1 Department of Pathology, Cambridge University, Tennis Court Road, Cambridge, CB2 1QP and 2 Department of Obstetrics and Gynaecology, The Rosie Hospital, Robinson Way, Cambridge CB2 2SW, UK
3 To whom correspondence should be addressed at: Department of Pathology, Cambridge University, Tennis Court Road, Cambridge, CB2 1QP, UK. Email: cgp22{at}cam.ac.uk
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Abstract |
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Key words: endometrious/gene array/VEGF-A/transcriptome
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Introduction |
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Endometriosis, the growth of ectopic endometrial fragments in the peritoneum, is a debilitating disease that causes pain with menstruation, pain with intercourse and other symptoms. In addition, endometriosis also causes infertility (Haney, 1990). Despite a significant recent increase of the disease coincident with the introduction of widespread fertility regulation, the cause of the problem remains unknown. Tissues that grow to greater than
1 mm3 need to establish a blood supply to prevent necrosis (Folkman, 2002
). Therefore, for endometriotic lesions to survive they must recruit a vasculature from the surrounding tissues (reviewed by Smith, 1997
). However, the processes that promote this angiogenesis are as yet uncertain. They may involve both endometrial and peritoneal factorsseveral examples of each have been proposed. Potential pro-endometriotic endometrial factors include increased proliferation capacity of endometrial endothelial cells (EC) (Wingfield et al., 1995
) and elevated expression of several molecules including: the pro-angiogenic integrin
v
3 (Hii et al., 1998
), hepatocyte growth factor and its c-Met receptor (Khan et al., 2003
), endoglin (Kim et al., 2001
), nitric oxide synthases (Wu et al., 2003
), urokinase receptor (Sillem et al., 1998
) and tissue inhibitor of metalloproteinases-2 (TIMP-2) (Sillem et al., 1998
). Potential peritoneal factors include: macrophage migration inhibitory factor (MIF) in peritoneal fluid of endometriotic women (Kats et al., 2002
), steroidally-regulated VEGF-A production by peritoneal macrophages (McLaren et al., 1996
), elevated tumour necrosis factor (TNF)-
production from peritoneal macrophages (Noda et al., 2003
; Richter et al., 2004
) and elevation of soluble TNF receptor (Steff et al., 2004
), angiogenin (Steff et al., 2004
) and PlGF (Suzumori et al., 2003
).
Previously, the programmed variation of the transcriptome of whole endometrium through the menstrual cycle has been characterized (Borthwick et al., 2003; Kao et al., 2003
). However, the effects of soluble factors produced by cells within the endometrium on endothelial gene expression, cell biology and angiogenesis have not been fully investigated. In this study, we have assessed the ability of the soluble factors secreted by endometrium collected from non-endometriotic and endometriotic women in the proliferative and secretory phases of the menstrual cycle to promote EC proliferation and angiogenesis and to modulate the EC transcriptome. Given the important role played by VEGF-A in angiogenesis, we have also assessed the concentration of VEGF-A in endometrial culture supernatants and in menstrual effluent from women with and without endometriosis.
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Materials and methods |
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EC proliferation and angiogenesis assays
Human umbilical vein EC(s) (HUVEC) were isolated by collagenase digestion as described (Jaffe et al., 1973) and cultured to passage 5 in LVEC medium. Once at passage 5, HUVEC were plated into 24-well plates at 70% confluence and supernatants from endometrial cultures added (20% V/V) for 24 h. Viable HUVEC numbers were then counted using a haemocytometer after trypan blue staining (0.2% Sigma, UK). Four wells were used for each experimental condition. The 20% V/V dilution of the endometrial cell culture supernatants was chosen empirically since it on average induced maximal HUVEC responses without inducing signs of stress in these cells (data not shown). To prevent confounding, medium of identical composition (LVEC medium described above) was used for culture of all endometrial explants and all HUVEC in this study. Since we have shown that quiescing HUEVC by transfer to low serum medium reduces the ability of these cells to mount transcriptional responses to growth factors (Johnson et al., 2003
), the HUVEC were not quiesced before incubation with the endometrial culture supernatants. In additional experiments, a blocking anti-VEGF-A monoclonal antibody (clone 26503, R&D Systems, UK) was added to HUVEC cultured as above, and relative cell numbers estimated using a tetrazolium compound (MTS) assay (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega) used according to the manufacturer's instructions.
