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Endocrinology Vol. 141, No. 3 995-1000
Copyright © 2000 by The Endocrine Society

ARTICLES

Suppression of Luteal Angiogenesis in the Primate after Neutralization of Vascular Endothelial Growth Factor1

Hamish M. Fraser, Sarah E. Dickson, Stephen F. Lunn, Christine Wulff, Keith D. Morris, Veronica A. Carroll and Roy Bicknell

Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology (H.M.F., S.E.D., S.F.L., C.W., K.D.M.), Edinburgh, United Kingdom EH3 9ET; the Department of Cancer Medicine, Imperial College School of Medicine, Hammersmith Campus (V.A.C.), London, United Kingdom W12 0NN; and Molecular Angiogenesis Laboratories, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital (R.B.), Oxford, United Kingdom OX3 9DS

Address all correspondence and requests for reprints to: Dr. H. M. Fraser, Medical Research Council, Reproductive Biology Unit, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9EW. E-mail: h.fraser{at}ed-rbu.mrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Manipulation of angiogenesis may have a profound effect on female reproductive function, but this has not yet been demonstrated by direct experiment in species with ovulatory cycles similar to those in women. To investigate whether angiogenesis could be inhibited in the primate corpus luteum, and the consequences of such inhibition on luteal function, marmosets were treated with an antibody to vascular endothelial growth factor (VEGF). Treatment commenced at the time of ovulation and was continued for 3 days (early luteal group) or 10 days (midluteal group). Bromodeoxyuridine was used to label proliferating cells, being administered 1 h before collecting ovaries from control and treated animals in the early or midluteal phase. Ovarian sections were stained using an antibody to bromodeoxyuridine, and a proliferation index was obtained; endothelial cell quantification was performed using factor VIII as an endothelial cell marker. Intense proliferation in the early luteal phase was suppressed by anti-VEGF treatment. This resulted in blockade of development of the normally extensive capillary bed, as in the animals treated until the mid-luteal phase the numbers of endothelial cells were reduced. The hormone-producing cells remained largely unaltered in the posttreatment corpus luteum, although the presence of lipid accumulation, and small pockets of cells showing basophilia and nuclear condensation were observed. Significantly, luteal function, as judged by secretion of progesterone, was markedly compromised by the treatment, being reduced by 60% in comparison with controls. It is concluded that VEGF-mediated angiogenesis is an essential component of luteal function in primates and therefore has the potential to be regulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS ESTABLISHED that inhibition of angiogenesis by broad spectrum or systematic targeting (1, 2, 3, 4) of the angiogenic pathway can have suppressive effects on tumor growth. However, comparatively little is known about the effect of inhibition of the highly active physiological angiogenesis that occurs in the reproductive tissues: the ovary, uterus, and placenta. The corpus luteum is essential for the establishment of pregnancy through the production of progesterone from lutein cells. Active angiogenesis occurs during early luteal development. It is believed that this angiogenesis is critical for establishment of the vasculature essential for transport of hormonal precursors of progesterone to and progesterone from the lutein cells (5). The role of angiogenesis in the rodent ovary has been addressed (6, 7), but the mechanisms that regulate luteal function in rodents and primates are markedly different (8). Our finding of the failure of TNP-470 to affect luteal function in Old World or New World primates (9) emphasizes the need for caution in extrapolating results from the rodent, in which this angiogenesis inhibitor prevented pregnancy (7). Thus, an effect of inhibition of angiogenesis on female reproductive function has not yet been demonstrated by direct experiment in species with ovulatory cycles similar to those in women.

The aim of this study was to investigate the effects of immunoneutralization of vascular endothelial growth factor (VEGF) on the marmoset corpus luteum of the normal cycle as a means of exploring the role of angiogenesis in luteal function of the primate. VEGF has been shown to be expressed in the corpus luteum of the human and nonhuman primate and is therefore a likely candidate for targeting (10, 11, 12).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
VEGF antibody
Mouse monoclonal anti-VEGF, VG76e, was raised by immunization with recombinant human VEGF-189. The pET146 expression plasmid was used to enable affinity purification of Escherichia coli-expressed recombinant protein. The antibody was produced from hybridomas grown in culture, and a highly purified preparation was obtained using protein A-Sepharose columns. Hybridomas were screened by Western blotting of VEGF-189. Using recombinant proteins, it was demonstrated that VG76e recognizes the 121, 165, and 189 isoforms of VEGF.

