Endostatin inhibits the growth of endometriotic lesions but does not affect fertility

Endostatin inhibits the growth of endometriotic lesions but does not affect fertility

Endostatin inhibits the growth of endometriotic lesions but does not affect fertility Christian M. Becker, M.D.,a,c,e David A. Sampson, B.A.,a Maria A...

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Endostatin inhibits the growth of endometriotic lesions but does not affect fertility Christian M. Becker, M.D.,a,c,e David A. Sampson, B.A.,a Maria A. Rupnick, M.D.,a,d,f Richard M. Rohan, Ph.D.a Jason A. Efstathiou, M.D.,a Sarah M. Short, Ph.D.,a George A. Taylor, M.D.,b Judah Folkman, M.D.,a and Robert J. D’Amato, M.D.a,g a

Department of Surgery, Vascular Biology Program, and b Department of Radiology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts; c Department of Obstetrics and Gynecology and d Department of Cardiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; e Department of Obstetrics and Gynecology, Charité— Campus Benjamin Franklin, Berlin, Germany; f Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; and g Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts

Objective: To determine whether endometriosis can be treated with the angiogenesis inhibitor endostatin and the effect of this treatment on fertility and reproduction. Design: Pharmacologic intervention in a surgically induced model of endometriosis and in female mice undergoing mating. Setting: Animal research facility. Animal(s): Eight-week-old, female C57BL/6 and SCID mice. Intervention(s): After implantation of autologous endometrium, mice received endostatin or the vehicle-matched control for 4 weeks. For the reproductive function study, mice receiving endostatin or vehicle were mated and reproductive functions were observed. Main Outcome Measure(s): Growth of endometriotic lesions after 4 weeks of treatment; estrous cycling, corpus luteum formation, serum hormone levels, and mating time as fertility measures; and pregnancy rates, length of pregnancy, fetal vitality, number, and outcome of litter as reproductive measures. Result(s): Endostatin suppressed the growth of endometriotic lesions by 47% compared with controls. Estrous cycling and corpus luteum formation were normal in both groups. Female mice receiving endostatin were as fertile as mice receiving vehicle, had normal pregnancies, and delivered the same number of pups. The offspring were healthy without teratogenic stigmata and reproduced normally themselves. Conclusion(s): Antiangiogenic therapy with endostatin may present a promising novel, nontoxic therapeutic option for patients with endometriosis. (Fertil Steril威 2005;84(Suppl 2):1144 –55. ©2005 by American Society for Reproductive Medicine.) Key Words: Endostatin, angiogenesis inhibitors, reproduction, endometriosis, toxicity

Endometriosis is caused by the implantation of endometrial tissue in sites outside the uterus. It is an estrogen-driven disease and, as such, afflicts women almost exclusively during their childbearing years. Current estimates suggest that 5%–21% of women with pelvic pain suffer from endometriosis (1). In addition, it is estimated that almost half of women with endometriosis are infertile (2). Surgical removal of lesions and hormonal suppression are currently the gold

Received November 18, 2004; revised and accepted April 9, 2005. Supported by a postdoctoral research exchange grant from the Max Kade Foundation to C.M.B., by a grant from the Sidney A. Swensrud Foundation to R.J.D., and by a National Institutes of Health/National Cancer Institute Program Project Grant (P01-CA45548). Presented at the 2002 Annual Meeting of the Society for Gynecologic Investigation, which was held in Los Angeles, California, on March 20 –23, 2002, and the 2004 Annual Meeting of the European Society for Human Reproduction and Embryology, which was held in Berlin, Germany, on June 27–30, 2004. Reprint requests: Robert D’Amato, M.D., Vascular Biology Program, Children’s Hospital Boston, Harvard Medical School, 300 Longwood Ave., Boston, MA (FAX: 617-730-0231; E-mail: robert.damato@childrens. harvard.edu).

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standards of therapy, but both approaches are associated with significant side effects. Although endometriosis has been recognized for many centuries, the pathogenesis of the disease remains enigmatic (3). The most favored hypothesis implicates retrograde menstruation into the abdominal cavity with subsequent adhesion and implantation of endometrium to the peritoneum (4). Metaplasia of mesothelial cells in the peritoneum is another popular explanation (5, 6). In any scenario, angiogenesis, the acquisition of new blood vessels, is essential for the establishment and growth of endometriotic tissue (7). Increased levels of proangiogenic factors have been demonstrated in endometriotic tissue and peritoneal fluid of women with endometriosis (8 –10). Endometriotic lesions and the surrounding tissue are highly vascularized (11, 12). Therefore, inhibition of angiogenesis may be an attractive new therapeutic option for patients with endometriosis (13). However, a challenge to this approach is that endometriosis typically affects women during their reproductive years and angiogenesis plays an important role in many reproductive processes such as ovulation, corpus luteum formation, and

Fertility and Sterility姞 Vol. 84, Suppl 2, October 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.