In vitro angiogenesis assays consisted of co-cultures of HUVEC and human dermal fibroblasts (Bishop et al., 1999). They were purchased pre-plated in 24-well tissue culture plates from TCS Cellworks (ZHA-1000; Botolph, UK) and cultured in the LVEC medium for 11 days with or without the addition of endometrial culture supernatants (20% V/V, replaced every 4 days). After 11 days cells were fixed in 2% paraformaldehyde for 20 min at 4°C, permeablized in 0.1% Triton X-100 and 0.1% Tween-20 for 30 min at room temperature, stained with UlexFITC (Sigma L9006, UK) at a concentration of 10 µg/ml and Hoescht Bisbenzimide 33342 (Calbiochem 382061, UK) at a concentration of 1 µg/ml. For each experimental condition, the number of branches and total length of capillary-like structures was measured by imaging 21 randomly chosen 1 mm2 fields using a confocal microscope (Lieca, Germany). Images were processed to quantify angiogenesis using the TCS Angiogenesis assessment software (TCS, Botolph, UK).
Gene array analysis
RNA was prepared from HUVEC using Trizol reagent (Gibco/BRL, UK) followed by clean up through an RNeasy spin column (Qiagen, UK) and ethanol precipitation. RNA integrity was assessed using an Agilent 2100 bioanalyser (Agilent, UK). 33P-labelled complex cDNA probes were prepared from the RNAs and hybridized to an array of 1000 cDNAs immobilized onto nylon filters as described (Evans et al., 2003
). Hybridization was quantified using a phosphoimager (Molecular Dynamics Inc., CA) and IMAGENE software (BioDiscovery, CA). After global and local normalization (Evans et al., 2003
), transcript abundance data were compared using the CyberT algorithm (version 7.03; sliding window = 301, Bayes estimate = 10). This algorithm is an unpaired t-test, modified by the inclusion of a Bayesian prior based on the variance of other transcripts in the data set (Long et al., 2001
). For further statistical analysis, the R statistical software system and GeneSpring Expression Analysis Software (Silicon Genetics, Redwood City, CA) were used.
Quantitative polymerase chain reaction analysis
The ABI PRISM 7700 Sequence Detection System (TaqMan) was used to perform real-time polymerase chain reactions according to the manufacturer's protocols. Ct values for each transcript were compared to those for cyclophilin A, which according to the gene array results remained relatively constant in abundance. Primers and probes for GSTP-1, VE-cadherin, IL1RL-1 and vimentin were proprietary oligonucleotides obtained from Applied Biosystems (ABI, UK) Assays-on-Demand and used a 5' FAM reporter and 3' non-fluorescent minor groove binder.
ELISA for VEGF-A
To quantify VEGF-A in endometrial culture supernatants an ELISA assay was used (R&D systems, UK) according to the manufacturer's instructions, and the VEGF-A concentration determined by measuring absorbance at 490 nm by comparison with a standard curve. For each measurement six replicates were performed.
Collection of menstrual effluent
Menstrual effluent was collected by volunteers by vaginal insertion of a menstrual cup (The KeeperTM, Eco Logique Inc., Canada) in the second 24 h of bleeding. The cup was left in situ for 4 h, the fluid sample collected and the volume measured. Menstrual cup samples were washed with an equal volume of PBS and centrifuged at 200 g for 10 min at 4°C. The supernatant was stored at 70°C for ELISA which was performed as described above.
Statistical analysis
All experimental data except those generated from gene arrays were analysed using the R statistical software package (http://rweb.stat.umn.edu/R/doc/html/index.html). Before comparisons were made, data were tested for normality using Chi squared tests and then for equal variance between groups using Bartlett's method. Based on these results, we were able to use multi-way analysis of variance (ANOVA). In all cases where ANOVA suggested a statistically significant effect, post hoc Tukey's Multiple Comparison Tests were used to assess the source of the effect.
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Results |
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To assess whether soluble factors derived from endometrium could promote angiogenesis in addition to promoting endothelial proliferation, endometrial culture supernatants were added (20% V/V) to co-cultures of HUVEC and human dermal fibroblasts for an 11 day period. These co-cultures spontaneously formed capillary-like structures (in vitro angiogenesis; Bishop et al., 1999). We found that endometrial culture supernatants promoted the acquisition of capillary length and branching (estimated by counting junction number) more effectively than medium alone. Medium alone (n=3) induced vessels with a mean length of 4.0±0.1 mm/mm2 and a mean branching of 12±1.5/mm2, respectively (mean±SEM). However, proliferative phase supernatants (n=4) induced vessels with a mean length of 6.5±0.1 mm/mm2 and a mean branching of 26.3±0.9/mm2 (mean±SEM) and secretory phase supernatants (n=2) induced vessels with a mean length of 5.8 and 6.0 mm/mm2 and a mean branching of 19 and 21/mm2 (Figure 1). Due to the small number of secretory phase endometrial supernatants, for statistical purposes the results for the two secretory phase and four proliferative phase endometrial supernatant groups were combined, and a Student's t-test indicated that both length and branching were significantly elevated when compared to the three control cultures (P
0.05). The promotion of EC proliferation and in vitro angiogenesis appears to be specific to endometrium, since supernatants from HUVEC themselves, primary vascular smooth muscle cells, Jurkat T lymphocytes and primary human macrophages cultured in identical conditions to the endometrium had no significant effect on HUVEC cell biology or in vitro angiogenesis (data not shown). To assess the possibility that differences in the cellular composition of the endometrial cultures were responsible for these effects, we analysed the cellular composition of disaggregated endometrium derived from three patients in the mid-proliferative and two in the mid-secretory phases of the menstrual cycle after culture for 24 h. These cultures contained variable proportions (711%) of cytokeratin+ ve epithelial cells. The cellular proportions were not significantly different (ANOVA; P>0.05) in the cultures derived from either proliferative or secretory phase endometrium.
