Molecular Human Reproduction, Vol. 9, No. 11, 663-669,
November 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium
Submitted on May 6, 2003; accepted on July 14, 2003
Department of Obstetrics and Gynecology, Heinrich-Heine-University Medical Center, Moorenstrasse 5, D-40225 Düsseldorf, Germany
1 To whom correspondence should be addressed. e-mail: Hirchenhain{at}med.uni-duesseldorf.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)–PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.
Key words: angiogenesis/angiopoietin/endometrium/gene expression
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Angiogenesis is the formation of new blood vessels from pre-existing ones, a physiological process that is normally seen in fetal development, wound healing and in the female reproductive tract (Findlay, 1986; Folkman and Shing, 1992). The critical role of angiogenesis in ovarian and endometrial function was demonstrated when the angiogenesis inhibitor, AGM-1470, substantially retarded growth of both the corpus luteum and the endometrium when administered chronically to non-pregnant, cycling mice (Klauber et al., 1997). In human endometrium, angiogenesis occurs periodically as part of the cyclical growth and shedding which take place during the menstrual cycle. During menstruation, repair of the ruptured blood vessels must occur; in the proliferative phase the rapid growth of endometrial tissue must be accompanied by angiogenesis; and finally in the secretory phase there is development and coiling of the spiral arterioles and growth of the subepithelial capillary plexus (Rogers and Gargett, 1998). While circulating estrogen and progesterone primarily regulate the overall control of endometrial growth and regression, it appears unlikely that steroids are direct regulators of vascular growth in this tissue.
Several groups, including ourselves, have previously shown that vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, and its receptors, VEGFR-1 (Flt-1), VEGFR-2 (KDR) and soluble Flt (for review see Neufeld et al., 1999), are expressed in human endometrium in a cycle-dependent fashion throughout the menstrual cycle (Shifren et al., 1996; Torry et al., 1996; Krüssel et al., 1999; Möller et al., 2001).
So far, very little is known about the expression of angiopoietins, another group of angiogenic growth factors, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. The prominent roles of angiogenic growth factors and their receptors have become evident from gene knock-out experiments in mice. Inactivation of Tie-2 and Ang-1 leads to disrupted vessel structure and embryonic lethality (Dumont et al., 1994; Sato et al., 1995; Suri et al., 1996). This occurs at a later developmental stage and with a phenotype distinct from those associated with inactivation of the vascular endothelial growth factor receptor tyrosine kinases (VEGFR-1 and VEGFR-2) which are also involved in vasculogenesis (Fong et al., 1995; Shalaby et al., 1995). Ang-1 is important in maintaining vessel integrity. The Ang-2 negative signal, acting through inhibition of Tie-2 signalling, leads to a loosening of cell–matrix and cell–cell contacts, allowing access to angiogenic inducers, e.g. VEGF. Thus, co-expression of Ang-2 and VEGF leads to angiogenesis. In the absence of angiogenic growth or survival signals, Ang-2 action results in a regression of vessel structures (reviewed in Hanahan, 1997; Yancopoulos et al., 2000). In mice and humans, Ang-2 is selectively expressed in ovary, uterus and placenta (Maisonpierre et al., 1997), the three tissues subject to physiological angiogenesis, whereas Ang-1 is widely expressed both in the embryo and in the adult (Davis et al., 1996).
