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a Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge, The Rosie Hospital, CB2 2SW, United Kingdom
b Metris Therapeutics Ltd., Strangeways Research Laboratory, Worts Causeway, Cambridge, CB1 8RN, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The receptors for VEGF—the fms-like tyrosine kinase, Flt-1 [12], and the kinase domain receptor, KDR [13]—are restricted in their expression patterns. While both receptors are present on endothelial cells obtained from many different tissues [14–16], Flt-1 but not KDR is highly expressed by trophoblast cells [17, 18]. Monocytes and peritoneal fluid macrophages respond to VEGF [19, 20], and melanomas have been shown to express VEGF receptors [21].
Angiogenesis is a complex process necessitating the interaction of numerous cell types that leads to the coordinated development of a complex three-dimensional vascular structure. Many factors are involved in angiogenesis, and it is the balance of stimulators such as VEGF and inhibitors like angiostatin that determines the net result [22, 23]. In the placenta there is profound angiogenesis as high-capacity transport develops between the maternal and fetal circulation. However, in the human, it is notable that little angiogenesis occurs in the maternal tissue while there is obvious capillary growth within the placental villi. This suggests that there are locally acting factors that regulate vascular growth.
We have previously shown that VEGF is highly expressed in the placenta and that early in gestation, maternal macrophages within the decidua contain large amounts of VEGF mRNA [17, 24]. We have also shown that villous and extravillous trophoblast (EVT) contain very high levels of mRNA encoding Flt-1 [17, 18]. In situ hybridization specific for Flt-1 shows a particularly strong signal over the villous and extravillous trophoblast cells with very little signal corresponding to the endothelial cells. The immunohistochemical localization of Flt-1, however, shows low-level uniform signal over each of these cell types. The probe used for the in situ hybridization detects both the membrane-bound and truncated, soluble forms of Flt-1 [25], while the antibody used in this study [18] reacts only with the C-terminus of the membrane-bound form. It is therefore possible that the trophoblast cells are producing a soluble form of Flt-1 lacking the cytoplasmic tyrosine kinase domain, which could act as a local antagonist for VEGF. We set out to determine whether soluble forms of Flt-1 were produced by the placenta and whether such molecules could play a role in regulating placental and decidual angiogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted in accordance with the conditions laid down by, and with the approval of, the ethical committee of Addenbrooke's Hospital (Cambridge, UK) N.H.S. Trust. Gestational dates were calculated from the first day of the last menstrual period and were ±7 days from those estimated at an ultrasound scan performed in the first trimester of pregnancy. First-trimester placental tissue was obtained after the surgical termination of pregnancy conducted between Weeks 8 and 12 of gestation, and third-trimester material was obtained from normal placentae at term after cesarean delivery. In the latter case, a thin slice of tissue was taken from the maternal surface of the placenta, which contained a significant amount of EVT and decidua, and also a 1-cm3 block was excised from deeper in the placental cotyledon, containing only villous tissue. A sample from each area was checked by histology. Samples were either snap-frozen or fixed overnight in 10% buffered formalin and embedded in paraffin wax. Maternal serum samples were collected in nonheparinized tubes and after clotting and centrifugation were aliquoted and frozen.
Binding of 125I-VEGF to Tissue Sections
Sections of frozen tissue were prepared, and binding assay of 125I-VEGF was conducted according to the method of Shweiki et al. [26]. Sections were preincubated with 0.2% gelatin and 1 mg/ml heparin in PBS for 30 min at room temperature. 125I-VEGF (Amersham International plc; 1500 Ci/mmol) was then added at a concentration of 100 pM in PBS. Serial control sections contained competitor VEGF (R&D Systems, Minneapolis, MN) at a concentration of 3 nM in the binding medium. After binding for 1 h at room temperature, sections were washed in cold PBS, fixed in 2.5% glutaraldehyde, and washed again. Sections were air dried, and autoradiographic emulsion (LM-1 emulsion; Amersham International plc, Buckinghamshire, UK) was applied to the slides, which were developed after 48 h.
