Molecular Human Reproduction, Vol. 5, No. 5, 452-458,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Expression of mRNA for vascular endothelial growth factor transmembraneous receptors Flt1 and KDR, and the soluble recetor sflt in cycling human endometrium
1 Department of Obstetrics and Gynecology, Heinrich-Heine-University Medical Center, Moorenstraße 5, D-40225 Düsseldorf, Germany, and 2 Department of GYN/OB, Reproductive Immunology Laboratory, Stanford University Medical Center, Palo Alto, CA, USA
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Abstract |
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The aim of this study was to quantify and localize the mRNA expression of the vascular endothelial growth factor (VEGF) receptors Flt1, KDR and sflt, in human endometrium throughout the menstrual cycle. Since neoangiogenesis is crucial during embryonic implantation, we postulate that endometrial receptivity to VEGF may be altered during the luteal phase in order to support implantation. Human endometrium was collected and specified as early proliferative (n = 3), mid-proliferative (n = 4), late proliferative (n = 3), early secretory (n = 2), mid-secretory (n = 4), and late secretory (n = 4). Competitive reverse transcription–polymerase chain reaction (RT–PCR) was performed to evaluate the mRNA values throughout the menstrual cycle. Additionally, four samples were separated into epithelial and stromal-enriched cell fractions and competitive RT–PCR was carried out to specify the distribution of the mRNA expression. While mRNA for the transmembraneous receptors Flt1 and KDR was shown to be present at almost constant values throughout the menstrual cycle, the soluble receptor, sflt, had a three-fold higher level of transcription during mid-proliferative and late proliferative when compared with early proliferative and the entire secretory phase. The expression of Flt1, KDR and sflt mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. In conclusion, the down-regulation of sflt, which functions as a soluble antagonist, during the luteal phase may act to sensitize the maternal endothelial receptors to angiogenetic stimuli secreted by the implanting embryo.
angiogenesis/cytokines/gene expression/growth factors/implantation
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Introduction |
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Infertility and pregnancy wastage affect one of every nine couples in Western Europe and in the US, and the molecular events of embryonic attachment to the endometrial epithelium and subsequent invasion and nidation into the stroma are not yet fully understood. Gaining knowledge about the factors involved in preimplantation embryo development and embryo–maternal cross-talk which result in the complex maturation of the embryo and eutopic implantation is crucial for reproductive medicine. The preimplantation embryo produces several factors during its development to signal its presence to the maternal organism (Chard, 1995
; Simón et al., 1995; Tazuke and Giudice, 1996; Huang et al., 1997a; Liu et al., 1997; Krüssel et al., 1998a,b,c). Embryonic implantation in both humans and mice is characterized by the following four phases: (i) preimplantation embryo development; (ii) apposition and attachment of the embryo to the maternal endometrium; (iii) trophoblast invasion into the endometrium; and (iv) nidation and placentation.
After invading the maternal endometrium, embryonic development is characterized by a dramatic growth of blood vessels coincident with decidualization, development of vascular membranes, and placenta formation (Ramsey and Donner, 1980; Moore, 1988). These active processes involve both angiogenesis, the growth of blood vessels by sprouting from a pre-existing endothelium (Klagsbrun and D'Amore, 1991; Folkman and Shing, 1992), and vasculogenesis, the in-situ formation of primordial vessels from haemangioblasts (Coffin and Poole, 1988; Schwartz et al., 1990). The vascular endothelial growth factor (VEGF) system is composed out of one agonist, VEGF, two transmembraneous receptors, the kinase insert domain-containing receptor (KDR or Flk-1) and the fms-like tyrosine kinase (Flt1), as well as one soluble receptor (sflt) that acts as an antagonist to VEGF (for review see Ferrara and Davis-Smyth, 1997). The agonist, VEGF, is a dimeric heparin-binding glycoprotein that has been purified as a vascular permeability factor from various tumour cell lines (Connolly et al., 1989; Keck et al., 1989) and that has been shown to increase the proliferative ability of vascular endothelial cells in vitro (Ferrara and Henzel, 1989). By alternative splicing of the mRNA, four different isoforms can be generated: the human VEGFs have been characterized as containing 121, 165, 189 and 206 amino acids (VEGF121, VEGF165, VEGF189, and VEGF206). Interestingly, all isoforms contain the exons 1–5 and 8 and differ only by various combinations of either no additional exon (VEGF121), or addition of exon 7 (VEGF165), exon 6 and exon 7 (VEGF189) or exon 6, exon 6' and exon 7 (VEGF206) (Houck et al., 1991; Shibuya, 1995; Neufeld et al., 1996). Biologically, VEGF121 and VEGF165 are secreted forms, whereas VEGF189 and VEGF206 appear to be mostly insoluble.
