Mol. Hum. Reprod. Advance Access originally published online on April 28, 2006
Molecular Human Reproduction 2006 12(6):367-375; doi:10.1093/molehr/gal027
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Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium
1Research Institute for Growth and Development (GROW), 2Department of Pathology and 3Department of Obstetrics and Gynaecology, University Hospital Maastricht and University Maastricht, Maastricht, The Netherlands
4 To whom correspondence should be addressed at: Philips Research, High Tech Campus 4 WAG 11, 5656 AA Eindhoven, The Netherlands. E-mail: chamindie.punyadeera{at}philips.com
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Abstract |
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Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6–8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1
and CA-IX in the menstrual and early proliferative phases. HIF1 was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase. Key words: angiogenesis/ECM/gene-expression profiling/human endometrium/hypoxia/menstrual cycle/neuropilin/estrogen/VEGF and VEGF receptor
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Introduction |
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Angiogenesis, or neovessel formation, is mandatory to support endometrial growth after menstruation and to provide a vascularized, receptive endometrium for implantation and placentation. The mechanisms that mediate vascular growth in the human endometrium and the time frame in which the various processes occur are still unclear. In contrast to pre-ovulatory follicles where vascular sprouts do demonstrate proliferative activity (Goodger and Rogers, 1995
), proliferative activity in vessels in the endometrium is confined to the existing vessels (Rogers and Gargett, 1998). Moreover, the cyclic changes in the growth rate of the endometrium during the menstrual cycle are not accompanied by cyclic patterns of endothelial cell proliferation (Goodger and Rogers, 1994; Rogers and Gargett, 1998). Recent studies suggest a role for vessel elongation during the mid-late proliferative phase of the menstrual cycle (Gambino et al., 2002).
One of the key angiogenesis inducers in the human endometrium is vascular endothelial growth factor-A (VEGF-A). This was demonstrated in xenotransplantation models where blocking VEGF-A action with antibodies dramatically reduced ectopic survival of endometrium tissue because of reduced revascularization (Nap et al., 2004, 2005). Despite the clear association with angiogenic activity, VEGF-A expression could not be correlated to any vascular events in the human endometrium. These may be determined by the cell-specific expression of the receptors for VEGF-A, VEGF-R2 and the co-receptor NRP-1 (Moller et al., 2001; Sugino et al., 2002; Germeyer et al., 2005).
VEGF-A is expressed in all cell types in the human endometrium and in all phases of the menstrual phase (Graubert et al., 2001). The expression of VEGF is elevated during menstruation, most likely as a result of hypoxia (Sharkey et al., 2000; Graubert et al., 2001; Lockwood et al., 2002). VEGF-A is also increased in the mid-luteal phase, which is most likely because of concerted actions of estradiol (E2) and progesterone (Lockwood et al., 2002; Sugino et al., 2002). Various investigators have shown that VEGF-A production is estrogen dependent. In vitro studies in cultures of human endometrial cells have demonstrated estrogen stimulation of VEGF-A expression (Charnock-Jones et al., 1993; Shifren et al., 1996; Bausero et al., 1998; Mueller et al., 2000; Koos et al., 2005; Herve et al., 2006). Ovariectomy in baboons and rhesus macaques resulted in very thin, atrophic endometrium and very low VEGF-A levels (Nayak and Brenner, 2002; Niklaus et al., 2003). Supplementation with E2 restored the endometrial thickness and histology to normal as well as the VEGF-A levels. These studies clearly indicate that VEGF-A production in human endometrium is stimulated by estrogen.
Because of ethical restraints, however, similar invasive studies cannot be performed in human subjects. We therefore employ explant cultures of human endometrium to obtain more insight into the steroid regulation of the expression of all VEGF ligands and receptors during menstruation and post-menstrual repair during the proliferative phase of the menstrual cycle. This model system was shown to retain physiologically relevant responses to estrogen, such as the induction of proliferation and expression of the progesterone receptor and cyclooxygenase-2 (Punyadeera et al., 2004, 2005).
Our results confirm that VEGF-A mRNA is the most abundantly expressed VEGF isoform in the pre-ovulatory human endometrium and that NRP-1 is the most abundantly expressed receptor. In addition, we found that the post-menstrual repair and subsequent growth of the human endometrium are accompanied by distinct concerted changes in the mRNA expression of VEGF receptors and ligands. Our data suggest that the disparity between VEGF-A mRNA and protein expression levels is due to changes in VEGF protein release and trapping of VEGF-A in the extracellular matrix (ECM). Finally, we show that VEGF-A production by cultured endometrial tissue is high during menstruation and decreases during the second half of the proliferative phase and that VEGF-A levels during menstruation and the early proliferative phase are decreased by estrogen. The observed temporal–spatial and cell-specific expression of VEGF-R2 and NRP-1 most likely determines which cell compartments are affected by VEGF-A.
