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Departments of Pathology and Obstetrics and Gynecology (M.L.H., D.S.C.-J., C.L.K.C., S.K.S.) and Pharmacology (T.-P.D.F.), Reproductive Molecular Research Group, Cambridge, United Kingdom CB2 1QP; Women’s Reproductive Health Research Center, Vanderbilt University (K.L.B.-T., K.G.O.), Nashville, Tennessee 37232-2519; and Medical Research Council, Biostatistics Unit, Institute of Public Health (B.D.M.T.), Cambridge, United Kingdom CB2 2SR
Address all correspondence and requests for reprints to: Dr. Louise Hull, Departments of Pathology and Obstetrics and Gynecology, Reproductive Molecular Research Group, Tennis Court Road, Cambridge, United Kingdom CB2 1QP. E-mail: mlh30{at}cam.ac.uk.
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Introduction |
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The cause of endometriosis is unknown, but it is thought that endometrium, shed at the time of menstruation, passes backward along the fallopian tubes and into the abdominal cavity (4, 5). Here the explants establish a blood supply and grow. Indeed, a characteristic clinical feature of these lesions is the profusion of blood vessels that surround the ectopic endometrial tissue. Although most women have retrograde menstruation, only 10% of women develop endometriosis. It may be that women with endometriosis have a greater ability to develop a blood supply to ectopic endometrium than women without the disease. There is evidence that vascular development is important in the pathogenesis of endometriosis. Peritoneal fluid from women with endometriosis has greater angiogenic activity than fluid aspirated from women without the disease (6), and at least in the estrogenic proliferative phase of the menstrual cycle (7), this fluid contains high levels of the angiogenic growth factor, vascular endothelial cell growth factor A (VEGF-A).
The development of new blood vessels is a closely regulated process that involves the coordinated interaction of many gene families (8). The VEGF family is particularly important (9). VEGF-A, acting through the kinase domain receptor (VEGFR2), induces differentiation, proliferation, and migration of endothelial cells (10). The formation of these cells into tubes requires the Fms-like tyrosine kinase receptor (flt-1), or VEGFR1 (11). VEGF-A stimulates new vessel growth in a variety of physiological and pathological conditions (12, 13), but if VEGF-A support is withdrawn, endothelial cells undergo apoptosis, and the vessels regress (14). However, endothelial cells are only responsive to VEGF-A and its withdrawal if they are not surrounded by pericytes (12, 14). Pericytes are smooth muscle 
-actin (SMA)-containing, mesenchymal cells that are attracted to immature blood vessels by platelet-derived growth factor synthesized by endothelial cells (15). Pericyte-derived angiopoetin-1 prevents endothelial cell apoptosis and maintains vessel integrity (16). When endothelial cells are surrounded by pericytes, they become resistant to the withdrawal of VEGF-A.
In view of the importance of blood vessels to the establishment and subsequent growth of endometriotic lesions, we sought to test the hypothesis that antiangiogenic agents may be effective in preventing ectopic endometrial growth. We chose to specifically antagonize VEGF-A because of the elevated levels of VEGF-A in peritoneal fluid of women with endometriosis and its importance in other angiogenesis-dependant conditions. To evaluate the likely success of this approach, we also wanted to determine the VEGF-A responsiveness of vessels that supply ectopic endometrial lesions.
To address these questions we quantified the number of blood vessels that supply the full thickness of human endometrium and determined whether the endothelial cells were associated with pericytes. Eutopic and ectopic endometria from women with ovarian endometriosis were compared in the same way. We then used an in vivo model of endometriosis in which human endometrium is implanted into nude mice (17, 18, 19) to test the effect of VEGF-A antagonists on lesion formation. Several groups have shown that lesions taken from this model are macroscopically and microscopically similar to endometriotic lesions in women (17, 18, 19). We initially determined that the endothelial cells in vessels supplying these lesions were pericyte free and thus responsive to VEGF-A withdrawal. Both soluble flt-1 (20), a competitive inhibitor of VEGF-A (21), and antihuman VEGF-A antibodies were shown to inhibit lesion formation in this model.
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Methods and Materials |
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Ulex europaeus agglutinin-1 (UEA-1) and biotinylated UEA-1 were obtained from Vector Laboratories, Inc. (Burlingame, CA); anti-SMA (clone 1A4) was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Rabbit immunoglobulins (Igs) and anti-von Willebrand factor (anti-vWF) were obtained from DAKO Corp. (Ely, UK). The isotype-matched mouse immunoglobulin G was obtained from Harlan Sera-Lab Ltd. (Loughborough, UK). MRC-OX43 was obtained from Covance Laboratories, Inc. (Maidenhead, UK).
