1 MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK and 2 Department of Reproductive Medicine, University of California at San Diego, USA
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Abstract |
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Key words: endothelial cells/factor VIII/IGFBP-3/monkey/proliferation
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Introduction |
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The study was designed to investigate the effects of TNP-470 on angiogenesis at the time of transformation of the pre-ovulatory follicle into the functional corpus luteum. Two species of non-human primate were employed. First, stump-tailed macaques were investigated to allow detailed analysis of the endocrinology in response to TNP-470 treatment. We chose this species because we have previously established its clinical relevance with respect to manipulation of the menstrual cycle in studies using gonadotrophin releasing hormone analogues (Fraser et al., 1997) and on the physiology of inhibin (Fraser et al., 1999
). In addition, we investigated the effects of TNP-470 in the marmoset monkey in which we have built up considerable information on the cellular and molecular control of luteal function (e.g. Young et al., 1997
; Duncan et al., 1998
; Fraser et al., 1998
). The establishment and function of the corpus luteum was determined by measuring circulating progesterone concentrations in both species. Cellular responses were studied in the marmoset by determining the number of mitotic cells, following bromodeoxyuridine (BrdU) administration as a marker, by examining the establishment of the microvascular network using factor VIII staining to identify endothelial cells, and by studying the capacity of the endothelial cells to express insulin-like growth factor-binding protein-3 (IGFBP-3) messenger ribonucleic acid (mRNA) which we have shown previously to be a marker for healthy endothelial cells in the marmoset corpus luteum (Fraser et al., 1998
).
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Materials and methods |
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Common marmoset monkeys were housed in cages in rooms at a temperature of 22.5°C, exposed to the same lighting conditions as described above. The animals were fed daily with a selection of fruit, SDS Mazuri (E) primate diet pellets, and high protein porridge with multivitamin supplements three times per week. Adult females having a body weight of approximately 350 g, with regular ovulatory cycles, were housed together with a younger sister or prepubertal female in cages measuring 1.15 m in height, 1.1 m in depth and 0.6 m wide. Each contained larch branches and a nest box with the floor of the cage filled with wood chippings to allow foraging. Blood samples were collected three times per week by femoral venepuncture without anaesthesia to confirm normal ovulatory cycles. Plasma was stored at 20°C until required for assay. Criteria for the occurrence of ovulation and normal luteal phase length (1822 days) were based on determination of plasma progesterone concentrations as described previously (Smith et al., 1990).
Treatments
The experiments were carried out in accordance with the Animals (Scientific Procedures) Act, 1986. TNP-470 (O-chloroacetylcarbamoyl fumagillol, also known as AGM-1470), has been administered to patients with Kaposi's sarcoma at doses ranging from 1070 mg/m2 given by i.v. infusion over a period of 1 h once per week (Dezube et al., 1998). The current clinical schedule is of 60 mg/m2 TNP-470 administered by i.v. infusion over a period of 1 h three times per week (Dezube et al., 1998
). In an attempt to ensure effective treatment, it was decided to administer 120 mg/m2 to the primates, calculated according to a published formula (Du Bois and Du Bois, 1916
). On a dose/kg basis, for a woman weighing 56 kg, the total daily dose would be 96 mg, i.e. 1.7 mg/kg. For the macaques, a dose of between 60 and 84 mg was used, equivalent to 6 mg/kg. Seven stump-tailed macaques, with regular ovulatory menstrual cycles, were treated with TNP-470 dissolved in 5% dextrose at a concentration of 4 mg/ml immediately before use. On three occasions 2 days apart, starting around the time of predicted ovulation, the macaques received a total volume of between 1521 ml, depending upon body weight, administered by slow i.v. injection in two separate doses 1 h apart. A second experiment was undertaken in which five macaques were given the same treatment, commencing 4 days prior to expected ovulation, a period covering final pre-ovulatory development.
Since the final results in macaques indicated an absence of effect of treatment on luteal function, it was decided to investigate the susceptibility of the primate corpus luteum to an increased (daily) dose of TNP-470 over a longer time period (10 days) and, to investigate the possibility that angiogenesis could be inhibited in the absence of an effect on progesterone secretion, a further experiment was undertaken using the marmoset. To synchronize timing of ovulation during the treatment cycle, eight animals were treated with 1 µg prostaglandin F2 analogue (Planate®; Coopers Animal Health Ltd, Crewe, Cheshire, UK) i.m., during the mid- to late luteal phase of the pre-treatment cycle to induce luteolysis. This treatment is normally followed by ovulation 1012 days later (Summers et al., 1985
). Four marmosets were treated with TNP-470 starting 11 days post-prostaglandin, the day of anticipated ovulation, at a dose of 18 mg/kg/day (equivalent to 120 mg/m2), by slow i.v. injection. This treatment was repeated for a further 9 days: the remaining four controls were treated with vehicle following the same schedule.
