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Department of Obstetrics and Gynecology, Faculty of Medicine (T.N., T.F., M.K., T.Y., Y.O., M.M., S.K., Y.T.), University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan 113-8655; and Department of Obstetrics and Gynecology, Xiehe Hospital, Tongii Faculty of Medicine (L.Z.), Huazhong University of Science and Technology, Jiefang Dadao, Hubei, Wuhan 430000, China
Address all correspondence and requests for reprints to: Tomoyuki Fujii, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: fujiit-tky{at}umin.ac.jp.
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Introduction |
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CTs are highly specialized epithelial cells comprising the villous structure of the placenta. At early pregnancy, CTs located at the tips of the villi proliferate extensively and form trophoblastic cell columns, which are eventually anchored to the maternal side, the decidua. Then, they breach the superficial portions of the uterine walls and remodel the vessels by replacing the endothelial lining, resulting in the completion of the physical connections between the fetus and the mother (1, 4). As such, CTs seem to play a pivotal role in the development of fetoplacental vasculature.
It must be admitted that extended vascular pruning or remodeling taking place in the uterine wall represent fundamental mechanisms of the development of the fetoplacental circulation. Although vascular remodeling occurring at the fetomaternal interface is executed by coordinated interplay between the fetus and mother, several recent lines of evidence have established essential roles played by CTs in this process. Specifically, angiogenic factors, such as vascular endothelial growth factor (VEGF) (5, 6, 7, 8) and placenta growth factor (PlGF) (9), are suggested to act locally via their receptors and thus control the vascular remodeling required for the establishment of the fetoplacental circulation.
In general, angiogenesis is driven by hypoxic conditions, as seen in a growing tumor (10), and diabetic retinopathy (11). Looking at placental development, which proceeds in an environment of relative low oxygen tension, we may speculate that low oxygen tension could serve to stimulate the production of angiogenic substances. Actually, the oxygen tension environment surrounding CTs drastically alters depending on the location in the placenta and the gestational age. As reported in previous work (12), local oxygen pressure in the placenta is lower in the villous side (18 mm Hg) compared with the maternal side (40 mm Hg) at 8–10 gestational weeks, but it increases up to 60 mm Hg after maternal blood is supplied into the intervillous space (later than 12 gestational weeks). Therefore, it can be hypothesized that local oxygen tension of the placenta controls the activity of CTs required for the vascular remodeling and then works as a major regulator for the reconstitution of uterine vascular structure. The mechanism underlying uterine vascular remodeling is closely related in the pathophysiology of preeclampsia because incomplete uterine vascular remodeling has been suggested in preeclamptic placenta (1, 2, 3). At present, however, little is known regarding a link between oxygen tension and the production of angiogenic substances by CTs. Furthermore, it is unclear how these angiogenic substances are involved in the process of vascular remodeling.
With this background in mind, we attempted to explore how oxygen tension regulates the expression of VEGF and PlGF using primary cell culture of CTs. We also focused on the regulation of the expression of soluble fms-like tyrosinekinase-1 (sFlt-1), which is a potent biological antagonist for both VEGF and PlGF (13, 14, 15), and thus how this could modify the effect of both angiogenic factors. As well as examining the relation of oxygen to the VEGF system regulation in CTs, it is important to observe any difference in response to oxygen tension between CTs and other cellular components comprising the placenta. To address this, we looked at the effects of oxygen in human umbilical vein endothelial cells (HUVECs) and villous fibroblasts (VFs) and showed that CTs in a reduced oxygen state enhance their capacity to produce sFlt-1.
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Materials and Methods |
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Isolation of CTs and VFs
The procedures to isolate CTs and VFs were performed as previously described (16). Briefly, the first trimester placenta tissues (5–10 wk of gestation) were obtained from the cases of legal abortion with consent. Minced villi tissues were digested in PBS (Sigma, St. Louis, MO) supplemented with 0.125% trypsin (GibcoBRL, Grand Island, NY), 0.42 mM MgSO4, and 20 U/ml DNase type 1 (Invitrogen, Carlsbad, CA) at 37 C for 20 min. After passing through a mesh (100-µm pore size), the collected cells were separated in three layered Percoll (Sigma) density gradients (4 ml of 40%, 4 ml of 25%, and 0% Percoll layers in a 15-ml conical tube). After the centrifugation at 800 x g for 20 min, the floating cells between 25 and 40% Percoll layer were collected. The cells were incubated with anti-CD9 antibody-bound (clone P1/33/2; Dako, Kyoto, Japan) magnetic beads (Cellection PanMouse IgG Kit; Dynal Biotech, Oslo, Norway) for 30 min at 4 C to exclude contaminating stromal cells.