VG76e was shown to be a blocking antibody by its inhibition of the growth stimulatory activity of VEGF on human umbilical vein endothelial cells (HUVEC), a gift from Prof. B. R. Binder, Vienna University (Vienna, Austria). The cells were cultured in medium 199 (Sigma, Poole, UK) with 20% heat-inactivated FBS (Sigma), 100 IU/ml penicillin/100 µg/ml streptomycin (Life Technologies, Inc., Paisley, UK), 250 ng/mL fungizone (Life Technologies, Inc.), 2 mM glutamine (Life Technologies, Inc.), 5 IU/ml heparin (Sigma), and 50 µg/ml endothelial cell growth supplement (ECGS) (Technoclone, Inc., Vienna, Austria). HUVEC (3 x 103) at passage 5 were plated in complete medium in 96-well plates and allowed to attach overnight. Growth was arrested by incubation with 100 µl reduced growth medium (medium 199 containing the supplements described above without ECGS and 2% FBS) for 24 h. Human VEGF (10 ng/ml; R&D Systems, Abingdon, UK) was added to wells with various concentrations of the anti-VEGF antibody (VG76e), a positive control anti-VEGF blocking antibody 2C3 (a gift from Prof. P. Thorpe, ME Medical Center, Portland, ME), or a negative control (mouse IgG, Sigma). Control wells received medium with or without VEGF. After 3 days of incubation, cell number was quantified colorometrically by the addition of 20 µl Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega Corp., Southampton, UK) at 492 and 620 nm reference filter. Background absorbance was determined with substrate alone and subtracted from all wells. Cell growth was calculated by subtracting absorbance in wells with cells grown without VEGF.

Animals
Common marmoset monkeys (Callithrix jacchus) were housed in cages in rooms at a temperature of 22.5 C as described previously (9). Adult females with a body weight of approximately 350 g, with regular ovulatory cycles, were housed together with a younger sister or prepubertal female. Blood samples were collected three times per week by femoral venipuncture without anesthesia, and plasma was stored at -20 C until required for progesterone assay to confirm normal ovulatory cycles. Criteria for the occurrence of ovulation and normal luteal phase length (18–22 days) were based on determination of plasma progesterone concentrations as described previously (13).

Treatment
The experiments were carried out in accordance with the Animals (Scientific Procedures) Act, 1986. To synchronize timing of ovulation during the treatment cycle, animals were treated with 1 µg PGF2{alpha} analog (Planate, Coopers Animal Health Ltd., Crewe, UK), im, during the mid- to late luteal phase of the pretreatment cycle to induce luteolysis. This treatment is normally followed by ovulation 10 days later (14). Four marmosets were treated with VEGF antibody at a concentration of 2.7 mg/ml starting 10 days after PG, i.e. the day of anticipated ovulation (luteal phase day 0), at a dose of 2 mg followed by 1 mg on days 1 and 2 (early luteal group). Four controls were treated with the same dose of mouse {gamma}-globulin (Sigma) following the same schedule.

Treatment was extended to the midluteal phase in an additional six marmosets; additional injections of 1 mg anti-VEGF were administered on days 3, 5, 7, and 9, with six animals receiving mouse {gamma}-globulin as controls (midluteal group). All treatments were given by slow iv injection.

Ovaries were obtained the day after final treatment, luteal day 3 or 10, after the animals had received 20 mg bromodeoxyuridine (BrdU: Roche Molecular Biochemicals, Essex, UK) in saline by slow iv injection. One hour later, the animals were sedated using 100 µl ketamine hydrochloride (Parke-Davis Veterinary, Pontypool, UK), im, and killed with an iv injection of 400 µl Euthetal (sodium pentobarbitone, Rhone Merieux, Harlow, Essex, UK). Ovaries were removed immediately, weighed, fixed in 4% paraformaldehyde for 24 h, dehydrated, and embedded in paraffin. In addition, a small portion of a representative corpus luteum was placed in 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. These specimens were fixed for 2 h, rinsed in the same buffer, postfixed in buffered 2% osmium tetroxide for 2 h, and embedded in Araldite after dehydration in ethyl alcohol. Semithin (1 µm) sections were stained with toluidine blue for light microscope analysis. When the midluteal group was examined, it was apparent that the density of the lutein cells was increased after treatment. To quantify this change, paraffin sections were stained with hematoxylin and eosin, and lutein cell density was determined in 12 corpora lutea/group.