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Materials and methods |
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Human endometrium tissue
Seventy-six endometrium biopsies were obtained during surgery for benign indications from women during menstruation and the proliferative phase of the menstrual cycle [cycle days (CDs) 1–15]. Samples were collected from hysterectomy specimens or during laparoscopy by a Pipelle catheter (Unimar, Prodimed, Neuilly-Enthelle, France) under sterile conditions. The hysterectomy specimens were examined macroscopically by a pathologist, and uteri with abnormalities such as polyps were excluded. Women were 20–45 years old, had no clinical symptoms of endometriosis, had regular ovulatory cycles and were not on hormonal treatment, oral contraception or any other medications. Endometrium tissues were dated according to the last menstrual period and histological evaluation by a pathologist (Noyes et al., 1975
). All women gave their written informed consent, according to a protocol approved by the Medical Ethical Committee of the academic hospital Maastricht. Owing to the limitation in the number of tissues collected, we were not able to use all biopsies for all experiments. The number of biopsies used in each experiment is documented in the following sections.
Explant cultures of human endometrium tissue
Human endometrium tissue (CDs 1–15; n = 47) was cut into pieces of 2–3 mm3. These endometrium tissue fragments were placed in Millicell-CM culture inserts (pore size of 0.4 µm, 30 mm diameter, Millipore, Molsheim, France) in 6-well plates containing Phenol Red-free Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium (1.2 ml) (Life Technologies, Grand Island, NY, USA) (Punyadeera et al., 2004). To standardize the explant culture procedure and to avoid well–well variations, equal number of endometrium tissue explants (n = 24) were placed on each well. The medium was supplemented with L-glutamine (1%), penicillin and streptomycin (1%, P/S), and this was used in all stages of explant preparations. Cultures were maintained for 20–24 h. It has been shown that under these culture conditions, collagenase activity remains low and that the tissue viability is not affected up to 24 h of culture (Cornet et al., 2002). To control for anaerobic conditions, the culture supernatant was assayed for lactate dehydrogenase. The treatments included (i) control (0.1% ethanol) or (ii) E2 (17ß-E2, 1 nM in 0.1% ethanol). The 17ß-E2 was a gift from Organon N.V. (Oss, The Netherlands).
RNA isolation and cDNA synthesis
The samples (n = 25) for RNA isolation and PCR analysis included CDs 2–5 (n = 7), CDs 6–8 (n = 4), CDs 9–10 (n = 6), CDs 11–12 (n = 3) and CDs 13–15 (n = 5). Total RNA was extracted from endometrium tissues using the SV total RNA isolation kit (Promega, Leiden, The Netherlands) according to the supplier’s protocol with slight modifications. The concentration of DNase-1 was doubled, and the incubation time was extended by 15 min to completely remove genomic DNA. Total RNA was eluted from the column in 50-µl RNase-free water. The quality of the RNA samples was determined by agarose gel electrophoresis and ethidium bromide staining.
Two micrograms of total RNA was incubated with random hexamers (1 µg, Promega) at 60°C for 10 min. The samples were chilled on ice for 5 min and supplemented with 5x first-strand buffer (4 µl, Promega), 10 mM dNTP mix (1 µl, Amersham Pharmacia, Uppsala, Sweden), RNAsin (10 U, Promega) and M-MLV reverse transcriptase (200 U, Promega) in a total volume of 20 µl. The samples were incubated at 42°C for 1 h after which the enzyme was inactivated by heating at 95°C for 5 min. MilliQ was added to a final volume of 100 µl, and the cDNA was stored at –20°C.
RT–PCR
All PCR reactions were performed using an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA). Calculations of absolute copy numbers were performed as previously described (Thijssen et al., 2004). Primers targeted against cyclophilinA, PlGF, VEGF-A/B/C/D, VEGF-R1/R2/R3 and NRP-1/2, have been described earlier (Thijssen et al., 2004).
Immunohistochemistry
Twenty-three endometrium samples were used for the semiquantitative assessment of VEGF-A, VEGF-R2, HIF1 and CA-IX staining. The endometrium samples were formalin fixed and embedded in paraffin. They were collected on CDs 2–5 (n = 7), CDs 6–8 (n = 4), CDs 9–10 (n = 5), CDs 11–12 (n = 3) and CDs 13–15 (n = 4). In addition, NRP-1 staining was performed on a subset to assess their localization. Serial (5 µm) tissue sections were cut, dewaxed in xylene and rehydrated through descending grades of ethanol. Endogenous peroxidase activity was quenched by immersion in 0.3% H2O2 in methanol for 20 min. After washing the slides in phosphate-buffered saline (PBS), a tissue antigen retrieval step was carried out for the VEGF-R2, NRP-1 and HIF1 antibodies by incubating the sections in Tris–EDTA buffer (pH 9.0) at 96°C for 20 min. In all staining procedures, non-specific binding was blocked with 5% bovine serum albumin (BSA) in PBS/Tween (0.02%) for 10 min at room temperature, and endogenous peroxidase activity was quenched by immersion in 0.3% H2O2 in methanol for 20 min. Primary antibodies were applied in 0.1% BSA in PBS/Tween (VEGF-A, clone A-20, 1:400; VEGF-R2, clone A-3, 1:600; NRP-1, clone C-19, 1:500, all from Santa Cruz Biotech (Santa Cruz, CA, USA); HIF1, clone 54, 1:120, BD Biosciences, Alphen aan den Rijn, The Netherlands; CA-IX, clone M75, 1:50, kindly provided by Dr B. Wouters). Antibody binding was visualized with the ChemMateTM DAKO EnVisionTM Detection Kit (DakoCytomation, Carpinteria, CA, USA) and diaminobenzidine. The NRP-1 antibody was visualized with biotinylated rabbit anti-goat IgG (1:400, DakoCytomation) and the avidin–biotin–HRP complex for 30 min (StrepABC kit, DakoCytomation). Antibody binding was visualized with diaminobenzidine. For the evaluation of tissue morphology, tissue sections were counterstained with diluted haematoxylin (1:16).