There are two known high affinity VEGF receptors, the kinase insert domain containing receptor (KDR or FLK-1) (Terman et al., 1991; Millauer et al., 1993) and the fms-like tyrosine kinase (Flt1) (Shibuya et al., 1990). Both receptors, KDR and Flt1, contain a single membrane spanning domain, seven extracellular immunglobulin-like domains and an intracellular kinase-insert domain, they share 33% identity in the amino acid sequence (Terman and Dougher-Vermazen, 1996). Binding of VEGF to either of the receptors induces autophosphorylation and signal transduction but initiation of a biological response appears to be facilitated through the binding of VEGF to KDR rather than to Flt1 (Waltenberger et al., 1994).
There are data from knock-out mice lacking specific components of the VEGF-system: VEGF+/– (Carmeliet et al., 1996, Ferrara et al., 1996), Flt1–/– (Fong et al., 1995), as well as KDR–/– (Shalaby et al., 1995), knock-out mice do not produce viable offspring. Embryos with functional inactivation of one VEGF allele (VEGF+/–) show several malformations in the vascular system resulting in lethality on days 11 and 12 of pregnancy, thus suggesting a dose-dependent regulation of fetal vascular development by VEGF. KDR is expressed on the surface of haemangioblasts and KDR–/– embryos fail to produce mature haematopoietic cells as well as endothelial cells. Therefore KDR seems to be involved in the differentiation of both cell lines derived from haemangioblasts. Flt1–/– embryos do produce endothelial cells, but these cells fail to develop into normal blood vessels. The specific roles of KDR and Flt1 in vascular development and function are, however, not totally clear.
There is also a soluble form of the Flt1 receptor called sflt (Kendall and Thomas, 1993) generated by alternative splicing of Flt1 mRNA (Figure 1a), which encodes a protein similar to the Flt1 protein but without the transmembraneous region and the intracellular kinase-insert domain. This soluble receptor acts as a specific high-affinity antagonist of VEGF function by competitively binding VEGF and, therefore, preventing the agonist–receptor interaction and the induction of a biological response.
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Transmembraneous VEGF receptors are expressed mostly on endothelial cells (Millauer et al., 1993
; Fong et al., 1995; Shalaby et al., 1995) and haemangioblasts (Jakeman et al., 1993), but Flt-1 could as well be detected in human trophoblast and choriocarcinoma cells (Charnock-Jones et al., 1994; Clark et al., 1996). The soluble receptor has initially been detected in endothelial cells (Kendall and Thomas, 1993). sflt mRNA and protein has only recently been detected in villous trophoblast cells of the human placenta and the protein could also be detected within the circulation of pregnant women, suggesting a role in blocking angiogenesis locally in the placenta and probably in the pathogenesis of pre-eclampsia (Clark et al., 1998). VEGF and its receptors have also been identified in several reproductive tissues, including corpus luteum, ovarian follicles, endometrial vessels and embryonic implantation sites in mice (Shweiki et al., 1993) as well as in giant trophoblast cells and early yolk sac (Jakeman et al., 1993) and in the human placenta (Houck et al., 1991), Fallopian tube and ovary (Gordon et al., 1996). Recently, VEGF was detected at both the mRNA and protein levels in the human endometrium throughout the menstrual cycle with a maximal expression in secretory endometrium during the luteal phase; the protein was localized in glandular epithelial cells (Shifren et al., 1996). This study, however, did not discriminate between the isoforms of VEGF. To gain information about the regulation and working mechanism of the VEGF system it is also necessary to examine not only the expression of the agonist, VEGF, but also of the receptors and the antagonist. The aim of our study, therefore, was to detect the relative mRNA levels of the VEGF receptors Flt1 and KDR, as well as the antagonist in endometrial biopsies obtained from women at various stages of the menstrual cycle. Additionally, to analyse the distribution of mRNA expression within the endometrium, we separated a number of endometrial biopsies into stromal and epithelial compartments as described before (Huang et al., 1998). These samples were examined separately for their Flt1, KDR, and sflt mRNA expression. |
Materials and methods |
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Patients
Patients who underwent hysteroscopy for non-malignant reasons were asked to participate in this study. Each participating patient signed an informed consent approved by the human investigations committee of the Stanford University Medical Center. Patients who decided to participate underwent endometrial biopsy using a Novak curette (Novak Inc, Palo Alto, CA, USA) before the hysteroscopy. One part of the tissue was fixed in 4% paraformaldehyde; the rest was washed in 0.9% sodium chloride solution in order to remove contaminating blood and was directly processed for RNA extraction. Menstrual phase was determined by the patient's history and dating was verified by histological examination of the endometrium according to the criteria of Noyes et al. (1950). Human endometrium was specified and samples were divided into six groups: early proliferative (n = 3), mid-proliferative (n = 4), late proliferative (n = 3), early secretory (n = 2), mid-secretory (n = 4), and late secretory (n = 4). RNA was extracted from the biopsy specimen of total endometrium.