The establishment and function of the corpus luteum were determined by measuring plasma progesterone concentrations in daily blood plasma samples as described previously (13). Only marmosets in which treatment was started at the time of presumptive ovulation (13) (plasma progesterone having reached >30 nmol/liter and found retrospectively to be sustained at luteal phase values) were included in the study. Analysis of differences in plasma progesterone concentrations was performed by repeated measures ANOVA followed by post-hoc Fisher’s protected least significant difference test (when a significant interaction was found).

Immunocytochemistry
Cellular responses were studied by determining the number of mitotic cells, after BrdU administration as a marker, and by examining the establishment of the microvascular network using factor VIII staining to identify endothelial cells. Tissue sections (5 µm) were cut onto Tespa-coated (Sigma) slides for immunocytochemistry and morphological examination. Sections were stained with hematoxylin and eosin and examined for morphological features of apoptosis as described previously (15). Localization of factor VIII was determined by immunocytochemistry as described previously (16); visualization was performed with nitro blue tetrazolium. These sections were not counterstained, so that quantitative image analysis could be performed; the corpora lutea were identified from the hematoxylin- and eosin-stained sections. Immunostaining for factor VIII to compare control and treated animals was carried out in separate runs for the early and midluteal groups. Factor VIII immunostaining was quantified using the Image Pro-Plus 3.0 (Media Cybernetics, Silver Spring, MD) image analysis program. Four to six randomly chosen areas of 13.3 x 104 µm2 for the early luteal sections, and 5.3 x 104 µm2 for the larger midluteal sections were studied; in total, these areas covered approximately two thirds of each corpus luteum. The captured gray scale image was thresholded and converted to a binary image, and the factor VIII-positive area was quantified. The mean of these areas per corpus luteum was taken as representative for that animal.

Proliferating cells were visualized in ovarian sections using a mouse monoclonal antibody to BrdU (Roche Molecular Biochemicals) as described previously (9). Cell proliferation was assessed by counting the number of BrdU-positive cells in six randomly chosen fields of 62,500 µm2/corpus luteum and expressed as a mean percentage of the total cells in these fields. For factor VIII quantification, proliferation index, ovarian weight, and luteal cell numbers, differences between groups were determined using a two-tailed unpaired t test; P < 0.05 was taken as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neutralizing antibody
The endothelial cell growth assay demonstrated that the monoclonal antibody to human VEGF (VG76e) inhibited VEGF-stimulated HUVEC growth in a dose-dependent manner (Fig. 1Go). These results indicate that VG76e is a blocking antibody that inhibits the growth stimulatory activity of VEGF.



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  		Figure 1. Effect of VEGF antibody, VG76e, on VEGF-mediated HUVEC growth. Results are given as a percentage of the control value, where cells were grown in the presence of VEGF alone. Results show the mean ± SD of three independent experiments for VG76e and two experiments for positive control VEGF antibody, 2C3, and control IgG. Both VEGF antibodies exhibited a marked inhibitory effect on VEGF-stimulated proliferation of HUVECs. A negative control antibody of irrelevant specificity had no effect on endothelial cell growth.

 
Cell proliferation and endothelial cell area
Treatment with anti-VEGF during the early luteal phase did not prevent the formation of clearly identifiable corpora lutea, and ovarian sections revealed the presence of recently formed corpora lutea in all control and treated marmosets. BrdU staining of proliferating cells was readily observed in all corpora lutea (Fig. 2aGo). It was clear that few (if any) of the hormone-producing cells had incorporated BrdU; positive staining was restricted to some cells surrounding the luminal microvessels and others whose appearance was consistent with that of capillary endothelial cells. We have shown previously that cells incorporating BrdU have the location and morphological appearance of endothelial cells (9). In addition, colocalization studies have demonstrated that more than 80% proliferating cells stain with endothelial cell markers (17). Proliferation was high within the corpora lutea from control marmosets and was markedly reduced by anti-VEGF treatment (Fig. 2bGo), as confirmed by the decreased proliferation index (Fig. 2cGo). Factor VIII immunostaining demonstrated the initiation of the development of the microvasculature in control corpora lutea (Fig. 2dGo), but although there was an indication of reduced capillary number associated with anti-VEGF treatment (Fig. 2eGo), quantification of factor VIII immunostaining showed no significant difference at this time (Fig. 2fGo).