VFs were removed due to their attaching to anti-CD9-bound magnetic beads in the previously mentioned process of CT isolation. The cell-magnetic bead complexes were incubated with DNase for 15 min at room temperature to detach the beads from these cells according to the manufacturer’s instructions. After removing beads by magnetic collector, the detached cells were cultured in medium 199 (Sigma) supplemented with 10% fetal bovine serum (FBS; Sigma) on culture flasks. VFs proliferated rapidly by changing medium every 3 d and were obtained with high purity 14 d after the isolation.
Culture of HUVECs
HUVECs in the first passage were obtained from CAMBREX Bio Science (Walkersville, MD) and maintained in endothelial cell basic medium supplemented with a growth factor mixture containing hydrocortisone, heparin, ß-fibroblast growth factor, VEGF, IGF-I, human epidermal growth factor, and FBS (0.2%) (EGM-2; CAMBREX Bio Science). All the experiments were conducted using the 3–5 times passed cells.
Cell lines
The choriocarcinoma-derived cell lines, BeWo, JAR, and JEG3, were obtained from American type culture collection. They were maintained in RPMI 1640 (Sigma) supplemented with 10% FBS.
Culture under different oxygen conditions
The freshly isolated CTs were resuspended in defined Keratinocyte SFM (GibcoBRL) at a concentration of 1 x 105 cells/ml and seeded on collagen type 4-coated 35-mm dishes (2 ml/dish). After 3 h of incubation in ambient oxygen culture condition (5% CO2-20% O2-75% N2) at 37 C, the medium was refreshed to remove the debris, and then CTs were exposed to different oxygen conditions.
BeWo, JAR, JEG3, VFs, and HUVECs in 80% confluency were detached from the culture flasks with 0.05% trypsin and 0.53 mM EDTA (GibcoBRL). After being washed twice with PBS, they were diluted to 4 x 104 cells/ml in RPMI 1640 plus 10% FBS (BeWo, JAR, and JEG3), medium 199 plus 10% FBS (VFs), and endothelial basic medium plus 1% FBS (HUVECs) and were seeded on 35-mm dishes (2 ml/dish) with no extracellular matrix coating.
The cells were prepared in two sets of culture dishes, one for cell culture in ambient oxygen condition and the other for cell culture in reduced oxygen conditions (5% CO2-2% O2-93% N2 or 5% CO2-8% O2-87% N2) at 37 C, with the oxygen concentration being correctly regulated in a BioLabo Multigas Incubator (Jujifield, Tokyo, Japan) for different periods.
The assessment of cell viability and cell number in culture
The cultured cells were detached from culture dishes with 0.05% trypsin-EDTA (GibcoBRL) at 72 h of culture under different oxygen conditions. The cells were stained with trypan blue (GibcoBRL), and the viable cells without staining were counted on a hemocytometer.
Enzyme immunoassays
The culture media under ambient and reduced oxygen conditions were collected at different incubation periods. Free VEGF (not bound to sFlt-1), free PlGF, and sFlt-1 concentrations in each culture media were measured using sandwich ELISA (Quantikine ELISA kit; R&D Systems, Minneapolis, MN). All the procedures were performed according to the manufacturer’s instructions. Total VEGF (the sum of free and sFlt-1-combined VEGF) concentrations were measured using competitive enzyme immunoassay (ChemiKine EIA kit; Chemicon International, Temecula, CA). The minimal detectable doses of assays were 5.0 pg/ml (free VEGF), 7.0 pg/ml (PlGF), 5.0 pg/ml (sFlt-1), and 195 pg/ml (total VEGF). When the concentrations were below the detectable dose, they were regarded as zero.
RNA isolation and reverse-transcriptase reaction
Total RNA of cells was isolated using RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The amount of the isolated RNA was assessed spectrophotometrically. The total RNA was reverse transcribed into cDNA using the Rever Tra Ace kit (Toyobo, Osaka, Japan) in the volume of 40 µl, including 8 µl of 5x reverse-transcriptase buffer, 4 µl deoxynucleotide triphosphate, 2 µl RNA inhibitor, 2 µl Rever Tra transcriptase, 2 µl random primer, and 22 µl sample RNA in a thermal cycler (30 C for 10 min, 42 C for 20 min, and 99 C for 5 min).
