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Peroxisome proliferator–activated receptor-gamma induces regression of endometrial explants in a rat model of endometriosis
FERTILITY AND STERILITY威 VOL. 82, SUPPL. 3, OCTOBER 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Peroxisome proliferator–activated receptor-gamma induces regression of endometrial explants in a rat model of endometriosis Dan I. Lebovic, M.D., M.A.,a Mustafa Kir, M.D.,b and Colleen L. Casey, M.D.a Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Michigan, Ann Arbor, Michigan
Received October 23, 2003; revised and accepted February 9, 2004. Supported in part by the University of Michigan Department of Obstetrics and Gynecology through the Ansbacher Fund for Resident/Fellow Education and Research. Reprint requests: Dan I. Lebovic, M.D., Department of Obstetrics and Gynecology, University of Michigan, 1500 E. Medical Center Drive, L4100 Women’s Hospital, Ann Arbor, Michigan 481090276 (FAX: 734-647-1006; E-mail: [email protected]). a Reproductive Endocrinology Division, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan. b Department of Obstetrics and Gynecology, Acibadem Hospital, Istanbul, Turkey. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 02.148
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Objective: To determine the effects of a thiazolidinedione, ciglitazone, in a rat model of endometriosis. Design: Prospective, randomized, placebo-controlled study. Setting: Experimental surgery laboratory in a university department. Animal(s): Twenty female Sprague-Dawley rats given endometriotic lesions by transplanting autologous uterine tissue to ectopic sites on the peritoneum. Intervention(s): Four weeks after surgery, 20 rats were randomly divided into two groups and treated with IP injections of vehicle every other day (control; n ⫽ 10) or ciglitazone (1 mg per rat; n ⫽ 10) and euthanized 4 weeks from the start of treatment. Main Outcome Measure(s): At the end of treatment, laparotomy was performed to photograph each explant and then they were measured and weighed. Histologic analysis was performed on the uterine allograft, ovary, and eutopic uterine tissue. Result(s): By histologic assessment, both groups maintained folliculogenesis and normal eutopic endometrial architecture. Treatment with ciglitazone significantly decreased the size of ectopic uterine tissues and the mean explant wet weight. The ciglitazone-treated group showed marked epithelial regression compared with the control group. Conclusion(s): We conclude that a PPAR-␥ ligand, ciglitazone, reduced the size of experimental endometriosis in the rat model of endometriosis. This animal model suggests that a thiazolidinedione drug may be helpful in women with endometriosis. (Fertil Steril威 2004;82(Suppl 3):1008 –1013. ©2004 by American Society for Reproductive Medicine.) Key Words: : Endometriosis, immune modulators, immunotherapy, PPAR-␥, rat model, thiazolidinedione
Endometriosis affects 1 in 10 reproductiveage women and has far greater prevalence in the pelvic pain and subfertile populations (1). The histologic criteria for diagnosing endometriosis are endometrial glands and stroma. Not included in this classification is the associated inflammatory response that is now recognized as the sine qua non for the disease (2, 3). Whether this altered immune response is the cause or effect of the disorder is not certain, although it appears to contribute to the persistence of the disease and associated symptoms. Endometriosis is a progressive disease. Surgical intervention or medical suppression of the endometriotic lesions often lead to only temporary relief. The recurrence rate is
approximately 5%–20% per year with a cumulative rate of 40% after 5 years (4 – 6). Two common medical treatment options are GnRH agonists and combination estrogen/ progestin oral contraceptives, which were first used for endometriosis more than 20 years ago (7, 8). More efficacious, bettertolerated, and more affordable treatment options must be developed. An ideal treatment would eliminate endometriotic lesions, prevent recurrence, and not impede ovulation. Immune-modulating drugs such as recombinant human tumor necrosis factor-␣ binding protein-1 (r-hTBP-1), interferon-␣2b, loxoribine, pentoxifylline, and peroxisome proliferator–activated receptor-␥ (PPAR-␥) li-
gands are candidate treatment options (9 –13). PPARs are a new class of immune modulators found in adipose tissue, liver, spleen, colon, adrenal gland, muscle tissue, macrophages, and endometrial epithelial and stromal cells (14 –16). Endogenous agonists regulate adipocyte differentiation and glucose homeostasis. Interestingly, recent reports indicate that the PPAR-␥ subtype can mediate anti-inflammatory effects (17–19). Surgically transplanted endometrial tissue in the rat provides an animal model to study the effects of experimental drugs on ectopic endometrial tissue (20). Rat endometriotic implants show histologic transformations similar to those seen in human endometriotic lesions. In the current study, we used the rat model of endometriosis to test if a thiazolidinedione (TZD), an activator of PPAR-␥, could impede the growth of ectopic uterine tissue.
