Monash University Department of Obstetrics and Gynecology, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, 3168 Australia
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: angiogenesis/endometrium/human/neutrophils/VEGF
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the adult new vessel growth occurs by four mechanisms: sprouting, intussusception, vessel elongation and incorporation of circulating endothelial progenitor cells into growing vessels (Asahara et al., 1997; Rogers and Gargett, 1999
). Classical angiogenesis or sprouting involves a series of steps including endothelial cell activation, breakdown of the basement membrane, migration, proliferation and tube formation of endothelial cells, followed by basement membrane deposition and investment with pericytes and smooth muscle cells to stabilise the newly formed vessel (Klagsbrun and D'Amore, 1991
). This process is important where neovascularization of an avascular tissue occurs, such as during the rapid growth of the corpus luteum following ovulation and when tumours become angiogenic and recruit host vasculature. Intussusception is the process where the lumen of a vessel is internally divided into two as proliferating endothelial cells migrate inwards, producing a network or an arcade of parallel vessels, as occurs in the developing lung (Burri and Tarek, 1990
). It is possible that the endometrial subepithelial capillary plexus develops by an intussusceptive mechanism. Elongation and vessel widening probably occur in growing tissues as existing vessels constantly restructure in response to the metabolic demands of the surrounding cells, a process also known as remodelling or pruning (Risau, 1997
). Such a process is likely to occur during endometrial growth (Rogers and Gargett, 1999
). Whether circulating endothelial progenitors have a role in human endometrial angiogenesis is yet to be determined, although there is evidence in a mouse model (Asahara et al., 1999
).
Vascular endothelial growth factor (VEGF) is a major regulator of developmental, physiological and pathological angiogenesis (Ferrara and DavisSmyth, 1997; Neufeld et al., 1999
). VEGF is expressed in a wide range of cells and tissues, including rodent, primate and human endometrium (Gordon et al., 1995
; Torry and Torry, 1997
; Smith, 1998
). A number of attempts have been made to relate VEGF expression in human endometrium with stages of the menstrual cycle (Torry and Torry, 1997
; Rogers and Gargett, 1999
). These studies have produced conflicting results, although it is now clear that the bulk of endometrial VEGF is glandular in origin (Shifren et al., 1996
; Zhang et al., 1998
; Gargett et al., 1999
). Since the majority of this VEGF is secreted apically (Hornung et al., 1998
) it is unlikely that glandular epithelial VEGF has an angiogenic role in human endometrium. In an attempt to relate VEGF expression in human endometrium with a marker of angiogenesis, we reported that neither total glandular nor stromal VEGF correlated with endometrial endothelial cell proliferation (Gargett et al., 1999
). However during the course of that study we observed foci of intense VEGF immunostaining associated with some blood vessels in most endometrial samples studied (Gargett et al., 1999
). The aims of the present study were to elucidate the relationship between focal microvessel VEGF and endometrial angiogenesis in each of the three layers of human endometrium and to identify the VEGF-expressing cells associated with endometrial microvessels.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue blocks comprising the entire endometrial layer and several mm of myometrium were fixed for 4 h in 10% phosphate buffered formalin (pH 7.4) for routine paraffin embedding. Serial sections (3 or 5 µm) were used for immunohistochemical analysis and staining with haematoxylin and eosin (H&E) for dating. An experienced histopathologist examined the endometrial samples for normal histology and dated them using established criteria for the normal menstrual cycle (Noyes et al., 1950). Samples were categorized into three groups: menstrual (n = 3), proliferative (n = 7) and secretory (n = 8).