Real-time PCR
Real-time PCR was carried out to semiquantify the amounts of mRNA expression of VEGF, PlGF, sFlt-1, and membrane-spanning Flt-1 (msFlt-1) in the cultured cells. The primer sequences, their location, and their annealing temperatures are summarized in Table 1
. VEGF primers amplified the sequence between exon 1 and exon 3, which was shared by all known splicing variants of VEGF (17). The specific primers for PlGF were designed to amplify the sequence common to all known PlGF splicing variants (PlGF, PlGF2, and PlGF3) (18, 19). To quantify sFlt-1 and msFlt-1 expression distinctively, the antisense primer sequences for them resided in unique regions to each mRNA, whereas the same sense primer was commonly used for both sFlt-1 and msFlt-1.
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, and the extension time depended on the PCR product size (1 sec per each 25 bp). ß-actin mRNA was quantified in each sample as an internal control to normalize the level of mRNA among samples. After the amplification program, PCR products were analyzed by melting curve to confirm the amplification specificity. The PCR product inserted in pCRII-TOPO plasmid vector was subcloned using the TOPO TA cloning kit (Invitrogen), and the accordance of its nucleotide sequence with the ever-reported one was confirmed by sequencing analysis.
Statistical analysis
Statistical analysis was performed using Wilcoxon’s test for paired data obtained from paired culture in which the cells isolated from the same placenta tissue were assigned to two different oxygen conditions (20 and 2% pair culture or 20 and 8% pair culture). All the values were presented as means ± SE, and P < 0.05 was considered significant. In the results of real-time PCR analysis, the mRNA amounts in samples obtained from culture groups in reduced oxygen conditions were shown as relative values to those under 20% O2 conditions after compensation with ß-actin mRNA amount in each sample. In each experiment, n equals the number of different individuals who supplied the placental tissues.
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Results |
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The effect of reduced oxygen on viable cell number in culture
The cell viabilities of cultured CTs and VFs under ambient oxygen condition were maintained at least up to 144 h in culture as confirmed using WST-8 assay in our previous work (16). We compared viable cell numbers among culture groups of different oxygen conditions using trypan blue exclusion test. In all oxygen conditions, the percentage of stained cells at 72 h in culture was less than 0.5%, regardless of cultured cell types. The cell numbers of CTs were 2.11 ± 0.29 x 105 cells/dish (20% O2, n = 12), 2.47 ± 0.38 x 105 cells/dish (8% O2, n = 6), and 2.90 ± 0.54 x 105 cells/dish (2% O2, n = 6; P < 0.05), indicating that the reduced oxygen condition promoted CT proliferation (Fig. 1A) in agreement with the previous report (20). VFs and HUVECs showed no significant change in the cell numbers under reduced oxygen condition (VFs: 2.23 ± 0.24 x 105 cells/dish in 20% O2 and 2.18 ± 0.26 x 105 cells/dish in 2% O2, n = 6; HUVECs: 2.29 ± 0.085 x 105 cells/dish in 20% O2 and 2.31 ± 0.11 x 105 cells/dish in 2% O2, n = 6; Fig. 1B).
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, A and B).
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). When CTs were cultured for 72 h in different oxygen conditions, sFlt-1 concentrations in CT culture media were 920 ± 180 pg/ml (20% O2, n = 18), 1223 ± 408 pg/ml (8% O2, n = 8, P < 0.05), and 1865 ± 457 pg/ml (2% O2, n = 10, P < 0.01; Fig. 3B). Thus, sFlt-1 concentrations increased with a decrease in O2 concentration. sFlt-1 was also detected in the culture media of both HUVECs and VFs at 72 h. However, sFlt-1 concentrations in the culture media of HUVECs and VFs did not differ between 20 and 2% O2 condition (HUVECs: 1120 ± 178 pg/ml, 20% O2, n = 8 and 909 ± 186 pg/ml, 2% O2, n = 8; VFs: 41 ± 16 pg/ml, 20% O2, n = 10 and 40 ± 16 pg/ml, 2% O2, n = 10; Fig. 3B).
