1FGo). To control for antibody specificity, preadsorption of both anti-EG-VEGF and anti-Bv8 antibodies with an excess of immunizing peptides and incubation with preimmune sera were used. No staining was observed in chorionic villi or mouse testis under these conditions (undersized pictures, Fig. 1Go, B and F).


Figure 1
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  		FIG. 1. EG-VEGF and Bv8 immunolocalization in human placental villous tissue during early pregnancy. A total of 18 placentas, from 4 to 12 wk gestation, were used (two different samples for each gestational age). A–C, Chorionic villi at 6, 9, and 12 weeks gestation immunostained (brown staining) with anti-EG-VEGF antibody as described in Materials and Methods. D and E, Chorionic villi at 6 and 9 wk gestation stained with anti-Bv8 antibody. F, Positive control for Bv8 staining (mouse testis). B and F, Undersized photographs, Negative controls treated with either antigen neutralized anti-EG-VEGF and anti-Bv8 antibodies or preimmune sera. Note that EG-VEGF immunoreactivity was stronger at 9 wk, compared with 6 and 12 wk gestation. All sections were counterstained with heamatoxylin. Ct, Cytotrophoblast; Ho, Hobfauer cells; EVT, extravillous trophoblast; St, syncytiotrophoblast; *, Leydig cells. Scale bar, 20 µm.

 
Western blot analysis
The pattern of EG-VEGF protein expression was then analyzed by Western blot on protein extracts from placental tissue homogenates (n = 21 placentas). As shown in Fig. 2AGo, under our experimental conditions, EG-VEGF antibody was able to detect 20 ng recombinant human EG-VEGF, and the signal was completely abolished in the presence of an excess of antigenic peptide.


Figure 2
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  		FIG. 2. EG-VEGF protein expression during the first trimester of pregnancy. A, The sensitivity of EG-VEGF antibody was tested by Western blot on indicated quantities of recombinant human (r) EG-VEGF. The specificity was tested in the presence of 9.6 µg/ml antigenic peptide corresponding to 20 times the concentration of EG-VEGF antibody used in this experiment. B, Representative Western blot for EG-VEGF protein expression in homogenates of placental villous tissues from 6 to 12 wk gestation. Protein Gß (37 kDa) was used as an internal control for protein loading. C, The histogram represents the mean relative OD of EG-VEGF protein signal normalized to protein Gß signal (n = 21 different placentas). Data are mean ± SE. *, P= 0.05.

 
Figure 2BGo shows a representative Western blot of EG-VEGF expression in placental extracts from 6 to 12 wk of gestation. EG-VEGF protein was detected as a major band of 12 kDa. Immunoreactivity was present since the sixth week of gestation, picked at 8 wk, and then gradually decreased. Quantification of three independent experiments revealed a significant difference in EG-VEGF expression levels between the 8th and 12th wk of gestation.

Differential localization of EG-VEGF and VEGF proteins in human chorionic villi during the first trimester of pregnancy
Because both VEGF and EG-VEGF are expressed in human placenta during the first trimester of pregnancy, we sought to compare their cellular localization within chorionic villi at different gestational ages. Using immunohistochemistry, we compared EG-VEGF and VEGF protein expression in placentas from 6 to 12 wk of gestation (Fig. 3Go). VEGF expression was restricted to the cytotrophoblast layer in the chorionic villi (Fig. 3AGo). In anchoring villi, VEGF expression was also observed in extravillous trophoblasts (Fig. 3BGo). In contrast to VEGF, EG-VEGF was mainly localized to the syncytiotrophoblast layer (Fig. 3DGo). Its expression was also found in the cytotrophoblast at advanced gestational ages (Fig. 3Go, E and F). However, no expression of EG-VEGF was ever observed in extravillous trophoblasts at all gestational ages examined (Fig. 3EGo). Such a differential pattern of expression for VEGF and EG-VEGF suggests possible complementary functions for the two factors in human placenta during the first trimester of pregnancy.