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Discussion |
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The transcriptome changes induced in EC by endometrium-derived factors may partly underlie the angiogenesis that is also induced by these factors, since many of the regulated EC transcripts encode promoters of angiogenesis (Table I). These include transcripts previously found to be up-regulated during in vitro angiogenesis (Kahn et al., 2000) such as CXC receptor-4, collagen IV
6 and tissue factor-inhibitor-2. Given that VEGF-A was secreted by the cultured endometrium, it is interesting that transcripts previously found to be up-regulated in EC by VEGF-A (Weston et al., 2002
; Yang et al., 2002
) were also up-regulated by endometrial supernatants, such as CXC receptor-4, MMP-2 tissue factor-inhibitor-2 and VE-cadherin. Several other transcripts regulated by the endometrial supernatants have clear associations with EC survival and proliferation. For example, angiopoietin-1 was down-regulated. Angiopoietin-1 usually functions to stabilize vessels, but is functionally inhibited by angiopoietin-2 during angiogenesis. Endoglin (up-regulated) is a component of the transforming growth factor beta receptor complex that is essential for vessel development (Zhang et al., 2002
). PECAM-1 and VE-cadherin (both up-regulated) are EC adhesion molecules that promote EC survival through protein kinase B (AKT) signaling. Other regulated signaling molecules include; ADP ribosylation factor-like-2, interleukin-1 receptor-like-1, interferon
receptor-2, MAP kinase-12, Rho-7 and SIVA. Several transcripts associated with the extracellular matrix were regulated in EC by endometrial supernatants. These may contribute to cyclical remodeling of both vessels and other elements of endometrium. For example, Plasminogen activator-inhibitor-1 and fibronectin-1 were up-regulated. MMP-2 and MMP-3 were also up-regulated and have previously been associated with cyclical endometrial remodelling (Ueda et al., 2002
) and endometriosis (Cox et al., 2001
).
Previous studies (summarized in the Introduction) have suggested that both endometrial and peritoneal factors promote the angiogenesis that underlies endometriosis. Our study investigated the role played by endometrial factors. We found that endometriosis had no effect on the capacity of endometrial culture supernatants to induce angiogenesis or gene expression changes in EC. In addition, we found no relationship between endometriosis and VEGF-A production by cultured endometrium or VEGF-A concentration or mass in menstrual effluent. This suggests that differences between the soluble pro-angiogenic factors secreted by the endometrium of endometriotic and non-endometriotic women may not be a major determinant of this disease. It is possible that the small size of our study (dictated by our stringent patient selection criteria and the relative rarity of unmedicated endometriotic patients) has caused us to miss a subtle role played by soluble endometrial factors. However, it is also possible that confounding and the lack of stringent patient selection may have caused some previous investigators to overestimate the role played by endometrial factors in this disease. The importance of stringent patient selection criteria and measures to reduce confounding has been highlighted in a recent study published in this journal of soluble ICAM-1 in endometriotic patients (Steff et al., 2004). Models of endometriosis in which human endometrium is transplanted into immunocompromized mice (Hull et al., 2003
) will provide powerful tools to dissect the relative roles of endometrial and peritoneal factors.
In conclusion, we have shown that endometrium cultured in vitro produces soluble factors, including VEGF-A, that promote angiogenesis. Endometrium, especially from the proliferative phase of the menstrual cycle, also promotes significant pro-angiogenic changes to the EC transcriptome. However, we were unable to detect any association between endometriosis and the effects of soluble endometrial factors on EC biology or gene expression. We believe that the endometrial factor-induced EC transcriptome changes identified here provide insight into the dynamic control of vessel structure on which both the eutopic endometrium and endometriotic lesions depend. They also provide potential therapeutic targets for the modulation of endometrial angiogenesis.
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Acknowledgements |
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Notes |
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Submitted on November 5, 2003; resubmitted on May 18, 2004; accepted on June 16, 2004.
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