Immunohistochemistry
Single immunostaining
VEGF immunostaining was performed with rabbit anti-human polyclonal anti-VEGF antibody (Sc-152; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) at 2 µg/ml using a modification of a previously described method (Lau et al., 1999). Briefly, sections were boiled for 10 min in 10 mmol/l Na-citrate buffer, pH 6 for antigen retrieval, endogenous peroxidase quenched and sections blocked with 10% normal goat serum. The primary antibody (2 µg/ml) was applied for 1 h at 37°C (Lau et al., 1999
) instead of 2 h as previously described to reduce the intensity of focal VEGF immunostaining, resulting in substantially diminished VEGF immunostaining of glands and stroma compared to previous studies (Gargett et al., 1999
). Sections were then incubated with biotinylated secondary antibody, streptavidin-HRP conjugate followed by AEC chromogen. For negative control sections an isotype matched IgG (2 µg/ml) was substituted for the primary antibody. A positive control section of human endometrium was included in every staining run as described (Lau et al., 1999
).
Other primary antibodies used were: monoclonal mouse anti- human CD68 IgG1 (clone KP1; Dako Ltd, High Wycombe, UK) at 7.2 µg/ml to identify monocytes and macrophages as previously described (Orre and Rogers, 1999); monoclonal mouse anti-human neutrophil elastase IgG1 (clone NP57; Dako) at 1.3 µg/ml to identify neutrophils (Song et al., 1996
); rabbit polyclonal anti-human CD3 IgG (Dako) at 3.3 µg/ml to identify human T lymphocytes; monoclonal mouse anti-human N-CAM IgG1 (Clone 123C3; Zymed, San Francisco, CA, USA) which recognizes CD56 antigen of NK cells and large granulated endometrial lymphocytes (Critchley et al., 1999
); monoclonal mouse anti-human tryptase IgG1 (Clone AA1; Dako) at 0.53 µg/ml to identify basophils and mast cells (Jeziorska et al., 1995
). A mouse monoclonal anti-human CD14 antibody (Clone RPA-M1; Zymed) was also used at 9.2 µg/ml to identify circulating monocytes.
Antigen retrieval for N-CAM immunostaining was carried out by boiling for 20 min in 10 mmol/l Na-citrate buffer, pH 6; for CD3 and tryptase by boiling for 10 min and for CD68 by trypsin (0.1%) digestion for 15 min at 37°C. Endogenous peroxidase was blocked with 3% H2O2 in 50% methanol for 10 min at room temperature (RT) and sections treated with protein blocking agent (PBA) (Lipshaw Immunon, Pittsburgh, PA, USA) for 10 min at RT. The primary antibodies were then incubated for 1 h at 37°C for anti-mast cell tryptase and anti-N-CAM, , 1 and 2 h at RT for anti-CD68, anti-CD3 and anti-neutrophil elastase respectively and overnight at 4°C for anti-CD14. This was followed by Dako LSAB+ biotinylated secondary antibody cocktail (specificities for rabbit, mouse and goat), streptavidin-HRP conjugate (Dako) each for 15 min at RT and then AEC chromogen (red) (Zymed) or DAB (brown) (Dako) for 510 min at RT. For negative controls, primary antibodies were substituted with an isotype matched IgG at the equivalent concentration of primary antibody. A positive control section of human tonsil was included in each staining batch.
Double immunostaining
Proliferating endothelial cells were detected by a standard double immunohistochemistry protocol using a monoclonal mouse anti-rat antibody to proliferating cell nuclear antigen (PCNA) (clone PC10; Novacastra, Newcastle, UK) and a mouse monoclonal anti-human antibody against the CD34 antigen (clone QBEND/10; Serotec, Oxford, UK), an endothelial cell marker, according to a previously published protocol (Goodger and Rogers, 1994). All other reagents were from Zymed. A sequential protocol was used with the anti-PCNA as the first primary antibody, followed by anti-CD34 after development of the first chromogen. Both primary antibodies were applied for 1 h at 37°C followed by incubation with biotinylated secondary antibody. A streptavidin-HRP conjugate with AEC chromogen (red) was used for PCNA staining and streptavidin-alkaline phosphatase conjugate with AP-blue chromogen was used for CD34 staining. Endogenous peroxidase was quenched with 3% H2O2 in 50% methanol. Positive and negative control sections were included in each staining run as previously described (Goodger and Rogers, 1994
).
