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RESEARCH |
Centre for Women’s Health Research, Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australia
Correspondence should be addressed to J Girling; Email: jane.girling{at}med.monash.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionAcknowledgementsReferences |
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Although neither epithelial, stromal nor total VEGF expression has been found to correlate with endometrial endothelial cell proliferation (Gargett et al. 1999), foci of intense VEGF immunostaining have been observed within the human endometrium; many of these foci were within blood vessels. Using immunocytochemical techniques on full-thickness endometrial sections, the focal VEGF was found to correlate both temporally and spatially with endometrial endothelial cell proliferation (Gargett et al. 2001). A significantly greater percentage of focal VEGF-expressing microvessels was found during the proliferative phase in comparison with the secretory phase, with the greatest numbers of immunopositive vessels within the subepithelial capillary plexus. There was also a significant correlation between focal VEGF-expressing microvessels and proliferating vessels in all three regions of the endometrium (subepithelial capillary plexus, functionalis and basalis). The focal VEGF associated with microvessels was found to correspond to marginating and adherent neutrophils and it was hypothesised that these leukocytes provide a source of VEGF for the endometrial vessels undergoing proliferation (Mueller et al. 2000, Gargett & Rogers 2001, Gargett et al. 2001).
The aim of this study was to investigate the role of intravascular neutrophils in oestrogen-mediated endometrial angiogenesis in mice. Previous research has shown that oestrogen stimulates endometrial endothelial cell proliferation within 24 h of administration in ovariectomised mice (Heryanto & Rogers 2002). Furthermore, the administration of VEGF receptor inhibitors or an anti-VEGF antibody prevented this oestrogen-mediated endometrial endothelial cell proliferation (Heryanto et al. 2003). This demonstrated that VEGF is the primary factor mediating the initial endothelial cell proliferation in response to oestrogen within the endometrium. However, the source of this VEGF, or the mechanisms leading to its release, are still unknown. Based on our observation in human endometrium where the appearance of VEGF-containing intravascular neutrophils correlated with endothelial cell proliferation, we hypothesised that treatment of ovariectomised mice with exogenous oestrogen would increase the number of marginating and adherent neutrophils present within endometrial blood vessels and that the induction of neutropenia would reduce the number of adherent neutrophils and consequently the amount the endothelial cell proliferation.
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Methods and Materials |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionAcknowledgementsReferences |
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Study 1: induction of neutropenia with an in-house anti-rat neutrophil serum (ANS)
On days 6 and 7 following ovariectomy, one group of mice (n = 5) was injected i.p. with a previously prepared in-house ANS (0.5 ml/mouse per day). (The serum was collected from rabbits immunised with rat neutrophils (Rogers et al. 1992). Serum absorbed against erythrocytes and lymphocytes had a leukoagglutination titre against rat neutrophils of approximately 1/160–1/320 and possessed antibodies cytotoxic against neutrophils in the presence of complement. ANS tested using immunohistochemistry against various purified populations of formalin-fixed, paraffin-embedded leukocytes showed >95% neutrophil staining and <5% lymphocyte or eosinophil staining. Serum absorbed against neutrophils and tested by immuno histochemistry showed approximately 90% reduced staining (Rogers et al. 1992).) On day 7 post-ovariectomy, these mice were also injected s.c. with 100 ng oestradiol (in 100 µl oil) before dissection 24 h later. A group of control mice (n = 5) received the oestradiol injection only before dissection 24 h later. A further group of ovari-ectomised mice was left untreated (no vehicle injections) and dissected on day 7 post-ovariectomy to confirm regression of endometrial tissues (n = 3).
Four hours prior to dissection, all mice were injected i.p. with 1 mg bromodeoxyuridine (BrdU) in 0.5 ml 0.9% normal saline (40 mg/kg body weight). At the time of dissection, mice were anaesthetised using Avertin (as for ovariectomy) and perfusion-fixed (via a cannula inserted into the left ventricle of the heart) with 10% buffered formalin after a saline washout at physiological pressure (110–130 mmHg). The uterine tissues were removed and further immersion-fixed in 10% buffered formalin for 4 h before processing into paraffin sections for immunohistochemistry.