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implantation of the embryo (14). Further, the formation of new blood vessels is indispensable during embryonic and fetal development. Angiogenesis inhibitors have also been shown to cause birth defects in animals and humans (15–17). Selective inhibition of angiogenesis in the endometriotic lesions without disruption of reproduction would therefore be optimal. Endostatin, a proteolytic fragment of collagen XVIII, is an endogenous antiangiogenic protein (18). It has been shown to inhibit endothelial proliferation, migration/invasion, and tube formation in vitro. Endostatin has also been repeatedly demonstrated to inhibit tumor angiogenesis and growth in various tumor models and is currently in clinical phase II/III cancer trials (19). We have previously reported that surgically induced endometriosis in mice is angiogenesis dependent and can be suppressed by antiangiogenic agents (20, 21). Recently, endostatin has been shown to inhibit established human endometriotic lesions in nude mice (22). In the present study, we demonstrate that endostatin can inhibit the establishment and growth of surgically induced endometriotic lesions in a mouse autograft model. However, once established, the lesions are not regressed by this treatment. Our results further indicate that endostatin therapy does not interfere with fertility and pregnancy in mice. The offspring of mothers treated with endostatin show no teratogenic birth defects, develop normally, and are fertile. Taken together, these results demonstrate a potent new therapeutic approach that may avoid the typical side effects of current drugs used to treat endometriosis. MATERIALS AND METHODS Animal Studies All animal work was performed in the animal facility at Children’s Hospital, Boston, in accordance with federal, local, and institutional guidelines. Eight-week-old female immune-compromised (SCID, Massachusetts General Hospital, Boston) and C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were caged in groups of five to ten mice with free access to chow and water and acclimated for a week. All surgical procedures and ultrasound imaging were performed under inhalative anesthesia with isoflurane (Baxter, Deerfield, IL), and mice were observed until fully recovered. All animal studies were repeated at least twice. Surgical Procedure Endometriosis-like lesions were induced using a surgical technique modified from Vernon and Wilson (23) and Cummings and Metcalf (24) as previously described by our group (25). Briefly, mice underwent laparotomy by midventral incision. Both uterine horns were ligated with small surgical titanium clips (Horizon, Research Triangle Park, NC), removed, and placed in a Petri dish containing warmed Dulbecco’s modified Eagle’s medium F-12 (Gibco, Grand Island, NY) supplemented with 100 U/mL of penicillin and Fertility and Sterility姞

100 ␮g/mL of streptomycin (Gibco). The uterine horns were opened longitudinally, and seven biopsies 2 mm in diameter were taken using a dermal biopsy punch (Miltex, Bethpage, NY). Four pieces of tissue were then sutured to the peritoneal wall and three onto the mesentery with a 7-0 braided silk suture (Ethicon, Somerville, NJ). The abdominal wall was closed with a 5-0 braided silk suture in a continuous fashion. Endostatin Treatment To investigate the role of antiangiogenic therapy during the initial establishment and growth of endometriotic lesions, we treated C57BL/6 mice with murine endostatin immediately after endometriosis surgery for 4 consecutive weeks. In two sets of experiments (n ⫽ 10/group/experiment), soluble recombinant murine endostatin was continuously administered via miniosmotic pumps (Durect, Cupertino, CA) placed subcutaneously (SC) into an interscapular pocket on the dorsum of the animals (26). For this, lyophilized murine endostatin (a generous gift from EntreMed Corporation, Rockville, MD) was reconstituted in sterile micro pure double-distilled water to a stock concentration of 128 mg/mL and diluted in sterile phosphate-buffered saline (PBS) immediately before further use. The mice received 20 mg/kg/day of endostatin via continuous infusion at a rate of 0.1 ␮L/hour. The pumps of control mice contained vehicle alone (PBS). After 4 weeks of treatment, mice were sacrificed and the original ventral midline incision was reopened. Implantation sites were localized by the presence of a lesion or by a suture alone. Lesions were counted and measured in two perpendicular diameters (D1, D2) to the nearest 0.1 mm using calipers. Lesion areas were determined using the formula for an ellipse (D1 ⫻ D2 ⫻ ␲/4). Then lesions were excised and preserved in 10% phosphate-buffered formalin at 4°C for histological analysis. Ovaries and residual uterus were also collected. After 10 hours, samples were transferred into a 3:1 PBS/ethanol solution, processed using standardized techniques, and embedded in paraffin for histological sectioning. To investigate the effects of endostatin on established endometriotic lesions, treatment was delayed until 4 weeks after surgical induction of endometriosis. Eight-week-old C57BL/6 mice (n ⫽ 8/group) underwent abdominal surgery for endometriosis as described previously. Endostatin administration began 4 weeks postoperatively via SC miniosmotic pumps as described previously. Mice in the treatment group received 20 mg/kg/day of murine endostatin, whereas the control group received vehicle alone (PBS). Eight weeks after the initial surgical procedure, mice were sacrificed and lesions were counted and measured. Estrous Cycling One of the goals of this study was to investigate the influence of endostatin on estrous cycling. To synchronize estrous cycling before surgery, female C57BL/6 mice were housed in groups of five for 1 week. During the treatment period, 1145