To examine the relationship between VEGF expressing cells and microvessels a double immunostaining protocol using anti-CD34 and anti-VEGF antibodies was developed and used on 3 µm sections. Antigen retrieval was achieved as described for VEGF and blocking with H2O2 and PBA as described for single immunostaining. A sequential protocol was used, where anti-CD34 (diluted 1/50 in PBS containing 1% bovine serum albumin) was applied to sections for 40 min at 37°C as the first primary antibody followed by streptavidin-alkaline phosphatase conjugate and AP-blue chromogen. After washing, sections were treated with double staining enhancer (Zymed), followed by anti-VEGF (2 µg/ml in 10% goat serum) for 1 h at 37°C, biotinylated secondary antibody, streptavidin-HRP and AEC chromogen as described for single immunostaining, except washes between steps were in high ionic strength PBS (0.5 mol/l NaCl, 10 mmol/l phosphate buffer, pH 7.4) to reduce background staining. Positive and negative control sections for both primary antibodies were included in each staining batch as described above.
Quantification of proliferating vessels and VEGF expressing vessels
An H&E section was used to identify the three layers of endometrium: subepithelium, functionalis and basalis (Figure 1A). The subepithelium contains the subepithelial capillary plexus, which is located in approximately the first 150 µm below the surface epithelium and was examined in one field of view on the digital camera beneath the surface epithelium using a 40X objective. The basalis was distinguished from the functionalis by the density of the stromal compartment, which is greater in the basalis (Figure 1A
). Sequential fields of view for each of the three endometrial layers were examined on serial sections immunostained with PCNA/CD34 and VEGF under 400X magnification using a Zeiss Axioscop microscope, (Carl Zeiss, Oberkochen, West Germany). The same fields of view (at least 10 per layer) from each section were captured using a digital video camera (Fujix, Fuji, Tokyo) and the images examined side by side using Analytical Imaging StationTM software (AIS; Image Research Inc., Ontario, Canada). Microvessel profiles were identified by blue CD34 immunostaining (Figure 1B
) and at least 100 were counted in each layer of each section. Vessels containing proliferating endothelial cell (EC) nuclei (proliferating vessels) that showed immunostaining distinctly greater than background as assessed visually were counted as positive (red in the PCNA/CD34 section, Figure 1B
) and reported as a percentage of total vessels counted. In the serial VEGF-immunostained section, identical vessels were identified and those with immunostained cells associated with the vessel wall (Figure 1C
) were counted (VEGF positive vessels) and reported as a percentage of total vessels. Finally, those vessels containing both PCNA positive EC nuclei and VEGF-immunostained cells were counted and reported as a percentage of proliferating vessels. Vessels in the functionalis layer were counted in sequential fields from just below the subepithelial capillary plexus, through the depth of the endometrium until the basalis layer was reached. In some samples where the surface epithelium was not preserved, vessel counts were only performed on the functionalis and basalis layers. Results were confirmed with CD34/VEGF double immunostained sections (Figure 1D
).
|
Statistical analysis
Analyses were performed using SPSS, version 6.1.3 (SPSS, Australasia, North Sydney, Australia). Data were not normally distributed as determined by KS-Lilliefors/Shapiro-Wilks test and were therefore analysed using non-parametric tests. The Kruskal-Wallis one-way analysis of variance was used to examine for differences between stages of the menstrual cycle for each of the endometrial layers and between layers within a given cycle stage. The Mann-Whitney U test (two tailed) was used to confirm differences between groups where the Kruskal-Wallis was significant. Correlations were performed using least squares regression analysis and Spearman Rank Correlation Coefficients (RS) were determined. Results were considered statistically significant when P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
It was clear that the VEGF-immunostained intravascular leukocytes were not T lymphocytes, since the majority of CD3+ cells were found in the stroma and not within the vasculature (Figure 6D, E) and were more prevalent during the secretory phase. Furthermore no overlap between VEGF and CD3 immunostaining was observed. Similarly, CD56+ lymphocytes were predominantly found in the stroma, with substantially increased numbers detected in the secretory compared with the proliferative phase, and did not correspond to the intravascular VEGF-immunostained cells (Figure 6F
, G). Few CD3+ or CD56+ lymphocytes were found in the subepithelial capillary plexus and the distribution gradient in the endometrium for the latter cell type was opposite to intravascular VEGF- immunostained cells.