Secondary antibodies
Biotinylated goat antirabbit immunoglobulins, biotinylated rabbit antimouse immunoglobulins, and goat anti-UEA-1 were obtained from Vector Laboratories, Inc. Biotinylated rabbit antigoat antibody was purchased from Zymed Laboratories, Inc. (San Francisco, CA). Fluorescent secondary antibodies were obtained from the same suppliers: fluorescein isothiocyanate (FITC)-streptavidin and tetramethylrhodamine isothiocyanate (TRITC)-rabbit antimouse immunoglobulins from DAKO Corp., streptavidin-TRITC from Sigma-Aldrich Corp., and FITC-donkey antirabbit immunoglobulins and Cy5-goat antimouse immunoglobulins from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Vectorsheild, the Vectorstain ABC Kit, and the Vectorstain Mouse on Mouse Kit were also purchased from Vector Laboratories, Inc. Goat and rabbit sera, trypsin, BSA, fructose, and diaminobenzidine (DAB) were purchased from Sigma-Aldrich Corp.
Other reagents
VEGF-A, basic fibroblast growth factor (bFGF), goat immunoglobulins, anti-VEGF and affinity-purified goat antihuman VEGF, and goat antimurine VEGF antibodies were purchased from R&D Systems (Abington, UK). Soluble flt-1 receptor was obtained from Metris Therapeutics Ltd. (Wokingham, UK). Phenol red-free DMEM/Ham’s F-12 medium and estradiol were obtained from Sigma-Aldrich Corp., Excyte from Miles, Inc. (Kankakee, IL), insulin-transferrin-selenium (ITS) from Collaborative Biomedical Products (Bedford, MA), and culture inserts from Millipore Corp. (Bedford, MA). Slow release estrogen pellets were purchased from Innovative Research (Sarasota, FL), and Metofane was obtained from Pittman-Moore (Toledo, OH. 133Xe was obtained from Amersham International (Little Chalfont, UK).
Experiment 1: morphometric analysis of endometrial and endometriotic blood vessels
The local ethical committee of Addenbrooke’s Hospital National Health Service Trust (Cambridge, UK) approved this study. To characterize the blood vessels supplying eutopic endometrium and to determine whether the nature of the vessels changed during the normal menstrual cycle, 16 women with regular menstrual cycles (25–30 d in length), undergoing hysterectomy for benign gynecological conditions were recruited to the study. Eight were in the follicular phase of the cycle, and eight were in the secretory phase, as judged by the date from the last menstrual period and histological examination of an endometrial biopsy (22). The median age of women in the proliferative phase was 42 yr (range, 35–46 yr), and that for women in the secretory phase was 40 yr (range, 34–43 yr). The indications for hysterectomy included painful (n = 6) or heavy (n = 6) periods, prolapse of the uterus (n = 2), chronic pelvic pain (n = 1), and prophylactic hysterectomy because of a family history of ovarian cancer (n = 1). No gynecological pathology was identified in any of the specimens.
To compare blood vessels in endometriotic lesions with blood vessels in uterine endometrium, eight additional patients were recruited to the study. These women were diagnosed as having peritoneal endometriosis involving the ovaries and were scheduled for total abdominal hysterectomy and bilateral salpingo-ophorectomy. All women had regular menstrual cycles, and none was taking medication. The median age of this group was 42 yr (range, 36–49 yr), and histological examination of the uterine endometrium confirmed that four women were in the proliferative phase of the cycle and four were in the secretory phase (22). Endometriosis was diagnosed histologically in all cases in the ovarian specimens.
Endothelial cells and pericytes were identified immunohistochemically. A 36-point grid was used to standardize the morphometric analysis of blood vessels. The proportion of the endometrium occupied by endothelial cells (the volume fraction) was determined by counting the endothelial cells coincident with the points on the overlaid grid. This method is unaffected by tissue swelling or shrinkage. The volume fraction of endothelial cells alone or in association with pericytes was estimated in superficial and deep uterine endometria and ectopic endometria. Ten different fields were counted, and the mean of the observations was used for further analysis.
Experiment 2: inhibition of blood vessel development by antiangiogenic agents
Sponge implantation model. To validate the efficacy of our anti-VEGF reagents in vivo, an implanted sponge model was used (23). Two sterile circular polyether sponge discs were implanted sc in the dorsum of anesthetized male Wistar rats (180–200 g). Cannulas attached to the center of the disc were exteriorized and plugged with sterile polyethylene stoppers. Rats were housed individually and given a normal diet and water ad libitum. Test substances in 50 µl sterile PBS were administered daily through the cannulae for 13 d. To exclude possible vasomotor effects, test substances were injected at least 16 h before 133Xe clearance measurements. Synergistic doses of VEGF-A (25 ng) and FGF (10 ng) were injected with or without either the soluble flt-1 (sflt-1) receptor (6.25 or 100 ng) or anti-VEGF monoclonal antibody (6.25 or100 ng). Soluble flt-1 or anti-VEGF antibody was also administered alone.
Sponge blood flow determination by a 133Xe clearance technique. Blood flow through the sponge implants was assessed using a 133Xe clearance technique (23). 133Xe (0.5µCi) was injected into each sponge. The washout of radioactivity from the sponge was measured for 10 min with a collimated scintillation detector.
Endothelial volume fraction estimation. The endothelial volume fraction was determined by counting the number of endothelial cells at the intersections of a 36-point grid in 6 random fields in each of the 2 sponges present in the 6–10 animals/group. Endothelial cells were identified immunohistochemically.