A blood sample was also collected and used to confirm neutropenia in treated animals. From the blood sample, a smear was prepared for immunohistochemistry using ANS as the primary antibody. The smears were fixed in absolute methanol for 10–20 min and then stained with haematoxylin for 2 min prior to immunostaining with the same protocol used for paraffin sections (described below). The number of ANS-positive cells within an area of 1 cm2 was determined from an even part of the blood smear.
Study 2: induction of neutropenia with anti-neutrophil monoclonal antibody Gr-1
To corroborate data obtained in Study 1, a similar study was performed using an anti-neutrophil monoclonal antibody, Gr-1, purified from hybridoma supernatants, generously donated by Dr M J Hickey and Associate Prof. P Tipping (Centre for Inflammatory Diseases, Monash University, Australia) to induce neutropenia. (The effect of the rodent Gr-1 on the depletion of circulating neutrophils in mice has previously been reported (Thakur et al. 1996). Administration of 150 µg of the specific monoclonal antibody RB6-8C5 (Gr-1) to mice caused a >95% reduction in circulating neutrophils; it took 4 days to restore circulating neutrophils to pre-treatment levels (Thakur et al. 1996).) A group of ovari-ectomised mice (n = 6) were injected i.p. with 150 µg Gr-1, followed 4 h later by an s.c. injection of 100 ng oestradiol in 100 µl oil. Mice were dissected 24 h after oestradiol treatment. A control group (n = 6) received i.p. injections of 0.9% saline in place of Gr-1 prior to oestradiol treatment. As in Study 1, all mice received an i.p. injection of BrdU 4 h prior to perfusion fixation and dissection. A blood sample was collected from these animals and a white blood cell differential count performed after staining with DiffQuik (performed by Dr M Hickey, Centre for Inflammatory Diseases, Monash University, Australia).
Immunohistochemistry
Neutrophil immunostaining
Neutrophils were identified by immunostaining with ANS. Deparaffinised endometrial sections (5 µm) were treated with 3% H2O2 in PBS and then treated with a protein blocking agent (PBA; Immunon Shandon, Pittsburgh, PA, USA). Sections were incubated in ANS (1:100 in 1% BSA/PBS) for 45 min at 37 °C. The sections were then incubated with biotinylated secondary antibody (goat anti-rabbit IgG; Zymed Lab. Inc., San Francisco, CA, USA) before immunostaining was visualised using steptavidin-horse-radish peroxidase followed by aminoethyl carbazole (AEC) chromogen. A negative control side was prepared by substituting the primary antibody with non-immune rabbit serum. A section of mouse spleen was used as a positive control. All the neutrophils present in a single cross-section of uterus were identified using a x 20 objective lens; to confirm whether the neutrophils were within a blood vessel and not just in the stroma, a x 40 or x 100 objective was used. Neutrophils within blood vessels were counted and expressed as a number per endometrial area (mm2) or per section.
VEGF immunostaining
VEGF immunostaining was performed as previously described (Gargett et al. 2001). For antigen retrieval, sections (5 µm) were microwaved (500 W microwave, 3 x 5 min on defrost) in 10 mmol/l sodium citrate buffer (pH 6). Endogenous peroxidase was quenched and non-specific binding blocked using PBA. (Sections were incubated with a polyclonal antibody rabbit anti-human VEGF; 2 µg/ml in 1% BSA/PBS) (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) for 1 h at 37 °C. Sections were then incubated in biotinylated secondary antibody (goat anti-rabbit IgG), streptavidin-horseradish peroxidase and immunoreactivity visualised with AEC chromogen. A negative control section was obtained by substituting the primary antibody with an rabbit IgG (2 µg/ml). Cells focally expressing VEGF in a single cross-section of uterus were identified using the x 20 objective lens; to confirm whether these cells were within a blood vessel or in the stroma, a x 40 or x 100 objective was used. VEGF-positive cells within blood vessels were counted and expressed as a number per endometrial area (mm2) or per section.