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  		Figure 2. Low power photomicrographs of early luteal phase marmoset corpora lutea showing the general distribution of BrdU incorporation (dark-staining nuclei) into endothelial cells of microvessels and capillaries in controls, showing high incorporation (a) and reduced incorporation (b) after anti-VEGF treatment. c, The proliferation index in corpora lutea from early luteal phase control (open bar) and anti-VEGF-treated (closed bar) marmoset corpora lutea. Values from treated animals were significantly lower (P < 0.01) than those in controls. d and e, Factor VIII localization (dark staining) in the endothelial cells of microvessels and capillaries of corpora lutea from control and anti-VEGF-treated marmosets, respectively. Scale bar, 100 µm. f, Quantification of factor VIII immunostaining in control (open bar) and anti-VEGF-treated (closed bar) marmoset corpora lutea. There was no significant difference between the groups. Data are the mean ± SEM.

 
In the midluteal phase group, treatment with anti-VEGF from day of ovulation again did not prevent formation of the fresh corpora lutea. In corpora lutea from control animals, the numbers of proliferating cells had declined, as expected by this stage of the luteal phase (Fig. 3aGo). Some proliferation was also evident in all the corpora lutea from the anti-VEGF-treated marmosets (Fig. 3bGo), and the proliferation index showed no significant treatment differences (Fig. 3cGo). However, whereas in control corpora lutea staining for factor VIII revealed that a fully developed microvasculature had been established (Fig. 3dGo), treatment with anti-VEGF resulted in corpora lutea with a much lower degree of vascularization and the absence of an extensive capillary bed, similar to that observed in the early luteal phase (Fig. 3eGo). Quantification of factor VIII immunostaining showed a marked suppression of endothelial cell area associated with treatment (Fig. 3fGo).



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  		Figure 3. Low power photomicrographs of midluteal phase marmoset corpora lutea showing the general distribution of BrdU incorporation (dark-staining nuclei) into endothelial cells of microvessels and capillaries in controls (a) and similar incorporation after anti-VEGF treatment (b). c, The proliferation index in corpora lutea from midluteal phase control (open bar) and anti-VEGF-treated (closed bar) marmoset corpora lutea. There was no significant difference between the groups. d, Factor VIII localization (dark staining) in the endothelial cells of microvessels and capillaries of corpora lutea in a control corpus luteum. e, Reduced staining, especially for incidence of capillary endothelial cells, after anti-VEGF treatment. Scale bar, 100 µm. f, Quantification of factor VIII immunostaining in control (open bar) and anti-VEGF-treated (closed bar) marmoset corpora lutea. Data are the mean ± SEM. Values from treated animals were significantly lower (P < 0.001) than those in controls.

 
Morphology
The weights of the ovaries of the early and midluteal control animals were similar, as were those of both treated groups. However, comparison of the combined weights of the controls (215 ± 11 mg) with those of the treated animals (158 ± 14 mg) revealed a significant reduction (P < 0.005) after anti-VEGF. Examination of sections stained with hematoxylin and eosin by two observers with expertise in identification of apoptotic cells, as described previously (15), revealed a virtual absence of apoptosis in both groups. Semithin sections stained with toluidine blue showed that the most prominent features in the control corpora lutea collected during the midluteal phase were the large polyhedral lutein cells, characterized by circular nuclei in cross-section with a nucleolus and large cytoplasmic volume (Fig. 4Go). Within the cytoplasm were mitochondria and lysosomes together with occasional, less basophilic inclusions typical of lipid droplets. In some tissue sections a few lutein cells showed varying degrees of basophilia, although it was not possible to determine whether cytoplasmic density was correlated with distinct differences in organelle or inclusion content. The lutein cells were supported by connective tissue, including fibroblasts, and there was an extensive blood supply characterized by the occurrence of endothelial cell nuclei and numerous lumina, often containing erythrocytes. In the corpora lutea from the midluteal anti-VEGF-treated marmosets, lutein cells appeared more densely packed (Fig. 4bGo), with a closer contact with neighboring lutein cells. Quantification of lutein cell number showed that numbers of lutein cells per area were significantly greater (P < 0.001) in the treated group (103.8± 5.5, for controls vs. 79.2 ± 3 after anti-VEGF). In addition, numerous clusters of basophilic cells, presumably lutein, were present principally in the central area of the corpus luteum. Higher magnification revealed that although the majority of the lutein cells had retained their integrity, they exhibited an increased occurrence of lipid droplets in the peripheral zone of the cytoplasm identified by their gray coloration and homogeneous appearance (Fig. 4dGo).