Figure 3
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  		FIG. 3. Comparison of VEGF and EG-VEGF immunolocalization in placental villous tissues. A total of 18 placentas, from 4 to 12 wk of gestation, were used (two different placental samples for each gestational age). A–C, Representative VEGF immunoreactivity at 6, 8, and 9 wk gestation, respectively. D–F, EG-VEGF immunoreactivity at the same gestational ages. Note that VEGF and EG-VEGF exhibit complementary sites of immunoreactivity in chorionic villi and that extravillous trophoblasts express VEGF but not EG-VEGF. All sections were counterstained with heamatoxylin. Ct, Cytotrophoblast; Ho, Hobfauer cells; EVT, extravillous trophoblast; St, syncytiotrophoblast. Scale bar, 20 µm.

 
EG-VEGF, Bv8, PKR1, PKR2, and VEGF mRNA expression during the first trimester of human pregnancy
Human placenta, like other endocrine tissues, has been reported to be an important site of EG-VEGF expression (19). However, to date no study has examined the pattern of its expression during the first trimester of pregnancy when the angiogenic process is the most active. This prompted us to examine the expression pattern of EG-VEGF, Bv8, and their common receptors, PKR1 and PKR2, mRNAs in human placentas from 4 to 12 wk of gestation, using real-time RT-PCR. EG-VEGF mRNA was detected at all gestational ages examined (n = 27, but its level was strongly increased (up to 6- to 7-fold) between the 8th and 10th wk of gestation, as compared with gestational ages before and after this period (P < 0.001) (Fig. 3AGo). In contrast, Bv8 mRNA expression was undetectable except between the 8th and 10th wk of gestation (data not shown). PKR1 and PKR2 were also expressed during the first trimester of pregnancy. PKR1 exhibited the same pattern of expression as EG-VEGF with levels peaking between 8 and 10 wk of gestation (Fig. 4CGo). In contrast, PKR2 mRNA levels did not vary much until the end of the first trimester (12 wk) when it increased 3-fold (Fig. 4DGo).


Figure 4
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  		FIG. 4. Patterns of placental EG-VEGF (A), VEGF (B), PKR1 (C), and PKR2 (D) mRNA expression during the first trimester of pregnancy. The expression levels of the selected mRNAs were quantified by real-time RT-PCR and normalized to the 18S rRNA levels, as described in Materials and Methods. The relative expression levels of each gene were plotted as function of gestational age. A total of 27 placentas (three different placental samples for each gestational age examined) from 4 to 12 wk of gestation were used. Data are expressed as mean ± SE; each bar is the mean of three determinations from three independent placentas of the same gestational age. Values overwritten with different letters are significantly different from each other (P < 0.05).

 
Because VEGF is considered the most important factor controlling human placental angiogenesis during the first trimester of pregnancy (28), we sought to examine its pattern of expression in the same samples. VEGF mRNA expression did not significantly change during the first trimester. A slight increase was, however, observed by 12 wk of gestation. These results indicate that placental EG-VEGF and VEGF exhibit quite different patterns of expression during early pregnancy (Fig. 4BGo).

To further evaluate the relative expression of EG-VEGF and Bv8 in human placenta, we used real-time RT-PCR to measure their mRNA levels in placental tissues from 7 to 10 wk of gestation (n = 12), a period of gestation when both factors seem to be expressed. A similar analysis was performed on primary cultures of trophoblast cells isolated from placentas with the same gestational age (n = 8) and cultured for 48 h. In both placental tissue (Fig. 5AGo) and primary trophoblast cultures, EG-VEGF mRNA was 5 times more abundant than Bv8 mRNA (Fig. 5BGo), thereby confirming the differences observed between the two factors at their protein levels.


Figure 5
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  		FIG. 5. Comparison of EG-VEGF/ Bv8 and PKR1/PKR2 mRNA expression in human placental tissue and in primary trophoblast cells. The expression levels of the selected mRNAs were quantified by real-time RT-PCR and normalized to the 18S rRNA levels, as described in Materials and Methods. Placentas (n = 20) were collected between 7 and 10 wk of gestation. 18S rRNA was used as internal standard for sample normalization. A and C, Placental tissue EG-VEGF mRNA levels normalized to Bv8 and PKR1 mRNA levels normalized to PKR2, respectively. B and D, Cultured trophoblast EG-VEGF mRNA levels normalized to Bv8 and PKR1 mRNA levels normalized to PKR2, respectively. Data are mean ± SE. *, P < 0.05. ns, Not statistically significant.