Immunohistochemistry and Antibodies
Sections serial to those used for 125I-VEGF binding were stained for CD34 to identify blood vessels. Briefly, sections were fixed for 5 min in 10% neutral buffered formalin, and anti-CD34 (Clone QBEND/10; Serotec, Raleigh, NC) was applied and detected with secondary biotin-streptavidin-horseradish peroxidase reagents from Zymed (Cambridge Biosciences, Cambridge, UK). Final detection was with diaminobenzidine. An anti-cytokeratin antibody (Clone MNF116; Dako Corp., Carpinteria, CA), as previously described, was used to identify epithelial cells and trophoblast [18]. There was no counterstaining of the tissue. In the Western blots, an anti-Flt-1 N-terminal antibody (fltNT) was used as described by Kendall et al. [27] and also a biotinylated goat polyclonal antibody directed against Flt-1 (BAF321; R&D Systems).
Cloning and Expression of Soluble Flt-1(sFlt-1) from the Placenta
A cDNA library prepared in 
gt11 from mRNA obtained from term placenta (Clontech, Palo Alto, CA) was screened using a probe specific for a portion of the extracellular domain of Flt-1. This probe (bases 1507 to 1779; Shibuya et al. [12]) is within an area showing minimal homology between Flt-1 and other related receptors and was obtained by polymerase chain reaction using the primers described by Charnock-Jones et al. [17]. Screening was performed as described in Charnock-Jones et al. [28]. Numerous hybridizing plaques were identified, and several of these were purified by further rounds of screening. Six of these clones, upon sequence analysis, proved to be similar to the clone described by Kendall and Thomas (EMBL accession number U01134 [25]) that encodes a soluble variant of Flt-1, termed sFlt-1 by us. The longest of these clones was fully sequenced using fluorescent dye terminators (ABI, The Perkin-Elmer Corp., Foster City, CA) and contained cDNA that encoded soluble Flt-1. The coding portion of this cDNA from the EagI site at position 189 to the linker-derived EcoRI site at the 3' end was subcloned into the baculoviral transfer vector pVL1392 (Pharmingen, San Diego, CA), and its orientation was confirmed by DNA sequencing. DNA was prepared from this plasmid using a Qiagen (Mississauga, ON, Canada) miniprep and then cotransfected into Sf9 cells with baculogold DNA (Pharmingen) to allow in vivo site-specific recombination to occur and the subsequent generation of recombinant viruses. The recombinant viruses were plaque purified in soft agar, and the plaques were identified by visual examination under strong illumination over a dark background. High-titer viral stocks were then prepared as described in the Pharmingen baculovirus manual. Protein expression was accomplished by infecting small-scale liquid cultures of approximately 100 ml of Sf9 cells at a density of approximately 2 x 106/ml with a high-titer viral stock at a multiplicity of infection of between 2 and 10. Cells were grown in complete TMN-FH medium (Pharmingen) or SF 900-II serum-free medium (Gibco/BRL, Gaithersburg, MD).
Protein Purification
Soluble receptor was purified using the method described by Kendall and Thomas [25]. Conditioned medium (45 ml) from the baculoviral-infected Sf9 cells was clarified and then passed through a 1-ml heparin-sepharose affinity column (Hi-trap column; Pharmacia Biotech, Milton Keynes, Bucks, UK) that had been previously equilibrated with 0.15 M NaCl in 10 mM sodium phosphate (pH 6.2). The column was washed with this buffer and also with the same buffer but containing 0.6 M NaCl. Soluble receptor was eluted from the column using 1.0 M NaCl in sodium phosphate buffer. The protein was desalted and concentrated further by centrifugal ultrafiltration (Centricon 30; Amicon Inc., Bedford, MA).
In Situ Hybridization
Primers specific for the novel 3' end of sFlt-1 were 5' AGAGGTGAGCACTGCAAC 3' and 5' ACTGCTATCATCTCCGAAC 3'. Use of these primers amplified a 215-base pair (bp) region of sFlt-1 from position 2215 to 2429 of the EMBL sequence (accession number U01134). Riboprobes were labeled with [33P]UTP and in situ hybridization performed as described by Clark et al. [18].