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  		Figure 4. Photomicrographs of toluidine blue-stained corpora lutea to show the appearance of hormone-producing lutein cells in the midluteal corpus luteum of control (a) and anti-VEGF-treated (b) marmosets. Note how the lutein cells of the treated animal are more closely packed, and the cluster of densely stained cells (arrows) presumably of luteal origin. Scale bars, 50 µm. Higher magnification of a control corpus luteum (c) shows the appearance of the lutein cell nucleus (N) and cytoplasm (C), with each cell being in contact with an endothelial cell (E). In the anti-VEGF corpus luteum (d) the lutein cells have maintained their integrity, but contain numerous lipid droplets (gray staining, arrowhead) in the peripheral zone. Scale bars, 20 µm.

 
Progesterone
Plasma progesterone concentrations were reduced (P < 0.001) by anti-VEGF treatment to approximately 40% of the normal values in both the early luteal (data not shown) and midluteal treatments (Fig. 5Go).



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  		Figure 5. Plasma progesterone concentrations in control ({circ}) and anti-VEGF-treated marmosets (•). Treatment started on the day of expected ovulation and is associated with a significant suppression (P < 0.001). Values are the mean ± SEM (n = 6/group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report of the successful inhibition of angiogenesis in the primate corpus luteum. To our knowledge, this is the first time physiological angiogenesis has been successfully targeted in a primate species. The high level of endothelial cell proliferation during the early luteal phase in the marmoset, with decreased levels of proliferation during the midluteal phase, agrees with studies using the proliferation marker Ki67 in the rhesus monkey (18) and human (16). We have shown that it is this intense angiogenesis in the early luteal phase that is susceptible to VEGF inhibition, as evidenced by the marked decrease in BrdU incorporation in the marmosets treated with antibody during the early luteal phase. An effect on endothelial cell area was not initially apparent after this short term treatment, but by the midluteal phase, this parameter was markedly inhibited in anti-VEGF-treated corpora lutea as a consequence of the inhibition of endothelial cell proliferation during the intense period of angiogenesis. Although larger blood vessels were established by the midluteal phase in treated animals, there was a marked overall reduction in the number of endothelial cells, suggesting the establishment of early luteal vasculature (possibly from preexisting vessels) and its survival through the midluteal phase, but indicating blockade of development of the normally extensive capillary bed. The absence of an effect on cell proliferation in the midluteal animals treated with anti-VEGF was surprising. However, by this stage, proliferation in controls has been much reduced over that seen during the early luteal phase, and this midluteal proliferation suggests that the rate is maintained either by low levels of VEGF that remain as a result of incomplete neutralization or has resulted from the action of other angiogenic factors in the corpus luteum.

The absence of marked morphological change in the lutein cells after treatment contrasts with observations on the characteristic effects of withdrawal of the tropic factor, LH, which we have described recently (17, 19). Such treatment also results in inhibition of early luteal angiogenesis, but this is secondary to the destruction of the lutein cells, in which it is assumed most of the principal angiogenic factors are produced (10, 11, 12, 17). The observed changes in the structure of the corpus luteum after neutralization of VEGF are likely to result from the specific inhibition of angiogenesis. The increased density of the lutein cells results primarily from the dearth of capillaries, whereas the occurrence of aggregates of subsets of lutein cells with more densely stained nuclei and cytoplasm, found principally in the central area of the corpus luteum, suggests a premature onset of cell death, an observation that would be compatible with a failure of blood vessel development to deliver essential survival factors. The increased presence of lipid droplets is more difficult to explain, but suggests either a lack of tropic hormone stimulus for steroid hormone release or a failure of the normal secretory process. The accumulation of lipid is often associated with the onset of degenerative change in steroid-producing cells, including the early stages of luteolysis in the marmoset (19). Despite the absence of major structural effects within lutein cells, secretion of the principal steroidal hormonal product of these cells is reduced by 60%. The most likely explanation for these findings is that the reduction in development of the microvasculature deprives the lutein cells of both the low density lipoprotein precursors necessary for progesterone production and the transport system for efficient release of their products into the bloodstream. Some effects of the treatment could also be related to an inhibition of the vascular permeability properties of VEGF, although this was not studied specifically. It is also conceivable that the treatment could have an inhibitory effect on pituitary LH secretion. However, we have recently shown that suppression of LH during the luteal phase results in characteristic and marked changes in the morphology of the lutein cells (19) that were not observed in this study, indicating that any change in LH secretion would be minor, making detection of any change unlikely using existing assays and with the sampling frequency employed.