Mast cells as revealed by mast cell tryptase immunostaining were also distributed in a manner opposite to intravascular VEGF-immunostained cells, with the majority found in the myometrium and endometrial basalis and none in the subepithelial capillary plexus. Furthermore, mast cells were only found in the stroma and were not associated with microvessels containing VEGF-immunostained cells (Figure 6H, I).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intravascular VEGF in human endometrium is located in neutrophils
A second major finding of this study is that the intravascular intensely stained VEGF expressing cells were identified as marginating and adherent neutrophils. Four lines of evidence support this finding. Firstly, in very thin serial sections and in double-stained sections, the intensely stained intravascular VEGF expressing cells co-localized only with neutrophil elastase but not with any of the other leukocyte markers. Secondly, the distribution of neutrophils matched the distribution of VEGF expressing vessels within the endometrial layers in both the proliferative and secretory stages of the menstrual cycle. In contrast, the distribution and location of all other leukocytes examined, CD68+ macrophages and CD14 monocytes, CD56+ lymphocytes, CD3+ T lymphocytes and mast cells did not match that of VEGF expressing vessels. Thirdly, none of the leukocytes examined were found in the microvessels except neutrophils and monocytes. Monocytes were excluded on the basis that fewer were located intravascularly in CD14 immunostained sections compared with the number of VEGF expressing vessels and CD68/VEGF double-immunostained cells were only found in the stroma. Fourthly, it is clear at the ultrastructural level that stationary neutrophils are in close contact with the endothelium, and at these sites of contact there is morphological evidence of EC activation.
In human endometrium, neutrophils are predominantly found in the subepithelial stroma during the menstrual stage, while smaller numbers have been observed at other cycle stages (this study) (Porapatich et al., 1987; Yeaman et al., 1998
). We and others have also observed neutrophils within microvessels predominantly in the proliferative stages (Song et al., 1996
). Often intravascular neutrophils are ignored in tissues, including human endometrium because they are considered to result from surgically-induced insult to microvascular beds (Porapatich et al., 1987
; Nolte et al., 1994
). However, the specific location of neutrophils in subepithelial capillary plexus and functionalis microvessels, their absence from myometrial vessels, their presence in biopsy material in the same locations, as well as the strong correlation observed between microvessel VEGF and proliferating vessels in both hysterectomy and biopsy specimens, would argue for a physiological role for intravascular neutrophils in endometrial angiogenesis. Other evidence suggests that there is a physiological level of leukocyte rolling (mainly neutrophils) in the absence of an inflammatory challenge or surgical trauma, which differs between vascular beds of different tissues (Nolte et al., 1994
). The strong association between VEGF containing intravascular neutrophils and proliferating vessels suggests a possible physiological role for marginating and adherent neutrophils in endometrial vessels of the subepithelial capillary plexus and in the functionalis to provide intravascular VEGF to growing vessels during the proliferative phase of the endometrial cycle. Since the subepithelial plexus reaches maximal blood flow during the early-mid secretory stage (Gannon et al., 1997
), significant growth of these vessels must occur during the proliferative and early secretory stages. However, it is less likely that marginating and adherent neutrophils are important in the significant growth and coiling of spiral arterioles that occurs during the secretory stage. It is also possible that the strong association between VEGF containing intravascular neutrophils and proliferating vessels is circumstantial. Although many proliferating vessels also contained VEGF-expressing cells, especially in the subepithelial capillary plexus and functionalis during the proliferative phase (Figure 4
), there were more vessels with VEGF-expressing neutrophils than proliferating vessels in most samples (Figures 2 and 3
), suggesting that not all marginating VEGF-expressing neutrophils are associated with endometrial angiogenesis. It is also possible that the different adhesion molecules expressed on activated and angiogenic vessels (Griffioen and Molema, 2000
) were responsible for the marginating neutrophils observed in our studies, but our data do not distinguish these possibilities. These observations may also be due to the limitation of immunohistochemistry in that it represents a snapshot of molecular events at the time of fixation, while angiogenesis and margination of neutrophils are both dynamic processes. Certainly further in-vivo and in-vitro experimentation is required to demonstrate that neutrophil VEGF actually has a role in endometrial angiogenesis and to elucidate the mechanisms involved.