To determine whether the anti-human HIF1 antibody could be applied on formalin-fixed paraffin sections, endometrial carcinoma cells were exposed to normoxic and hypoxic conditions for 8 h, cells were then scraped, embedded in agar and processed routinely.
Paraffin-embedded tissue sections of term placenta and endometrium were used as positive controls. In all staining procedures, negative controls were included which were incubated with IgGs of the same isotype and the same concentration as the primary antibody.
Analysis of immunohistochemical staining
Endometrium tissue sections were examined under a light microscope, and a semiquantitative staining index (SI) was assessed for VEGF-A, VEGF-R2 and NRP-1 as previously described (Pijnenborg et al., 2005). A score was given for the percentage of cells (0 = 0%; 1 = <10%; 2 = 10–50% and 3 = >50%) and the intensity (0 = absent; 1 = weak; 2 = moderate and 3 = strong). The SI (min = 0, max = 9) is the product of the percentage and the intensity.
VEGF ELISA and LDH assay
The amount of VEGF in the culture supernatant was measured with the Quantikine VEGF ELISA kit (R&D Systems, Abingdon, UK) according to the manufacturer’s protocol. Lactate dehydrogenase in the supernatant was measured on a Synchronix 20 pro (Beckman Coulter, Fullerton, CA, USA) using the LD-P assay kit (Beckman Coulter) according to the supplier’s instructions.
Western blot analysis
For western blot analysis, tissues were homogenized in liquid nitrogen and lysed in RIPA buffer (50 mM Tris–HCl buffer pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% NP-40; 0.25% sodium deoxycholate) with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). After centrifugation to remove cell debris, protein was quantified using the 2D Plus kit (Biorad, Veenendaal, The Netherlands). The samples were mixed with 4x Laemmli sample buffer and heated at 95°C for 5 min and then electrophoresed through standard sodium dodecyl sulphate (SDS)–polyacrylamide gels and transferred to nitrocellulose membranes (PROTRAN, Schleicher&Schuell, Den Bosch, The Netherlands). Membranes were blocked with Tris-buffered saline (TBS) containing 5% non-fat dried milk and 0.05% Tween 20 and probed with anti-VEGF (1:1000, monoclonal antibody AB-6, Calbiochem, La Jolla, CA, USA), anti-NRP-1 (1:1000) and anti-VEGF-R2 (1:1000, goat polyclonal antibody C-20, Santa Cruz Biotech). After overnight incubation at 4°C, blots were probed with peroxidase-conjugated secondary antibodies (1:10 000, Amersham Pharmacia Biotech, Piskataway, NJ, USA), and bound secondary antibody was detected with a chemiluminescent peroxidase substrate (SuperSignal® West Pico chemiluminescence substrate, PIERCE, Rockford, IL, USA). The same blot was stripped with 62.5 mM Tris base, 2% SDS and 0.7% ß-mercaptoethanol for 1 h at room temperature and reprobed with an antibody against ß-actin (1:5000, Sigma Aldrich, Zwijndrecht, The Netherland). Binding was visualized with peroxidase-conjugated sheep anti-mouse antibodies (1:10 000, Amersham Pharmacia Biotech) and the SuperSignal® West Pico substrate.
Statistical analysis
Statistical tests were carried out using the SPSS 10 (SPSS, Chicago, IL, USA) statistical analysis package. RT–PCR data are given as mean values ± SEM. When evaluating the collective relative changes for the ligands and the receptors, we defined five phases: (i) CDs 1–5, (ii) CDs 6–8, (iii) CDs 9–10, (iv) CDs 11–12 and (v) CDs 13–15 (Figure 1). The Mann–Whitney rank sum test was used to analyse the differences between these phases. The Mann–Whitney U-test was used to analyse the differences in immunohistochemical staining indices. Correlations between CD and expression levels were calculated using the Spearman rank correlation test. The independent Student’s t-test was used to compare the mean VEGF output by the explants between phases. The Mann–Whitney rank sum test was used to analyse the effect of E2 on VEGF output. All values are two sided, and P-values <0.05 were considered significant.
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Results |
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Messenger RNA levels of VEGF receptors and their ligands in pre-ovulatory human endometrium
The main goal of this study was to get more insight into VEGF signalling during the proliferative phase of the menstrual cycle. Therefore, the mRNA expression levels of all known ligands and receptors involved in orchestrating VEGF signalling were analysed using qRT–PCR. Gene expression was normalized to cyclophilinA because there were no significant differences in the mRNA expression of this gene throughout the menstrual cycle, and there was no correlation between cyclophilinA expression level and CD (correlation coefficient 0.119, P = 0.570).