Endometrial samples from four additional patients were separated into epithelial and stromal cells as described before (Huang et al., 1998; Raga et al., 1998) and the RNA was extracted from the epithelial and stromal compartment separately. It was shown previously that endometrial stromal cells separated by this method contained <0.1% epithelial or endothelial cells (Irwin et al., 1989; Simón et al., 1993; Huang et al., 1998; Raga et al., 1998).
RNA extraction
The extraction of RNA from tissue samples was carried out as described previously (Chomczynski and Sachi, 1987) with the RNA–STAT-60 reagent (Tel-Test `B' Inc, Friedenswood, TX, USA). Briefly, tissue samples were washed three times in phosphate-buffered saline (PBS; GibCo BRL, Grand Island, NY, USA) to remove blood contamination. 100 mg tissue was homogenized in 1 ml of RNA–STAT-60 reagent. Total RNA was separated from DNA and proteins by adding chloroform and precipitated using isopropanol. The precipitate was washed twice in 75% ethanol, air-dried and re-diluted in diethylpyrocarbonate-(DEPC) treated dH2O. The amount and the purity of the extracted RNA was quantified by spectrophotometry in a GeneQuant RNA/DNA calculator (Pharmacia Biotech Ltd, Cambridge, UK).
Primers for reverse transcription–polymerase chain reaction (RT–PCR)
The cDNA sequences for ß-actin (Ponte et al., 1984), Flt1 (Shibuya et al., 1990), KDR (Terman et al., 1991), and sflt (Kendall and Thomas, 1993) were obtained from the GenBank Database of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (NIH, Internet address: http://www2.ncbi.nlm.nih. gov/cgi-bin/genbank). One set of primer-sequences was selected with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN, USA) and synthesized by the `protein, amino acid and nucleic acid facility', Stanford University Medical Center, Stanford, CA, USA. To ensure that the amplified product resulted from cDNA rather than contaminating genomic DNA, primers were designed to cross intron/exon boundaries. The human ß-actin primers that were used to amplify an internal standard were obtained from Clontech Laboratories Inc (Palo Alto, CA, USA). The primer-sequences, locations on the cDNA and the sizes of the amplified fragments are listed in Table I.
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Reverse transcription
For each mRNA to be detected, 20 µl RT-mastermix were prepared (4 µl 25 mM MgCl2 Solution, 2 µl 10x PCR-Buffer, 2.5 µl DEPC-treated H2O [dist.], 2 µl dATP, 2 µl dCTP, 2 µl dGTP, 2 µl dTTP, 1 µl Oligo d(T)16, 1 µl RNase Inhibitor, 0.5 µl MuLV reverse transcriptase [all Perkin-Elmer, Foster City, CA], and 1 µg RNA diluted in 1µl DEPC-treated H2O) and filled into a 0.5 ml thin walled PCR-tube (Applied Scientific, South San Francisco, CA, USA). RT-mastermix in PCR tubes was covered with 50 µl of light white mineral oil (Sigma, St Louis, MO, USA) and kept on ice until the RT.
The RT reaction was carried out in a DNA Thermal Cycler 480 by using a program with the following parameters: 42°C for 15 min; 99°C for 5 min; 4°C. After the reaction was complete, samples were stored at –20°C until the PCR.