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Reduced oxygen increased msFlt-1 mRNA commonly in CTs and HUVECs
Real-time PCR analysis revealed that the amount of msFlt-1 mRNA in CTs slightly but significantly increased with a decrease in O2 concentration (1.82 ± 0.48-fold, 2% O2, n = 10, P < 0.05 and 1.20 ± 0.07-fold, 8% O2, n = 8, P = not significant) relative to 20% O2 condition (Fig. 4). The mRNA of msFlt-1 increased under reduced oxygen condition in HUVECs (1.54 ± 0.06-fold relative to 20% O2, n = 6, P < 0.05) but not in VFs (Fig. 4). It should be noted that the degree of the increase in the msFlt-1 mRNA amount in CTs exposed to hypoxia was far smaller than that in sFlt-1.
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). Using the same ELISA kit, free VEGF in the culture media from VFs was detectable and significantly increased under lower O2 concentration (27 ± 7.8 pg/ml, 20% O2, n = 10 vs. 51 ± 11.5 pg/ml, 2% O2, n = 10, P < 0.01; Fig. 5A). The concentrations of total VEGF in CT culture media increased significantly with a decrease in O2 concentration (246 ± 41 pg/ml, 20% O2, n = 18; 359 ± 70 pg/ml, 8% O2, n = 8, P < 0.05; and 472 ± 58 pg/ml, 2% O2, n = 10, P < 0.01; Fig. 5B).
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). Likewise, the amounts of VEGF mRNA in HUVEC and VF culture for 72 h in 2% O2 significantly increased compared with the amounts of VEGF mRNA cultured in 20% O2 (4.7 ± 1.45-fold for HUVECs, n = 8, P < 0.05; and 1.74 ± 0.41-fold for VFs, n = 10, P < 0.01; Fig. 5C).
Reduced oxygen decreased free PlGF levels in the culture media of CTs
The ELISA kit for PlGF used in this study detects only free PlGF and not PlGF bound to sFlt-1 (21). Free PlGF concentrations in the culture media of CTs increased with time in culture (1115 ± 166 pg/ml at 48 h, 1498 ± 140 pg/ml at 96 h, and 2146 ± 614 pg/ml at 144 h, n = 6; Fig. 6A). Free PlGF concentrations in the culture media of CTs cultured for 72 h reduced with a decrease in O2 concentration (1293 ± 123 pg/ml, 20% O2, n = 18; 1089 ± 124 pg/ml, 8% O2, n = 8, P < 0.01; and 667 ± 70 pg/ml, 2% O2, n = 10, P < 0.01; Fig. 6B). On the other hand, no significant difference in free PlGF concentrations was observed in the culture media of HUVECs cultured for 72 h in 20% O2 (290 ± 35 pg/ml, n = 8) vs. 2% O2 (295 ± 39 pg/ml, n = 8; Fig. 6B). PlGF was not detected in VF culture media under both 20 and 2% O2 conditions (Fig. 6B). Real-time PCR analysis revealed that the amounts of PlGF mRNA in CTs were not altered among the 20% O2, 8% O2 (0.88 ± 0.06-fold relative to 20% O2, n = 8), and 2% O2 (1.31 ± 0.34-fold relative to 20% O2, n = 10) conditions (Fig. 6C). Likewise, the amounts of PlGF mRNA in HUVECs were not altered between the 20 and 2% O2 conditions (1.11 ± 0.31-fold relative to 20% O2, n = 8; Fig. 6C).
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Discussion |
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It is conceivable that sFlt-1 might antagonize the activity of VEGF and PlGF by binding with them (13, 14, 15). In the present study, VEGF mRNA increased under a reduced oxygen condition in cells including CTs, HUVECs, and VFs, a finding in keeping with the observations in other tissues and neoplasms (10). In view of sFlt-1 being a VEGF antagonist, it is reasonable to speculate that the actual angiogenic potential of the VEGF system is determined by the balance between VEGF and sFlt-1. Notably, in this study, free VEGF was not detectable in the culture media of CTs regardless of oxygen level despite an apparent increase in the total VEGF concentrations in a reduced oxygen milieu, suggesting that sFlt-1 production, concurrently increased in a reduced oxygen milieu, may overrule the increase in VEGF production and, thereby, negate the biological effects of VEGF.