MATERIALS AND METHODS Animals Female Sprague-Dawley rats (200 –250 g) were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Unit for Laboratory Animal Medicine. They were caged in a controlled environment with 12-hour light/dark cycles. Food (standard pellet diet) and water were provided ad libitum. The animals were sexually mature and demonstrated normal estrous cycle changes in uterine histology (data not shown). The animals were allowed to acclimatize to these conditions for at least 1 week. The University of Michigan Committee on Use and Care of Animals
approved the experiments, and all investigations complied with the 1996 National Academy of Science’s Guide for Care and Use of Laboratory Animals.
Study Drug Ciglitazone (GR-205) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA) and was brought into solution (20% ethanol, 35 mM NaHCO33, NaOH, pH 9.6) at a concentration of 10 g/L. Five hundred microliter aliquots were frozen at ⫺20°C. Although we used an IP route of administration based on prior rodent studies, this drug has high oral bioavailability (21).
Induction of Experimental Endometriosis in Rats Twenty-two rats were used in the experimental induction of endometriosis. When the rats were 3 months of age, ectopic endometrium was induced surgically as described by Vernon and Wilson (20). Briefly, animals were anesthetized with isofluorane, and after a midventral incision to expose the uterus, a 1.0-cm length distal segment was resected from the left uterine horn. The segment was placed in phosphate-buffered saline at 37°C and split longitudinally, and a 5 ⫻ 5 mm piece was sectioned. This piece of uterine tissue was transplanted without removing the myometrium onto the inner surface of the right abdominal wall with the epithelial lining apposed to the peritoneal surface. The explanted endometrial tissue margins were secured with nonadsorbable polypropylene sutures (Prolene 6-0; Ethicon, Piscataway, NJ) at two edges. The midline abdominal incision was closed with chromic catgut sutures (3-0; Ethicon). The skin incision was closed with horizontal mattress
FIGURE 1 Gross appearance of uterine autografts at the conclusion of treatment. Immediately postmortem appearance of endometriotic explant (circled) after 4 weeks of vehicle control treatment (A) or ciglitazone (B). Note the cystic morphology of the control group as opposed to the pellet-like residual explant in the ciglitazone treatment group. The two blue-dyed sutures on either end of the explant can be seen in both panels. *, adhesion; scale bar, 5 mm.
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
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sutures of absorbable polyglactin 910 (coated Vicryl 4-0; Ethicon, Inc.). Animals were allowed 4 weeks for recovery and development of the disease. Four weeks after transplantation of endometrial tissue, two rats were euthanized to evaluate the surgical technique. The explants were examined for size and viability. The surface area of the engrafted tissue was weighed and measured using a caliper.
Experimental Design Four weeks after surgery, 20 rats were randomly allocated into two groups and given one IP injection every other day for 4 consecutive weeks with the following agents: control solution (vehicle, 20% ethanol, 35 mM NaHCO3 NaOH, pH 9.6; 100 L; n ⫽ 10) or ciglitazone (1 mg ciglitazone, 100 L, n ⫽ 10) every other day over a 4-week period. This dose and treatment schedule were based on a previous study in a mouse model of endometriosis (13). During the treatment period, any adverse treatment effects (such as weight loss, alopecia) were monitored. Vaginal smears were performed on all rats after drug treatments to ensure that their reproductive cycles were not disrupted at the end of the study: 30% of the control group were in either proestrus or estrus while 50% of the ciglitazone group were in one of these
stages. At the end of treatment, the animals were euthanized with CO2 and the uterine tissue implant was evaluated before an ovary and 0.5-mm segment of right uterine horn were removed and placed into 10% neutral buffered formalin and processed for hematoxylin and eosin staining.
Evaluation of Ectopic Uterine Tissue After euthanasia with CO2, the rats were immediately fixed in the supine position on a platform, the abdomen was opened, and all three dimensions (length ⫻ width ⫻ height in millimeters) of ectopic uterine tissue were measured in situ using a caliper. The spherical volume of each ectopic uterine tissue was calculated using the prolate ellipsoid formula: V (mm3) ⫽ 0.52 ⫻ A ⫻ B ⫻ C, where A, B, and C denote width, length, and height, respectively. Tissues were photographed using a digital camera and then excised and weighed (in milligrams) before being placed in 10% neutral buffered formalin and processed for hematoxylin and eosin staining.
Evaluation of Persisting Epithelium in Endometrial Autografts The persistence of epithelial cells in uterine autografts was evaluated by a previously published method as follows:
FIGURE 2 Histology of ovary, eutopic uterus, and ectopic uterine tissue. Hematoxylin and eosin staining of ovarian tissue, eutopic endometrium, and ectopic uterine explant from the two treatment groups: control (A, C, E) and ciglitazone, Ciglit (B, D, F). Left-side panels (A and B) show representative ovarian antral follicles. The middle panels (C and D) reveal normal appearing endometrium with associated luminal epithelium (*) and stromal cells (ST). Right-side panels (E and F) show the endometriotic implant cyst lumen (LU) with epithelial cell lining (*) for the control group (E) and degeneration of the ciglitazone group explant (F). Scale bar, 100 m.