Construction of the competitive cDNA fragments
Competitive cDNA fragments were designed and constructed utilizing a method described previously (Krüssel et al., 1998b). A `floating' primer was designed for each cDNA to be modified (sflt, Flt1, KDR) from a sequence complementary to the cDNA between the 3' and 5' primer binding sites followed by the reverse complementary 3'-binding site (Figure 2a). After 30 cycles of PCR with an RT from a luteal phase endometrial biopsy (45 s at 95°C, 45 s at 56°C, 60 s at 72°C) with the regular 5'-primer and the 3'-`floating'-primer, the PCR product was purified from an agarose gel with an extraction kit (Boehringer Mannheim, Mannheim, Germany) and quantified by spectrophotometry (GeneQuant, Pharmacia, Cambridge, UK). The resulting cDNA fragments had a deletion between the 3' and 5' primer binding sites (Figures 2b and 2c) compared to the cDNA to be detected (target-cDNA). The identities of all PCR products were confirmed by sequencing (data not shown). Sequences of the `floating' primers and sizes of the competitive cDNA fragments resulting from the PCRs described above are listed in Table I
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Competitive polymerase chain reaction
RT products were mixed with 80 µl PCR mastermix containing 4 µl 25 mM MgCl2 Solution, 8 µl 10x PCR buffer, 0.5 µl Polymerase GoldTM (all Perkin-Elmer, Foster City, CA, USA), 60.5 µl DEPC-treated H2O, and 2 µl 3' + 5' primer-mix (5 µM each) for either Flt1, KDR, sflt or ß-actin. Each reaction mix contained additional competitive cDNA for either Flt1, KDR or sflt diluted in DEPC treated H2O to a volume of 5 µl. The amount of competitive cDNA for each PCR is described in Table II
. The reaction-mix was covered with 50 µl light white mineral oil and heated to 99°C for 9 min to denature all proteins and to activate the Polymerase Gold. After completion of n cycles (Table II) of 94°C for 45 s, 56°C for 45 s and 72°C for 60 s, followed by a final extension step at 72°C for 5 min, reactions were cooled down to 4°C. PCR products were stored at –20°C until 2% agarose gel electrophoresis was carried out in the presence of ethidium bromide. After completion of electrophoresis, the agarose gel was analysed on the GelDoc 1000 system (Bio-Rad Laboratories, Hercules, CA, USA). DNA size calculation and densitometry was carried out by using the Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA, USA).
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Data analysis
Statistical analysis was performed by analysis of variance with post-hoc t-test. The analysis was carried out using the Statistical Package for Social Science (SPSS Inc, Chicago, IL, USA); P < 0.05 was considered to be statistically significant.
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Results |
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We detected the mRNA for ß-actin (positive control), both transmembraneous receptors, Flt1 and KDR, as well as the mRNA of the soluble receptor, sflt, in every endometrial biopsy throughout the menstrual cycle (Figure 3
). These mRNAs were also detected within the endometrial biopsies separated into endometrial epithelial cells and endometrial stromal cells.
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Flt1, KDR and sflt mRNA throughout the menstrual cycle
Both transmembraneous receptors, KDR and Flt1, are expressed at an almost constant level throughout the entire menstrual cycle of regular cycling women (Figure 4
). Flt1 is expressed at the lowest level in the early proliferative phase shortly after the onset of the menstrual bleeding and the expression rises to an almost constant level throughout the remaining cycle stabilizing at 2–3-fold higher than during the early proliferative phase. KDR is expressed at an almost constant level throughout the entire menstrual cycle, there are no significant changes in the expression levels.
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), Flt1 (
), and KDR (
) as mean ± SD in endometrial biopsies of regular cycling women throughout the menstrual cycle. The sflt expression in the mid-proliferative phase is significantly different from that in the early proliferative, early secretory, mid-secretory, and late secretory phases (P < 0.05, analysis of variance with post-hoc t-test)The soluble receptor or receptor antagonist sflt shows a significant pattern within its mRNA expression throughout the cycle. The sflt mRNA is expressed in a 1.5–3-fold higher level during the mid-proliferative phase when compared with the early proliferative phase and the entire secretory phase (P < 0.05, analysis of variance with post-hoc t-test).
Flt1, KDR and sflt mRNA in endometrial epithelial and stromal cells
Flt1, KDR and sflt mRNA were detected in isolated endometrial epithelial and endometrial stromal cell fractions from each of the four patients examined (Figure 5). We were not able to draw any quantitative conclusions from the results due to the fact that endometrial specimens from only four patients (two in the follicular, and two in the luteal phase) were included in this analysis.