mice were housed separately to avoid cycle arrest from group housing. Endostatin treatment was begun immediately after endometriosis surgery. Two weeks into the 4-week treatment period, estrous cycling was evaluated by cytological analysis of daily vaginal smears for 2 weeks. Exams were performed each morning. Autoclaved, wetted, blunt toothpicks were used to gently scrape the superficial cell layer of the vaginal canal. Cells were transferred to a drop of Evan’s blue in 0.9% saline on a glass slide for staining, sealed under a glass coverslip, and examined under a microscope (Carl Zeiss, Thornwood, NY). Each slide was analyzed by two independent investigators who were blinded to the form of treatment and the previous data for each mouse. Hormone Serum Levels After 4 weeks of treatment, immediately before sacrifice, blood samples were collected by retro-orbital bleeding under anesthesia using microhematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA) into Microtainer serum separator tubes (Becton Dickinson, Franklin Lakes, NJ). These tubes were centrifuged at 8,000 rpm for 15 minutes. Serum was immediately collected and stored at ⫺80°C. Hormone levels were assayed by the Endocrine Services Laboratory, Oregon Regional Primate Research Center, under the direction of David L. Hess, Ph.D., according to previously published methods (27). Briefly, samples were thawed and serum was extracted with diethyl ether. The extract was chromatographed on LH-20 Sephadex microcolumns. The estradiol (E2) fraction was collected and assayed by radioimmunoassay according to established protocol (27). Samples are reported as median with interquartile range (n ⫽ 5/group) and compared using a nonparametric (Mann–Whitney) test. Corpus Luteum Counts To investigate the effect of continuous endostatin treatment on follicular development, we counted the number of corpora lutea per ovary as an indicator of recent ovulation. Ovaries from 12, randomly chosen mice (3 mice/group from 2 experiments) were paraffin embedded and sectioned (4 – 6 ␮m). Four consecutive sections were put on each slide, and every other slide was stained with hematoxylin-eosin. The number of corpora lutea/ovary were independently counted by three investigators. The highest number of corpora lutea per ovary on each slide was recorded, and the average numbers of all slides were compared using Student’s t-test. Tumor Model We previously showed that human endostatin inhibits tumor growth of human pancreatic cancer in mice. We used this tumor as an indicator of biologic activity of endostatin while investigating the effect of endostatin on fertility and reproduction. Human BxPC-3 pancreatic carcinoma cells were grown and maintained as described (26). Cells were grown in 175-cm2 cell culture flasks in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/ 1146

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streptomycin/glutamine (Invitrogen Gibco, Carlsbad, CA) at 37°C, 10% CO2. When confluent, cells were washed with sterile PBS, trypsinized, washed again, and resuspended at a cell concentration of 50 ⫻ 106 cells/mL in additive-free RPMI 1640 medium and kept on ice. Female SCID mice were shaved on the dorsal side, and the skin was cleaned with ethanol before injection. A suspension of 5 ⫻ 106 cells/mL in 100 ␮L was then injected SC into the dorsum about 1 cm left of the midline. Animals were weighed, and tumors were measured every 7 days in two diameters using a dial caliper. Tumor volumes were calculated using the formula a2 ⫻ b ⫻ 0.52, where a is the shorter and b is the longer diameter. When tumors reached a volume of approximately 100 mm3, mice were divided into four groups. Mice received human endostatin (a generous gift from EntreMed Corporation, Rockville, MD) (20 mg/kg; n ⫽ 8) or vehicle (n ⫽ 8) via either SC osmotic pumps (Durect) or SC injections. Pumps were inserted via a small incision and placed into a SC pocket on the right lateral aspect of the animals’ dorsa. The pumps were changed every week for 4 weeks. Those animals in the injection arm of the study received daily SC injections of human endostatin (100 mg/ kg, n ⫽ 5) or vehicle (n ⫽ 5) for the same period. Mating Three days after initiation of treatment, female mice were added to a cage containing a male SCID mouse. All males had been housed separately 3 days before, and female bedding had been added to the male cages to enhance the males’ libido. The females were checked each morning for a vaginal mucous plug, which indicated that mating had occurred. A mucous plug was considered “successful mating,” and the female was not examined further to avoid unnecessary stress. Over the course of pregnancy, the following parameters were assessed: mating time describes the time between male encounter and the occurrence of a mucous plug; pregnancy rate was measured by ultrasound 15 days after the mucous plug was observed, and the number of viable fetuses was counted during this procedure; length of pregnancy was measured from the time of successful mating to delivery; number of total and viable litter was counted on the day of birth; weight of individual pup was measured by weighing the entire litter on the day of birth and calculating the mean weight. Ultrasound Imaging Using the day of successful mating as the initiation point, possible pregnancy dates were calculated (usually 19 –21 days). To rule out loss of offspring due to problems during delivery or postnatal cannibalism, we counted the number of vital fetuses during calculated pregnancy day 15 using ultrasound techniques. Under inhalation anesthesia, we used a 15-MHz linear transducer (Sequoia Systems, Siemens Med-