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
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a score of 3 indicates a well-preserved epithelial layer; a score of 2, moderately preserved epithelium with leukocyte infiltrate; a score of 1, a poorly preserved epithelium (occasional epithelial cells only); and a score of 0, no epithelium (11). An individual other than the authors evaluated three sections from each explant in a blinded fashion. The bars in Figure 4 represent an average value for each group.
FIGURE 3 Effects of ciglitazone on the volume and weight of ectopic uterine tissue. Spherical volumes and weights of uterine explants of vehicle (Ctrl)- and ciglitazone (Cig)-treated rats are depicted in the left and right panels, respectively. There were 10 rats per treatment group. Each bar represents the mean ⫾ SD. a, P⬍.006 (Kruskal-Wallis test); b, P⬍.003 (Kruskal-Wallis test).
Histology The formalin-fixed endometriotic-like explants, ovaries, and eutopic uterine tissues were embedded in paraffin wax blocks, and 5-m sections were prepared (three sections per sample). The sections were heat-dried and stained with hematoxylin and eosin.
Statistical Analysis
The data are expressed as the mean ⫾ SD. We used the nonparametric Kruskal-Wallis test to analyze the data (JMP version 5.0; Cary, NC). Significant differences were accepted when two-tailed analyses yielded P⬍.017 (Bonferroni correction ␣ of 0.05/3 for the three comparison outcomes).
RESULTS Surgical Outcome in the Rat Endometriosis Model Four weeks from the time of uterine transplantation, two rats were euthanized to examine the endometriotic explants. In these animals, the explant appeared as well vascularized, unilocular cystic structures containing clear, serous fluid. The explants had an average spherical volume of 20 mm3 and a weight of 25 mg. Representative in vivo images of the explants after 1 month of respective treatment revealed a cystic implant in the vehicle control group (Fig. 1A) in contrast to the degenerative, pellet appearance in a majority of the ciglitazone-treated cohort of rats (Fig. 1B).
Histology for Ovary, Uterine Tissue, and Uterine Explant Neither vehicle control nor ciglitazone had any observable adverse effect (i.e., weight loss, grooming changes) or organ-specific changes such as follicular atresia in the ovaries (Fig. 2A, 2B) or tissue necrosis in eutopic uterine tissue (Fig. 2C, 2D) as verified by histologic comparison. Multiple ovarian and eutopic uterine tissue sections were examined. Progesterone concentrations from serum obtained at the time of euthanasia revealed a higher mean level in the ciglitazone group (7.9 ⫾ 1.3 pmol/L, P⬍.05, Student’s t-test) as compared with the control group (4.1 ⫾ 0.6 pmol/L), although both means were within baseline values for Sprague-Dawley female rats (22). The control cystic implants (Fig. 2E and insert) were similar to the eutopic endometrium because they both contained endometrial epithelium and stromal cells. The inner lining of the cysts contained simple columnar epithelium. The uterine autograft from ciglitazone-treated rats showed marked epithelial changes and overall regression of the explant (Fig. 2F and insert). FERTILITY & STERILITY威
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
Effects of Ciglitazone on the Ectopic Endometrial Tissue Volume and Weight There was a significant difference (P⬍.006) in posttreatment spherical volume between control (19.7 ⫾ 13.6 mm3) and ciglitazone (5.9 ⫾ 5.1 mm3) treatment groups (Fig. 3, left panel). Similarly, there was a significant difference (P⬍.003) in explant weight between control (24.0 ⫾ 13.4 mg) and ciglitazone-treated (6.7 ⫾ 3.9 mg) rats (Fig. 3, right panel).
Effect of Ciglitazone on the Ectopic Endometrial Epithelium Semiquantitative evaluation of the persistence of endometrial epithelial cells in the uterine explants (Fig. 4) showed a significantly lower score in the ciglitazone group (1.1 ⫾ 1.0) compared with the control group (2.4 ⫾ 1.0; P⬍.016).