RNase Protection Assay
The method used was essentially that of Charnock-Jones et al. [29]. An sFlt-1 probe was generated that corresponded to bp 2014 to 2297 of the sFlt-1 sequence (EMBL accession number U01134). This spans the region where sFlt-1 and the membrane-bound form differ, so that the full-length probe was protected for sFlt-1 while a smaller 205-bp fragment of RNA was protected for full-length Flt-1. The antisense probe was labeled with a total of 60 µCi of [
-32P]UTP (800 Ci/mmol; Amersham International plc) using the MAXIscript in vitro transcription kit (Ambion, Austin, TX). The resulting probe was treated with RNase-free DNase to remove the template before being purified by preparative PAGE. For each 30-µg sample of placental RNA, 100 000 cpm of probe was mixed and precipitated before addition of the hybridization buffer (RPAII kit; Ambion). This mix was heat denatured and then hybridized overnight at 45°C. Excess probe was digested with RNase A, and the hybridized probe was ethanol precipitated and loaded onto a 6% polyacrylamide sequencing gel.
Placental RNA samples examined were from the surface and deep regions of term placenta and from the villi of first-trimester placenta collected as already described. Five samples from each group were examined. Results were expressed as a ratio of sFlt-1 to full-length Flt-1 mRNA.
Villus Culture
First-trimester villi from four patients were sorted and washed thoroughly with PBS. Each of the villus samples was cultured in a 60-mm Petri dish for 24 h; the medium was changed, and the samples were cultured for a further 48 h in 6 ml of serum-free, phenol red-free Dulbecco's Modified Eagle's medium (DMEM)/F12. Medium alone was subjected to the same conditions as a control. Samples of the villi were fixed for histological examination. Placental tissue obtained at term was cultured in a similar manner and purified as described above and by Kendall et al. [27].
Covalent Cross-Linking
125I-VEGF (900 pM) was incubated overnight at room temperature with recombinant sFlt-1, trophoblast-conditioned medium, or heparin-sepharose affinity-purified serum. Control experiments were undertaken with 25 ng of VEGF (Amgen Inc., Thousand Oaks, CA), which competed with the 125I-VEGF. Complexes were cross-linked for 5 min at room temperature with 5 mM bis(sulfosuccinimidyl)suberate (Pierce Chemical Company, Rockford, IL) dissolved in 5 mM sodium citrate (pH 5.0). The reaction was terminated with 1.0 M Tris-HCl (pH 6.8) containing 10% SDS and 8% glycerol. Samples were reduced by the addition of ß-mercaptoethanol and subjected to electrophoresis on 4–15% gradient polyacrylamide gels (Bio-Rad, Cambridge, MA).
Inhibition of Binding of 125I-VEGF to Bovine Aortic Endothelial (BAE) Cells
BAE cells, obtained from the laboratory of Dale Redmer of North Dakota State University (Fargo, ND) and cultured in DMEM/F12 with 10% fetal calf serum, were cultured to confluence in 24- or 48-well plates, and all binding experiments were conducted in triplicate at 4°C. Cells were rinsed in wash buffer (PBS containing 1 mg/ml BSA and 10-7 M KI). 125I-VEGF at a final concentration of 20 pM was added to the binding medium (DMEM/F12; 25 mM Hepes; 1 mg/ml BSA; 10-7 M KI; 10 µg/ml heparin). To ascertain the level of nonspecific binding, identical incubations were performed in the presence of 2 nM unlabeled VEGF (R&D Systems). Cells were incubated for 2 h and rinsed three times with wash buffer before being solubilized in 0.5 M NaOH, and the levels of bound 125I-VEGF were determined by gamma counting (Packard, Meriden, CT).