Of all the ligands that were analysed, VEGF-A was found to be the most abundant transcript present in the peri- and post-menstrual human endometrium (Figure 1A). VEGF-B expression was 10-fold lower than VEGF-A, whereas VEGF-C and VEGF-D levels were 15- and 20-fold lower, respectively. Of the analysed receptor transcripts, NRP-1 was found to be the most abundantly expressed co-receptor, and its expression level was similar to VEGF-A, whereas NRP-2 expression was four-fold lower. Of the VEGF receptors, VEGF-R2 had the highest mRNA copy number, which was still 10-fold lower than NRP-1.
As indicated in the Materials and methods, we defined five phases based on the expression profiles: (i) CDs 2–5, (ii) CDs 6–8, (iii) CDs 9–10, (iv) CDs 11–12 and (v) CDs 13–15 (Figure 1B and C) The first two phases (CDs 2–8) were characterized by high expression of VEGF ligands and receptors. In the third phase (CDs 9–10), a dramatic reduction was observed for all these factors. In phase 4 (CDs 11–12), the transcript levels of all factors, except VEGF-D, were increased again. In phase 5 (CDs 13–15), the expression of most factors dropped again to similar levels as in phase 3. A complete overview of all significant changes between the five phases is summarized in Table I. From these data, it is evident that the most prominent changes in mRNA expression occur between phases 2 and 3, 3 and 4 and 4 and 5.
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VEGF-A, VEGF-R2 and NRP-1 protein expression in pre-ovulatory human endometrium
The expression of three of the abundantly expressed factors in the human endometrium (VEGF-A, NRP-1 and VEGF-R2) was further studied at the protein level. VEGF-A protein was observed in all cell types, glandular epithelial cells, luminal epithelial cells, stromal fibroblasts and vascular endothelial cells (Figure 2A). The VEGF-R2 was also expressed in all cell types: the vascular endothelium, stroma and the glandular and luminal epithelium (Figure 2B). Positive staining for NRP-1 was observed in endothelial cells and the surrounding stroma. Later in the proliferative phase, NRP-1 staining was also observed in the glandular epithelium (Figure 2C).
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A semiquantitative SI was used to assess the area and the intensity of staining in glandular epithelium, stroma and endothelium separately. VEGF staining was observed in all cell types; however, no significant changes were observed in VEGF staining in glandular epithelium, stroma and endothelial cells (Figure 3A). VEGF-R2 staining was significantly lower in endothelial cells of endometrium from CD 6 to CD 8 compared to those from CD 2 to CD 5 (Figure 3B). No changes were detected in glands or stroma. Surprisingly, the significantly reduced expression levels of VEGF-A and VEGF-R2 mRNA on CDs 9–10 were not reflected at the protein level. We then compared the VEGF-A mRNA expression, protein expression and protein localization in a small subset of patients from which sufficient material was obtained. Again, a discrepancy was observed between mRNA (Figure 4A) and protein (Figure 4B) expression levels. Western blot analysis indicated that in the menstrual and early proliferative phase, VEGF-A protein levels are high, with VEGF165 and VEGF121 being the major isoforms, whereas in later phases, the levels rapidly decrease and are limited to the larger isoforms (Figure 4B). In addition, immunostaining in the tissue still revealed protein in the late proliferative phase (Figure 4C), whereas the output of VEGF-A by cultured explants of human endometrium significantly decreased during the proliferative phase (Figure 4D). On the basis of these observations, we hypothesized that part of the VEGF-A must be sequestered in the tissue, most likely by binding to the ECM through the heparin proteoglycan-binding domains. To show this, we solubilized the pellet generated after protein extraction directly in the Laemmli buffer. This pellet contains the insoluble ECM proteins and part of the membrane fraction. As expected, this fraction also contained significant amounts of VEGF-A (Figure 4E).
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Ct. Beta-actin was used to confirm equal protein loading in the western blot analysis. IHC was performed on paraffin sections. (D) VEGF concentrations in culture supernatants of human endometrium explants. In untreated cultures, VEGF production was significantly reduced in cycle days (CDs) 13–15 compared with CDs 1–5 (*P < 0.05). In the estradiol (E2)-treated cultures, VEGF concentrations were lower in CD 6–8 (*P < 0.05), CD 11–12 (**P < 0.01) and CD 13–15 (***P < 0.001) cultures compared with the CD 1–5 cultures. E2 treatment significantly reduced VEGF levels in the culture supernatants of CD 1–5 and CD 6–8 cultures (
P < 0.05) compared with the untreated cultures. (E) Western blot analysis using an anti-VEGF antibody on the pellet fraction of protein extracts.
Apart from the decrease in VEGF-A output in cultured explants, we also observed that treatment of these explants with E2 significantly reduced the overall release of VEGF-A (Figure 4D). When analysing the groups separately, the release of VEGF-A was significantly reduced in explants obtained from CD 1 to CD 5 and CD 6 to CD 8 but not in explants of CDs 11–12 and CDs 13–15.
When comparing the mRNA and protein expression levels of VEGF-R2 (Figure 5A–C) and NRP-1 (Figure 5E–G), we again observed a discrepancy. Changes in the expression at the mRNA level were clearly not reflected at the protein level as assessed with IHC and western blot analysis.