DISCUSSION In this study, we used the rat model of surgically induced endometriosis to test the effect of a TZD PPAR-␥ agonist, ciglitazone, on the growth of ectopic uterine tissue. In contrast to vehicle control–treated rats, ciglitazone treatment for 1 month was found to be effective in inducing regression of the endometriotic implants. The 52% reduction in explant spherical volume, 70% lower wet weight, and 54% diminu1011
FIGURE 4 Effect of ciglitazone on the persisting epithelium score. Semiquantitative evaluation of persisting epithelium in uterine tissue allografts of vehicle (Ctrl)- and ciglitazone (Cig)-treated animals. There were 10 rats per treatment group. Each bar represents the mean ⫾ SD. a, P⬍.016 (Kruskal-Wallis test).
are mediated by diminished vascular endothelial growth factor production (38). Furthermore, acting through PPAR-␥, TZDs inhibit proinflammatory cytokines (13, 14, 39) as well as NF-B (18), an important nuclear transcription factor for the production of many cytokines (40). Taken together, since human endometrial epithelial and stromal cells contain PPAR-␥ (14, 39), we felt it would be useful to evaluate the influence of a PPAR-␥ ligand, ciglitazone, in a rat endometriosis model as a prelude to further studies in primates. The results in the present study have shown that ciglitazone significantly reduced the uterine explant burden in the rat model and, at the same time, did not seem to interfere with ovarian folliculogenesis. It may thus be possible to use TZDs to suppress existing endometriotic lesions without suppressing ovulation. Further studies need to definitively assess ovarian function using TZDs and to determine the reproductive safety profile. Investigation with this class of drug in the baboon model for endometriosis would be useful for prevention and treatment outcomes including assessment of the presence of ovulation. The data in this study suggest that TZDs, such as the commercially available rosiglitazone or pioglitazone, may be effective in the treatment of human endometriosis.
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
tion of persisting epithelium score confirmed ciglitazone to be a significant (P⬍.016) modulator of endometriotic implant growth. Moreover, the ciglitazone dose used for effective structural regression of rat endometriotic implants did not seem to disrupt estrous cycling or folliculogenesis or affect the rats’ physical well-being. The pathogenesis of endometriosis appears to involve a process of subclinical peritoneal chronic graft-versus-host immune response, marked by an accumulation of leukocytes including peritoneal macrophages (23–25), T cells, natural killer cells, and soluble products produced by these cells (2, 26 –31). Immunomodulatory agents such as r-hTBP-1, interferon-␣2b, loxoribine, and pentoxifylline have been used in animal models of endometriosis, effectively causing regression of uterine explant lesions (9 –12). A randomized controlled study in the baboon endometriosis model showed that r-hTBP-1 significantly inhibited the development of endometriotic lesions (32). Women with endometriosis have increased numbers of activated peritoneal fluid macrophages (23–25) along with macrophage-derived cytokines (26 –31). One such cytokine, interleukin-1 beta is thought to serve as a mediator between peritoneal fluid macrophages and the promotion of endometriotic implant growth and angiogenesis (33, 34). Considerable evidence suggests that PPAR-␥ ligands, such as TZDs, are potent cell growth inhibitors (35, 36) and inducers of apoptosis (35, 37). They have anti-angiogenic effects that 1012 Lebovic et al.
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15. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell 1995;83: 835–9. 16. Lemberger T, Desvergne B, Wahli W. Peroxisome proliferator–activated receptors: a nuclear receptor signaling pathway in lipid physiology. Ann Rev Cell Dev Biol 1996;12:335– 63. 17. Hornung D, Waite LL, Ricke EA, Bentzien F, Wallwiener D, Taylor RN. Nuclear peroxisome proliferator–activated receptors alpha and gamma have opposing effects on monocyte chemotaxis in endometriosis. J Clin Endocrinol Metab 2001;86:3108–14. 18. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator–activated receptor-gamma is a negative regulator of macrophage activation. Nature 1998;391:79 – 82. 19. Welch JS, Ricote M, Akiyama TE, Gonzalez FJ, Glass CK. PPARgamma and PPARdelta negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages. Proc Natl Acad Sci USA 2003;100:6712–7. 20. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684 –94. 21. Matthaei S, Stumvoll M, Kellerer M, Haring HU. Pathophysiology and pharmacological treatment of insulin resistance. Endocrinol Rev 2000; 21:585– 618. 22. Smith MS, Freeman ME, Neill JD. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 1975;96:219 –26. 23. Hill J, Faris H, Schiff I, Anderson D. Characterization of leukocyte subpopulations in the peritoneal fluid of women with endometriosis. Fertil Steril 1988;50:216 –22. 24. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1983;35:696 – 8. 25. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333–7. 26. Akoum A, Kong J, Metz C, Beaumont MC. Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis. Fertil Steril 2002;77:989 –94. 27. Keenan J, Chen T, Chadwell N. IL-1, TNF-␣, and IL-2 in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol 1995;34:381–5. 28. Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, et al. Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 2000;85:824 –9.