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ical Solutions, Malvern, PA) to count the number of fetuses in B-mode by identifying both cardiac and skeletal structures. Doppler ultrasound imaging of the fetal heart and aorta was performed, and mice were counted as vital when blood flow could be displayed. Endostatin Levels in Serum of Treated Pregnant Mice Serum levels of endostatin were measured. Blood was collected by retro-orbital bleeding through microhematocrit capillary tubes (Fisher Scientific) into Microtainer tubes (Becton Dickinson) and spun at 8,000 rpm for 15 minutes at room temperature. Serum was collected in standard Eppendorf tubes and frozen at ⫺20°C until further use. Human endostatin levels in the serum of mice were measured by enzyme-linked immunosorbent assay (ELISA; Cytimmune Sciences, College Park, MD) following the manufacturer’s instructions. Migration Assay Primary human umbilical vein endothelial cells (HUVEC; Cambrex/Biowhittaker, San Diego) were maintained according to the supplier’s directions. Cells were grown to subconfluence and used between passages 4 and 7. Cell migration assays were performed using modified Boyden chambers (6.5-mm diameter, 10-␮m thickness, 8-␮m pores; TranswellCostar, Cambridge, MA) coated with 10 ␮g/mL fibronectin in PBS overnight at 4°C and rinsed with PBS. Subconfluent cells were trypsinized (0.01% trypsin/5 mM ethylenediaminetetraacetic acid (EDTA), neutralized with trypsin neutralization solution (Cascade Biologics, Portland, OR), washed, and resuspended in endothelial basal medium (Cambrex, East Rutherford, NJ) with 0.1% BSA in the presence or absence of human endostatin (40, 200, 1,000 ng/mL). In a second experiment, we added 600 ng/mL human endostatin at various doses (60, 200, 600 ng/mL). Cells were maintained in suspension for 30 minutes, added to the top of each migration chamber, and allowed to migrate for 4 hours in the presence or absence of vascular endothelial growth factor (VEGF) (5 ng/mL) in the lower chamber. Adherent cells were fixed and stained using the Hema-3 Stain System (Fisher Diagnostics, Middletown, VA) following the manufacturer’s instructions. Nonmigratory cells were removed with a cotton swab, and the number of migratory cells per membrane was captured using bright-field microscopy connected to a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI). Migrated cells from the captured image were counted using National Institutes of Health image software. Each determination represents the average of three individual wells. Values were compared using Student’s t-test and presented as mean ⫾ SD. Migration was normalized to percent migration, with migration to VEGF alone representing 100% migration. Each experiment was repeated a minimum of three times. Fertility and Sterility姞

Statistical Analysis The cross-sectional area (CSA) was calculated for each lesion according to the formula for an ovoid: D1 ⫻ D2 ⫻ ␲/4. The measures of the seven lesions for each animal were averaged and taken as the animal mean. Repeated-measures analysis of variance indicated no significant differences among within-animal lesion areas. These data were used to calculate a mean ⫾ SD for each treatment group. The number of established lesions (⬎0 mm2), the mean CSA for established lesions, and the total disease burden (mean CSA for all seven implants/mouse, including lesions ⫽ 0 mm2) were compared among groups using one-way analysis of variance (ANOVA). When the overall ANOVA indicated a significant F-test, pairwise comparisons among treatments were conducted using post hoc Dunnett t-tests. Student’s t-test was performed when comparing the corpora lutea/ovary and the fertility and pregnancy data. The Mann–Whitney nonparametric test was used to compare serum hormone levels. Proportions and categorical data were compared using the Pearson ␹2 test. Two-tailed values of P⬍.05 were considered statistically significant. RESULTS Confirmation of the Biological Activity of the Endostatin The biological activity of the endostatin used in these studies was confirmed by inhibition of endothelial migration in vitro and tumor suppression in vivo. Human endostatin significantly inhibited migration of human umbilical vein endothelial cells (HUVEC) in response to VEGF at 200 ng/mL in the Boyden chamber assay (P⬍.04) (Fig. 1A). To verify in vivo activity of the human endostatin used in these studies, female SCID mice were injected with human pancreatic tumor cells SC. When tumors reached volumes of approximately 100 mm3, endostatin or vehicle treatment was initiated for 30 days. Tumor growth was inhibited in mice receiving endostatin compared with controls. Endostatin inhibited tumor growth similarly by both routes of delivery. Tumor growth was inhibited by 43% (P⫽.03) when delivered continuously via a miniosmotic pump at 20 mg/kg (Fig. 1B), and inhibited by 44% (P⫽.01) when administered via daily injections (100 mg/kg) (Fig. 1C). These results show that the human endostatin used in this experiment was detectable. Serum endostatin levels were measured in mice receiving endostatin or vehicle alone by each route of delivery. The sensitivity of the assay is 2 ng/mL. No cross reactivity was noted. Both the intraassay and interassay variations were less than 10% for the human endostatin enzyme immunoassay. The mean endostatin concentration in animals receiving continuous endostatin via miniosmotic pumps (20 mg/kg) was 23.1 ng/mL (⫾7.3). For mice injected with the drug (100 mg/kg, with the last injection approximately 2– 4 hours before serum sampling), the endostatin concentration was well above the assay’s limit of 500 ng/mL. Values were extrapolated to approximately 750 and 1,000 ng/mL using the 1147

standard curve for endostatin. In contrast, the control mice did not have any detectable endostatin level.

FIGURE 1 Endostatin is biologically active. (A) Human endostatin (hES) significantly inhibits migration of human umbilical vein endothelial cells (HUVEC) at 200 ng/mL in vitro. (B) Human endostatin inhibits growth of BxPC-3 tumors in vivo. Endostatin (red) or vehicle (black) is administered continuously via SC miniosmotic pumps (20 mg/kg) or as daily SC injections (C) (100 mg/kg), compared with controls. Error bars indicate SD; asterisk indicates P⬍.04.