Cell proliferation detection (BrdU immunostaining)
Sections (5 µm) were microwaved (500 W microwave, 4 x 5 min on defrost) in 10 mmol/l sodium citrate buffer (pH 6) and treated with 0.1 mol/l HCl for 45 min for antigen retrieval. Endogenous peroxidase was quenched and non-specific binding blocked with PBA. The sections were then incubated with a polyclonal antibody sheep anti-BrdU; 10 µg/ml in 1% BSA/PBS) (BioDesign, Saco, ME, USA) for 1 h at room temperature. After incubating sections with biotinylated secondary antibody (donkey anti-sheep IgG) and streptavidin-horseradish peroxidase, BrdU immunostaining was visualised with AEC. A negative control section was obtained by substituting the primary antibody with an sheep IgG (10 µg/ml). A section of mouse intestine was included in each staining run as a positive control. The BrdU immunostaining was used to determine the number of proliferating endothelial cells as a percentage of the total number of endothelial cells.
Statistics
Values are presented as means±S.E. All statistical tests were performed using SPSS for Windows, Version 11.0.0 (SPSS Inc., Chicago, IL, USA). The effects of treatment were analysed using t-tests or one-way ANOVA after log transformation (if necessary) of data to conform to the assumptions of ANOVA. If homogeneity of variance could not be obtained, the equivalent non-parametric test was used. A P value <0.05 was considered significant.
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).
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). Slides stained with non-immune rabbit serum did not show any specific staining in individual cells.
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). In four out of the five mice treated with oestrogen and ANS, no intravascular neutrophils were observed.
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Endometrial intravascular focal VEGF
Cells strongly positive for VEGF (focal cells) were observed in the stroma and within endometrial vessels (Fig. 3). No positive staining was observed in sections after the primary antibody was replaced with the rabbit IgG.
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Endometrial endothelial cell proliferation
In Study 1, there was significantly less endothelial cell proliferation in mice treated with oestrogen and ANS than in mice treated with oestrogen only (t(7) = 3.4, P = 0.01, 42% reduction, Fig. 5). There was no endothelial cell proliferation in untreated ovariectomised mice. There was no evidence of a reduction in luminal epithelial cell proliferation in ANS-treated mice (oestrogen only: 32.8 ± 2.98%, oestrogen + ANS: 38.2 ± 1.28%; Mann–Whitney U(8) = 6.0, P = 0.18).
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). There was no evidence of less luminal epithelial cell proliferation in ANS-treated mice (oestrogen only: 30.8 ± 5.81%, oestrogen + Gr-1: 36.0 ± 2.13%; t(10) = 0.85, P = 0.41). |
Discussion |
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While the current study identifies a role for circulating neutrophils in oestrogen-mediated endometrial angiogenesis, it does not investigate the mechanisms by which neutrophils induce an angiogenic response. Previous research has demonstrated that VEGF is the primary factor mediating oestrogen-driven endothelial cell proliferation in ovariectomised mice (Heryanto et al. 2003); however, the source of this VEGF is unknown. Potentially, the intravascular neutrophils identified as having a role in endometrial angiogenesis in the current studies may provide a VEGF source. Current data suggest that VEGF secreted by neutrophils may be essential for control of angiogenesis and vascular permeability during inflammatory processes (Cassatella 1999) and in other models of angiogenesis (e.g. angiogenesis induced in the normally avascular cornea (Taichman et al. 1997, Edelman et al. 1999)). Neutrophils are known to constitutively express VEGF within their specific (secondary) granules (Gaudry et al. 1997); they also express mRNA for two of the most common VEGF splice variants, VEGF 121 and VEGF 165 (Webb et al. 1998). This VEGF is released by activated neutrophils in response to inflammatory mediators such as lipo-polysaccharide or tumour-necrosis factor-
(Webb et al. 1998, McCourt et al. 1999, Scapini et al. 1999). In the study by Gargett et al.(2001), endometrial microvessels that contained a VEGF-positive neutrophil often also stained positively for VEGF. It was hypothesised that the VEGF present in endothelial cells was due to degranulation by neutrophils. However, neutrophils by no means provide the whole explanation; in the present study, neutropenia only partially reduced endometrial endothelial cell proliferation in the mouse. Other mechanisms, including the role and identity of other endometrial intravascular VEGF-positive cells (discussed further below), will have to be explored.