On day 10 after commencement of TNP-470, each marmoset received by slow i.v. infusion, 20 mg BrdU (Boehringer Mannheim, Essex, UK) in saline. One hour later, the animals were sedated using 100 µl ketamine hydrochloride (Parke-Davis Veterinary, Pontypool, Gwent, UK) i.m. and euthanased with an i.v. injection of 400 µl sodium pentobarbitone (Euthetal®; Rhone Merieux, Ireland). Ovaries were removed rapidly, weighed, and fixed immediately in 4% paraformaldehyde for 24 h. After fixation, the corpora lutea of the cycle were identified macroscopically, the ovaries were bisected as closely as possible through the centre of the corpora lutea, and the tissues dehydrated and embedded in paraffin.
Hormone assays
Circulating progesterone and oestradiol concentrations were measured as described previously for the stump-tailed macaque (Fraser and Sandow, 1985; Fraser et al., 1997
) and marmoset (Smith et al., 1990
).
Immunocytochemistry
Tissue sections (5 µm) were cut onto Tespa-coated (Sigma, Poole, Dorset, UK) slides for immunocytochemistry and morphological examination. Sections were stained with haematoxylin and eosin and examined for morphological features of apoptosis as described previously (Young et al., 1997). Localization of factor VIII as a marker for endothelial cells was determined by immunocytochemistry as described for the human corpus luteum (Rodger et al., 1997
) except that visualization was with nitro blue tetrazolium (NBT) in buffer (100 mol/l Tris pH 9.5, 100 mol/l NaCl and 50 mol/l MgCl2). These sections were not counterstained, so that quantitative image analysis could be performed, the corpora lutea being identified from the haematoxylin and eosin-stained sections. Factor VIII immunostaining was quantified using the Image Pro-Plus 3.0® (Media Cybernetics, Silver Spring, MD, USA) image analysis program. Sections were examined at x400 magnification. The image was converted to grey scale and the area of dark objects on a white background was counted at a threshold of 70. For each corpus luteum, six randomly chosen areas, each of 27 860 µm2, were analysed and their mean taken as being representative for that animal. Differences between groups were determined using a two tailed unpaired t-test, P < 0.05 being taken as the level of significance.
Proliferating cells were visualized in ovarian sections using a mouse monoclonal antibody to BrdU (Boehringer Mannheim). Sections were dewaxed in histoclear and rehydrated in decreasing concentrations of industrial methylated spirits. Antigen retrieval was accomplished by microwaving sections for 4x5 min in 0.01 M citrate buffer. Using Sequenza racks (Life Sciences International, Hampshire, UK), sections were treated for 30 min with normal rabbit serum block (NRS; Dako, High Wycombe, Bucks, UK), then incubated in mouse monoclonal anti-BrdU (3 µg/ml in TBS) at 4°C, overnight. Control sections were treated with mouse IgG (Vector Laboratories, Peterborough, UK) (3 µg/ml in TBS) in place of primary antibody. Secondary antibody, rabbit anti-mouse (26 µg/ml in blocking serum), was applied for 30 min at room temperature. BrdU binding was visualized using mouse APAAP (1 µg/ml in blocking serum) incubation for 30 min, and the NBT detection system (Boehringer Mannheim). Sections were briefly counterstained with haematoxylin and mounted for analysis. Cell proliferation was assessed by counting the number of BrdU positive cells in six randomly chosen fields per corpus luteum and expressed as a mean percentage of the total cells in these fields. Differences between groups were determined using a two tailed unpaired t-test, P < 0.05 being taken as the level of significance.
In-situ hybridization
Paraffin sections (5 µm) were mounted onto poly-L-lysine coated glass slides for IGFBP-3 mRNA localization undertaken by in-situ hybridization as described previously (Fraser et al., 1998). In addition to the ovaries from control and treated animals, ovaries from follicular phase marmosets known to contain spontaneously regressing corpora lutea were included in the same run. Following hybridization with antisense (two slides per ovary) and sense probes, and washing, dry slides were dipped in Kodak NTB-2® liquid emulsion (Eastman Kodak, Rochester, NY, USA) and exposed for 3.5 weeks at 4°C in light-tight slide boxes. Slides were developed with D-19 developer (Eastman Kodak) for 3.5 min at 4°C, fixed, and the sense and one antisense stained with haematoxylin (Richard-Allan, Richland, MI, USA), while the remaining antisense slide was stained with both haematoxylin and eosin to allow a more reliable identification of cell type. Identified endothelial cells that were not associated with a perceptible vascular lumen were classified as capillary endothelial cells: when a lumen was evident, they were classified as microvessels and included venules and arterioles. The localization of expression to microvessels, capillaries and lutein cells of the corpora lutea was scored as follows: no expression above tissue background , low expression +, moderate expression ++, high expression +++.