S-200 Gel-Filtration Chromatography
125I-Labeled VEGF (Amersham International plc) was incubated at a final concentration of 15 pM with 400 µl of serum obtained from healthy male or female volunteers and from pregnant women at different stages of gestation. Samples of recombinant sFlt-1 and villus culture supernatant were also examined. After incubation overnight at room temperature, samples were analyzed by gel-filtration chromatography on a 12.5-ml S-200 Sephadex column (dimensions of 0.8 cm x 25 cm; Pharmacia and Upjohn, Kalamazoo, MI) as described by Hill et al. [30]. The column was equilibrated in PBS and eluted in the same buffer. Fractions (400 µl) were collected and the elution position of the 125I-VEGF was determined by gamma counting (Packard). As controls, 125I-VEGF was also run on the column in the absence of serum, as well as after incubation with serum and a large excess of unlabeled VEGF (5 nM).
125I-VEGF Binding in Serum
Serum samples from nonpregnant women and from women in the third trimester of pregnancy were investigated (n = 3 in each group). Serum (5 ml) was mixed with an equal volume of 10 mM sodium phosphate (pH 6.2) with 0.15 M NaCl and passed through a 1-ml heparin-sepharose affinity column as described above for recombinant sFlt-1. The 0.6 M wash and 1.0 M eluted fractions were collected (6 ml of each), desalted, and concentrated 6-fold in Centricon 50 tubes (Amicon Inc.). Cross-linking and PAGE were conducted as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On term placentae, 125I-VEGF bound within large and small villi in a pattern compatible with localization to blood vessels (Fig. 2A; n = 3). A serial section immunostained for CD34 localized the blood vessels in the placenta; some light nonspecific staining was evident around the large villus in Figure 2B. Within the large villus (Fig. 2A), silver grains were particularly obvious in association with the blood vessels, in addition to there being some diffuse grains present within the stroma. A high-power photograph of a villus revealed that 125I-VEGF binding was not over the trophoblast but within the villous core (Fig. 2C). In Figure 2D, a darkfield and a brightfield image have been merged. This clearly shows that the silver grains are associated with the blood vessels but also that there is some binding over the stroma. Over the decidua (Fig. 2E), diffuse but specific binding was detected. A serial control section to Figure 2E, which had the addition of 3 nM unlabeled VEGF in the binding mix, confirms that the diffuse silver grains in the decidua represent specific binding (Fig. 2F). The level of 125I-VEGF binding in Figure 2F is representative of background levels found on control sections for Figures 1 and 2.
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Cloning of sFlt-1 from the placenta was performed, and 6 of the 15 clones obtained from a placental cDNA library were sFlt-1 as opposed to full-length Flt-1. For sFlt-1, a 5' sequence additional to that published by Kendall and Thomas (EMBL accession number U01134) was obtained (5' CGGAGCGCGCCAG 3'). At the 3' end, a new poly A termination site was found. The translation termination site for sFlt-1 ends at bp 2313 in the Kendall and Thomas sequence, and the new poly A tail was found from bp 2331 onward.
In situ hybridization with a probe specific for sFlt-1 was used to confirm the presence of this mRNA in the placenta. Throughout pregnancy the EVT cells, which are present within the maternal decidual tissue, were highly positive for sFlt-1 (Fig. 3, A–D). Within the villous trophoblast, sFlt-1 mRNA was detected throughout pregnancy, but at varying levels both between placentae and within any one placenta (Fig. 3, E and F).
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An RNase protection assay that could discriminate between mRNAs encoding sFlt-1 and Flt-1 was used to determine the relative levels of these mRNAs (data not shown). The tissues investigated included first-trimester villus and term placenta. In the term placenta, surface samples containing villus, decidua, and EVT, as well as deeper samples with only villi, were examined. Both Flt-1 forms were readily detectable, suggesting that there was a significant amount of mRNA present to encode these proteins. In all samples the full-length Flt-1 band was stronger than that for sFlt-1. The ratio of sFlt-1 to full length was approximately 0.45 in all three sampling areas.