The aim of our study was to detect the relative mRNA expression levels of Ang-1, Ang-2 and the receptor Tie-2 in human endometrium throughout the menstrual cycle. Additionally, we separated a number of endometrial biopsies into stromal and epithelial compartments to analyse the cellular mRNA distribution. Furthermore we analysed by immunohistochemistry the protein distribution within the endometrium.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Endometrial biopsies
Patients who underwent hysteroscopy for non-malignant reasons, mainly for unexplained infertility, were asked to participate in this study. All patients included had regular menstrual cycles and normal hormonal profiles; patients with polycystic ovaries were excluded. Each participating patient signed an informed consent approved by the local ethical board. Biopsies were taken by curettage before the hysteroscopy. By using NaCl distention medium instead of CO2 for the hysteroscopy, we were able to flush the uterine cavity, such that the biopsy did not diminish the visibility. Specimens were washed briefly in phosphate-buffered saline (PBS) to remove contaminating blood and mucus. One part of the tissue was fixed in 4% paraformaldehyde; the other part was directly processed for RNA extraction. Menstrual phase was determined by patient’s history and dating was verified by histological examination of the endometrium according to the criteria of Noyes et al. (1950). Subjects were from the early proliferative (days 4–7, n = 4), mid proliferative (days 8–11, n = 12), late proliferative (days 12–14, n = 3), early secretory (days 15–18, n = 3), mid secretory (days 19–22, n = 5) and late secretory (days 23–28, n = 3) phases. Endometrial samples (mid proliferative phase) from six additional patients were digested with collagenase for 2–4 h and separated into epithelial and stromal cell fractions by passing the cell suspension through a 40 µm nylon cell strainer (Falcon, Becton Dickinson) essentially as described before (Irwin et al., 1989). The RNA was extracted from both cell types immediately after harvesting. The purity of the cell fractions was typically 92–96% as determined by immunohistochemical analysis of aliquots of cells plated on Labtec chamber slides (Falcon) with antibodies against cytokeratin (Dako) or vimentin (Dako).
RNA extraction
Total RNA was isolated from tissue specimens using the Trizol reagent (Gibco BRL) according to the manufacturer’s instructions. The amount and purity of the extracted RNA was quantified by spectrophotometry using an Eppendorf Biophotometer.
Reverse transcription
For each mRNA sample, 10 µl RT mastermix was prepared [4 µl 25 mmol/l MgCl2 solution, 2 µl 10xPCR buffer II, 1 µl dNTP mix (20 mmol/l each), 1 µl Oligo d(T)16, 1 µl RNase Inhibitor, 1 µl MuLV Reverse Transcriptase (all Perkin–Elmer)] and added to 1 µg total RNA diluted in 10 µl DEPC-treated H2O in a 0.5 ml PCR-Softtube (Biozym Diagnostik). RT mix was incubated at room temperature for 10 min, before starting the reaction in a Thermocycler (Uno II, Biometra) by using a program with the following parameters: 42°C for 1 h, 94°C for 5 min, 4°C for 
. After the reaction was complete, samples were diluted with 80 µl DEPC- treated H2O and stored at –20°C.
Primers for PCR
The cDNA sequences were obtained from the GenBank database of the National Center for Biotechnology Information (NCBI) of the National Institute of Health (NIH); http://www.ncbi.nlm.nih.gov/. Primer sequences were selected with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, USA). Primers were designed to cross intron/exon boundaries to ensure that the amplified product resulted from cDNA rather than contaminating genomic DNA. The identities of all PCR products were confirmed by cycle sequencing. The primer sequences, locations on the cDNA and the sizes of the amplified fragments are listed in Table I.
|
Construction of the competitive cDNA fragments
Competitive cDNA fragments were constructed utilizing a method described previously (Krüssel et al., 1998). A ‘floating’ primer was designed for each cDNA to introduce an internal deletion, resulting in a competitive fragment with the same primer binding characteristics as the target cDNA. Competitive cDNA was extracted from the agarose gel with an extraction kit (QIAEX II; Qiagen) and quantitated by spectrophotometry. The identities of all competitor fragments were confirmed by cycle sequencing. Sequences of the ‘floating’ primers and sizes of the resulting competitor fragments are listed in Table I.
Competitive PCR
5 µl RT product (that is 1/20 of RT reaction) was mixed with 0.02 amol competitive cDNA for either Ang-1 or Ang-2 in 5 µl sterile H2O and with 40 µl PCR mastermix containing 5 µl 10x reaction buffer; 0.2 µl Taq DNA Polymerase (Pharmacia Biotech); 2 µl dNTP mix (50 mmol/l each, Eppendorf); 2 µl of 5' and 3' specific primers (5 µmol/l each) and 31.8 µl sterile H2O. The PCR was initiated by heating up to 94°C for 3 min, followed by 32 cycles denaturation at 94°C for 45 s, annealing at 53°C for Ang-1 or at 57°C for Ang-2 and extension at 72°C for 45 s. The reaction was terminated at 72°C for 5 min and cooled down to 4°C. PCR for Tie-2 was with 0.04 amol competitor, an annealing temperature of 55°C and 30 cycles. In each experiment a negative control reaction in which no cDNA was added was included. Preliminary experiments were performed to determine the optimal numbers of PCR amplification cycles for different primer pairs to ensure that the amplification was in the exponential phase and had not reached the plateau. PCR products were visualized on a 2% agarose gel. The densitometrical analysis of bands was carried out on the GelDoc 1000 System (Bio-Rad Laboratories, USA) by using the Molecular Analyst Software (Bio-Rad). Densitometry values were used to calculate the ratios between target and competitor bands (t/c ratio).