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Results |
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Discussion |
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With respect to timing and duration of treatment, the experiment was designed to target the intense angiogenesis which occurs in the corpus luteum during the early stages of development (Christenson and Stouffer, 1996; Rodger et al., 1997
). Accordingly, treatment was started at the time of predicted ovulation and covered the period of the early luteal phase in the macaque and was extended to the mid-luteal phase in the marmoset. The studies in the marmoset allowed the investigation of the effects of a more frequent and prolonged dose schedule of TNP-470 and the examination of its potential consequences on cell proliferation and the luteal vasculature. Progesterone secretion was not impaired and the number of corpora lutea formed was normal. Furthermore, staining for factor VIII illustrated the establishment of the luteal vascular tree despite treatment with the putative angiogenesis inhibitor. As expected, BrdU incorporation demonstrated a high level of proliferation of endothelial cells during the mid-luteal phase and this too was unaffected by treatment with TNP-470. Finally, expression of IGFBP-3 mRNA, which we have previously shown to be a marker for endothelial cells in the functioning corpus luteum of the marmoset and human (Fraser et al., 1998
), was also unchanged. Collectively, these results suggest that TNP-470 does not have negative effects on the structure and function of the normal corpus luteum of the marmoset, in contrast to the findings in the mouse (Klauber et al., 1997
). At this early stage of experiments to inhibit angiogenesis in the primate corpus luteum, we are forced to conclude that the differences between the present results and those in the mouse may reflect inter-species variance, and/or differential susceptibility to the anti-angiogenic effect of TNP-470.
The latter possibility could arise from the dose of TNP-470 employed in the present study being insufficient to prevent luteal angiogenesis, or the route of administration being suboptimal. The clinical dose of TNP-470 is 60 mg/m2 administered by 1 h i.v. infusion three times per week (Dezube et al., 1998). The dose chosen for the macaques was twice that with respect to surface area and 3.5 times that of the human dose when expressed on a drug weight to body weight ratio. Also, a dose of 6 mg/kg by 1 h infusion in macaques was anti-angiogenic with respect to suppression of ocular angiogenesis (J. Folkman and M. O'Reilly, personal communication). The stump-tailed macaques used in the present study were treated using two i.v. injections 1 h apart, rather than by infusion over 1 h, because this enabled the treatment to be administered without surgical intervention or sedation. Studies in the mouse employed the s.c. route at a dose of 30 mg/kg every second day. The marmosets in the present study were treated with 18 mg/kg daily, so approaching the murine dose. It should be noted that TNP-470 is metabolized very rapidly and that its pharmacodynamic properties are incompletely understood (Cretton-Scott et al., 1996
). Thus, differences in the pattern of exposure to the drug may result from the different routes of administration. However, at present, a preparation of TNP-470 that can be administered by the s.c. route to larger animals and humans is not available.
This is the first report of an attempt to block angiogenesis in the primate corpus luteum. Although inhibitory effects were not observed, we believe that the approach used to study angiogenesis in the marmoset will be of value to future experiments on the effects of angiogenesis inhibitors on luteal function in the primate. BrdU incorporation proved an accurate method of identifying proliferating cells in the corpus luteum. This technique has been used previously to quantify luteal proliferation in the sheep (Jablonka-Shariff et al., 1993) and rat (Gaytan et al., 1996
) but, to our knowledge, this is the first report of its use with respect to the primate ovary. The high level of proliferation during the mid-luteal phase in the marmoset agrees with results using the proliferation marker Ki67 in the rhesus monkey and human (Christenson and Stouffer, 1996
; Rodger et al., 1997
). The localization of IGFBP-3 mRNA in the marmoset ovary confirms and extends our recent observation that production of this mRNA is associated with the functioning corpus luteum in the primate (Fraser et al., 1998
), and contrasts with the situation in the rodent where its expression is temporally associated with luteolysis (Erickson et al., 1993
). This binding protein is likely to be of importance in the modulation of IGF activity within the corpus luteum. The observation that the expression of the IGFBP-3 mRNA is associated with the endothelial cells of the functioning corpus luteum only, also indicates a role yet to be defined in endothelial cell proliferation and survival.
The potential of using angiogenesis inhibitors as anti-fertility agents is currently generating considerable interest (Klauber et al., 1997; Ferrara et al., 1998
). Furthermore, while they are being developed primarily for treatment of vascular solid tumours, they are also likely to find a role in control of rheumatoid arthritis, retinal neovascularization and psoriasis (Folkman, 1995
). It is essential, therefore, that their potential effects upon the reproductive system and its function in such patients be determined. However, the current results show that with the treatment regimens employed, TNP-470 has no significant effect on the expression of the differentiated state of the primate corpus luteum.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 4, 1998; accepted on April 8, 1999.