Experiment 3: antagonism of ectopic endometrial growth using antiangiogenic molecules in an experimental model of endometriosis
Preparation of human endometrial explants. Ethical approval for the use of human tissue was obtained from Vanderbilt University’s institutional review board and committee for the protection of human subjects. The Vanderbilt institutional animal care and use committee approved this animal study. Endometrial tissue was obtained by endometrial suction curettage (Pipelle, Unimar, Inc., Wilton, CT) from volunteers (age, 21–45 yr) with regular menstrual cycles on d 9–12 of their cycle. To ensure the endometrium was proliferative, a serum progesterone level less than 1.5 ng/ml was required. Endometrial biopsies were immediately washed in prewarmed phenol red-free DMEM/Ham’s F-12.
Endometrial biopsies were sectioned into uniform 1- to 2-mm3 pieces, and 8–10 fragments were suspended as explant cultures in serum-free DMEM/Ham’s F-12 medium supplemented with 1% ITS, 0.1% Excyte, and 10 nM estradiol and incubated overnight at 37 C in 95% air/5% CO2.
Nude mouse model of endometriosis. This model, with minor modifications, has been described previously (17). Five-week-old athymic (nude) ovariectomized mice (20–25 g) were purchased from Taconic Laboratories (Germantown, NY) and housed in individually ventilated cages. Animals were anesthetized using Metofane and were operated on in a sterile environment. Twenty-four to 96 h before the injection of human tissue, estradiol-releasing pellets (1.5 mg/60-d release) were implanted sc just below the scapula.
In vitro treatment and in vivo injection of human endometrial tissue. Human endometrial tissue, maintained for 24 h in culture, was washed with sterile PBS before injection. Each animal received a single injection of 8–10 endometrial fragments in 200 µl sterile PBS as described by Zamah et al. (24). Endometrial fragments were injected sc using tuberculin syringes and 18-gauge needles at a site on the ventral midline below the umbilicus. Immediately after tissue injections, mice received an sc injection of the appropriate experimental or control protein in 100 µl PBS.
In experiment 1, explant cultures additionally received 50 µg/ml goat IgG (group 1), 50 µg/ml affinity-purified antihuman VEGF antibody (group 2), 50 µg/ml affinity-purified antimurine VEGF antibody (group 3), and 25 µg/ml each of affinity-purified antihuman and antimurine antibodies (group 4).
Explant cultures used in experiment 2 received vehicle (group 1), 50 µg/ml of soluble flt-1 in PBS (group 2), VEGF-A (250 ng/ml) and FGF (100 ng/ml; group 3), or VEGF-A (250 ng/ml), FGF (100 ng/ml), and soluble flt-1 receptor (50 µg/ml; group 4).
Groups of mice in the first experiment received nonspecific goat IgGs matched to antihuman VEGF antibody (5 µg; group 1), antihuman VEGF antibody (5 µg; group 2), antimurine VEGF-A antibody (5 µg; group 3), or both antihuman and anti-murine VEGF (2.5 µg each; group 4).
In the second experiment the four treatment groups received PBS (group 1), soluble flt-1 (5 µg; group 2), VEGF (25 ng) and FGF (10 ng; group 3), or VEGF (25 ng), FGF (10 ng), and soluble flt-1 (5 µg; group 4). For the next 9 d, mice received the same sc injections at approximately the same time of day. On d 11, 24 h after the last injection, the mice were killed.
Examination of lesions. The peritoneum and visceral organs of animals were examined using a dissecting microscope. All potential lesions were removed and immediately placed in 10% ice-cold buffered formalin. Hematoxylin and eosin staining of these lesions revealed the histological characteristics of endometriosis. Any tissues removed that did not contain both glandular and stromal elements (i.e. fibrous adhesions) were excluded from the study.
Immunohistochemistry
Endothelial cells and pericytes were identified immunohistochemically in human eutopic and ectopic endometrial tissue, sponge explants from Wistar rats, and human endometrial explants from the nude mouse model of endometriosis. All tissues were formalin fixed, paraffin embedded, then cut into 5-µm sections. Before staining, all tissues were dewaxed and rehydrated. Antigen retrieval was performed by pressure cooking at maximum pressure in 0.1 M sodium citrate (human endometrial and Wistar rat sponge sections) or by enzymatic digestion with 0.1% trypsin in a 0.1% CaCl/PBS solution (nude mouse explants).
Nonspecific binding was blocked with 10% goat serum (Wistar rat and some nude mouse sections) or 5% rabbit serum (human endometrial sections) in 0.1% BSA in PBS. Mouse-on-mouse blocking reagent was used for anti-SMA antibody staining of nude mouse lesions.
UEA-1 was used to identify human endothelial cells. UEA-1 preincubated with a 200-fold molar excess of fructose was the negative control. In human endometrial samples, unconjugated UEA-1 (10 µg/ml) was detected with goat anti-UEA antibody (10 µg/ml), a biotinylated rabbit antigoat antibody (25 µg/ml), then an FITC-streptavidin conjugate (7.5 µg/ml). Biotinylated UEA-1 (10 µg/ml) was used to stain nude mouse lesions, and this was detected with streptavidin-TRITC (31.2 µg/ml) or the Vectastain ABC system with DAB as the final substrate.