Neutrophils have been shown to constitutively express VEGF in their specific granules (Gaudry et al., 1997), which are released on activation (Gaudry et al., 1997
), as well as produce VEGF mRNA on activation (Webb et al., 1998
; Scapini et al., 1999
). It is possible that in endometrial microvessels adherent, activated neutrophils release their VEGF-containing granules (Williams and Solomkin, 1999
). Some of this VEGF appeared to bind VEGF receptors on juxtaposed EC, where it would directly stimulate proliferation, while the majority may be released into the vessel lumen thus contributing to the level of circulating VEGF (Salven et al., 1999
). Neutrophils may also contribute to endometrial angiogenesis through their ability to alter vascular permeability, since angiogenic vessels are frequently hyperpermeable (Dvorak et al., 1995
). Adherent neutrophils may increase endothelial permeability through their action on adherens junction proteins (Tinsley et al., 1999
) or through the local release of VEGF, also known as vascular permeabilizing factor (Ferrara and DavisSmyth, 1997
). Both mechanisms would account for the observations made in the present study, where apart from the menstrual stage, the majority of VEGF expressing neutrophils were found in close association with the microvasculature. This further emphasises that neutrophils could elicit their putative effect on endometrial EC proliferation within the vasculature. Another possibility for the association of VEGF containing neutrophils with proliferating vessels in human endometrium is the adhesion of neutrophils to proliferating EC, which have recently been shown to express E-selectin (Detmar et al., 1998
). There is also some, although conflicting, evidence showing that oestrogen modulates adhesion molecule expression on EC (Cid et al., 1994
; Caulin-Glasser et al., 1996
) and, in artificially cycling mice neutrophil influx into the uterus is promoted by oestrogen and inhibited by progesterone (Tibbetts et al., 1999
).
Role of intravascular neutrophil VEGF in endometrial angiogenesis
The intravascular VEGF expressing neutrophils associated with proliferating vessels mainly occurred during the proliferative phase of the endometrial cycle. Therefore we propose the novel hypothesis that marginating and adherent neutrophils provide an intravascular source of VEGF to endothelial cells for vessels undergoing angiogenesis by elongation or intussusception. Intravascular neutrophils have been implicated in several models of angiogenesis, particularly associated with acute and chronic inflammation. In an elegant mouse model of chronic airway inflammation, McDonald and co-workers have shown that endothelial cell proliferation and vessel growth and enlargement was associated with leukocytes adherent to and transmigrating through microvessel walls (Thurston et al., 1998; Murphy et al., 1999
). These investigators showed that vessel growth was not by classical angiogenesis but rather through microvascular remodelling where endothelial cells proliferate within intact vessel walls (Murphy et al., 1999
). However, the role of leukocyte VEGF in vessel growth was beyond the scope of their studies. In another study involving chronic inflammation in a variety of human mucosal lesions, neutrophils within or emigrating from microvessels stained strongly for VEGF (Taichman et al., 1997
). While this study did not examine for endothelial cell proliferation in these vessels, the authors speculated that neutrophil derived VEGF was an efficient way of delivering VEGF to endothelial cells and concluded that they had a role in angiogenesis associated with inflammation (Taichman et al., 1997
). Intravascular neutrophils and monocytes also play an important role in the neovascularization associated with diabetic retinopathy, where large numbers of these cells have been documented in retinal microvessels (Schroder et al., 1991
; McCleod et al., 1995
). In a rat model of injury-induced corneal inflammation, migratory VEGF expressing neutrophils and monocytes successively entered the avascular cornea over 4 days contributing to the significant neovascularization that was observed (Edelman et al., 1999
). The similarity of the above studies and our own observations in human endometrium raises the question as to whether endometrial angiogenesis is a pseudo inflammatory mediated response.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asahara, T., Murohara, T., Sullivan, A. et al. (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science, 275, 964967.