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Immunohistochemical staining and western blot analysis showed abundant NRP-1 expression. Similarly, IHC showed good expression of VEGF-R2 in the endometrium; however, western blot analysis showed very low levels of VEGF-R2 protein. It is then tempting to speculate that similar to VEGF-A, part of the VEGF-R2 proteins might be confined to the insoluble fraction. Indeed, this was supported by the western blot analysis of the pellet, which revealed comparable levels of VEGF-R2 (Figure 5D).
Expression of hypoxia markers HIF1 and CA-IX in pre-ovulatory human endometrium
To determine whether the expression of VEGF ligands and receptors corresponds to episodes of hypoxia, we analysed the expression of HIF1 and CA-IX by immunohistochemistry (IHC). Sections prepared from agar/paraffin-embedded ECC1 cells and stained with the anti-HIF1 antibody showed induction of HIF1 expression after exposure to hypoxia, showing antibody activity in paraffin-embedded tissues (Figure 6A). In the tissues, HIF1 was strongly expressed in the nuclei and cytoplasm of both glandular and stromal endometrium cells (Figure 6B). The SI was high in endometrium biopsies from CD 2 to CD 5 and significantly decreased (P < 0.05) on CDs 9–10 (Figure 6C). After CD 10, the expression levels of HIF1 increased again.
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CA-IX was predominantly localized in the cytoplasm of glandular epithelial cells, whereas stromal cells were negative (Figure 6D). The expression of CA-IX was highest in endometrium biopsies collected on CDs 2–5 and CDs 6–8 and decreased (P < 0.05) thereafter (Figure 6E). Endometrium collected on CDs 11–15 showed no immunopositivity for CA-IX, indicating that there is no chronic hypoxia in these tissues.
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Discussion |
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The main goal of this research was to gain more insight into the role of VEGF-A in the repair, growth and maturation of the vasculature in human endometrium after menstruation. We evaluated the expression of all known ligands and receptors involved in VEGF signalling in menstrual and proliferative phase human endometrium. Because VEGF-A and its receptors were among the most abundantly expressed genes, we investigated these proteins in more detail.
Two distinct periods with elevated mRNA expression of the VEGF receptors and their ligands were apparent: from CD 2to CD 8 and from CD 11 to CD 12. These observations are in agreement with the findings of Maas et al. (2001) who demonstrated with chorioallantoic membrane (CAM) assays that early-proliferative phase endometrium (CDs 2–7) is more angiogenic compared to endometrium tissue from the late proliferative phase (CDs 8–14). Others showed that vessel growth in the mid-late proliferative phase (CDs 8–15) is mostly due to vessel extension rather than endothelial cell proliferation (Gambino et al., 2002). Collectively, these data suggest that during and shortly after menstruation, the induction of an angiogenic response is aimed at repairing the damaged vessels in the functional layer and revascularization of the ischemic endometrium, whereas in the late proliferative phase, vessel elongation (morphogenesis) and maturation (stabilization) are promoted.
We observed VEGF-A protein in multiple cell types, including the glandular epithelium, corroborating other research findings (Charnock-Jones et al., 1993; Torry et al., 1996; Gargett et al., 1999; Moller et al., 2001; Sugino et al., 2002). Thus far, it has been postulated that VEGF-A plays an important role in angiogenesis and vascular remodelling of blood vessels. However, there are also indications that VEGF-A is involved in epithelial cell function. Recently, it was shown that VEGF-A induces tube morphogenesis of renal epithelial cells, a process which was shown to be VEGF-R2 and NRP-1 dependent (Karihaloo et al., 2005). In the same system, VEGF-A was shown to induce cell proliferation and increase cell survival in an autocrine manner (Villegas et al., 2005). This implies that in the epithelial compartment, the expression of VEGF and VEGF-R2, and later in the proliferative phase NRP-1, may have functional consequences for gland development.
Further evaluation of VEGF protein expression revealed a disparity between the mRNA and protein expression levels. For this discrepancy, there are several explanations. First, it is known that essential proteins and transcription factors are often present at levels that are not readily predicted by mRNA levels (Gygi et al., 1999). This is most likely because of substantial variations in post-translational processing and indicates that proper expression profiling requires analysis of both mRNA and protein levels. Second, VEGF is a major secretory product of the glandular epithelium which is mostly apically released into the lumen of the glands (Hornung et al., 1998; Gargett et al., 1999). This may mask major changes that occur at the mRNA level because secreted VEGF protein molecules are likely to remain undetected with IHC or western blotting. Indeed, we observed in tissue explants that the VEGF release decreased throughout the proliferative phase and did not increase again after CDs 9–10. Moreover, we found that a significant amount of VEGF-A was trapped in the non-soluble protein fraction, which could account for the immunohistochemical detection of VEGF-A protein in late-proliferative endometrium when mRNA levels are low. This could also explain the observed discrepancy in mRNA and protein expression levels of VEGF-R2 and NRP-1.