Murine Endostatin Inhibits Endometriosis in a Mouse Model Treatment with either murine endostatin or PBS via SC miniosmotic pumps was initiated immediately after implantation of the endometrial tissue. Lesions were measured after 4 weeks of treatment at necropsy. Endostatin therapy decreased disease establishment, which was defined as the number of detectable lesions. Ninety-seven percent of endometriotic lesions in the control mice were detectable compared with 60% in the endostatin group (P⬍.001). Comparing the size of the established lesions in each group revealed that endostatin significantly inhibited the growth of endometriotic lesions compared with vehicle-treated mice in two experiments by 47% (Fig. 2A; endostatin 3.0 ⫾ 0.7 mm2; control 5.7 ⫾ 2.2 mm2;

FIGURE 2 Endostatin inhibits endometriotic lesions. (A) Four weeks of endostatin treatment inhibits growth of surgically induced endometriotic lesions in mice. (B) Growth of established endometriotic lesions is not significantly suppressed by endostatin treatment (black squares: vehicle-treated; gray squares: endostatin-treated). Error bars indicate SD; asterisk indicates P⬍.001.

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P⬍.001). If all nonestablished lesions were counted as a 0, endostatin inhibits the size of endometriotic lesions by 63% (endostatin group 2.1 vs. 5.6 mm2) (data not shown). Endostatin Does Not Regress Established Lesions To study the effect of antiangiogenic therapy with endostatin on established lesions, treatment was delayed until 4 weeks after lesion implantation and then continued for 4 weeks. Results were compared with the responses to treatment initiated on day 1 after surgery. When treatment was begun on postoperative day 1, the mean lesion burden in the endostatin group (1.15 ⫾ 1.26 mm2; n ⫽ 10) was significantly reduced by 79% compared with controls (5.6 ⫾ 2.36 mm2; n ⫽ 18; (⬍.001) (Fig. 2B). However, when treatment was initiated on postoperative day 30, there was no statistical difference between control (6.1 ⫾ 1.5 mm2; n ⫽ 15) and endostatin-treated groups (5.22 ⫾ 2.1 mm2; n ⫽ 8) (Fig. 2B). The disease burden in the delayed endostatin group was significantly greater than in the mice treated immediately after surgery (P⬍.001). Results were similar when comparing the number of established lesions and the size of those established lesions among the groups. This suggests that endostatin inhibits disease development in this model but does not regress established lesions. Endostatin Does Not Affect Estrous Cycling The estrous cycle lasts between 1.5 and 5 days and consists of four stages (diestrus, proestrus, estrus, and metestrus) of differing duration (Fig. 3A). The proportions of leukocytes and epithelial cells in the vaginal cytology vary depending on the stage (28). If cycling is interrupted, it usually arrests in the estrous stage. As shown in Figure 3B, all mice in both the endostatin and vehicle groups repeatedly passed through the various stages of the estrous cycle. The length of the cycles was the same in both groups (data not shown). These results indicate that endostatin treatment does not suppress ovarian function in these animals. Endostatin Does Not Suppress E2 and P Levels in Serum After 4 weeks of treatment, blood samples were collected and centrifuged. Serum E2 and progesterone (P) levels were measured by radioimmunoassay. Assay detection limits are 10 pg/mL for E2 and 0.3 ng/mL for P. The intraassay coefficient of variation (CV) for E2 is 6.8% and 5.7% for P. Median E2 levels did not differ significantly between the endostatin- and vehicle-treated groups (41.5 pg/mL, interquartile range [IQR] 29.5; vs. 11 pg/mL, IQR 17; P⫽.08). Individual E2 levels were, for the most part, within the physiologic range in both groups (10 – 60 pg/mL) (29). Median P serum levels were also not statistically different in both groups (endostatin group: 1,405 pg/mL, IQR 3,945; vs. placebo group: 1,130 pg/mL, IQR 375; P⫽.48). Values were within or slightly below normal range (2,000 – 40,500) (29). Fertility and Sterility姞