The supernatants from placental villus cultures were examined to determine whether there was a soluble factor present that bound VEGF in a similar manner to recombinant sFlt-1. First-trimester villus was cultured in serum-free medium as described above, and the resulting medium was incubated with 125I-VEGF overnight and cross-linked. Samples were reduced and examined after gel electrophoresis (Fig. 4). 125I-VEGF bands, which could be displaced with unlabeled VEGF, were found at approximately 250 and 160 kDa. Villi were thus found to produce one or more soluble factors capable of binding VEGF. Recombinant sFlt-1 was also cross-linked with 125I-VEGF, and this resulted in bands similar in molecular mass to those from the villus cultures (Fig. 4).
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The binding of 125I-VEGF to BAE cells was used to assay for the inhibitory activity of factors binding VEGF (Fig. 5). The heparin-sepharose affinity-purified recombinant sFlt-1 inhibited binding of 125I-VEGF to the BAE cells. Supernatants from separate cultures of first-trimester villi were added to the VEGF binding assay at different concentrations. In all three cases the villus supernatants showed dose-dependent inhibition of binding, although there was some variation in the activity of the culture supernatants.
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Western blotting of the supernatants from villus cultures was undertaken (n = 4). Western blot analysis was performed using an anti-Flt-1 N-terminal antibody (FltNT) as described by Kendall et al. [27]. Figure 6A shows the presence of a soluble protein that reacts with the Flt-1 antibody and has a molecular mass of approximately 100 kDa. Villus-conditioned medium was purified by heparin-sepharose affinity and ion-exchange chromatography using the protocol described by Kendall et al. [27] for sFlt-1 (n = 2; Fig. 6B). This gave a band of slightly higher molecular mass to the recombinant sFlt-1. The recombinant sFlt-1 band was not detected when the antibody was preabsorbed with an excess of the peptide against which the antibody was raised. In a similar experiment, the band from the purified villus supernatant was not detected after preabsorption of the Flt-1 antibody with peptide. Using a polyclonal antibody raised against the extracellular domain of Flt-1 (BAF321; R&D Systems), a similar band could be readily detected in culture supernatants obtained using delivered placental material (Fig. 6C). In this instance the greater apparent difference in size compared to the recombinant sflt is due to the use of a different-strength polyacrylamide gel. In Figure 6C the resolution around 100 kDa has been maximized.
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Since sFlt-1 was released into the villus supernatants, which in vivo would correspond to the maternal circulation, we investigated whether there were any VEGF-binding proteins that were unique to the serum of pregnant women. An S-200 Sephadex column was used to separate 125I-VEGF and 125I-VEGF complexes formed in samples of serum (n = 3 from each group), cultured villus supernatant (n = 3), and recombinant sFlt-1 (n = 2). 125I-VEGF in PBS was analyzed in a similar manner to provide a baseline, and results showed that free VEGF was detected around fraction 30 with another small peak occurring around fraction 15. Binding at fraction 15 was found to occur consistently but was of a low affinity as it was competed for by other binding proteins (Fig. 7). Ligand blotting studies revealed that 125I-VEGF binds to purified BSA (data not shown), and since the 125I-VEGF is supplied with BSA as a carrier, it is probable that the binding seen at faction 15 is due to BSA. The three samples of male serum and nonpregnant female serum also gave a peak around fraction 15 as indicated by the arrow labeled BSA/VEGF (Fig. 7, male). In serum samples from early pregnancy (Week 12) and at term, 125I-VEGF bound to a larger serum protein, and this was eluted from the column around fractions 10–11 (Fig. 7, arrow). This binding competed with the BSA/VEGF peak. At term, one of the three samples had very high levels of binding in this fraction. Samples from cultured first-trimester villus supernatant gave a peak at fractions 10–11, and recombinant sFlt-1 gave a peak at fraction 11. Control medium (DMEM/F12) for the villus culture experiments gave only a small peak at fraction 15.