Multiplex PCR
PCR was carried out for 35 cycles (45 s at 94°C, 45 s at 53°C, 45 s at 72°C) including 5'- and 3'-specific primers (5 µmol/l each) for both Ang-1 and Ang-2.
Immunohistochemistry
Immunohistochemistry was conducted using specific goat polyclonal antibodies raised against human Ang-1 and Ang-2 (sc-9360 and sc-7015; Santa Cruz Biotechnology, Inc., USA). Paraffin sections (5 µm) of endometrium from all stages of the menstrual cycle (n = 19) were dewaxed in xylene and rehydrated through descending grades of ethanol. Endogenous peroxidase activity was quenched by immersion in 3% H2O2 for 10 min. Sections were then incubated with blocking solution containing 10% rabbit serum (Vector Laboratories, USA) and 1.5% bovine serum albumin (BSA) in PBS for 20 min at room temperature. Primary antibodies were applied diluted at 1:100 in 1.5% BSA–PBS overnight at 4°C. Antibody localization was detected by sequential application of biotinylated rabbit anti-goat IgG (Vector) in PBS, and an avidin–biotin complex conjugated to horse-radish peroxidase (Vector). The substrate used was diaminobenzidine (Vector), which forms an insoluble brown precipitate, and nuclei were counterstained blue with Mayer’s haemalum solution (Merck, Germany).
A similar protocol was utilized for detection of Tie-2 and von Willebrand Factor (vWF), which were localized using a rabbit polyclonal antibody (sc-324, Santa Cruz for Tie-2; and A-0082, Dako, Denmark for vWF), goat serum and a biotinylated goat anti-rabbit IgG (Vector).
A negative control was included for each tissue section by substitution of the primary antibody with a matching concentration of either goat or rabbit IgG.
Statistical analysis
All PCR experiments were repeated three times. Results were expressed as the mean ± SEM. Analyses were performed using SPSS, Version 10.1 (SPSS, USA). To determine if there were significant differences in mRNA expression levels at different stages of the menstrual cycle, non-parametric tests were used (Kruskal–Wallis, followed by Mann–Whitney U-test to detect differences between groups). A value of P < 0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We detected mRNA for the angiopoietins and the receptor Tie-2 in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. The expression levels of the angiopoietins were comparable, as judged from the same amount of competitor (0.02 amol) to be used in the competitive PCR reactions (Figure 1). Analysis of the target/competitor ratios revealed a significantly (P = 0.044, Kruskal–Wallis) different Ang-1 expression during the course of the cycle. Ang-1 expression was elevated in the early, mid and late secretory phase and the mid proliferative phase compared with the late proliferative phase (Figure 2). When we compare the data for the proliferative (n = 19) and secretory (n = 11) phases, there is a statistically significant (P = 0.027) increase of Ang-1 expression in the secretory phase (Figure 3).
|
|
|
Analysis of the expression levels of Ang-2 revealed a significant difference between early and mid secretory phase (Figure 2). Tie-2 expression showed only minor differences during the endometrial cycle (Figure 2), which did not reach statistical significance. There were, however, considerable individual variations in the mRNA levels within biopsies from the same phase of the menstrual cycle.
To confirm these results, another PCR experiment was performed, in which the relative expression levels of both angiopoietin genes were compared in a multiplex PCR reaction (Figure 4 and Figure 5). The lower Ang-2 versus Ang-1 ratios in the secretory phase were indicative of either reduced Ang-2 expression or, more likely in the light of our above result, an increased Ang-1 expression.
|
|
Figure 5 also shows a comparison of the multiplex and the competitive PCR methods used. The values for the Ang-2/Ang-1 ratio for the competive PCR were obtained by comparing the mean target/competitor ratios for Ang-2 and Ang-1 for each individual biopsy. Both PCR experiments gave the same result, indicating the methodical accuracy.