Ovaries of Endostatin-Treated Mice Have Similar Numbers of Corpus Lutea Gross morphology was identical in ovaries of both endostatinand vehicle-treated mice (Fig. 4A). All ovaries contained follicles at various stages according to the classification proposed by Pedersen and Peters (30). All ovaries studied contained corpora lutea, which indicates that therapy with endostatin did not prevent ovulation (Fig. 4B). When comparing the corpus luteum (CL) counts per histological slide, we found no statistical difference in the numbers between vehicle- and endostatin-treated mice in either experiment (endostatin: 2.5 CL/section ⫾ 1.6, CV 66.8%; control: 2.1 ⫾ 1.3, CV 63.8%; P⫽.2). In addition, the cross-sectional area of the CL was also similar between the groups (endostatin: 0.1038 ⫾ 0.069 mm2; control: 0.1019 ⫾ 0.073 mm2; P⫽.9). Endostatin Does Not Affect the Fertility of Female Mice Daily vaginal checks showed that endostatin therapy did not delay the time for mating as indicated by the presence of mucous plugs. Mating, defined as the time between male encounter and the presence of a mucous plug in the female’s vagina, was similar in all groups (endostatin pumps, 2.6 ⫾ 1.4 days vs. control pumps, 2.0 ⫾ 1.0 days; P⫽.35; endostatin injections, 2.8 ⫾ 1.5 days vs. control injections, 1.9 ⫾ 0.9 days; P⫽.14) (Fig. 5A). Pregnancy rates with live births were not reduced by treatment with endostatin. In the animals treated via miniosmotic pumps they ranged between 60% (control) and 88% (endostatin). In the injected mice, the pregnancy rate was 80% for the control mice, whereas all endostatin mice became pregnant (Fig. 5B). Treatment with endostatin did not alter the duration of pregnancy. The normal duration of pregnancy in mice ranges between 19 and 21 days. As shown in Figure 5C, endostatin pump mice were pregnant for a mean of 20.7 ⫾ 1.4 days and control mice for 20.2 ⫾ 0.4 days (P⫽.36). Those mice receiving endostatin via daily injections delivered after 19.7 ⫾ 0.6 days and controls after 20.1 ⫾ 0.4 days (P⫽.29). Endostatin Treatment Does Not Affect Offspring In the present study, there were no teratogenic effects apparent in the offspring of mice that were treated with endostatin. All pups were vital and healthy and had no obvious abnormalities. Mice frequently cannibalize part of their litter. To be able to count the number of vital mice, we used Doppler ultrasound imaging of the fetal heart and aorta 2 days before the expected delivery date. The mean number of vital fetuses as measured by ultrasound in the pump groups was 5.8 ⫾ 1.3 (endostatin) and 4.9 ⫾ 1.7 (control). In injected female mice, we counted 5.0 ⫾ 1 vital fetuses in utero compared with 4.9 ⫾ 2.3 fetuses in the control group (P⫽.64) (Fig. 5D). When counted 21 days postpartum, we found 4.9 ⫾ 1.9 (endostatin) and 4.6 ⫾ 1.7 healthy pups in the pump group. Injected mice treated with endostatin had a mean of 1149

FIGURE 3 Endostatin does not affect estrous cycling. (A–D) Methylene blue staining of vaginal smears at different stages of the estrous cycle (clockwise from upper left panel: diestrus (A), proestrus (B), estrus (C), metestrus (D)) Magnification, ⫻400. (E) Number of full cycles individual mice went through over a period of 2 weeks as determined by daily vaginal smear.

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5.0 ⫾ 1.0 pups, and controls had 4.7 ⫾ 2.2 (P⫽.84) healthy pups 3 weeks after delivery. This comparison demonstrates that there was no significant perinatal loss in either the endostatin or the control groups. 1150

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The mean weight of pups on the day of delivery was measured in the pump group and was not significantly different between endostatin- and PBS- (control) treated animals (endostatin: 1.5 ⫾ 0.2 g; control: 1.4 ⫾ 0.2 g; P⫽.36).

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FIGURE 4 Endostatin does not inhibit CL formation. (A) Hematoxylin-eosin staining of a representative ovary found in both endostatin- and vehicle-treated groups. Magnification, ⫻100. Note two large CL at the right lower end of the ovary (highlighted with arrows and dotted lines). (B) Mean number of CL per tissue section of ovaries in endostatin-treated or vehicle-treated mice. Error bars indicate SD.

The pups were weighed again 21 days postpartum. The offspring from the endostatin-treated mothers weighed slightly but significantly more than the control group (9.8 ⫾ 1.0 g vs. 8.9 ⫾ 1.5 g; P⬍.02). The gender distribution was equal in both groups (Table 1). Finally, we were interested in potential dysmelia caused by the inhibition of angiogenesis during embryogenesis as described in human studies (15, 17). None of the mice exhibited any gross abnormalities. We did, however, find significantly longer tail lengths in the endostatin group compared with the controls (6 ⫾ 0.2 cm vs. 5.1 ⫾ 0.4 cm, respectively; P⬍.001). When the offspring reached maturity, they were mated among each other. All female offspring from endostatin-treated mothers were able to successfully mate with males from the same litter. DISCUSSION Endometriosis primarily afflicts women of childbearing age and is associated with infertility rates of 30%–50%, approximately twice that of the general population (31–34). Patients also suffer from dysmenorrhea, dyspareunia, and pelvic pain that can be debilitating. Current therapies are aimed at surgical removal of endometriotic lesions and at suppression of ovarian steroid production. The outcome is still highly unsatisfactory because of a high recurrence rate of symptoms and the inability to improve fecundability. Therefore, new interventions that address the disease pathophysiology without causing limiting side effects or impeding reproduction are urgently needed. Angiogenesis is essential to the pathogenesis of many diseases such as malignant tumors, diabetic retinopathy, rheumatoid arthritis, and psoriasis (35). An imbalance between pro- and antiangiogenic factors induces endothelial cell sprouting leading to the development of new blood vessels that support the surrounding cells (36). Levels of proangiogenic factors, such as VEGF, interleukin-8 (IL-8), and placental growth factor are elevated in the peritoneal fluid, macrophages, endometriomas, and endometriotic tissue of these patients (8 –10, 37). The increased expressions of these angiogenic factors have been implicated in the advancement of the disease by promoting angiogenesis in the lesions. Therefore, others and we have proposed that inhibiting angiogenesis may offer a novel therapeutic approach for treating endometriosis (22, 38, 39).