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29. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, et al. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996;81:3112– 8. 30. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. 31. Mori H, Sawairi M, Nakagawa M, Itoh N, Wada K, Tamaya T. Expression of interleukin-1 (IL-1) beta messenger ribonucleic acid (mRNA) and IL-1 receptor antagonist mRNA in peritoneal macrophages from patients with endometriosis. Fertil Steril 1992;57:535– 42. 32. D’Hooghe TM, Cuneo N, Nugent N, Chai D, Deer F, Mwenda J. Recombinant human TNF binding protein-1 (r-hTBP-1) inhibits the development of endometriosis in baboons: a prospective, randomized, placebo- and drug-controlled study. Fertil Steril 2001;76(Suppl 3):S1 (abstract O-1). 33. Lebovic DI, Baldocchi RA, Mueller MD, Taylor RN. Altered expression of a cell-cycle suppressor gene, Tob-1, in endometriotic cells by cDNA array analyses. Fertil Steril 2002;78:849 –54. 34. Lebovic DI, Bentzien F, Chao VA, Garrett EN, Meng YG, Taylor RN. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1. Mol Hum Reprod 2000;6:269 –75. 35. Yee LD, Sabourin CL, Liu L, Li HM, Smith PJ, Seewaldt V, et al. Peroxisome proliferator–activated receptor gamma activation in human breast cancer. Int J Oncol 1999;15:967–73. 36. Houston KD, Copland JA, Broaddus RR, Gottardis MM, Fischer SM, Walker CL. Inhibition of proliferation and estrogen receptor signaling by peroxisome proliferator–activated receptor gamma ligands in uterine leiomyoma. Cancer Res 2003;63:1221–7. 37. Heaney AP, Fernando M, Melmed S. PPAR-gamma receptor ligands: novel therapy for pituitary adenomas. J Clin Invest 2003;111:1381– 8. 38. Panigrahy D, Singer S, Shen LQ, Butterfield CE, Freedman DA, Chen EJ, et al. PPARgamma ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis. J Clin Invest 2002;110:923–32. 39. Wanichkul T, Han S, Huang RP, Sidell N. Cytokine regulation by peroxisome proliferator–activated receptor gamma in human endometrial cells. Fertil Steril 2003;79(Suppl 1):763–9. 40. Lebovic DI, Chao VA, Martini JF, Taylor RN. IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter. J Clin Endocrinol Metab 2001;86:4759 – 64.
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Peroxisome proliferator–activated receptor-gamma induces regression of endometrial explants in a rat model of endometriosis Dan I. Lebovic, M.D., M.A.,a Mustafa Kir, M.D.,b and Colleen L. Casey, M.D.a Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Michigan, Ann Arbor, Michigan
Received October 23, 2003; revised and accepted February 9, 2004. Supported in part by the University of Michigan Department of Obstetrics and Gynecology through the Ansbacher Fund for Resident/Fellow Education and Research. Reprint requests: Dan I. Lebovic, M.D., Department of Obstetrics and Gynecology, University of Michigan, 1500 E. Medical Center Drive, L4100 Women’s Hospital, Ann Arbor, Michigan 481090276 (FAX: 734-647-1006; E-mail: [email protected]). a Reproductive Endocrinology Division, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan. b Department of Obstetrics and Gynecology, Acibadem Hospital, Istanbul, Turkey. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 02.148
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Objective: To determine the effects of a thiazolidinedione, ciglitazone, in a rat model of endometriosis. Design: Prospective, randomized, placebo-controlled study. Setting: Experimental surgery laboratory in a university department. Animal(s): Twenty female Sprague-Dawley rats given endometriotic lesions by transplanting autologous uterine tissue to ectopic sites on the peritoneum. Intervention(s): Four weeks after surgery, 20 rats were randomly divided into two groups and treated with IP injections of vehicle every other day (control; n ⫽ 10) or ciglitazone (1 mg per rat; n ⫽ 10) and euthanized 4 weeks from the start of treatment. Main Outcome Measure(s): At the end of treatment, laparotomy was performed to photograph each explant and then they were measured and weighed. Histologic analysis was performed on the uterine allograft, ovary, and eutopic uterine tissue. Result(s): By histologic assessment, both groups maintained folliculogenesis and normal eutopic endometrial architecture. Treatment with ciglitazone significantly decreased the size of ectopic uterine tissues and the mean explant wet weight. The ciglitazone-treated group showed marked epithelial regression compared with the control group. Conclusion(s): We conclude that a PPAR-␥ ligand, ciglitazone, reduced the size of experimental endometriosis in the rat model of endometriosis. This animal model suggests that a thiazolidinedione drug may be helpful in women with endometriosis. (Fertil Steril威 2004;82(Suppl 3):1008 –1013. ©2004 by American Society for Reproductive Medicine.) Key Words: : Endometriosis, immune modulators, immunotherapy, PPAR-␥, rat model, thiazolidinedione