The present results are in agreement with a recent report in the immature rat in which VEGF was inhibited while follicle development and multiple ovulations were induced by PMSG and hCG, respectively (6). However, in the rat model, as VEGF antagonist treatment was initiated before the onset of follicular hyperstimulation, it was not possible to dissociate the luteal inhibition from the consequences of inhibition of angiogenesis in the developing follicle.

Because angiogenesis inhibitors are currently being developed primarily for treatment of vascular solid tumors and are also likely to have a role in the control of rheumatoid arthritis, retinal neovascularization, and psoriasis it is essential that their potential effects on the reproductive system and its function be determined. In addition, the potential of using such inhibitors as antifertility agents is currently generating considerable interest. However, it is unlikely that all angiogenesis inhibitors identified in studies using nonprimate species will influence fertility, as shown by our findings in the marmoset, in which TNP-470 was administered in a protocol similar to that used in the present study but had no inhibitory effect on luteal angiogenesis (9).

As we have shown that luteal inadequacy is associated with inhibition of angiogenesis, this suggests that the clinical incidence of these conditions may in part be the result of defects in the angiogenic process. They may therefore prove to be amenable to treatment with angiogenic growth factors such as VEGF. The potential to inhibit angiogenesis in the female reproductive tract should have important implications for clinical practice, but the development of safe and effective strategies will require detailed investigation of suitable animal models, as described in this study. Finally, manipulation of VEGF in this fashion should provide a novel approach to investigate the precise physiological role of VEGF in ovarian angiogenesis.


   Acknowledgments
 
We thank the staff of our Primate Unit for animal care; M. Millar, S. MacPherson, and P. Largue for expert support with histology; and F. Pitt and I. Swanston for RIAs.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft for financial support (to C.W.). Back