By separating epithelial and stromal cell fractions from six additional endometrial biopsies, we could show that both the endometrial epithelial cells as well as the stromal cell fraction, including immune and endothelial cells, expressed mRNA for Ang-1, Ang-2 and Tie-2 (Figure 6). The minor differences in expression are not statistically significant.
|
We examined the distribution of Ang-1, Ang-2 and Tie-2 protein in proliferative (n = 11) and secretory (n = 7) endometrium by immunohistochemistry (Figure 7). Staining showed interindividual differences, but nevertheless a clear pattern emerged. Staining for Ang-1 was detected in stromal cells, in glandular and luminal epithelium as well as in endothelial cells in both proliferative and secretory phase endometrium (Figure 7a, b).
|
Ang-2 immunoreactivity was detected in glandular epithelium, and in the stromal compartment of proliferative and secretory phase endometrium (Figure 7c, d). Both Ang-1 and Ang-2 proteins were found predominantly in the apical part of glandular epithelial cells.
Tie-2 staining was found in glandular epithelium and endothelial cells throughout the cycle, whereas staining of stromal cells was very low and heterogeneous (Figure 7e, f). As a control for staining of endothelial cells, we used an antibody against von Willebrand factor, which, as expected, selectively stained endometrial blood vessels (Figure 7g).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The regulation of human endometrial angiogenesis is still poorly understood, despite its unique role in the female reproductive cycle. Many studies have focused on the endometrial expression of vascular endothelial growth factor (VEGF) and its receptors (Charnock-Jones et al., 1993; Shifren et al., 1996; Torry et al., 1996; Krüssel et al., 1999; Sharkey et al., 2000), but the functional significance in the endometrium is still uncertain, taking into account that the bulk of VEGF is of glandular origin and secreted into the uterine cavity (Hornung et al., 1998). The endometrium is a complex tissue with many different cell types in dynamic fluctuations. Therefore, Maas et al. (2001) studied the overall angiogenic activity of endometrium in the chick chorioallantoic membrane (CAM) assay. They found an angiogenic potential throughout the menstrual cycle, with a significantly increased VDI (vascular density index) in the early proliferative, early and late secretory phases as compared to the late proliferative phase. According to the hypothesis that changes in the balance of inducers and inhibitors of angiogenesis may activate the angiogenic switch (Hanahan and Folkman, 1996), it is highly unlikely that only one factor is responsible for the angiogenic potential of a specific tissue.
So far, there are only few studies dealing with angiopoietin expression in the endometrium. In this study we found a significant increase in Ang-1 mRNA expression during the secretory phase of the endometrial cycle. This finding was confirmed, although not quantitatively, at the protein level. On the other hand we found only minor differences in the mRNA and protein expression of Ang-2. Since we found neither mRNA nor protein levels of Tie-2 changed during the cycle, we assume that angiogenesis is regulated by the availability of the ligands rather than the receptor.
The observed Ang-1 mRNA increase during the secretory phase may reflect the need for both stabilization and maturation of the newly developed vessels. The secretory phase is characterized by a significant growth and coiling of spiral arterioles (Kaiserman-Abramof and Padykula, 1989), which supply the subepithelial capillary plexus, where the blood flow reaches a maximum during the early and mid-secretory phases in preparation for the implanting embryo (Gannon et al., 1997). Ang-1 is assumed to function by mediating the dialogue between the endothelium and surrounding pericytes and vascular smooth muscle cells, so as to promote the stability of blood vessels (Yancopoulos et al., 2000). We propose that the Ang-1/Tie-2 system is important for endometrial vessel development during the postovulatory phase, when the spiral arterioles are developing. Our results are supported by a recent publication by Hewett et al. (2002), who found that endometrial Ang-1 expression (mRNA and protein) was down-regulated in patients with menorrhagia. Our data concerning the Ang-1 protein distribution are mostly in good agreement with their results. Ang-1 staining was detected in the glandular epithelium, stroma and blood vessels in the proliferative endometrium. The staining was more intense in the secretory phase, especially in the stroma surrounding the blood vessels (Hewett et al., 2002). Contrary to our result, they found Ang-1 to be down-regulated in glandular epithelium during the secretory phase.