Becker. Endostatin in endometriosis and reproduction. Fertil Steril 2005.

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In this study, we show that endostatin, a 20-kDa to 22-kDa fragment of collagen XVIII, suppresses the establishment of endometriotic lesions in a mouse autograft model (18). Murine endostatin inhibited growth of surgically induced endometriosis repeatedly by approximately 50% when therapy was initiated immediately after surgery. On the other hand, we were not able to regress established lesions with this regimen. These results are similar to our previous report where we demonstrated that the cyclooxygenase-2 inhibitor celecoxib inhibited the growth of surgically induced endo1151

FIGURE 5 Endostatin does not negatively influence fertility or pregnancy. Mice were treated either continuously via SC miniosmotic pumps or via daily SC injections. Error bars indicate SD. (A) Mating time. (B) Pregnancy rate. (C) Duration of pregnancy. (D) Litter size (black squares: vehicle-treated mice at ultrasound 2–3 days prepartum; lined squares: control mice at 21 days postpartum; gray squares: endostatin-treated mice at ultrasound 2–3 days prepartum; lined gray squares: endostatin-treated mice 21 days postpartum).

Becker. Endostatin in endometriosis and reproduction. Fertil Steril 2005.

metriotic lesions in our model but did not regress established lesions (25). In a recently published study, the effects of various angiogenesis inhibitors on established endometriotic lesions in another mouse model were studied (22). Human endometrial tissue was inserted into the abdominal cavity of nude mice under constant estrogen stimulation. Treatment with human endostatin, initiated 3 weeks after implantation, reduced the number of identifiable endometriotic lesions compared with controls. There are many differences in both models that make it difficult to compare results. In the study by Nap et al. (22), human tissue was implanted in immunocompromised nude mice, whereas we autotransplanted murine endometrium in immunocompetent mice. It is postulated in humans that the immune system plays an important role in the pathogenesis of endometriosis (40). Other differences between the nude mice model and our model, which may 1152

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account for the heterogeneous results, are, respectively, the presence of continuous versus fluctuating serum estrogen levels and the surgical versus nonsurgical induction of endometriosis. Endometriosis can only be diagnosed after tissue biopsy during surgery. It is clinically reasonable to initiate adjuvant therapy with endostatin after surgical resection or coagulation of endometriotic lesions. Therefore, treatment after surgical resection to prevent recurrence is a realistic therapeutic approach. The precise mechanism for the antiangiogenic effect of endostatin has been elusive (41). Direct interference of endostatin with the VEGF receptor-2 signaling pathway has been demonstrated (42). Down-regulation of VEGF isoforms may also be involved (43). In addition, VEGF has been shown to be an endothelial survival factor (44, 45), and VEGF is

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TABLE 1 Weight (g) per pup on postpartum days 1 and 21 and the tail length of pups on postpartum day 21 from endostatin- or vehicle-treated mothers. Mean weight (g) per pup At birth Control 1.40 ⫾ 0.2 Endostatin 1.5 ⫾ 0.2

21 days Increase postpartum (%) 8.9 ⫾ 1.5 9.8 ⫾ 1.0a

637.1 647.7

Average tail length (cm) 21 days postpartum Control Endostatin

5.77 ⫾ 0.35 6.03 ⫾ 0.16b

Note: Values are means ⫾ SD. a P⬍.02. b P⬍.001. Becker. Endostatin in endometriosis and reproduction. Fertil Steril 2005.

up-regulated in endometriotic tissue and increased in the peritoneal fluid of patients with endometriosis (10). It is conceivable that angiogenesis plays a major role during the initial stages of disease when peritoneal vessels connect to the transplanted endometrium. The VEGF levels were higher in red endometriotic lesions that were considered more active as compared with less active black lesions in humans (8). Hypoxia, one of the cardinal stimuli of VEGF expression, would be expected in the transplanted tissue (46). The expression of hypoxia-inducible transcription factor alpha (HIF-1 alpha) mRNA is reduced in microvascular endothelial cells by endostatin treatment (42). Hypoxia may lead to VEGF-induced sprouting of endothelial cells and formation of new blood vessels from the peritoneum to the transplanted endometriotic lesions. Once the blood supply is established, HIF-1 alpha and, subsequently, VEGF are down-regulated because of decreasing hypoxic conditions in the lesion (47). This may be the reason why antiangiogenic therapy with endostatin may not be as efficient in established lesions because of the lower VEGF production at this stage. In this study, we show that, while inhibiting the growth of endometriotic lesions, endostatin therapy does not impede fertility. Mice treated with endostatin had regular estrous cycles as shown by daily vaginal cytology, and E2 and P levels were in the normal range. The fact that the serum levels of these hormones varied similarly within each of the two groups further supports that the conclusion that endostatin did not affect estrous cycling. The E2 and P levels in mice have been reported to vary depending on estrous stage and age of the animal (29). It is necessary to point out that the number of serum samples used in this study for measuring Fertility and Sterility姞