Bausero, P., Cavaillé, F., Méduri, G. et al. (1998) Paracrine action of vascular endothelial growth factor in the human endometrium: production and target sites, and hormonal regulation. Angiogenesis, 2, 167182.
Bulmer, J.N., Lunny, D.P., and Hagin, S.V. (1988) Immunohistochemical characterization of stromal leukocytes in nonpregnant human endometrium. Am. J. Reprod. Immunol., 17, 8390.[ISI]
Burri, P.H. and Tarek, M.R. (1990) A novel mechanism of capillary growth in the rat pulmonary microcirculation. Anat. Rec., 228, 3545.[ISI][Medline]
Caulin-Glasser, T., Watson, C.A., Pardi, R. et al. (1996) Effects of 17ß-estradiol on cytokine-induced endothelial cell adhesion molecule expression. J. Clin. Invest., 98, 3642.
Cid, M.C., Kleinman, H.K., Grant, D.S. et al. (1994) Estradiol enhances leukocyte binding to tumor necrosis factor (TNF)-stimulated endothelial cells via an increase in TNF-induced adhesion molecules E-selectin, intercellular adhesion molecule type, 1, and vascular adhesion molecule type 1. J. Clin. Invest., 93, 1725.[ISI][Medline]
Coussens, L.M., Raymond, W.W., Bergers, G. et al. (1999) Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev., 13, 13821397.
Critchley, H.O.D., Jones, R.L., Lea, R.G. et al. (1999) Role of inflammatory mediators in human endometrium during progesterone withdrawal and early pregnancy. J. Clin. Endocrinol. Metab., 84, 240248.
Detmar, M., Brown, L.F., Schön, M.P. et al. (1998) Increased microvessel density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J. Invest. Dermatol., 111, 16.[Abstract]
Dvorak, H.F., Brown, L.F., Detmar, M. et al. (1995) Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am. J. Path., 146, 10291039.[Abstract]
Edelman, J.L., Castro, M.R., and Wen, Y. (1999) Correlation of VEGF expression by leukocytes with the growth and regression of blood vessels in the rat cornea. Invest. Ophthalmol. Vis. Sci., 40, 11121123.[Abstract]
Fanger, H. and Barker, B.E. (1961) Capillaries and arterioles in the normal endometrium. Obstet. Gynecol., 17, 543550[ISI][Medline]
Ferrara, N. and DavisSmyth, T. (1997) The biology of vascular endothelial growth factor. Endocrine Rev., 18, 425.
Freeman, M.R., Schneck, F.X., Gagnon, M.L. et al. (1995) Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res., 55, 41404145.[Abstract]
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, 132139.[ISI][Medline]
Gargett, C.E., Lederman, F., Lau, T.M. et al. (1999) Lack of correlation between vascular endothelial growth factor production and endothelial cell proliferation in the human endometrium. Hum. Reprod., 14, 20802088.
Gaudry, M., Bregerie, O., Andrieu, V. et al. (1997) Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood, 90, 41534161.