Whether our observation, that Ang-2 mRNA expression is significantly different in the early versus the mid secretory phase (Figure 2), is of any physiological importance, remains to be shown. We were not able to confirm this finding at the protein level. From our data it seems that there is no major difference in Ang-2 protein expression levels during the cycle. In accordance with our results, Hewett et al. (2002) found Ang-2 mRNA to be expressed at similar levels as assessed by RNase protection assay normalized to ß-actin, in both normal and menorrhagic endometrium. In contrast, Krikun et al. (2000) showed by immunohistochemistry that expression of Ang-2 and Tie-2 was absent in the glands, low in stromal cells, and intense in the endothelial cells. Furthermore, they found no effect of the stage of menstrual cycle on the expression of Ang-2 or Tie-2. Their data, however, do not clearly show how many biopsies they have examined and how they reach this conclusion. Li et al. (2001) found Ang-2 expression, assessed by in-situ hybridization, predominantly in natural killer cells in the late secretory phase, which strongly implicates a role of these cells in vascular remodeling. In agreement with our semiquantitative RT–PCR data these authors found relatively steady mRNA levels of Ang-2 throughout the cycle. Contrary to our results, they found steady Ang-1 levels, but significantly altered Tie-2 expression during the course of the endometrial cycle. This might be explained by the different methodologies; they used a semiquantitative RT–PCR method comparing the expression level of the target gene with levels of ß-actin. Our method used with specifically designed competitors is generally considered more accurate, since it eliminates tube-to-tube variation and the possible influence of different primers (Krüssel et al., 1998). More importantly, in our study the multiplex PCR resulted in nearly the same Ang-2 versus Ang-1 expression ratio as the competitive PCR. This adds further significance to our findings, since these are independent methods.
From our Ang-2 data we speculate that there is a baseline Ang-2 level that is sufficient for angiogenesis to occur, provided that there are other inducers available in sufficient amounts (Hanahan and Folkman, 1996). Alternatively, it may be the local increase of Ang-2 availability, as suggested from the ‘patchy’ Ang-2 expression found in natural killer cells (Li et al., 2001), rather than a generalized up-regulation, which results in angiogenesis. With respect to the local availability of a factor, the work of Gargett et al. (2001) is of importance. In a recent immunohistochemical analysis of full thickness endometrium, they were for the first time able to demonstrate a strong correlation between focal VEGF, found in marginating and adherent neutrophils inside the vessel lumen, and endothelial cell proliferation. We and others (Krikun et al., 2000; Hewett et al., 2002) were, however, not able to find spatially restricted Ang-2 expression loci, which one would assume to be in the vicinity of blood vessels.
We could show that both the endometrial epithelial cells as well as the stromal cell fraction expressed the mRNA for Ang-1, Ang-2 and Tie-2. Surprisingly, mRNA expression levels showed only minor differences between the cell types, since e.g. Tie-2 protein expression seemed much higher in the glandular epithelium than in the stromal cells. This may be explained by the fact that the Tie-2-expressing endothelial cells may contribute to the stromal cell fraction. Alternatively, post-transcriptional regulatory events are resulting in a differential protein expression. It would be important to clarify whether the glandular expression of angiopoietins results in an apical secretion into the endometrial cavity, as has been shown before for VEGF (Hornung et al., 1998), or if the proteins are secreted towards the underlying stroma, possibly acting on the subepithelial capillary plexus. The predominantly apical localization of the proteins, however, is suggestive of an apical secretion.
Despite extensive examination of the distribution of mRNA and protein of a wide variety of angiogenic factors, including the angiopoietins, no pattern has emerged that allows a conclusive explanation of endometrial angiogenesis. Certainly further in-vivo and in-vitro experimentation is required to demonstrate the specific role of angiopoietins in endometrial angiogenesis and to elucidate the mechanisms involved.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Charnock-Jones, D.S., Sharkey, A.M., Rajput-Williams J. Burch, D., Schofield, J.P., Fountain, S.A., Boocock, C.A. and Smith, S.K. (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]
Davis, S., Aldrich, T.H., Jones, P.F., Acheson, A., Compton, D.L., Jain, V., Ryan, T.E., Bruno, J., Radziejewski, C., Maisonpierre, P.C. et al. (1996) Isolation of angiopoietin-1, a ligand for the Tie-2 receptor, by secretion-trap expression cloning. Cell, 87, 1161–1169.[CrossRef][Web of Science][Medline]
Dumont, D.J., Gradwohl, G., Fong, G.H., Puri, M.C., Gertsenstein, M., Auerbach, A. and Breitman, M.L. (1994) Dominant-negative and targeted null mutations in the endothelial tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev., 8, 1897–1909.