The antibody MRC-OX43 (1:25) was used to identify Wistar rat endothelial cells in sponge explants. Rabbit Igs ensured primary antibody specificity. Biotinylated antirabbit secondary antibody was applied and identified with Vector Laboratories, Inc., ABC Kit and DAB.
vWF (5.7 µg/ml) was used to differentiate human endothelial cells (that stain positively for both vWF and UEA-1) from murine endothelial cells (that only stain positively for vWF) in explants taken from nude mice. Rabbit Igs (5.7 µg/ml) served as a negative control. The secondary antibody was either biotinylated goat antirabbit (7.5 µg/ml) detected by the Vectastain ABC system or donkey antirabbit FITC (15 µg/ml).
An SMA (clone 14A) mouse monoclonal antibody (2.5 µg/ml) identified pericytes. The negative control was an isotype-matched mouse IgG (2.5 µg/ml). In human endometrium, anti-SMA was detected with a TRITC-conjugated rabbit antimouse antibody (7.5 µm/ml; Vector Laboratories, Inc.). In nude mouse lesions either goat antimouse-Cy5 (14 µg/ml) or a Mouse on Mouse kit was used.
Quenching of endogenous peroxidases (with 3% hydrogen peroxidase in methanol) was performed before antibody detection with Vector Laboratories, Inc., ABC or Mouse on Mouse kits. Sections were counterstained with Corezzi’s hematoxylin, then rehydrated and mounted in Depex. Microscopy was performed with an Ultraphot microscope (Carl Zeiss, New York, NY).
In human eutopic and ectopic tissue, concurrent double fluorescent staining with UEA-1 and SMA was used to identify endothelial cells and pericytes. A triple-fluorescent stain was performed on sections from nude mouse lesions whereby UEA-1 lectin and vWF antibodies were concurrently applied, followed by anti-SMA antibodies. Fluorescently stained sections were mounted and counterstained in Vectorshield with Dapi. A TCS-NT confocal microscope (Leica Corp., Deerfield, IL) identified the fluorescent markers.
Statistical analysis
To provide a statistically integrated approach that accounts for multiple testing, adjusts for the effects of other variables, and addresses the correlation between measurements from an individual, linear mixed effects models (25) were fitted to the data from the 16 women without endometriosis. Both the total vessel volume fraction of the endothelial cells (i.e. the sum of the volume fraction of the endothelial cells with and without pericyte association) and the proportion of pericyte-free vessels were compared in the tissue layers in both phases of the menstrual cycle. This modeling approach was then repeated after including the 8 eutopic endometrial samples (basal and superficial) from the women with endometriosis with the 16 control samples. Initially, the interactions between cycle phase and endometrial layer were tested. However they were found to be nonstatistically significant, so the final models included only the main effects of cycle phase and endometrial layer. Linear mixed effects models were applied to investigate the effects of basal, superficial, and ectopic tissues from the 8 women with endometriosis on the total vessel volume fraction and the proportion of pericyte-free vessels. Statistical significance was assessed at the 5% level.
For analysis of 133Xe clearance from the rat sponge implants, the data were log-transformed to diminish the impact of extreme 133Xe clearance measurements (25), and a linear mixed effects model was fitted, assuming different slope and intercept parameters for the groups over time. Data for endothelial cell counts from morphometric analysis of immunostained sections were analyzed using a Poisson regression modeling approach.
Logistic regression models were used to analyze the effect of exposure of antihuman VEGF antibodies and antimurine antibodies on the presence or absence of lesions in nude mice. In this experiment all mice had only one lesion; therefore, a modeling approach that analyses binary data was used (25).
Several of the mice were shown to have more than one lesion in the experiment in which mice were exposed to soluble flt-1 with or without VEGF/FGF supplementation. To incorporate this information, a Poisson regression model was used to analyze the count data (25).
Statistical significance was determined at the 5% level, and 95% confidence intervals (CI) for the odds ratios and rate ratios are reported.
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Results |
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All P values in the subsequent text refer to the linear effects model. The volume fraction of endometrium occupied by blood vessels was determined in the histologically normal endometrium taken from 16 women throughout the menstrual cycle. The vessel volume fraction in the superficial zone of the endometrium was significantly lower than that found in the basal part of the endometrium (P < 0.0001; Fig. 1
, I and J). The fraction of blood vessels did not change throughout the cycle in either the superficial or basal zone of the endometrium (P < 0.66). However, examination of eutopic and ectopic endometria showed an increased blood vessel volume fraction in the endometriotic lesions compared with the patients’ own superficial eutopic endometrium (P < 0.002).
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). In ectopic endometrium (n = 8) the proportion of vessels without pericyte association was significantly higher than that in both basal (P < 0.003) and superficial eutopic (P < 0.045) endometria from the same eight women. There was no significant difference in the proportion of pericyte-free cells between normal eutopic endometrium (n = 16) and eutopic endometrium from women with endometriosis (n = 8) in either the superficial (P < 0.99) or basal (P < 0.80) layers (Fig. 1, H–J).