Goodger, A.M. and Rogers, P.A.W. (1994) Endometrial endothelial cell proliferation during the menstrual cycle. Hum. Reprod., 9, 399405.[Abstract]
Gordon, J.D., Shifren, J.L., Foulk, R.A. et al. (1995) Angiogenesis in the human female reproductive tract. Obstet. Gynecol. Surv., 50, 688697.[Medline]
Griffioen, A.W. and Molema, G. (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol. Rev., 52, 237267.
Harmey, J.H., Dimitriadis, E., Kay, E. et al. (1998) Regulation of macrophage production of vascular endothelial growth factor (VEGF) by hypoxia and transforming growth factor ß-1. Ann. Surg. Oncol., 5, 271278.[Abstract]
Hornung, D., Lebovic, D.I., Shifren, J.L. et al. (1998) Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells. Fertil. Steril., 69, 909915.[ISI][Medline]
Jeziorska, M.A., Salamonsen, L.A., and Woolley, D.E. (1995) Mast cell and eosinophil distribution and activation in human endometrium throughout the menstrual cycle. Biol. Reprod., 53, 312320.[Abstract]
Johannisson, E. (1990) Endometrial morphology during the normal cycle and under the influence of contraceptive steroids. In D'Arcangues, C., Fraser, I.S., Newton, J.R. et al. (eds) Contraception and Mechanisms of Endometrial Bleeding. Cambridge University Press, Cambridge, UK. pp. 5380.
Kaiserman-Abramof, I.R. and Padykula, H.A. (1989) Angiogenesis in the postovulatory primate endometrium: the coiled arteriolar system. Anat. Rec., 224, 479489.[ISI][Medline]
Klagsbrun, M. and D'Amore, P.A. (1991) Regulators of angiogenesis. Ann. Rev. Physiol., 53, 217239.[ISI][Medline]
Lau, T.M., Affandi, B., and Rogers, P.A.W. (1999) The effects of levonorgestrel implants on vascular endothelial growth factor expression in the endometrium. Mol. Hum. Reprod., 5, 5763.
Li, X.F., Gregory, J., and Ahmed, A. (1994) Immunolocalisation of vascular endothelial growth factor in human endometrium. Growth Factors, 11, 277282.[ISI][Medline]
Markee, J.E. (1940) Menstruation in intraocular endometrial transplants in the rhesus monkey. Contrib. Emb., 177, 219308.
McCleod, D.S., Lefer, D.J., Merges, C. et al. (1995) Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid. Am. J. Pathol., 147, 642653.[Abstract]
Murphy, T.J., Thurston, G., Ezaki, T. et al. (1999) Endothelial cell heterogeneity in venules of mouse airways induced by polarized inflammatory stimulus. Am. J. Pathol., 155, 93103.
Neufeld, G., Cohen, T., Gengrinovitch, S. et al. (1999) Vascular endothelial growth factor (VEGF) and its receptors. FASEB J., 13, 912.
Nolte, D., Schmid, P., Jäger, U. et al. (1994) Leukocyte rolling in venules of striated muscle and skin is mediated by P-selectin, not by L-selectin. Am. J. Physiol., 267, H1637-H1642.
Noyes, R.W., Hertig, A.T., and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 325.[ISI][Medline]
Orre, M. and Rogers, P.W. (1999) Macrophages and microvessel density in tumors of the ovary. Gynecol. Oncol., 73, 4750.[ISI][Medline]
Peek, M., Landgren, B.M., and Johannisson, E. (1992) The endometrial capillaries during the normal menstrual cycle: a morphometric study. Hum. Reprod., 7, 906911.[Abstract]
Porapatich, C., Rojas, M., and Silverberg, S.G. (1987) Polymorphnuclear leukocytes in the endometrium during the normal menstrual cycle. Int. J. Gynecol. Pathol., 6, 230234.[ISI][Medline]
Risau, W. (1997) Mechanisms of angiogenesis. Nature, 368, 671674.