Findlay, J.K. (1986) Angiogenesis in reproductive tissues. J. Endocrinol., 111, 357–366.
Folkman, J. and Shing, Y. (1992) Angiogenesis. J. Biol. Chem., 267, 10931–10934.
Fong, G.H., Rossant, J., Gertsenstein, M. and Breitmann, M.L. (1995) Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature, 376, 66–70.[CrossRef][Medline]
Gannon, B.J., Carati, C.J. and Verco, C.J. (1997) Endometrial perfusion across the normal human menstrual cycle assessed by laser Doppler fluxmetry. Hum. Reprod., 12, 132–139.
Gargett, C.E., Lederman, F., Heryanto, B., Gambino, L.S. and Rogers, P.A.W. (2001) Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils. Hum. Reprod., 16, 1065–1075.
Hanahan, D. and Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86, 353–364.[CrossRef][Web of Science][Medline]
Hanahan, D. (1997) Signaling vascular morphogenesis and maintenance. Science, 277, 48–50.
Hewett, P., Nijjar, S., Shams, M., Morgan, S., Gupta, J. and Ahmed, A. (2002) Down-regulation of Angiopoietin-1 expression in menorrhagia. Am. J. Pathol., 160, 773–780.
Hornung, D., Lebovic, D.I., Shifren, J.L., Vigne, J.L. and Taylor, R.N. (1998) Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells. Fertil. Steril., 69, 909–915.[CrossRef][Web of Science][Medline]
Irwin, J.C., Kirk, D., King, R.J.B., Quigley, M.M. and Gwatkin, R.B.L. (1989) Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil. Steril., 52, 761–768.[Web of Science][Medline]
Kaiserman-Abramof, I.R. and Padykula, H.A. (1989) Angiogenesis in the postovulatory primate endometrium: the coiled arteriolar system. Anat. Rec., 224, 479–489.[CrossRef][Medline]
Klauber, N., Rohan, R.M., Flynn, E. and D’Amato, R.J. (1997) Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470. Nat. Med., 3, 443–446.[CrossRef][Web of Science][Medline]
Krikun, G., Schatz F., Finlay T., Kadner, S., Mesia, A., Gerrets, R. and Lockwood, C.J. (2000) Expression of angiopoietin-2 by human endometrial endothelial cells: regulation by hypoxia and inflammation. Biochem. Biophys. Res. Commun., 275, 159–63.[CrossRef][Web of Science][Medline]
Krüssel, J.S., Huang, H.Y., Simon, C., Behr, B., Pape, A.R., Wen, Y., Bielfeld, P. and Polan, M.L. (1998) Single blastomeres within human preimplantation embryos express different amounts of messenger ribonucleic acid for ß-actin and interleukin-1 receptor type I. J. Clin. Endocrinol. Metab., 83, 953–59.
Krüssel, J.S., Casan, E.M., Raga, F., Hirchenhain, J., Wen, Y., Huang, H.Y., Bielfeld, P. and Polan, M.L. (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.
Li, X.F., Charnock-Jones, D.S., Zhang, E., Hiby, S., Malik, S., Day, K., Licence, D., Bowen, J.M., Gardner, L., King, A. et al. (2001) Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells. J. Clin. Endocrinol. Metab., 86, 1823–34.
Maas, J.W.M., Groothuis, P.G., Dunselman, G.A.J., de Goeij, A.F.P.M., Struyker Boudier, H.A.J. and Evers, J.L.H. (2001) Endometrial angiogenesis throughout the human menstrual cycle. Hum. Reprod., 16, 1557–1561.
Maisonpierre, P.C., Suri, C., Jones, P.F., Bartunkova, S., Wiegand, S.J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T.H., Papadopoulos, N. et al. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science, 277, 55–60.
Möller, 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.