The proportion of immature vessels was also determined for the 16 control women. There was no evidence of any difference in the total vessel volume fraction or the proportion of immature vessels between control endometrium (n = 16) and eutopic endometrium taken from women with endometriosis (n = 8). When eutopic endometrium from women with endometriosis was included in the analysis (n = 24), a significantly higher proportion of vessels were pericyte free in the superficial layer of the endometrium compared with the basal layer (P = 0.04; Fig. 1, H–J).
Experiment 2: inhibition of blood vessel development by antiangiogenic agents
The rat sponge model validated the in vivo efficacy of the anti-VEGF-A reagents used in these experiments. VEGF-A and bFGF stimulated angiogenesis in the implanted sponge model. There was a significantly higher 133Xe clearance (linear mixed effects model; P < 0.001; Fig. 2A) in sponges from rats treated with VEGF-A/FGF compared with untreated controls, and this was matched by increased endothelial cell staining in these sponges (Fig. 2, B and E). The growth factor-induced increase in blood flow was virtually abolished by treatment with low dose soluble flt-1 (linear mixed effects model; P < 0.001). Anti-VEGF-A antibody also reduced this effect in a dose-dependent manner (Fig. 2A). This was paralleled by a reduction in the density of endothelial cells in the sponge when soluble flt-1 or VEGF-A antibody was administered in either a low (by Poisson regression analysis, P < 0.06; Fig. 2C) or high (by Poisson regression analysis, P < 0.001; Fig. 2D) dose compared with the VEGF-A/bFGF-stimulated tissue.
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The staining specificity of UEA-1 for human endothelial cells, that of vWF for human and mouse endothelial cells, and that of SMA for pericytes were demonstrated in human endometrium and nude mouse peritoneum (Fig. 3). Macroscopically, multiple blood vessels were seen surrounding transplanted human endometrial explants in nude mice (Fig. 5A). Examination of the human endometrial explants removed from mice that received estradiol alone showed that most of the endothelial cells forming the blood vessels were of murine origin (i.e. they were vWF positive, but negative for UEA-1; Fig. 4, A–F and J). A small number of endothelial cells located centrally in the lesions stained with UEA-1, indicating their human origin (Fig. 4, A–C). Very few of the vessels in the explants had an SMA-positive pericytic covering (Fig. 4, G–I and K), further confirming the similarity of these lesions to those found in women with the disease.
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VEGF-A and its transcript were shown to be present in human endometrial lesions in nude mice by immunohistochemical analysis and in situ hybridization (data not shown). The nude mouse model of endometriosis was used to determine whether anti-VEGF-A agents could inhibit explant formation by antagonizing the action of VEGF-A. Initially, an antibody raised against human VEGF-A (anti-VEGF-A antibody) was shown to reduce the growth of the endometrial lesions (Table 1).
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Soluble flt-1 receptor antagonism of VEGF-A in the nude mouse model of endometriosis
Soluble flt-1 also significantly reduced the number of endometrial explants in nude mice (by Poisson regression analysis, P = 0.002; RR, 0.28; 95% CI, 0.12–0.61; Table 2). The additional injection of VEGF-A and FGF did not alter this effect (by Poisson regression analysis, P = 0.65). Endometrial lesions could not be identified in the majority of nude mice treated with soluble flt-1. However, histological examination of the small number of lesions seen (Fig. 5B) revealed a cystic-like space centrally that was surrounded by a rim of dense, mesothelial-like cells. The glandular epithelium had partially degenerated, and necrotic debris was present in the cystic space. Staining for UEA-1, vWF, and SMA was greatly reduced, demonstrating a virtual absence of the microvasculature (Fig. 5, E, F, I, J, M, and N).
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Like lesions taken from nude mice, human endometriosis has a vascular supply consisting of endothelial cells without an extensive pericytic layer. Human endometriosis is therefore likely to respond to antiangiogenic treatment in a similar way to the response seen in nude mice. Other in vivo models have demonstrated the response of immature vessels to VEGF-A antagonism. In the rat retina, such immature vessels regress when exposed to hyperoxia. The presence of VEGF-A prevents this regression, promoting the survival and growth of these pericyte-free vessels (12). In a tumor model, if VEGF-A is withdrawn, this effect is lost, and vasculature disruption is seen (14).
VEGF-A regulates the proliferation and survival of endothelial cells by the VEGFR-2 receptor (10). Both sflt-1 and the anti-VEGF-A antibody bind VEGF-A, preventing the ligand from accessing the VEGFR-2 receptor. The soluble flt-1 receptor may have additionally acted in a dominant negative manner to block VEGF-A action (26) as the Ig-like domains of the soluble VEGFR-1 can form heterodimers with VEGFR-2. In this study antagonism of just VEGF-A was sufficient to prevent the growth of explants despite the fact that human endometrium expresses other members of the VEGF family of genes as well as angiotensin-1 and -2 (27). This presumably reflects the overriding potency of VEGF-A in the growth of endometrial blood vessels, at least at ectopic sites.
Increased proliferation of endothelial cells may only partially account for VEGF-A’s ability to maintain an immature vasculature. Endothelial cell survival is also promoted by VEGF-A due to its antiapoptotic actions. VEGF-A increases the expression of the antiapoptotic genes, Bcl-2, A-1 (28), XIAP, and survivin (29); activates the prosurvival phosphoinositol 3-kinase/Akt-1 pathway (30); and inhibits Ets- and TNF-induced apoptosis (31). It may be that sflt-1 predominantly antagonized VEGF-A’s antiapoptotic actions, thereby decreasing the survival of vascular endothelial cells in nude mouse explants.