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125I-VEGF binding was conducted in serum samples that had been purified by heparin affinity (according to Kendall and Thomas [25]). In all three serum samples from pregnant women, the 1.0 M NaCl fraction contained a VEGF-binding protein that when complexed with 125I-VEGF gave a molecular mass just over 250 kDa (Fig. 8, lane 1). This protein was not present in the nonpregnant samples (lane 2), and binding to it could be competed for with unlabeled VEGF (lane 3). The control 0.6 M fraction from pregnant serum had a small amount of bound 125I-VEGF at the same molecular mass as the 1.0 M fraction (lane 4). It is likely that this is the same binding protein and that a small amount was eluted from the column in the washes. No binding was evident with the 0.6 M fraction from nonpregnant samples (lane 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ligand binding was performed on first- and third-trimester human placental sections. 125I-VEGF was found to principally bind to the fetal blood vessels within the villi (Figs. 1 and 2). Under the conditions used it is likely that the 125I-VEGF would have bound to both Flt-1 and KDR. Although estimates of the Kd values for these receptors of VEGF vary dramatically, there does seem to be a consensus that the Flt-1 high-affinity sites have a dissociation constant of between 1 and 16 pM and that the lower-affinity KDR sites are between 10 and 100 times that of Flt-1 [16, 34–36]. Both Flt-1 and KDR-like immunoreactivities have been localized to the fetal endothelial cells of the placental villi throughout pregnancy [18, 33], and the binding sites reported here confirm these results and show that fetal endothelial cells have specific high-affinity receptors for VEGF. This is in agreement with what is widely found in other tissues [37].
When investigating the localization of Flt-1 mRNA in the maternal decidual tissue, we previously used an in situ hybridization probe specific for the extracellular domain of Flt-1 and detected very strong signal over the EVT cells throughout pregnancy [17, 18]. Immunohistochemical localization with antibodies directed against the intracellular carboxyl terminus of Flt-1 indicated that some of the Flt-1 produced by these cells may be full-length membrane-bound receptor [18, 31, 32]. However, a striking feature of the 125I-VEGF binding studies is the lack of detectable binding in association with the EVT or any other specific cell within the decidua. Some diffuse but specific binding, which could be displaced by excess unlabeled VEGF, was detected over the maternal decidual tissue. It is possible that some 125I-VEGF associates with a soluble VEGF receptor that then interacts with the heparan sulfates present in the extracellular matrix. The in situ hybridization described in this study using a probe specific for sFlt-1 revealed that EVT cells contain large amounts of mRNA encoding this soluble VEGF receptor. It is therefore unlikely that VEGF will have any stimulatory effect on this population of cells. It is also unlikely that VEGF has appreciable effects on villous trophoblast in vivo since there is no specific binding of 125I-VEGF to these cells and they also produce soluble Flt-1.
The decidua is a site where large amounts of VEGF are produced by the macrophages in Nitabuch's stria during the first trimester of pregnancy [17] but where the process of vascular transformation is required rather than angiogenesis. A pool of sFlt-1 in the decidua may therefore be important for reducing the effects of VEGF on maternal endothelial cells within this tissue.
Significant quantities of both full-length Flt-1 and sFlt-1 mRNA were present in the placenta and were readily detectable by RNase protection assays, although there was more mRNA encoding full-length Flt-1. As yet it is not known whether there are differences in the half-lives of these two species or whether the ratio observed for the mRNA is reflected in the protein levels. The Flt-1 mRNAs identified to date arise by alternative splicing [25]. Understanding the mechanisms by which this splicing is controlled will have important consequences for the biological actions of VEGF and hence angiogenesis.
In situ hybridization using a probe specific for the novel 3' terminus of sFlt-1 revealed that the mRNA for sFlt-1 was present within villous trophoblast throughout pregnancy, although, interestingly, there was variability in the intensity within and between placentae. Analysis by an RNase protection assay of superficial and deep samples of placenta (i.e., tissue samples with and without substantial quantities of EVT cells present) showed that the ratio of Flt-1 to sFlt-1 mRNA remained the same, indicating that the villous trophoblast is a significant source of sFlt-1 mRNA throughout pregnancy. Because the size of the placenta, and thus the quantity of villous trophoblast, increase dramatically during pregnancy, it is likely that there will be an increase in the total sFlt-1 production.