Neufeld, G., Cohen, T., Gengrinovitch, S. and Poltorak, Z. (1999) Vascular endothelial growth factor (VEGF) and its receptors. FASEB J., 13, 9–22.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
Rogers, P.A.W. and Gargett, C.E. (1998) Endometrial angiogenesis. Angiogenesis, 2, 287–294.[CrossRef][Medline]
Sato, T.N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W. and Qin Y. (1995) Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature, 376, 70–74.[CrossRef][Medline]
Shalaby, F., Rossant, J., Yamaguchi, T.P., Gertsenstein, M., Wu, X.F., Breitman, M.L. and Schuh, A.C. (1995) Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature, 376, 62–66.[CrossRef][Medline]
Sharkey, A.M., Day, K., McPherson, A., Malik, S., Licence, D., Smith, S.K. and Charnock-Jones, D.S. (2000) Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia. J. Clin. Endocrinol. Metab., 85, 402–409.
Shifren, J.L., Tseng, J.F., Zaloudek, C.J., Ryan, I.P., Meng, Y.G., Ferrara, N., Jaffe, R.B. and Taylor, R.N. (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.
SuriC., Jones P.F., Patan S., Bartunkova, S., Maisonpierre, P.C., Davis, S., Sato, T.N. and Yancopoulos, G.D. (1996) Requisite role of Angiopoietin-1, a ligand for the Tie-2 receptor, during embryonic angiogenesis. Cell, 87, 1171–1180.[CrossRef][Web of Science][Medline]
Torry, D.S., Holt, V.J., Keenan, J.A., Harris, G., Caudle, M.R. and Torry, R.J. (1996) Vascular endothelial growth factor expression in cycling human endometrium. Fertil. Steril., 66, 72–80.[Web of Science][Medline]
Yancopoulos, G.D., Davis, S., Gale N.W., Rudge, J.S., Wiegand, S.J. and Holash, J. (2000) Vascular-specific growth factors and blood vessel formation. Nature, 407, 242–248.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. E Girling and P. A W Rogers Regulation of endometrial vascular remodelling: role of the vascular endothelial growth factor family and the angiopoietin-TIE signalling system Reproduction, December 1, 2009; 138(6): 883 - 893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Abramovich, A. R. Celin, F. Hernandez, M. Tesone, and F. Parborell Spatiotemporal analysis of the protein expression of angiogenic factors and their related receptors during folliculogenesis in rats with and without hormonal treatment Reproduction, February 1, 2009; 137(2): 309 - 320. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Lee, Y. Liu, P.-Y. Ng, K.-F. Lee, C.-L. Au, E. H.-Y. Ng, P.-C. Ho, and W. S.-B. Yeung Aberrant expression of angiopoietins-1 and -2 and vascular endothelial growth factor-A in peri-implantation endometrium after gonadotrophin stimulation Hum. Reprod., April 1, 2008; 23(4): 894 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Girling, F. L. Lederman, L. M. Walter, and P. A. W. Rogers Progesterone, But Not Estrogen, Stimulates Vessel Maturation in the Mouse Endometrium Endocrinology, November 1, 2007; 148(11): 5433 - 5441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Hur, J. Y. Lee, H.-S. Moon, and H. W. Chung Angiopoietin-1, angiopoietin-2 and Tie-2 expression in eutopic endometrium in advanced endometriosis Mol. Hum. Reprod., July 1, 2006; 12(7): 421 - 426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. N. Jabbour, R. W. Kelly, H. M. Fraser, and H. O. D. Critchley Endocrine Regulation of Menstruation Endocr. Rev., February 1, 2006; 27(1): 17 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sugino, T. Suzuki, A. Sakata, I. Miwa, H. Asada, T. Taketani, Y. Yamagata, and H. Tamura Angiogenesis in the Human Corpus Luteum: Changes in Expression of Angiopoietins in the Corpus Luteum throughout the Menstrual Cycle and in Early Pregnancy J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6141 - 6148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Nayak, C. J. Kuo, T. A. Desai, S. J. Wiegand, B. L. Lasley, L. C. Giudice, and R. M. Brenner Expression, localization and hormonal control of angiopoietin-1 in the rhesus macaque endometrium: potential role in spiral artery growth Mol. Hum. Reprod., November 1, 2005; 11(11): 791 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Voskas, N. Jones, P. Van Slyke, C. Sturk, W. Chang, A. Haninec, Y. O. Babichev, J. Tran, Z. Master, S. Chen, et al. A Cyclosporine-Sensitive Psoriasis-Like Disease Produced in Tie2 Transgenic Mice Am. J. Pathol., March 1, 2005; 166(3): 843 - 855. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||