 
Expression of PKR1 and PKR2 in human placental tissue and cultured trophoblast cells during the first trimester of pregnancy
We then compared the mRNA expression levels of the EG-VEGF/Bv8 receptors PKR1 and PKR2 in both placental tissue (n = 13) and primary trophoblast culture (n = 6). The placentas used in this experiment were from 7 to 10 wk of gestation. Real-time RT-PCR was used to quantify the two receptor transcripts. Our results show that PKR1 and PKR2 transcripts are expressed at the same level in whole placental tissue (Fig. 5CGo), whereas PKR1 mRNA was 80 times more abundant than PKR2 mRNA in cultured trophoblast cells (Fig. 5DGo). These data strongly suggest that, within human placenta, there is a cellular specificity for PKR1 and PKR2 expression.

Effect of oxygen tension on EG-VEGF and PKR1 expression in human trophoblast cells
Low oxygen tension is a key parameter that controls gene expression during the first trimester of pregnancy. Moreover, the highest level of expression of EG-VEGF and its receptor PKR1 is observed during the hypoxic period of human placentation. This observation prompted us to hypothesize that EG-VEGF and PKR1 mRNA might be regulated by oxygen tension in human trophoblast cells. To test this hypothesis, we incubated trophoblast cells isolated from 7- to 10-wk-old placentas for 24 h under either 20% O2 or 3% O2 and measured EG-VEGF and PKR1 mRNA abundance using either semiquantitative or quantitative RT-PCR. As shown in Fig. 6AGo, a significant increase in both EG-VEGF (160% of normoxic control) and PKR1 (200% of normoxic control) mRNA levels was observed under hypoxic conditions (3% O2). The graphs in Fig. 6Go, C and D, show the mean ± SE increase in EG-VEGF and PKR1 mRNA levels observed in six independent experiments. To confirm that reduced oxygen tension was responsible for the increase in EG-VEGF mRNA, hypoxia-mimicking drugs DFO and CoCl2 were also used. The results in Fig. 6BGo show treatment of primary cultures of human trophoblasts for 24 h with 200 µM CoCl2 or DFO. Both molecules resulted in a 2-fold increase in PKR1 mRNA and a 2- to 4-fold increase in EG-VEGF mRNA levels.


Figure 6
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  		FIG. 6. Effect of hypoxia on EG-VEGF and PKR1 mRNA expression in human trophoblast cells. Cytrophoblasts were isolated from 10 different placentas collected at 7–10 wk of gestation and cultured for 24 h to allow attachment and for another 24 h under either normoxic or hypoxic conditions. A, RT-PCR amplification products for EG-VEGF and PKR1 under either 20 or 3% O2 from three independent experiments. B, Effects of hypoxia-mimicking drugs, CoCl2 (Co++) and DFO on EG-VEGF and PKR1 mRNA expression. Variations [in percent of control (Ctl)] of EG-VEGF/18S or PKR1/18S mRNA to rRNA ratios are shown in C and D, respectively. The 20% O2 condition was used as a control for normalization. E, Effect of DRB and cycloheximide (CHX) on basal and hypoxia-induced EG-VEGF mRNA expression in trophoblast cells. F, Representative Western blot profile of EG-VEGF protein in total homogenates of trophoblast cells cultured under normoxic (20%) or hypoxic (3% O2, CoCl2, or DFO) conditions. G, Histograms represent the mean ± SE of ratio of EG-VEGF to Gß protein expression from four different placentas after incubation under normoxic or hypoxic conditions. Protein Gß immunoreactivity was used as an internal control for protein loading. EG-VEGF expression was increased under 3% O2, CoCl2, and DFO conditions by 33, 26, and 56%, respectively.*, P < 0.001.