Received October 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kim KJ, Winer J, Armanini M 1993 Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362:841–844[CrossRef][Medline]
  2. Olson TA, Mohanraj D, Roy, Ramakrishnan S 1997 Targeting of the tumor vasculature: inhibition of tumor growth by a vascular endothelial growth factor (VEGF)-toxin conjugate. Int J Cancer 73:865–870[CrossRef][Medline]
  3. O’Reilly MS., Boehm T, Shing Y, Fukai N, Vasios G, et al 1997 Endostation: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277–285[CrossRef][Medline]
  4. Skobe M, Rockwell P, Goldstein N, Vosseler S, Fusenig NE 1997 Halting angiogenesis suppresses carcinoma cell invasion. Nat Med 3:1222–1227[CrossRef][Medline]
  5. Redmer DA, Reynolds LP 1996 Angiogenesis in the ovary. Rev Reprod 1:182–192[Abstract]
  6. Ferrara N, Chen H, Davis-Smyth T, Hans-Peter G, Nguyen T-N, et al 1998 Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nat Med 4:336–340[CrossRef][Medline]
  7. Klauber N, Rohan RM, Flynn E, D’Amato RJ 1997 Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470. Nat Med 3:443–446[CrossRef][Medline]
  8. McCracken JA, Custer, EE, Lamasa JC 1999 Luteolysis: a neuroendocrine-mediated event. Physiol Rev 79:263–323[Abstract/Free Full Text]
  9. Fraser HM, Dickson SE, Morris KD, Erickson GF, Lunn SF 1999 The effect of the angiogenesis inhibitor TNP-470 on luteal establishment and function in the primate. Hum Reprod 14:2054–2060[Abstract/Free Full Text]
  10. Ravindranath N, Little-Ihrig L, Phillips H, Ferrara N, Zeleznik AJ 1992 Vascular endothelial growth factor messenger ribonucleic acid expression in the primate ovary. Endocrinology 131:254–260[Abstract]
  11. Kamat BR, Brown LF, Manseau EJ, Senger DR, Dvorak HF 1995 Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Am J Pathol 146:157–165[Abstract]
  12. Gordon JD, Mesiano S, Zaloudek CJ, Jaffe RB 1996 Vascular endothelial growth factor localization in human ovary and fallopian tubes: possible role in reproductive function and ovarian cyst formation. J Clin Endocrinol Metab 81:353–359[Abstract]
  13. Smith KB, Lunn SF, Fraser HM 1990 Inhibin secretion during the ovulatory cycle and pregnancy in the common marmoset monkey. J Endocrinol 126:489–495[Abstract]
  14. Summers PM, Wennink J, Hodges JK 1985 Cloprostenol-induced luteolysis in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 73:133–138[Abstract]
  15. Young FM, Illingworth PJ, Lunn SF, Harrison DJ, Fraser HM 1997 Cell death during luteal regression in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 111:109–119[Abstract]
  16. Rodger FE, Young FM, Fraser HM, Illingworth PJ 1997 Endothelial cell proliferation follows the mid-cycle luteinizing hormone surge, but not human chorionic gonadotrophin rescue, in the human corpus luteum. Hum Reprod 12:1723–1729[Abstract/Free Full Text]
  17. Dickson SE, Fraser HM Inhibition of early luteal angiogenesis by gonadotropin-releasing hormone antagonist treatment in the primate. J Clin Endocrinol Metab, in press
  18. Christenson LK, Stouffer RL 1996 Proliferation of microvascular endothelial cells in the primate corpus luteum during the menstrual cycle and simulated early pregnancy. Endocrinology 137:367–374[Abstract]
  19. Fraser HM, Lunn SF, Harrison DJ, Kerr JB 1999 Luteal regression in the primate: different forms of cell death during natural luteolysis and after GnRH antagonist or prostaglandin treatment. Biol Reprod 61:1468–1479[Abstract/Free Full Text]