The fact that multiple isoforms can be trapped in the ECM may have implications for VEGF-A function. Ruhrberg and coworkers (2002) showed that the expression of VEGF-A isoform lacking the heparin-binding domain causes a specific decrease in capillary branch formation (Ruhrberg et al., 2002). This process is dependent on matrix metalloproteinase (MMP) processing (Lee et al., 2005). In the endometrium, all vessel branch points are generated during the first 7 days after the initiation of menstruation (Gambino et al., 2002). Vessel growth in the second week is most likely to occur by vessel elongation (Gambino et al., 2002). It has been demonstrated that before menstruation, VEGF-A levels increase in the uterine cavity (Licht et al., 2003). These observations suggest that the ECM-bound fraction constitutes an additional pool of VEGF-A molecules, next to the VEGF-A proteins induced by the hypoxic conditions during menstruation. Progesterone withdrawal due to regression of the corpus luteum causes rapid production and activation of MMPs before the onset of menstruation (Freitas et al., 1999; Goffin et al., 2003), which digest the ECM and release the VEGF-A. Thus, at the same time vessel regression is induced, vessel repair mechanisms are already being prepared.
The actions of VEGF-A are mainly mediated through both VEGF-R2 and NRP-1. Like VEGF-A, we observed VEGF-R2 immunostaining in most cell types, which confirms the findings by others (Meduri et al., 2000; Moller et al., 2001; Sugino et al., 2002). In contrast with the observation by others (Krussel et al., 1999; Sugino et al., 2002), we observed reduced mRNA levels on CDs 9–10 and reduced protein levels in endothelium of CDs 6–8. However, heterogeneous staining in the tissues and variation between individuals on the CDs complicate evaluation of immunostaining, and these results should therefore be interpreted with caution. Next to VEGF-R2, NRP-1 was second abundantly expressed receptor transcript in the human endometrium. NRP-1 protein levels were also high throughout the proliferative phase. The high expression of NRP-1 in endothelial and stromal cells indicates that these cells are probably the main targets for VEGF-A. Our findings are in concert with the recent findings of Germeyer et al., who demonstrated that NRP-1 mRNA is located predominantly in endothelium and occasionally in the stroma (Germeyer et al., 2005). Interestingly, endothelial cells of NRP-1 knockout mice were shown to lack the capacity to elongate (Gerhardt et al., 2004), whereas NRP-1 alone was shown to mediate human umbilical vein endothelial cell migration (Wang et al., 2003). NRP-1 protein levels remained high during the proliferative phase, even after VEGF-A levels were dramatically reduced. It is tempting to suggest that NRP-1 mediates the vessel elongation process during the mid- and late proliferative phases as described by Gambino et al. (2002). Recently, soluble isoforms of NRP-1 have been described, which were able to antagonize the actions of VEGF-A (Rossignol et al., 2000). However, we did not detect this 90-kDa isoform in western blotting.
Because hypoxia has been shown to markedly up-regulate the expression of VEGF in cultured epithelium and stroma cells from human endometrium (Sharkey et al., 2000), we also evaluated the protein expression of HIF1 and CA-IX. We observed the highest expression of HIF1 and CA-IX in the biopsies from CD 2 to CD 5 which rapidly decreases thereafter. The expression of CA-IX completely disappeared around CDs 9–10, but the expression of HIF1 increased again in the late-proliferative phase biopsies. Because no CA-IX was observed, it is highly unlikely that this increase can be attributed to chronic hypoxia. These observations indicate that the expression of VEGF in the late proliferative phase may not be triggered by hypoxia. It is tempting to suggest that VEGF production is mediated by E2-induced HIF1. This possibility is supported by various investigators who have shown in vivo and in vitro that estrogen can increase the expression of VEGF production and release by endometrium cells (Shifren et al., 1996; Huang et al., 1998; Mueller et al., 2000; Nayak and Brenner, 2002; Niklaus et al., 2003; Charnock-Jones et al., 2004). Recently, it was shown in the rat uterus that E2 is able to induce VEGF production via induction of HIF1 mRNA and protein production and recruitment of the HIF1 protein to the VEGF promoter (Kazi et al., 2005). Even though we observed high levels of VEGF-A mRNA in the late-proliferative phase endometria, VEGF-A protein levels were dramatically reduced. Moreover, treatment with E2 reduced VEGF-A levels in the culture supernatants of explants collected from menstrual and early-proliferative phase endometrium but not mid- and late-proliferative phase endometrium. This is probably due to the fact that mid- and late-proliferative phase endometrium had already been exposed to E2 in vivo. Collectively, these findings indicate that in human endometrium estrogens overrule the effects of HIF1 on VEGF-A production in the late proliferative phase.
In conclusion, the post-menstrual repair and subsequent growth of the human endometrium is accompanied by two phases of increased production of angiogenic factors. Enhanced expression of angiogenesis factors during the first wave is probably the result of hypoxia and promotes vascular growth, remodelling and repair, whereas elevated angiogenesis expression levels in the second wave are likely to promote vessel stabilization and maturation. VEGF-A protein levels are prominent during menstruation and the early proliferative phase and decrease in the late proliferative phase. E2 inhibited VEGF-A output by explants of human endometrium.
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Acknowledgements |
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We thank Dr Brad Wouters of the Maastricht Radiation Oncology (Maastro) Lab, GROW Research Institute, University of Maastricht for the CA-IX antibody.