 
We then examined the effects of transcription and translation inhibitors on the hypoxic regulation of EG-VEGF expression. In the presence of the RNA polymerase inhibitor DRB (50 µg/ml), the hypoxic induction of EG-VEGF was completely abolished, whereas it was preserved in the presence of cycloheximide, a potent inhibitor of translation (Fig. 6EGo). These data suggest that hypoxia regulates EG-VEGF gene expression at the transcriptional level. To determine whether oxygen tension has an effect on EG-VEGF protein levels, trophoblast cells were isolated from 7- to 10-wk-old placentas and incubated for 24 h under either 20% O2 or 3% O2 or in the presence of hypoxia-mimicking drugs, CoCl2 and DFO. Figure 6FGo shows that all three hypoxic conditions increased EG-VEGF protein. Statistical significance, however, was only reached under DFO treatment (Fig. 6GGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates expression of EG-VEGF and its G protein-coupled receptors PKR1 and PKR2 in human placenta during the first trimester of pregnancy and shows that EG-VEGF and VEGF have distinct spatiotemporal patterns of expression. Furthermore, we report that the expression of EG-VEGF and PKR1 peaks at a specific time during the first trimester of pregnancy (8–10 wk gestation), corresponding to the hypoxic period of placental development that precedes the establishment of the fetomaternal vascular connection. In isolated trophoblast cells, we have also demonstrated that both EG-VEGF and PKR1 are up-regulated by hypoxia. Taken together the data generated by this work reveal that EG-VEGF and its receptor PKR1 may play an important role in human placentation during the hypoxic period of placental development.

To date, four studies (19, 21, 29, 30) have reported a constitutive expression of EG-VEGF in human placentas from term pregnancies, and two of them (21, 29) have shown an expression of Bv8 in the same tissue. In the present study, we examined the expression of both factors in human placentas during the first trimester of pregnancy when angiogenesis is high. Our data show that EG-VEGF is expressed at all gestational ages examined, whereas Bv8 is only barely detectable between the 8th and 10th wk of gestation. Moreover, EG-VEGF mRNA levels are five times higher than Bv8 mRNA levels during this period. This differential pattern of expression is similar to that observed in the human endometrium (31) and corpus luteum (32).

The immunolocalization experiments performed in the present study have revealed interesting patterns of distribution. The results substantiate the conclusions of the RT-PCR experiments and show that Ir-EG-VEGF is present in the syncytiotrophoblasts. The strong expression of EG-VEGF in the syncytiotrophoblast suggests that this factor is probably synthesized by this endocrine component of the placenta and that its expression is associated with the degree of trophoblast differentiation.

Given the established importance of VEGF in human placentation (33, 34, 35) and the partial similarity of its biological functions with those of EG-VEGF, we compared their pattern of expression during the first trimester of pregnancy. Interestingly, we observed that these factors were expressed in adjacent but distinct compartments. In contrast to EG-VEGF, which was localized to the syncytiotrophoblast layer, VEGF was mainly present in the cytotrophoblasts and extravillous trophoblasts. This finding is reminiscent of human ovaries, in which VEGF and EG-VEGF are rarely colocalized in the same cell type during the follicular and luteal phases (30). Based on its specific pattern of expression and its absence of colocalization with VEGF, EG-VEGF appears to be a novel growth factor likely to play complementary biological roles to those of VEGF.

EG-VEGF/PK1 and Bv8/PK2 are equally potent ligands for two structurally related G protein-coupled receptors named PKR1 and PKR2 (22, 35, 36, 37). These two receptors are expressed in gastrointestinal organs, endocrine glands, central nervous system, and many other tissues, suggesting a wide range of biological actions for EG-VEGF and Bv8 (22, 37). Different patterns of PKR1 and PKR2 expression have been observed in different cell types. For example, endothelial cells of the bovine corpus luteum appear to express both PKR1 and PKR2 (32), whereas endocrine granulosa and thecal cells preferentially express PKR1 (38). In first-trimester human placentas, our data show that PKR1 and PKR2 are expressed at the same levels, whereas in primary trophoblast culture, only PKR1 appears to be expressed. These findings clearly suggest that PKR1 and PKR2 may have different cellular localizations within the placental villi. We propose that PKR2 is likely to be present in endothelial cells of fetal blood vessels because, in other tissues, this receptor has been reported to be specifically localized to endothelial cells (21, 30, 38). The data presented herein clearly show PKR1 expression in trophoblast cells. However, it is difficult to know precisely which type of trophoblast cell expresses PKR1 because our cytotrophoblast cell preparation contains a small amount of syncytial trophoblast cells. Because of the lack of commercially available antibodies to PKR1 and PKR2, we cannot make any firm conclusions regarding the localization of these receptors within the placental villi at this time. Nevertheless, the expression of PKR1 in trophoblast cells suggests a direct effect of EG-VEGF on nonendothelial cells. This finding suggests that EG-VEGF may have an important role in human placentation. Therefore, the previously characterized endocrine tissue-specific angiogenic actions of EG-VEGF (19, 20, 21) may very well be one among several other functions played by this factor in endocrine tissues.