To determine whether the placenta is capable of secreting a protein with the characteristics of a soluble VEGF receptor, first-trimester villi and placental tissue obtained at delivery were cultured in serum-free media and the supernatants analyzed. Results from the gel-filtration chromatography, cross-linking, and the BAE binding assay were consistent with the presence of biologically active soluble receptor (Figs. 4, 5, and 7). To conclusively demonstrate the presence of sFlt-1, an antibody to the N-terminus of Flt-1 or one raised against the entire extracellular domain of Flt-1 was used. Western blotting with both Flt-1 antibodies identified a soluble protein in the villus supernatants (Fig. 6). Furthermore, a protein could be purified using the same conditions as for recombinant sFlt-1, and this protein was immunoreactive with two Flt-1 antibodies and was of a molecular mass consistent with its being sFlt-1 [25, 27].
Recombinant forms of sFlt-1 have been shown to inhibit the action of VEGF in vitro [25], and, furthermore, artificially engineered VEGF-receptor chimeric proteins have been shown to block VEGF-induced retinal neovascularization [38]. This indicates that soluble VEGF-binding proteins can play a role in the regulation of VEGF action in vivo.
In the placenta, the villi are in direct contact with maternal blood. Proteins secreted into the supernatant in vitro may therefore be detectable in maternal circulation. We investigated whether VEGF-binding activity could be identified in the serum of pregnant women. Using the gel-filtration assay described above, serum from women in the first trimester of pregnancy and at term had binding activity that was not detectable in the serum from nonpregnant women or males (Fig. 7). Consistent with this activity's being sFlt-1 was the purification of a binding protein, using the method for purifying sFlt-1 [25], from the serum of pregnant women at term that was not detected in serum from nonpregnant women (Fig. 8). The presence of a VEGF-binding protein in serum has implications for regulating the levels of bioavailable VEGF during pregnancy [39]. Although the placenta produces sFlt-1 and is thus likely to be an important source of this protein, there is the possibility that sFlt-1 production may be up-regulated in other organs during pregnancy.
It has been suggested that 2-macroglobulin, present in high levels in human serum, binds and inactivates VEGF [40]; however, the column fractionation and cross-linking studies conducted here did not detect this binding in any of the serum samples tested (male; nonpregnant and pregnant female). In addition, after column fractionation of the serum, the binding fractions were immunoblotted and no immunoreactivity was found with an antibody against 2-macroglobulin.
The balance between positive and negative effectors in the regulation of angiogenesis is now recognized as having considerable importance [23]. Thus, agents that specifically antagonize the actions of VEGF are of great interest. It is not currently known whether any other tissues produce significant amounts of sFlt-1. In the serum samples from nonpregnant women and males, no significant VEGF-binding activity could be detected. If additional tissues do produce VEGF-binding proteins, then their contribution to serum levels, at least in normal subjects, is significantly lower than that seen during pregnancy. We have found mRNA encoding sFlt-1, by reverse transcription-polymerase chain reaction, in tissue samples from the kidney, renal vein, adrenal, aorta, and pulmonary artery (data not shown). The possibility thus exists that sFlt-1 may locally mediate the action of VEGF in various tissues. Of considerable interest would be whether the production of either locally acting or circulating soluble receptors for VEGF are associated with various pathological conditions.
In conclusion, we have shown that the human placenta is a rich source of a soluble VEGF receptor that antagonizes the action of VEGF. This receptor has been characterized and shown to be sFlt-1, which had previously been detected only at low levels in cultured HUVEC [27]. This protein is present in maternal serum and so may influence the maintenance functions of VEGF on endothelial cells [4] in addition to blocking angiogenesis locally in the decidua.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D. Stephen Charnock-Jones, Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge, Box 223, The Rosie Hospital, Robinson Way, CB2 2SW, UK. FAX: 44 1223 215327; dscj1{at}cam.ac.uk
Accepted: August 6, 1998.