Rogers, P.A.W., Au, C.L., and Affandi, B. (1993) Endometrial microvascular density during the normal menstrual cycle and following exposure to long-term levonorgestrel. Hum. Reprod., 8, 13961404.[Abstract]
Rogers, P.A.W. and Gargett, C.E. (1999) Endometrial angiogenesis. Angiogenesis, 2, 287294.
Salven, P., Orpana, A., and Joensuu, H. (1999) Leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor. Clin. Cancer Res., 5, 487491.
Scapini, P., Calzetti, F., and Cassatella, M.A. (1999) On the detection of neutrophil-derived vascular endothelial growth factor (VEGF). J. Immunol. Meth., 232, 121129.[ISI][Medline]
Schroder, S., Palinski, W. and Schmid-Schönbein, G.W. (1991) Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am. J. Pathol., 139, 81100.[Abstract]
Sheppard, B.L. and Bonnar, J. (1980) The development of vessels of the endometrium during the menstrual cycle. In Diczfalusy, E., Fraser, I.S. and Webb, F.T.G. (eds), Endometrial Bleeding and Steroidal Contraception, Pitman Press, Bath, UK pp. 6577
Shifren, J.L., Tseng, J.F., Zaloudek, C.J. et al. (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, 31123118.[Abstract]
Smith, S.K. (1998) Angiogenesis, vascular endothelial growth factor and the endometrium. Hum. Reprod. Update, 4, 509519.
Song, J.Y., Russell, P., Markham, R. et al. (1996) Effects of high dose progestogens on white cells and necrosis in human endometrium. Hum. Reprod., 11, 17131718.[Abstract]
Taichman, N.S., Young, S., Cruchley, A.T. et al. (1997) Human neutrophils secrete vascular endothelial growth factor. J. Leukoc. Biol., 62, 397400.[Abstract]
Thurston, G., Murphy, T.J., Baluk, P. et al. (1998) Angiogenesis in mice with chronic airway inflammation. Strain-dependent differences. Am. J. Pathol., 153, 10991112.
Tibbetts, T.A., Conneely, O.M., and O'Malley, B.W. (1999) Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus. Biol. Reprod., 60, 11581165.
Tinsley, J.H., Wu, M.H., Ma, W.Y. et al. (1999) Activated neutrophils induce hyperpermeability and phosphorylation of adherens junction proteins in coronary venular endothelial cells. J. Biol. Chem., 274, 2493024934.
Torry, D.S. and Torry, R.J. (1997) Angiogenesis and the expression of vascular endothelial growth factor in endometrium and placenta. Am. J. Reprod. Immunol., 37, 2129.[ISI][Medline]
Webb, N.J., Myers, C.R., Watson, C.J. et al. (1998) Activated human neutrophils express vascular endothelial growth factor (VEGF). Cytokine, 10, 254257.[ISI][Medline]
Williams, M.A. and Solomkin, J.S. (1999) Integrin-mediated signaling in human neutrophil functioning. J. Leukoc. Biol., 65, 725736.[Abstract]
Yamamoto, S., Konishi, I., Tsuruta, Y. et al. (1997) Expression of vascular endothelial growth factor (VEGF) during folliculogenesis and corpus luteum formation in the human ovary. Gynecol. Endocrinology, 11, 371381.[ISI][Medline]
Yeaman, G.R., Collins, J.E., Currie, J.K. et al. (1998) IFN- is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils. J. Immunol., 160, 51455153.
Zhang, L., Scott, P.E., Turley, H. et al. (1998) Validation of anti-vascular endothelial growth factor (Anti-VEGF) antibodies for immunohistochemical localization of VEGF in tissue sections. Expression of VEGF in the human endometrium. J. Pathol., 185, 402408.[ISI][Medline]
Submitted on November 15, 2000; accepted on March 1, 2001.