In the current study, although neutropenia in ovari-ectomised mice significantly reduced endometrial endothelial cell proliferation after an oestrogen stimulus, the data concerning intravascular VEGF immunostaining were equivocal. In Study 1, when neutrophils were depleted using ANS, neutropenia significantly reduced the number of intravascular VEGF-positive cells. In Study 2, where Gr-1 was used to induce neutropenia, there was no significant change in the number of intravascular VEGF-positive cells. Several hypotheses can be proposed to explain the discrepancy between studies. There was considerable variation within the results concerning intravascular VEGF-positive cells in the study using Gr-1, raising the possibility that one or two outliers have skewed the data. Other VEGF-positive cells may be adhering to endometrial vessels, or the variation may be due to the difference in specificity between the two antibodies used (both discussed further below). Alternatively, the mechanism by which neutrophils reduce oestrogen-induced endothelial cell proliferation may not be due to their VEGF contents. It should be pointed out, however, that the discrepancy between studies concerning VEGF immunostaining does not negate the reduced endothelial cell proliferation observed in both neutropenia studies, even if alternative mechanisms of neutrophil action have to be considered.
The presence of VEGF-positive cells, even after the induction of neutropenia, suggests that other VEGF-positive leukocytes are adhering to endometrial microvessels; several other leukocytes are known to express VEGF and are potential candidates for these alternative cells. VEGF immunoreactivity has been detected in tissue macrophages present in ectopic endometrium (endometriosis) and in activated peritoneal fluid macrophages (McLaren et al. 1996); however, VEGF-positive macrophages were not detected in human endometrium through the menstrual cycle (Gargett et al. 2001). Murine granulated metrial gland cells (uterine natural killer (uNK) cells) were found to express VEGF, although uNK only appear during pregnancy in mice (Wang et al. 2003). In human uNK cells, VEGF-B (Chen et al. 2002) and -C (Li et al. 2001) are expressed. Eosinophils also constitutively express VEGF (Horiuchi & Weller 1997), but are absent from the human endometrium during most of the menstrual cycle (Salamonsen & Lathbury 2000). To determine the type and temporal distribution of intravascular neutrophils and other leukocytes in endometrial tissue after oestrogen treatment in ovariectomised mice, it will be necessary to use specific leukocyte markers to examine various time points over the first 24–48 h after oestrogen administration when endothelial cell proliferation reaches maximum levels (Heryanto & Rogers 2002).
Another possible reason for the variation in results between Studies 1 and 2 is the specificity of the antibodies used to induce neutropenia. The ANS used is a polyclonal serum raised against rat neutrophils (Rogers et al. 1992). Although the ANS was tested against various purified populations of leukocytes (<5% staining of lymphocytes or eosinophils), it is possible that small populations of mouse leukocytes other than neutrophils have been bound by the ANS. In contrast, Gr-1 is a monoclonal antibody specific to mouse neutrophils. Thus the significant reduction in focal VEGF staining in ANS-treated animals, in contrast to the lack of significant reduction in Gr-1-treated animals, may be due to removal of both neutrophils and some other VEGF-positive leukocytes from circulation. This would support the hypothesis outlined in the previous paragraph that VEGF-positive leukocytes other than neutrophils are also adhering to endometrial microvessels and stimulating proliferation.
In Study 1, the numbers of circulating neutrophils in animals treated with and without ANS were determined by counting the number of neutrophils within a set area of a blood smear. Although this showed that neutropenia had been induced (73.2% reduction), this is not the most accurate method to use. Leukocytes within a blood smear may not be evenly distributed on the slide; in addition, different leukocyte types may not be randomly distributed on the slide (Dacie & Lewis 1995). However, despite the potential lack of accuracy of the actual percentage reduction of circulating neutrophils, the count of neutrophils present within endometrial blood vessels indicates that our induction of neutropenia had been successful and the endometrium was not exposed to neutrophil-derived factors. In Study 2, the appropriate differential white blood cell count was conducted; a 95% reduction in circulating neutrophils was obtained.
To summarise, oestrogen treatment increases the number of intravascular neutrophils and intravascular VEGF-positive cells, and increases endothelial cell proliferation, within the endometrium of ovariectomised mice. Neutropenia significantly reduced endothelial cell proliferation in ovariectomised mice treated with exogenous oestrogen; the effects on numbers of intravascular VEGF-positive cells were less conclusive. These results support a role for neutrophils in endometrial angiogenesis, but also suggest the involvement of other circulating leukocytes.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionAcknowledgementsReferences |
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