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Discussion |
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There is increasing evidence of a role for VEGF during the normal menstrual cycle in humans and primates. Shifren et al. have detected VEGF on mRNA and on protein values in human endometrium throughout the menstrual cycle, with increasing levels during proliferative phase and a maximal expression in the secretory phase (Shifren et al., 1996
). Torry et al. found comparable results and could furthermore differentiate between the VEGF isoforms: the secretory isoforms VEGF121 and VEGF165 were the predominantly detectable mRNAs (Torrey et al., 1996). The mRNA for VEGF189 could only be detected in a very low quantity and the mRNA for VEGF206 remained undetectable. VEGF has been detected immunohistochemically in the cytoplasm of glandular epithelial cells and in the endometrial stroma associated with the walls of small blood vessels in primates (Greb et al., 1997). They also demonstrated that VEGF protein and mRNA levels are influenced by the steroidal milieu: staining for VEGF protein and expression of VEGF189 mRNA in primate endometrium was highly depending on the concentration of progesterone and was greatly reduced in anovulatory cycles or in animals treated with the progesterone antagonist mifeprestone (RU486). Other studies have demonstrated that cyclic expression of VEGF mRNA is related to the proliferation of blood vessels both in ovaries from various species (Phillips et al., 1990; Ravindranath et al., 1992) and in the rat uterus (Cullinan-Bove and Koos, 1993). These studies suggest a role for VEGF as a mediator of the cyclic growth of blood vessels in the female reproductive tract.
There is also evidence for an important role of VEGF during early embryonic development and implantation: within a few days after implantation, VEGF mRNA can be detected in giant trophoblast cells in the rat (Jakeman et al., 1993) and in the mouse (Breier et al., 1992). This suggests that VEGF may be intimately involved in the induction of vascular growth in the decidua, placenta, and vascular membranes. There is, however, only little knowledge about the very early period of implantation, for example, whether or not the blastocyst produces VEGF. Also, little is known about the expression of transmembraneous and soluble VEGF receptors.
This study is the first to demonstrate the presence of both transmembraneous receptors, Flt1 and KDR, as well as the soluble receptor sflt in cycling human endometrium. Both transmembraneous receptors are transcribed at almost constant levels in the endometrium throughout the menstrual cycle and they appear to be expressed in both epithelial and stromal cells. Therefore, it is likely that embryonic VEGF could act directly by binding to endometrial stromal cells or to endometrial epithelial cells after the invasion of the trophoblast and induce neoangiogenesis at the implantation site.
The soluble receptor is transcribed at significantly lower levels (approximately three times lower) during the mid-secretory phase than during the mid- and late proliferative phases of the menstrual cycle. The biological activity of VEGF depends not only on the synthesis of mature protein but also on the availability of these proteins to the transmembraneous receptors KDR and Flt1. It is clear that sflt acts as a receptor antagonist by binding to VEGF and, hence, competitively prevents the binding of VEGF to the transmembraneous receptors. Therefore, high levels of sflt reduce the biological response to a given VEGF stimulus. We postulate that the down-regulated sflt expression shown during the mid-secretory phase (the time of embryonic implantation) may act to sensitize the endometrium to angiogenetic stimuli secreted by the implanting embryo. Since our experiments provide only information on the transcriptional level, we cannot conclude how much of the mRNA is translated into protein or whether at all functional proteins are expressed. Further investigations are underway to examine this particular point.
In summary, we demonstrated cyclic changes in the expression of the VEGF antagonist sflt in human endometrium. These changes may not only influence the endometrial angiogenesis during the normal menstrual cycle but may also affect the endometrial receptiveness to an implanting embryo.
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Acknowledgments |
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The authors wish to thank Professor Dr Jürgen Hucke and Dr Filip De Bruyne from the Heinrich-Heine-University Department of OB/GYN for their support. This study was supported in part by grant-# HD31575 from the National Institutes of Health to MLP. JSK was a postdoctoral research fellow supported by the `Deutsche Forschungsgemeinschaft', grant-# Kr 1659/1–1.
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Notes |
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3 To whom correspondence should be addressed
* This work was presented in part at the 45th meeting of the Society for Gynecologic Investigation, Atlanta, GA, USA
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References |
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Submitted on October 1, 1998; accepted on February 12, 1999.
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