Endometriosis affects 1 in 10 reproductiveage women and has far greater prevalence in the pelvic pain and subfertile populations (1). The histologic criteria for diagnosing endometriosis are endometrial glands and stroma. Not included in this classification is the associated inflammatory response that is now recognized as the sine qua non for the disease (2, 3). Whether this altered immune response is the cause or effect of the disorder is not certain, although it appears to contribute to the persistence of the disease and associated symptoms. Endometriosis is a progressive disease. Surgical intervention or medical suppression of the endometriotic lesions often lead to only temporary relief. The recurrence rate is
approximately 5%–20% per year with a cumulative rate of 40% after 5 years (4 – 6). Two common medical treatment options are GnRH agonists and combination estrogen/ progestin oral contraceptives, which were first used for endometriosis more than 20 years ago (7, 8). More efficacious, bettertolerated, and more affordable treatment options must be developed. An ideal treatment would eliminate endometriotic lesions, prevent recurrence, and not impede ovulation. Immune-modulating drugs such as recombinant human tumor necrosis factor-␣ binding protein-1 (r-hTBP-1), interferon-␣2b, loxoribine, pentoxifylline, and peroxisome proliferator–activated receptor-␥ (PPAR-␥) li-
gands are candidate treatment options (9 –13). PPARs are a new class of immune modulators found in adipose tissue, liver, spleen, colon, adrenal gland, muscle tissue, macrophages, and endometrial epithelial and stromal cells (14 –16). Endogenous agonists regulate adipocyte differentiation and glucose homeostasis. Interestingly, recent reports indicate that the PPAR-␥ subtype can mediate anti-inflammatory effects (17–19). Surgically transplanted endometrial tissue in the rat provides an animal model to study the effects of experimental drugs on ectopic endometrial tissue (20). Rat endometriotic implants show histologic transformations similar to those seen in human endometriotic lesions. In the current study, we used the rat model of endometriosis to test if a thiazolidinedione (TZD), an activator of PPAR-␥, could impede the growth of ectopic uterine tissue.
MATERIALS AND METHODS Animals Female Sprague-Dawley rats (200 –250 g) were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Unit for Laboratory Animal Medicine. They were caged in a controlled environment with 12-hour light/dark cycles. Food (standard pellet diet) and water were provided ad libitum. The animals were sexually mature and demonstrated normal estrous cycle changes in uterine histology (data not shown). The animals were allowed to acclimatize to these conditions for at least 1 week. The University of Michigan Committee on Use and Care of Animals
approved the experiments, and all investigations complied with the 1996 National Academy of Science’s Guide for Care and Use of Laboratory Animals.
Study Drug Ciglitazone (GR-205) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA) and was brought into solution (20% ethanol, 35 mM NaHCO33, NaOH, pH 9.6) at a concentration of 10 g/L. Five hundred microliter aliquots were frozen at ⫺20°C. Although we used an IP route of administration based on prior rodent studies, this drug has high oral bioavailability (21).
Induction of Experimental Endometriosis in Rats Twenty-two rats were used in the experimental induction of endometriosis. When the rats were 3 months of age, ectopic endometrium was induced surgically as described by Vernon and Wilson (20). Briefly, animals were anesthetized with isofluorane, and after a midventral incision to expose the uterus, a 1.0-cm length distal segment was resected from the left uterine horn. The segment was placed in phosphate-buffered saline at 37°C and split longitudinally, and a 5 ⫻ 5 mm piece was sectioned. This piece of uterine tissue was transplanted without removing the myometrium onto the inner surface of the right abdominal wall with the epithelial lining apposed to the peritoneal surface. The explanted endometrial tissue margins were secured with nonadsorbable polypropylene sutures (Prolene 6-0; Ethicon, Piscataway, NJ) at two edges. The midline abdominal incision was closed with chromic catgut sutures (3-0; Ethicon). The skin incision was closed with horizontal mattress
FIGURE 1 Gross appearance of uterine autografts at the conclusion of treatment. Immediately postmortem appearance of endometriotic explant (circled) after 4 weeks of vehicle control treatment (A) or ciglitazone (B). Note the cystic morphology of the control group as opposed to the pellet-like residual explant in the ciglitazone treatment group. The two blue-dyed sutures on either end of the explant can be seen in both panels. *, adhesion; scale bar, 5 mm.
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
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sutures of absorbable polyglactin 910 (coated Vicryl 4-0; Ethicon, Inc.). Animals were allowed 4 weeks for recovery and development of the disease. Four weeks after transplantation of endometrial tissue, two rats were euthanized to evaluate the surgical technique. The explants were examined for size and viability. The surface area of the engrafted tissue was weighed and measured using a caliper.