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C. Stocco, C. Telleria, and G. Gibori
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Endocr. Rev., February 1, 2007; 28(1): 117 - 149.
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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.
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H. M Fraser, H. Wilson, C. Wulff, J. S Rudge, and S. J Wiegand
Administration of vascular endothelial growth factor Trap during the 'post-angiogenic' period of the luteal phase causes rapid functional luteolysis and selective endothelial cell death in the marmoset.
Reproduction, October 1, 2006; 132(4): 589 - 600.
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A. Tropea, F. Miceli, F. Minici, F. Tiberi, M. Orlando, M. F. Gangale, F. Romani, S. Catino, S. Mancuso, P. Navarra, et al.
Regulation of Vascular Endothelial Growth Factor Synthesis and Release by Human Luteal Cells in Vitro
J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2303 - 2309.
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P Froment, F Gizard, D Defever, B Staels, J Dupont, and P Monget
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J. Endocrinol., May 1, 2006; 189(2): 199 - 209.
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A Martelli, P Berardinelli, V Russo, A Mauro, N Bernabo, L Gioia, M Mattioli, and B Barboni
Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period
J. Mol. Endocrinol., February 1, 2006; 36(1): 107 - 119.
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A. Matsuoka-Sakata, H. Tamura, H. Asada, I. Miwa, T. Taketani, Y. Yamagata, and N. Sugino
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Reproduction, February 1, 2006; 131(2): 351 - 360.
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N. Sugino, T. Suzuki, A. Sakata, I. Miwa, H. Asada, T. Taketani, Y. Yamagata, and H. Tamura
Angiogenesis in the Human Corpus Luteum: Changes in Expression of Angiopoietins in the Corpus Luteum throughout the Menstrual Cycle and in Early Pregnancy
J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6141 - 6148.
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H. M. Fraser, H. Wilson, K. D. Morris, I. Swanston, and S. J. Wiegand
Vascular Endothelial Growth Factor Trap Suppresses Ovarian Function at All Stages of the Luteal Phase in the Macaque
J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5811 - 5818.
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F. Xu and R. L. Stouffer
Local Delivery of Angiopoietin-2 into the Preovulatory Follicle Terminates the Menstrual Cycle in Rhesus Monkeys
Biol Reprod, June 1, 2005; 72(6): 1352 - 1358.
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S. A. Pauli, H. Tang, J. Wang, P. Bohlen, R. Posser, T. Hartman, M. V. Sauer, J. Kitajewski, and R. C. Zimmermann
The Vascular Endothelial Growth Factor (VEGF)/VEGF Receptor 2 Pathway Is Critical for Blood Vessel Survival in Corpora Lutea of Pregnancy in the Rodent
Endocrinology, March 1, 2005; 146(3): 1301 - 1311.
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N. Lambeng, Y. Wallez, C. Rampon, F. Cand, G. Christe, D. Gulino-Debrac, I. Vilgrain, and P. Huber
Vascular Endothelial-Cadherin Tyrosine Phosphorylation in Angiogenic and Quiescent Adult Tissues
Circ. Res., February 18, 2005; 96(3): 384 - 391.
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H. M. Fraser, H. Wilson, J. S. Rudge, and S. J. Wiegand
Single Injections of Vascular Endothelial Growth Factor Trap Block Ovulation in the Macaque and Produce a Prolonged, Dose-Related Suppression of Ovarian Function
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1114 - 1122.
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H. M. Fraser, J. Bell, H. Wilson, P. D. Taylor, K. Morgan, R. A. Anderson, and W. C. Duncan
Localization and Quantification of Cyclic Changes in the Expression of Endocrine Gland Vascular Endothelial Growth Factor in the Human Corpus Luteum
J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 427 - 434.
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P D Taylor, S G Hillier, and H M Fraser
Effects of GnRH antagonist treatment on follicular development and angiogenesis in the primate ovary
J. Endocrinol., October 1, 2004; 183(1): 1 - 17.
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N. Ferrara
Vascular Endothelial Growth Factor: Basic Science and Clinical Progress
Endocr. Rev., August 1, 2004; 25(4): 581 - 611.
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A. J Rowe, C. Wulff, and H. M Fraser
Angiogenesis and microvascular development in the marmoset (Callithrix jacchus) endometrium during early pregnancy
Reproduction, July 1, 2004; 128(1): 107 - 116.
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A. Zippo, A. De Robertis, M. Bardelli, F. Galvagni, and S. Oliviero
Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro
Blood, June 15, 2004; 103(12): 4536 - 4544.
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F. Baffert, G. Thurston, M. Rochon-Duck, T. Le, R. Brekken, and D. M. McDonald
Age-Related Changes in Vascular Endothelial Growth Factor Dependency and Angiopoietin-1-Induced Plasticity of Adult Blood Vessels
Circ. Res., April 16, 2004; 94(7): 984 - 992.
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T. Sakurai, K. Tamura, S. Okamoto, T. Hara, and H. Kogo
Possible Role of Cyclooxygenase II in the Acquisition of Ovarian Luteal Function in Rodents
Biol Reprod, September 1, 2003; 69(3): 835 - 842.
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N. Ferrara, G. Frantz, J. LeCouter, L. Dillard-Telm, T. Pham, A. Draksharapu, T. Giordano, and F. Peale
Differential Expression of the Angiogenic Factor Genes Vascular Endothelial Growth Factor (VEGF) and Endocrine Gland-Derived VEGF in Normal and Polycystic Human Ovaries
Am. J. Pathol., June 1, 2003; 162(6): 1881 - 1893.
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D. R. Danforth, L. K. Arbogast, S. Ghosh, A. Dickerman, R. Rofagha, and C. I. Friedman
Vascular Endothelial Growth Factor Stimulates Preantral Follicle Growth in the Rat Ovary
Biol Reprod, May 1, 2003; 68(5): 1736 - 1741.
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J.C. Martinez-Chequer, R.L. Stouffer, T.M. Hazzard, P.E. Patton, and T.A. Molskness
Insulin-Like Growth Factors-1 and -2, but not Hypoxia, Synergize with Gonadotropin Hormone to Promote Vascular Endothelial Growth Factor-A Secretion by Monkey Granulosa Cells from Preovulatory Follicles
Biol Reprod, April 1, 2003; 68(4): 1112 - 1118.
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