The secretion of sFlt-1-unbound (free) PlGF was diminished in response to reduced oxygen in CTs but not HUVECs, whereas PlGF mRNA amounts were not altered by reduced oxygen both in CTs and HUVECs. Due to an appropriate method to measure total PlGF being unavailable, it remains to be seen how hypoxia influences the production of PlGF in CTs. Although this needs to be addressed by future studies, given a marked increase in sFlt-1 production in CTs under a reduced oxygen milieu, the observed reduction in free PlGF could be a reflection of enhanced sFlt-1 production, as also could be the case with VEGF. We found that BeWo, a choriocarcinoma cell line, down-regulated PlGF mRNA amount under reduced oxygen, which was in accordance with the previous reported observation (23). BeWo demonstrates a different response to oxygen concentration milieu from CTs; e.g. BeWo keeps proliferating in ambient oxygen milieu, whereas primary CTs cease to proliferate. The observed discrepancy in the effect of reduced oxygen on PlGF mRNA between primary CTs and BeWo might be due to the difference in the cell characters.
In general, the VEGF system is activated under a hypoxic condition, a reasonable mechanism to hold oxygen tension constant (10). In this study, we observed that HUVECs and VFs stimulated VEGF expression that was unassociated with the up-regulation of sFlt-1 under a reduced oxygen condition, which is in support of the known concept that hypoxia augments angiogenesis by mechanisms involving the VEGF system. These cell properties of HUVECs and VFs seem to be appropriate to develop the vascular network in the villous stroma depending on oxygen demand from the growing fetus. When looking at CTs, however, the response of CTs to reduced oxygen is paradoxical because CTs increase sFlt-1 production in a reduced oxygen milieu. This doesn’t seem to fit the hypothesis that VEGF-related angiogenesis is driven by hypoxia. Vascular remodeling in the uterine wall is a specific vascular event to placental development. CTs reconstitute the uterine vascular structure into low-resistance and high-conductance vessels. Considering that maternal endothelial cells are replaced by CTs, uterine vascular remodeling, despite progressing in the relatively hypoxic fetomaternal interface of early gestation, seems to put some restriction on angiogenesis by maternal endothelial cells. In this viewpoint, there might be some discrepancy between the well-accepted notion of angiogenesis and the uterine vascular remodeling. When based on our results, one possible interpretation of this paradox is that sFlt-1, produced by CTs in a reduced oxygen environment, might block angiogenesis by maternal endothelial cells and prepare an appropriate condition for CTs to progress uterine vascular remodeling. Although we just demonstrated that CTs had a property to up-regulate sFlt-1 expression by reduced oxygen and that unbound VEGF was undetectable in the media of CTs cultured under a reduced oxygen condition despite an apparent increase in total VEGF production, this line of reasoning might offer a basic paradigm for the establishment of a placental circulation.
Recently, concentrations of sFlt-1 in peripheral blood in patients with preeclampsia were reported to be higher than those in normal cases (21, 25, 26). Furthermore, pregnant rats given a large amount of sFlt-1 exhibited preeclampsia-like symptoms, and the same symptoms were observed even in case of nonpregnant rats when given sFlt-1 (21). Therefore, it seems logical to assume that sFlt-1 is closely interrelated to the pathology of preeclampsia. The predominant source of sFlt-1 elevation in preeclampsia is estimated to be placenta. CTs isolated from preeclamptic placenta secreted more abundant sFlt-1 than CTs from normal placenta (27). sFlt-1 mRNA level was significantly higher in placentas from preeclamptic patients (28). However, the mechanism of how sFlt-1 expression is elevated in preeclamptic placenta has not been elucidated. Preeclampsia is characterized by incomplete uterine vasculature and, thereby, reduced blood flow into the intervillous space, resulting in placental hypoxia (3, 4, 29). Based on present findings, it is conceivable that CTs in preeclampsia are exposed to abnormally low oxygen concentrations and, thus, produce excessive amounts of sFlt-1, leading to an elevation in sFlt-1 concentrations as is observed in clinical settings. In the same context, our findings regarding the response of VEGF and PlGF expression in CTs to reduced oxygen support the previous study in which VEGF mRNA was increased and PlGF mRNA was not altered in preeclamptic placenta (28).
In conclusion, we provided evidence that CTs possess a unique property in that they enhance their ability to produce sFlt-1, an antagonist for both VEGF and PlGF, in the face of reduced oxygen. Despite an increase in VEGF production in response to reduced oxygen, a concomitant even greater increase in sFlt-1 might override the effects of VEGF and, thereby, rather attenuate the ultimate capacity of CTs to stimulate angiogenesis. This unique response of VEGF system to oxygen tension in CTs might be closely involved in the mechanism of uterine vascular remodeling. Besides, the present findings might give some help for an understanding of the pathogenesis of preeclampsia.
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Footnotes |
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Received April 26, 2004.
Accepted for publication July 21, 2004.
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