Received: June 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin and the binding is inhibited by heparin. J Biol Chem 1993; 268:7685–7691.This article has been cited by other articles:
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  T. W. Bonagura, G. J. Pepe, A. C. Enders, and E. D. Albrecht Suppression of Extravillous Trophoblast Vascular Endothelial Growth Factor Expression and Uterine Spiral Artery Invasion by Estrogen during Early Baboon Pregnancy Endocrinology, October 1, 2008; 149(10): 5078 - 5087. [Abstract] [Full Text] [PDF]
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 A. Muehlenbachs, M. Fried, J. Lachowitzer, T. K. Mutabingwa, and P. E. Duffy From the Cover: Natural selection of FLT1 alleles and their association with malaria resistance in utero PNAS, September 23, 2008; 105(38): 14488 - 14491. [Abstract] [Full Text] [PDF]
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 M. Plaisier, E. Streefland, P. Koolwijk, V.W.M. van Hinsbergh, F.M. Helmerhorst, and J.J.H.M. Erwich Angiogenic Growth Factors and Their Receptors in First-Trimester Human Decidua of Pregnancies Further Complicated By Preeclampsia or Fetal Growth Restriction Reproductive Sciences, September 1, 2008; 15(7): 720 - 726. [Abstract] [PDF]
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 C. C. Zhou, S. Ahmad, T. Mi, S. Abbasi, L. Xia, M.-C. Day, S. M. Ramin, A. Ahmed, R. E. Kellems, and Y. Xia Autoantibody From Women With Preeclampsia Induces Soluble Fms-Like Tyrosine Kinase-1 Production via Angiotensin Type 1 Receptor and Calcineurin/Nuclear Factor of Activated T-Cells Signaling Hypertension, April 1, 2008; 51(4): 1010 - 1019. [Abstract] [Full Text] [PDF]
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 J. S. Gilbert, M. J. Ryan, B. B. LaMarca, M. Sedeek, S. R. Murphy, and J. P. Granger Pathophysiology of hypertension during preeclampsia: linking placental ischemia with endothelial dysfunction Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H541 - H550. [Abstract] [Full Text] [PDF]
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 O. Nevo, A. Many, J. Xu, J. Kingdom, E. Piccoli, S. Zamudio, M. Post, A. Bocking, T. Todros, and I. Caniggia Placental Expression of Soluble fms-Like Tyrosine Kinase 1 is Increased in Singletons and Twin Pregnancies with Intrauterine Growth Restriction J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 285 - 292. [Abstract] [Full Text] [PDF]
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 C. P. Thomas, J. I. Andrews, and K. Z. Liu Intronic polyadenylation signal sequences and alternate splicing generate human soluble Flt1 variants and regulate the abundance of soluble Flt1 in the placenta FASEB J, December 1, 2007; 21(14): 3885 - 3895. [Abstract] [Full Text] [PDF]
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J. S. Gilbert, S. A. Babcock, and J. P. Granger Hypertension Produced by Reduced Uterine Perfusion in Pregnant Rats Is Associated With Increased Soluble Fms-Like Tyrosine Kinase-1 Expression Hypertension, December 1, 2007; 50(6): 1142 - 1147. [Abstract] [Full Text] [PDF]
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 Y. Hattori, S. Yamamoto, and N. Matsuda Sympathetic Control of VEGF Angiogenic Signaling: Dual Regulations by {alpha}2-Adrenoceptor Activation? Circ. Res., September 28, 2007; 101(7): 642 - 644. [Full Text] [PDF]
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 C. J. Lockwood, P. Toti, F. Arcuri, E. Norwitz, E. F. Funai, S.-T. J. Huang, L. F. Buchwalder, G. Krikun, and F. Schatz Thrombin Regulates Soluble fms-Like Tyrosine Kinase-1 (sFlt-1) Expression in First Trimester Decidua: Implications for Preeclampsia Am. J. Pathol., April 1, 2007; 170(4): 1398 - 1405. [Abstract] [Full Text] [PDF]
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C. C. Zhou, S. Ahmad, T. Mi, L. Xia, S. Abbasi, P. W. Hewett, C. Sun, A. Ahmed, R. E. Kellems, and Y. Xia Angiotensin II Induces Soluble fms-Like Tyrosine Kinase-1 Release via Calcineurin Signaling Pathway in Pregnancy Circ. Res., January 5, 2007; 100(1): 88 - 95. [Abstract] [Full Text] [PDF]
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