This study also shows that most of the endothelial cells that constitute the blood vessels in explants are derived from the host mouse. The origin of these endothelial cells is less clear. They could be derived from proliferating blood vessels situated close to the lesion. If so, these cells are probably recruited by human VEGF-A secreted from the lesion itself, because antihuman, but not antimurine, VEGF-A antibody blocked the growth of the explants. It may be that ischemia in the implanted human endometrial explant up-regulates the transcription of human VEGF-A (32). These findings argue against the local release of murine VEGF-A in response to trauma or from murine macrophages attracted to the explant site. This aspect of the model may differ from the human situation, in which activated macrophages have a role in the increased levels of VEGF-A found in the peritoneal fluid of endometriotic patients (7).
Alternatively, bone marrow-derived endothelial progenitor cells (33), attracted by VEGF-A and inflammatory chemokines in the deposited endometrium (34), may have contributed to the new vessels. Elevated levels of VEGF-A can recruit both peripheral endothelial progenitor cells and hemopoietic endothelial cell precursors. Using Id mutant mice, Lyden et al. (35) showed that VEGF-A recruits different populations of endothelial cell progenitors during tumor angiogenesis. Indeed, approximately 90% of the endothelial cells present in explanted tumors 2 d after implantation were bone marrow derived. Circulating endothelial precursor cells, induced to enter the peripheral circulation by VEGF-A, were as effective in this regard as cells derived from the bone marrow. These VEGF-A-mobilized endothelial-like cells expressed the endothelial markers VEGFR2 and VE-cadherin.
A further possibility for the action of VEGF-A may arise from its ability to determine the differentiation state of peripheral CD14+ monocytes (36). In the presence of GM-CSF and IL-4, these cells differentiate into immature dendritic cells expressing the markers, CD1a, human leukocyte antigen-DR, and CD86. Further lineage progression is mediated by TNF. However, VEGF-A impairs this progression by down-regulating the expression of CD1a and inducing the cells to express endothelial markers, including vWF, VEGFR1, VEGFR2, CD105, and VE-cadherin (37).
In the cancer model (35), antibodies to VEGFR1 did not inhibit blood vessel numbers, but did reduce the accumulation of myelo-monocytic cells around the vessels. Anti-VEGFR2, on the other hand, did inhibit blood vessel growth. In this study both anti-VEGF-A and soluble flt-1 inhibited blood vessel growth. These findings suggest that anti-VEGF agents may be successful in treating endometriosis by both blocking blood vessel growth and at the same time inhibiting the accumulation of macrophages at the explant site.
In women with endometriosis, VEGF-A was only found to be elevated in peritoneal fluid in the proliferative phase of the cycle (7). It may be that VEGF-A’s influence on blood vessel growth in endometriosis is dependent on the steroidal environment. Lesion formation in the nude mouse model of endometriosis was dramatically reduced when mice were exposed to progesterone (17, 24). Antihuman antibody was less effective in reducing lesion formation in the nude mouse model when the tissue explants had been exposed to progesterone (data not shown). This suggests that anti-VEGF-A therapy may be more effective if given to women in the proliferative, rather than the secretory, phase of the menstrual cycle.
As shown in this study, a large number of the blood vessels supplying endometrial explants in women are immature. Thus, anti-VEGF-A agents have the potential to disrupt the vasculature of endometriotic lesions in women. These studies show for the first time that antiangiogenic agents are likely to be effective agents in the treatment of endometriosis. They provide a new opportunity for managing this debilitating disease. Further studies are now needed to demonstrate the efficacy of this approach in clinical practice.
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Acknowledgments |
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Footnotes |
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M.L.H. and D. S.C.-J. are joint first authors.
Abbreviations: bFGF, Basic fibroblast growth factor; CI, confidence interval; DAB, diaminobenzidine; FITC, fluorescein isothiocyanate; ITS, insulin-transferrin-selenium; Ig, immunoglobulin; sflt-1, soluble flt-1; SMA, anti-smooth muscle -actin; UEA-1, Ulex europaeus agglutinin-1; VEGF-A, vascular endothelial cell growth factor A; VEGFR, vascular endothelial cell growth factor receptor; vWF, von Willebrand factor.
Received December 9, 2002.
Accepted February 26, 2003.