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Notes |
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* These authors contributed equally to this work.
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References |
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Bausero P, Cavaille F, Meduri G, Freitas S and Perrot-Applanat M (1998) Paracrine action of vascular endothelial growth factor in the human endometrium: production and target sites, and hormonal regulation. Angiogenesis 2,167–182.[Medline]
Charnock-Jones DS, Sharkey AM, Rajput-Williams J, Burch D, Schofield JP, Fountain SA, Boocock CA and Smith SK (1993) Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol Reprod 48,1120–1128.[Abstract]
Charnock-Jones DS, Kaufmann P and Mayhew TM (2004) Aspects of human fetoplacental vasculogenesis and angiogenesis. I. Molecular regulation. Placenta 25,103–113.[CrossRef][Web of Science][Medline]
Cornet PB, Picquet C, Lemoine P, Osteen KG, Bruner-Tran KL, Tabibzadeh S, Courtoy PJ, Eeckhout Y, Marbaix E and Henriet P (2002) Regulation and function of LEFTY-A/EBAF in the human endometrium. mRNA expression during the menstrual cycle, control by progesterone, and effect on matrix metalloprotineases. J Biol Chem 277,42496–42504.
Freitas S, Meduri G, Le Nestour E, Bausero P and Perrot-Applanat M (1999) Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium. Biol Reprod 61,1070–1082.
Gambino LS, Wreford NG, Bertram JF, Dockery P, Lederman F and Rogers PA (2002) Angiogenesis occurs by vessel elongation in proliferative phase human endometrium. Hum Reprod 17,1199–1206.
Gargett CE, Lederman FL, Lau TM, Taylor NH and Rogers PA (1999) Lack of correlation between vascular endothelial growth factor production and endothelial cell proliferation in the human endometrium. Hum Reprod 14,2080–2088.
Gerhardt H, Ruhrberg C, Abramsson A, Fujisawa H, Shima D and Betsholtz C (2004) Neuropilin-1 is required for endothelial tip cell guidance in the developing central nervous system. Dev Dyn 231,503–509.[CrossRef][Web of Science][Medline]
Germeyer A, Hamilton AE, Laughlin LS, Lasley BL, Brenner RM, Giudice LC and Nayak NR (2005) Cellular expression and hormonal regulation of neuropilin-1 and –2 messenger ribonucleic acid in the human and rhesus macaque endometrium. J Clin Endocrinol Metab 90,1783–1790.
Goffin F, Munaut C, Frankenne F, Perrier D’Hauterive S, Beliard A, Fridman V, Nervo P, Colige A and Foidart JM (2003) Expression pattern of metalloproteinases and tissue inhibitors of matrix-metalloproteinases in cycling human endometrium. Biol Reprod 69,976–984.
Goodger AM and Rogers PA (1994) Endometrial endothelial cell proliferation during the menstrual cycle. Hum Reprod 9,399–405.
Goodger AM and Rogers PA (1995) Blood vessel growth in the endometrium. Microcirculation 2,329–343.[Medline]
Graubert MD, Ortega MA, Kessel B, Mortola JF and Iruela-Arispe ML (2001) Vascular repair after menstruation involves regulation of vascular endothelial growth factor-receptor phosphorylation by sFLT-1. Am J Pathol 158,1399–1410.
Gygi SP, Rochon Y, Franza BR and Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19,1720–1730.
Herve MA, Meduri G, Petit FG, Domet TS, Lazennec G, Mourah S and Perrot-Applanat M (2006) Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus. J Endocrinol 188,91–99.
Hornung D, Lebovic DI, Shifren JL, Vigne JL and Taylor RN (1998) Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells. Fertil Steril 69,909–915.[CrossRef][Web of Science][Medline]
Huang JC, Liu DY and Dawood MY (1998) The expression of vascular endothelial growth factor isoforms in cultured human endometrial stromal cells and its regulation by 17beta-oestradiol. Mol Hum Reprod 4,603–607.
Karihaloo A, Karumanchi SA, Cantley WL, Venkatesha S, Cantley LG and Kale S (2005) Vascular endothelial growth factor induces branching morphogenesis/tubulogenesis in renal epithelial cells in a neuropilin-dependent fashion. Mol Cell Biol 25,7441–7448.
Kazi AA, Jones JM and Koos RD (2005) Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor {alpha} and hypoxia-inducible factor 1 (HIF-1) to the vascular endothelial growth factor (VEGF) promoter. Mol Endocrinol 19,2006–2019.
Koos RD, Kazi AA, Roberson MS and Jones JM (2005) New insight into the transcriptional regulation of vascular endothelial growth factor expression in the endometrium by estrogen and relaxin. Ann N Y Acad Sci 1041,233–247.[CrossRef][Web of Science][Medline]
Krussel JS, Casan EM, Raga F, Hirchenhain J, Wen Y, Huang HY, Bielfeld P and Polan ML (1999) Expression of mRNA for vascular endothelial growth factor transmembraneous receptors Flt1 and KDR, and the soluble receptor sflt in cycling human endometrium. Mol Hum Reprod 5,452–458.