EG-VEGF levels appeared to greatly vary during early pregnancy, progressively increasing until the 10th week of gestation and rapidly dropping afterward. These dramatic changes appear to be correlated with the hypoxic developmental period of the placenta because the junction of the fetal and maternal vascular networks is known to occur between the 10th and 12th week of gestation. This correlation is supported by the transcriptionally controlled up-regulation of EG-VEGF mRNA and concomitant increase of its protein levels that were observed in primary cultures of trophoblasts after 24 h of culture under reduced O2 tension. This regulation was not unexpected as a functional hypoxia-response element (TACGTGCGGC) able to bind the hypoxia-inducible factor-1{alpha} has been identified in the human EG-VEGF promoter (19). However, we cannot totally exclude that other factors presenting peaks in their expression during the first trimester of pregnancy may also participate in the regulation of EG-VEGF expression. Human choriogonadotropin and progesterone are two such candidates because they have been recently shown to stimulate EG-VEGF mRNA expression in human luteinized granulosa cells (32) and human endometrial tissue (31), respectively.

Regulation of placental growth factor expression by oxygen tension is well established and has been described for several angiogenic factors, including VEGF (39, 40), TGFß3 (41), and the soluble form of VEGF receptor-1 (s-flt) (42, 43). In the present study, we also observed up-regulation of PKR1 receptor expression under hypoxic conditions. This is the first report indicating PKR1 regulation by oxygen tension. A GenBank screen of the human PKR1 promoter revealed the presence of one putative HIF-1 binding site, suggesting that the oxygen effect on PKR1 expression might occur through a pathway that involves HIF-1. This finding further supports the idea that EG-VEGF and PKR1 may play an important role in normal human placentation and perhaps in pathologies such as preeclampsia. A recent study by Chung et al. (30) reported no change in EG-VEGF expression in placentas from preeclamptic patients. In this study, changes in EG-VEGF expression have been examined during the second and third trimester of pregnancy. We now know that the origin of preeclampsia takes place during the first trimester of pregnancy and that any changes in protein expression after the establishment of the disease are considered to be consequences of the disease rather than causes of its development. Our data show that EG-VEGF expression is at its lowest levels by the end of the first trimester, suggesting that its greatest role should occur within that trimester. Moreover, given the correlation between the pattern of EG-VEGF and PKR1 expression with the hypoxic period of placental development and knowing that failure in placental angiogenesis is thought to contribute to preeclampsia development, one can speculate on the potential implication of this factor and/or its receptors in the development of preeclampsia. However, only a prospective study examining the expression of EG-VEGF and/or PKR1 during the first trimester of pregnancy in women who go on to develop preeclampsia will allow to provide an answer to this question. Future studies are also required to determine the biological activities triggered by EG-VEGF in the human placenta and identify whether dysregulation of EG-VEGF expression may result in placental pathologies.


   Acknowledgments
 
We are indebted to Professor J. P. Schaal (Head, Department of Obstetrics and Gynaecology, University Hospital of Grenoble) for authorizing collection of placentas in his department. We thank Dr. Eric Amblard (Clinique Mutualiste, Grenoble) for providing us placental samples. We also thank Dr. Alison Holloway for carefully reading the manuscript and Michelle Keramidas for her invaluable help in the setting up of the RT-PCR techniques.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale (EMI 01-05) and Commissariat à l’Energie Atomique (Département de la Science du vivant/Département réponse et dynamique cellulaires/Laboratoire d’Angiogenèse). N.A. was supported by Young Investigator fellowships from the Fondation pour la Recherche Médicale and the Institut National de la Santé et de la Recherche Médicale.

First Published Online December 29, 2005

Abbreviations: DFO, Deferoxamine; DRB, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole; EG-VEGF, endocrine gland-derived VEGF; HBSS, Hanks’ balanced salt solution; Ir-EG-VEGF, immunoreactive EG-VEGF; PBS-T, PBS-Tween 20; PK, prokineticin; PKR, PK receptor; VEGF, vascular endothelial growth factor.

Received July 20, 2005.

Accepted for publication December 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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