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M. W. Laschke, C. Schwender, C. Scheuer, B. Vollmar, and M. D. Menger Epigallocatechin-3-gallate inhibits estrogen-induced activation of endometrial cells in vitro and causes regression of endometriotic lesions in vivo Hum. Reprod., October 1, 2008; 23(10): 2308 - 2318. [Abstract] [Full Text] [PDF]
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 H. M. Fraser, H. Wilson, A. Silvestri, K. D. Morris, and S. J. Wiegand The Role of Vascular Endothelial Growth Factor and Estradiol in the Regulation of Endometrial Angiogenesis and Cell Proliferation in the Marmoset Endocrinology, September 1, 2008; 149(9): 4413 - 4420. [Abstract] [Full Text] [PDF]
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 M. L. Hull, C. R. Escareno, J. M. Godsland, J. R. Doig, C. M. Johnson, S. C. Phillips, S. K. Smith, S. Tavare, C. G. Print, and D. S. Charnock-Jones Endometrial-Peritoneal Interactions during Endometriotic Lesion Establishment Am. J. Pathol., September 1, 2008; 173(3): 700 - 715. [Abstract] [Full Text] [PDF]
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 S. Defrere, J.C. Lousse, R. Gonzalez-Ramos, S. Colette, J. Donnez, and A. Van Langendonckt Potential involvement of iron in the pathogenesis of peritoneal endometriosis Mol. Hum. Reprod., July 1, 2008; 14(7): 377 - 385. [Abstract] [Full Text] [PDF]
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A. Van Langendonckt, J. Donnez, S. Defrere, G. A.J. Dunselman, and P. G. Groothuis Antiangiogenic and vascular-disrupting agents in endometriosis: pitfalls and promises Mol. Hum. Reprod., May 1, 2008; 14(5): 259 - 268. [Abstract] [Full Text] [PDF]
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 O. Fainaru, A. Adini, O. Benny, I. Adini, S. Short, L. Bazinet, K. Nakai, E. Pravda, M. D. Hornstein, R. J. D'Amato, et al. Dendritic cells support angiogenesis and promote lesion growth in a murine model of endometriosis FASEB J, February 1, 2008; 22(2): 522 - 529. [Abstract] [Full Text] [PDF]
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C. M. Becker, N. Rohwer, T. Funakoshi, T. Cramer, W. Bernhardt, A. Birsner, J. Folkman, and R. J. D'Amato 2-Methoxyestradiol Inhibits Hypoxia-Inducible Factor-1{alpha} and Suppresses Growth of Lesions in a Mouse Model of Endometriosis Am. J. Pathol., February 1, 2008; 172(2): 534 - 544. [Abstract] [Full Text] [PDF]
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 G. Zhang, N. Dmitrieva, Y. Liu, K. A. McGinty, and K. J. Berkley Endometriosis as a neurovascular condition: estrous variations in innervation, vascularization, and growth factor content of ectopic endometrial cysts in the rat Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R162 - R171. [Abstract] [Full Text] [PDF]
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A. Van Langendonckt, C. Punyadeera, R. Kamps, G. Dunselman, L. Klein-Hitpass, L.J. Schurgers, J. Squifflet, J. Donnez, and P. Groothuis Identification of novel antigens in blood vessels in rectovaginal endometriosis Mol. Hum. Reprod., December 1, 2007; 13(12): 875 - 886. [Abstract] [Full Text] [PDF]
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G. Sha, D. Wu, L. Zhang, X. Chen, M. Lei, H. Sun, S. Lin, and J. Lang Differentially expressed genes in human endometrial endothelial cells derived from eutopic endometrium of patients with endometriosis compared with those from patients without endometriosis Hum. Reprod., December 1, 2007; 22(12): 3159 - 3169. [Abstract] [Full Text] [PDF]
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J. Gilabert-Estelles, L.A. Ramon, F. Espana, J. Gilabert, V. Vila, E. Reganon, R. Castello, M. Chirivella, and A. Estelles Expression of angiogenic factors in endometriosis: relationship to fibrinolytic and metalloproteinase systems Hum. Reprod., August 1, 2007; 22(8): 2120 - 2127. [Abstract] [Full Text] [PDF]
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 M. W. Laschke and M. D. Menger In vitro and in vivo approaches to study angiogenesis in the pathophysiology and therapy of endometriosis Hum. Reprod. Update, July 1, 2007; 13(4): 331 - 342. [Abstract] [Full Text] [PDF]
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M. Oktem, I. Esinler, D. Eroglu, N. Haberal, N. Bayraktar, and H. B. Zeyneloglu High-dose atorvastatin causes regression of endometriotic implants: a rat model Hum. Reprod., May 1, 2007; 22(5): 1474 - 1480. [Abstract] [Full Text] [PDF]
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 H. Masuda, T. Maruyama, E. Hiratsu, J. Yamane, A. Iwanami, T. Nagashima, M. Ono, H. Miyoshi, H. J. Okano, M. Ito, et al. Noninvasive and real-time assessment of reconstructed functional human endometrium in NOD/SCID/{gamma}Formula immunodeficient mice PNAS, February 6, 2007; 104(6): 1925 - 1930. [Abstract] [Full Text] [PDF]
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 A. E. Roberts, L. K. Arbogast, C. I. Friedman, D. E. Cohn, P. T. Kaumaya, and D. R. Danforth Neutralization of Endogenous Vascular Endothelial Growth Factor Depletes Primordial Follicles in the Mouse Ovary Biol Reprod, February 1, 2007; 76(2): 218 - 223. [Abstract] [Full Text] [PDF]