Experimental Design Four weeks after surgery, 20 rats were randomly allocated into two groups and given one IP injection every other day for 4 consecutive weeks with the following agents: control solution (vehicle, 20% ethanol, 35 mM NaHCO3 NaOH, pH 9.6; 100 L; n ⫽ 10) or ciglitazone (1 mg ciglitazone, 100 L, n ⫽ 10) every other day over a 4-week period. This dose and treatment schedule were based on a previous study in a mouse model of endometriosis (13). During the treatment period, any adverse treatment effects (such as weight loss, alopecia) were monitored. Vaginal smears were performed on all rats after drug treatments to ensure that their reproductive cycles were not disrupted at the end of the study: 30% of the control group were in either proestrus or estrus while 50% of the ciglitazone group were in one of these
stages. At the end of treatment, the animals were euthanized with CO2 and the uterine tissue implant was evaluated before an ovary and 0.5-mm segment of right uterine horn were removed and placed into 10% neutral buffered formalin and processed for hematoxylin and eosin staining.
Evaluation of Ectopic Uterine Tissue After euthanasia with CO2, the rats were immediately fixed in the supine position on a platform, the abdomen was opened, and all three dimensions (length ⫻ width ⫻ height in millimeters) of ectopic uterine tissue were measured in situ using a caliper. The spherical volume of each ectopic uterine tissue was calculated using the prolate ellipsoid formula: V (mm3) ⫽ 0.52 ⫻ A ⫻ B ⫻ C, where A, B, and C denote width, length, and height, respectively. Tissues were photographed using a digital camera and then excised and weighed (in milligrams) before being placed in 10% neutral buffered formalin and processed for hematoxylin and eosin staining.
Evaluation of Persisting Epithelium in Endometrial Autografts The persistence of epithelial cells in uterine autografts was evaluated by a previously published method as follows:
FIGURE 2 Histology of ovary, eutopic uterus, and ectopic uterine tissue. Hematoxylin and eosin staining of ovarian tissue, eutopic endometrium, and ectopic uterine explant from the two treatment groups: control (A, C, E) and ciglitazone, Ciglit (B, D, F). Left-side panels (A and B) show representative ovarian antral follicles. The middle panels (C and D) reveal normal appearing endometrium with associated luminal epithelium (*) and stromal cells (ST). Right-side panels (E and F) show the endometriotic implant cyst lumen (LU) with epithelial cell lining (*) for the control group (E) and degeneration of the ciglitazone group explant (F). Scale bar, 100 m.
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a score of 3 indicates a well-preserved epithelial layer; a score of 2, moderately preserved epithelium with leukocyte infiltrate; a score of 1, a poorly preserved epithelium (occasional epithelial cells only); and a score of 0, no epithelium (11). An individual other than the authors evaluated three sections from each explant in a blinded fashion. The bars in Figure 4 represent an average value for each group.
FIGURE 3 Effects of ciglitazone on the volume and weight of ectopic uterine tissue. Spherical volumes and weights of uterine explants of vehicle (Ctrl)- and ciglitazone (Cig)-treated rats are depicted in the left and right panels, respectively. There were 10 rats per treatment group. Each bar represents the mean ⫾ SD. a, P⬍.006 (Kruskal-Wallis test); b, P⬍.003 (Kruskal-Wallis test).
Histology The formalin-fixed endometriotic-like explants, ovaries, and eutopic uterine tissues were embedded in paraffin wax blocks, and 5-m sections were prepared (three sections per sample). The sections were heat-dried and stained with hematoxylin and eosin.
Statistical Analysis
The data are expressed as the mean ⫾ SD. We used the nonparametric Kruskal-Wallis test to analyze the data (JMP version 5.0; Cary, NC). Significant differences were accepted when two-tailed analyses yielded P⬍.017 (Bonferroni correction ␣ of 0.05/3 for the three comparison outcomes).
RESULTS Surgical Outcome in the Rat Endometriosis Model Four weeks from the time of uterine transplantation, two rats were euthanized to examine the endometriotic explants. In these animals, the explant appeared as well vascularized, unilocular cystic structures containing clear, serous fluid. The explants had an average spherical volume of 20 mm3 and a weight of 25 mg. Representative in vivo images of the explants after 1 month of respective treatment revealed a cystic implant in the vehicle control group (Fig. 1A) in contrast to the degenerative, pellet appearance in a majority of the ciglitazone-treated cohort of rats (Fig. 1B).