hormone levels was small, making it difficult to draw a final conclusion about the influence of endostatin treatment on E2 and P. However, we found equal numbers of CL in the ovaries from all groups of mice, confirming the fact that endostatin did not affect reproductive processes. Also, no “trapped oocytes” were noted, thus ovulation occurred. The CL is one of the most highly vascularized organs (48). During follicle development, blood vessels within the thecal cell layer are prevented from entering the granulosa cell layer by the existence of a basement membrane. After ovulation, the follicle collapses, the integrity of the basement membrane is disrupted, and new blood vessels sprout into a newly forming CL (49). Vascular endothelial growth factor and an endocrine gland–specific VEGF (EG-VEGF) play a major role during luteal angiogenesis (50 –52). Indeed, increased permeability of blood vessels plays a role in CL formation (53). Endostatin has been shown to down-regulate VEGF expression in aortic explants and various cancer cell lines (43). In addition, an antipermeability effect of endostatin has been described (53, 54). Endostatin also inhibits VEGF-induced permeability in the Miles assay (personal communication, October 2003, S. Soker and J. Folkman). It is possible that endostatin effects in this model are mediated through inhibition of the permeability effect of VEGF rather than its proliferative and prosurvival effects on endothelial cells. It is also conceivable that potential antipermeability effects on the CL may be rescued by the expression of EG-VEGF, which may not be responsive to endostatin therapy. Endometriosis is a chronic disease of women during their childbearing years. Therefore, continuous therapy is likely to be necessary. However, it must not cause teratogenic effects if a child is conceived during treatment. Pregnancy itself often improves endometriosis-associated abdominal pain, but symptoms frequently recur postpartum. Antiangiogenic therapy during pregnancy has been connected with birth defects. Thalidomide, a sedative that was used to treat morning sickness, has been shown to cause dysmelia and other birth defects in humans if taken during early pregnancy (15, 17). Only after these initial reports of thalidomide’s teratogenic potential in humans were studies undertaken to show similar effects in animals (55, 56). These adverse effects have been associated with the inhibition of blood vessel formation during embryonic development (57). In another study, our laboratory demonstrated that the antiangiogenic compound TNP-470, a fumagillin-analog, injected during embryonic development resulted in complete failure of embryonic growth (16). Furthermore, injection of TNP-470 and soluble VEGF receptor-1 (Flt-1) into the preovulatory follicle decreased systemic luteal P levels and inhibited ovulation (58). However, in the same study, we found that application of the endogenous angiogenesis inhibitor angiostatin, a 38-kDa fragment of plasminogen, did not alter ovulation or the development or lifespan of the CL in monkeys (58, 59), 1153

which suggests a different mechanism for this molecule. In contrast, gene therapy using an adenovirus vector to deliver angiostatin caused impaired ovarian function with suppressed CL development (38). In the study presented here, we have discovered that therapy with endostatin is able to inhibit endometriotic lesions with no adverse effects on pregnancy or the offspring of treated mice. Endostatin therapy did not alter the duration of pregnancy or the size of the litter at birth and 21 days postpartum. In contrast, thalidomide therapy decreased the size of mice pup tails (56). In our study, we did not find this feature. In fact, we found that the offspring of endostatintreated mice had significantly longer tails on day 21 than the controls. In addition, the weight of these pups on the day of delivery and on postpartum day 21 was higher in the treatment groups compared with controls, which is probably correlated with the difference in tail length. It suggests that intrauterine and postpartum development is not inhibited by endostatin. Finally, we found that all mice were able to conceive after reaching maturity, which supports our hypothesis that endostatin does not interfere with reproductive processes. Endostatin has been shown in phase I/II cancer trials to be virtually nontoxic (60), although fertility and pregnancy are not toxicity endpoints in this population. The absence of negative effects of endostatin on reproduction in our system suggests that endostatin may have different effects on the angiogenic mechanisms involved in these physiological processes as opposed to pathological processes such as endometriosis and cancer. Such an agent may be expected to have fewer side effects than angiogenesis inhibitors with broader effects. In summary, our results show that endostatin significantly inhibits the growth of endometriotic lesions. At the same time, we are able to demonstrate that endostatin, while being effective against endometriosis, does not suppress various reproductive functions in female mice. Finally, endostatin therapy does not negatively influence pregnancy and is not associated with teratogenic adverse effects on the progeny of mice treated with this drug. As endostatin is already under investigation in clinical trials for cancer and other diseases, it may eventually serve as a promising novel therapeutic approach for patients with endometriosis. REFERENCES 1. Cramer DW, Missmer SA. The epidemiology of endometriosis. Ann NY Acad Sci 2002;955:11–22; discussion 34-6, 396 – 406. 2. American Society for Reproductive Medicine. Endometriosis and infertility. Fertil Steril 2004;81:1441– 6. 3. Murphy AA. Clinical aspects of endometriosis. Ann NY Acad Sci 2002;955:1–10; discussion 34-6, 396 – 406. 4. Sampson J. Peritoneal endometriosis due to the menstrual dissemination of endothelial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927;14:422– 69. 5. Cullen TS. Adenomyoma of the round ligament. Bull Johns Hopkins Hosp 1896;7:112.

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