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R. Grummer Animal models in endometriosis research Hum. Reprod. Update, September 1, 2006; 12(5): 641 - 649. [Abstract] [Full Text] [PDF]
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C. M. Becker, R. D. Wright, R. Satchi-Fainaro, T. Funakoshi, J. Folkman, A. L. Kung, and R. J. D'Amato A Novel Noninvasive Model of Endometriosis for Monitoring the Efficacy of Antiangiogenic Therapy Am. J. Pathol., June 1, 2006; 168(6): 2074 - 2084. [Abstract] [Full Text] [PDF]
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K. L. Bruner-Tran, Z. Zhang, E. Eisenberg, R. C. Winneker, and K. G. Osteen Down-Regulation of Endometrial Matrix Metalloproteinase-3 and -7 Expression in Vitro and Therapeutic Regression of Experimental Endometriosis in Vivo by a Novel Nonsteroidal Progesterone Receptor Agonist, Tanaproget J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1554 - 1560. [Abstract] [Full Text] [PDF]
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S. Defrere, A. Van Langendonckt, R. G. Ramos, M. Jouret, M. Mettlen, and J. Donnez Quantification of endometriotic lesions in a murine model by fluorimetric and morphometric analyses Hum. Reprod., March 1, 2006; 21(3): 810 - 817. [Abstract] [Full Text] [PDF]
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Y.-J. Lin, M.-D. Lai, H.-Y. Lei, and L.-Y. C. Wing Neutrophils and Macrophages Promote Angiogenesis in the Early Stage of Endometriosis in a Mouse Model Endocrinology, March 1, 2006; 147(3): 1278 - 1286. [Abstract] [Full Text] [PDF]
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M.W. Laschke, A. Elitzsch, B. Vollmar, P. Vajkoczy, and M.D. Menger Combined inhibition of vascular endothelial growth factor (VEGF), fibroblast growth factor and platelet-derived growth factor, but not inhibition of VEGF alone, effectively suppresses angiogenesis and vessel maturation in endometriotic lesions Hum. Reprod., January 1, 2006; 21(1): 262 - 268. [Abstract] [Full Text] [PDF]
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M. Bhanoori, K. Arvind Babu, N.G. Pavankumar Reddy, K. Lakshmi Rao, K. Zondervan, M. Deenadayal, S. Kennedy, and S. Shivaji The vascular endothelial growth factor (VEGF) +405G>C 5'-untranslated region polymorphism and increased risk of endometriosis in South Indian women: a case control study Hum. Reprod., July 1, 2005; 20(7): 1844 - 1849. [Abstract] [Full Text] [PDF]
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R. Matsuura-Sawada, T. Murakami, Y. Ozawa, H. Nabeshima, J.-i. Akahira, Y. Sato, Y. Koyanagi, M. Ito, Y. Terada, and K. Okamura Reproduction of menstrual changes in transplanted human endometrial tissue in immunodeficient mice Hum. Reprod., June 1, 2005; 20(6): 1477 - 1484. [Abstract] [Full Text] [PDF]
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H. A. Harris, K. L. Bruner-Tran, X. Zhang, K. G. Osteen, and C. R. Lyttle A selective estrogen receptor-{beta} agonist causes lesion regression in an experimentally induced model of endometriosis Hum. Reprod., April 1, 2005; 20(4): 936 - 941. [Abstract] [Full Text] [PDF]
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M.L. Hull, A. Prentice, D.Y. Wang, R.P. Butt, S.C. Phillips, S.K. Smith, and D.S. Charnock-Jones Nimesulide, a COX-2 inhibitor, does not reduce lesion size or number in a nude mouse model of endometriosis Hum. Reprod., February 1, 2005; 20(2): 350 - 358. [Abstract] [Full Text] [PDF]
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C. Print, R. Valtola, A. Evans, K. Lessan, S. Malik, and S. Smith Soluble factors from human endometrium promote angiogenesis and regulate the endothelial cell transcriptome Hum. Reprod., October 1, 2004; 19(10): 2356 - 2366. [Abstract] [Full Text] [PDF]
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 N. Ferrara Vascular Endothelial Growth Factor: Basic Science and Clinical Progress Endocr. Rev., August 1, 2004; 25(4): 581 - 611. [Abstract] [Full Text] [PDF]
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K. J. Berkley, N. Dmitrieva, K. S. Curtis, and R. E. Papka Innervation of ectopic endometrium in a rat model of endometriosis PNAS, July 27, 2004; 101(30): 11094 - 11098. [Abstract] [Full Text] [PDF]
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A. Van Langendonckt, F. Casanas-Roux, J. Eggermont, and J. Donnez Characterization of iron deposition in endometriotic lesions induced in the nude mouse model Hum. Reprod., June 1, 2004; 19(6): 1265 - 1271. [Abstract] [Full Text] [PDF]
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A. W. Nap, A. W. Griffioen, G. A. J. Dunselman, J. C. A. Bouma-Ter Steege, V. L. J. L. Thijssen, J. L. H. Evers, and P. G. Groothuis Antiangiogenesis Therapy for Endometriosis J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1089 - 1095. [Abstract] [Full Text] [PDF]
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R. Varma, T. Rollason, J. K Gupta, and E. R Maher Endometriosis and the neoplastic process Reproduction, March 1, 2004; 127(3): 293 - 304. [Abstract] [Full Text] [PDF]
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