Histology for Ovary, Uterine Tissue, and Uterine Explant Neither vehicle control nor ciglitazone had any observable adverse effect (i.e., weight loss, grooming changes) or organ-specific changes such as follicular atresia in the ovaries (Fig. 2A, 2B) or tissue necrosis in eutopic uterine tissue (Fig. 2C, 2D) as verified by histologic comparison. Multiple ovarian and eutopic uterine tissue sections were examined. Progesterone concentrations from serum obtained at the time of euthanasia revealed a higher mean level in the ciglitazone group (7.9 ⫾ 1.3 pmol/L, P⬍.05, Student’s t-test) as compared with the control group (4.1 ⫾ 0.6 pmol/L), although both means were within baseline values for Sprague-Dawley female rats (22). The control cystic implants (Fig. 2E and insert) were similar to the eutopic endometrium because they both contained endometrial epithelium and stromal cells. The inner lining of the cysts contained simple columnar epithelium. The uterine autograft from ciglitazone-treated rats showed marked epithelial changes and overall regression of the explant (Fig. 2F and insert). FERTILITY & STERILITY威
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
Effects of Ciglitazone on the Ectopic Endometrial Tissue Volume and Weight There was a significant difference (P⬍.006) in posttreatment spherical volume between control (19.7 ⫾ 13.6 mm3) and ciglitazone (5.9 ⫾ 5.1 mm3) treatment groups (Fig. 3, left panel). Similarly, there was a significant difference (P⬍.003) in explant weight between control (24.0 ⫾ 13.4 mg) and ciglitazone-treated (6.7 ⫾ 3.9 mg) rats (Fig. 3, right panel).
Effect of Ciglitazone on the Ectopic Endometrial Epithelium Semiquantitative evaluation of the persistence of endometrial epithelial cells in the uterine explants (Fig. 4) showed a significantly lower score in the ciglitazone group (1.1 ⫾ 1.0) compared with the control group (2.4 ⫾ 1.0; P⬍.016).
DISCUSSION In this study, we used the rat model of surgically induced endometriosis to test the effect of a TZD PPAR-␥ agonist, ciglitazone, on the growth of ectopic uterine tissue. In contrast to vehicle control–treated rats, ciglitazone treatment for 1 month was found to be effective in inducing regression of the endometriotic implants. The 52% reduction in explant spherical volume, 70% lower wet weight, and 54% diminu1011
FIGURE 4 Effect of ciglitazone on the persisting epithelium score. Semiquantitative evaluation of persisting epithelium in uterine tissue allografts of vehicle (Ctrl)- and ciglitazone (Cig)-treated animals. There were 10 rats per treatment group. Each bar represents the mean ⫾ SD. a, P⬍.016 (Kruskal-Wallis test).
are mediated by diminished vascular endothelial growth factor production (38). Furthermore, acting through PPAR-␥, TZDs inhibit proinflammatory cytokines (13, 14, 39) as well as NF-B (18), an important nuclear transcription factor for the production of many cytokines (40). Taken together, since human endometrial epithelial and stromal cells contain PPAR-␥ (14, 39), we felt it would be useful to evaluate the influence of a PPAR-␥ ligand, ciglitazone, in a rat endometriosis model as a prelude to further studies in primates. The results in the present study have shown that ciglitazone significantly reduced the uterine explant burden in the rat model and, at the same time, did not seem to interfere with ovarian folliculogenesis. It may thus be possible to use TZDs to suppress existing endometriotic lesions without suppressing ovulation. Further studies need to definitively assess ovarian function using TZDs and to determine the reproductive safety profile. Investigation with this class of drug in the baboon model for endometriosis would be useful for prevention and treatment outcomes including assessment of the presence of ovulation. The data in this study suggest that TZDs, such as the commercially available rosiglitazone or pioglitazone, may be effective in the treatment of human endometriosis.
PPAR-␥ effects in rat endometriosis model. Fertil Steril 2004.
tion of persisting epithelium score confirmed ciglitazone to be a significant (P⬍.016) modulator of endometriotic implant growth. Moreover, the ciglitazone dose used for effective structural regression of rat endometriotic implants did not seem to disrupt estrous cycling or folliculogenesis or affect the rats’ physical well-being. The pathogenesis of endometriosis appears to involve a process of subclinical peritoneal chronic graft-versus-host immune response, marked by an accumulation of leukocytes including peritoneal macrophages (23–25), T cells, natural killer cells, and soluble products produced by these cells (2, 26 –31). Immunomodulatory agents such as r-hTBP-1, interferon-␣2b, loxoribine, and pentoxifylline have been used in animal models of endometriosis, effectively causing regression of uterine explant lesions (9 –12). A randomized controlled study in the baboon endometriosis model showed that r-hTBP-1 significantly inhibited the development of endometriotic lesions (32). Women with endometriosis have increased numbers of activated peritoneal fluid macrophages (23–25) along with macrophage-derived cytokines (26 –31). One such cytokine, interleukin-1 beta is thought to serve as a mediator between peritoneal fluid macrophages and the promotion of endometriotic implant growth and angiogenesis (33, 34). Considerable evidence suggests that PPAR-␥ ligands, such as TZDs, are potent cell growth inhibitors (35, 36) and inducers of apoptosis (35, 37). They have anti-angiogenic effects that 1012 Lebovic et al.
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