Lee S, Jilani SM, Nikolova GV, Carpizo D and Iruela-Arispe ML (2005) Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J Cell Biol 169,681–691.
Licht P, Russu V, Lehmeyer S, Wissentheit T, Siebzehnrubl E and Wildt L (2003) Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function. Fertil Steril 80,1228–1233.[CrossRef][Medline]
Lockwood CJ, Krikun G, Koo AB, Kadner S and Schatz F (2002) Differential effects of thrombin and hypoxia on endometrial stromal and glandular epithelial cell vascular endothelial growth factor expression. J Clin Endocrinol Metab 87,4280–4286.
Maas JW, Groothuis PG, Dunselman GA, de Goeij AF, Struyker Boudier HA and Evers JL (2001) Endometrial angiogenesis throughout the human menstrual cycle. Hum Reprod 16,1557–1561.
Meduri G, Bausero P and Perrot-Applanat M (2000) Expression of vascular endothelial growth factor receptors in the human endometrium: modulation during the menstrual cycle. Biol Reprod 62,439–447.
Moller B, Rasmussen C, Lindblom B and Olovsson M (2001) Expression of the angiogenic growth factors VEGF, FGF-2, EGF and their receptors in normal human endometrium during the menstrual cycle. Mol Hum Reprod 7,65–72.
Mueller MD, Vigne JL, Minchenko A, Lebovic DI, Leitman DC and Taylor RN (2000) Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta. Proc Natl Acad Sci USA 97,10972–10977.
Nap AW, Griffioen AW, Dunselman GA, Bouma-Ter Steege JC, Thijssen VL, Evers JL and Groothuis PG (2004) Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 89,1089–1095.
Nap AW, Dunselman GA, Griffioen AW, Mayo KH, Evers JL and Groothuis PG (2005) Angiostatic agents prevent the development of endometriosis-like lesions in the chicken chorioallantoic membrane. Fertil Steril 83,793–795.[CrossRef][Web of Science][Medline]
Nayak NR and Brenner RM (2002) Vascular proliferation and vascular endothelial growth factor expression in the rhesus macaque endometrium. J Clin Endocrinol Metab 87,1845–1855.
Niklaus AL, Aberdeen GW, Babischkin JS, Pepe GJ and Albrecht ED (2003) Effect of estrogen on vascular endothelial growth/permeability factor expression by glandular epithelial and stromal cells in the baboon endometrium. Biol Reprod 68,1997–2004.
Noyes RW, Hertig AT and Rock J (1975) Dating the endometrial biopsy. Am J Obstet Gynecol 122,262–263.[Medline]
Pijnenborg JM, van de Broek L, Dam de Veen GC, Roemen GM, de Haan J, van Engeland M, Voncken JW and Groothuis PG (2005) TP53 overexpression in recurrent endometrial carcinoma. Gynecol Oncol 100,397–404.
Punyadeera C, Dunselman G, Marbaix E, Kamps R, Galant C, Nap A, Goeij A, Ederveen A and Groothuis P (2004) Triphasic pattern in the ex vivo response of human proliferative phase endometrium to oestrogens. J Steroid Biochem Mol Biol 92,175–185.[CrossRef][Web of Science][Medline]
Punyadeera C, Dassen H, Klomp J, Dunselman G, Kamps R, Dijcks F, Ederveen A, de Goeij A and Groothuis P (2005) Oestrogen-modulated gene expression in the human endometrium. Cell Mol Life Sci 62,239–250.[CrossRef][Web of Science][Medline]
Rogers PA and Gargett CE (1998) Endometrial angiogenesis. Angiogenesis 2,287–294.[CrossRef][Medline]
Rossignol M, Gagnon ML and Klagsbrun M (2000) Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms. Genomics 70,211–222.[CrossRef][Medline]
Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, Fujisawa H, Betsholtz C and Shima DT (2002) Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 16,2684–2698.
Sharkey AM, Day K, McPherson A, Malik S, Licence D, Smith SK and Charnock-Jones DS (2000) Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia. J Clin Endocrinol Metab 85,402–409.
Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, Jaffe RB and Taylor RN (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 81,3112–3118.
Sugino N, Kashida S, Karube-Harada A, Takiguchi S and Kato H (2002) Expression of vascular endothelial growth factor (VEGF) and its receptors in human endometrium throughout the menstrual cycle and in early pregnancy. Reproduction 123,379–387.[Abstract]
Thijssen VL, Brandwijk RJ, Dings RP and Griffioen AW (2004) Angiogenesis gene expression profiling in xenograft models to study cellular interactions. Exp Cell Res 299,286–293.[CrossRef][Web of Science][Medline]
Torry DS, Holt VJ, Keenan JA, Harris G, Caudle MR and Torry RJ (1996) Vascular endothelial growth factor expression in cycling human endometrium. Fertil Steril 66,72–80.[Web of Science][Medline]
Villegas G, Lange-Sperandio B and Tufro A (2005) Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells. Kidney Int 67,449–457.[CrossRef][Web of Science][Medline]
Wang L, Zeng H, Wang P, Soker S and Mukhopadhyay D (2003) Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J Biol Chem 278,48848–48860.
Submitted on January 25, 2006; accepted on February 16, 2006.
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