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doi:10.1002/jog.12711
J. Obstet. Gynaecol. Res. Vol. 41, No. 3: 489 495, March 2015
Regression of experimental endometriotic implants in a rat model with the angiotensin II receptor blocker losartan Bulent Cakmak1, Turker Cavusoglu2, Utku Ates2, Ayfer Meral3, Mehmet Can Nacar1 and Oytun Erbas¸4 Departments of 1Obstetrics and Gynecology, and 4Physiology, School of Medicine, Gaziosmanpasa University, Tokat, 2 Department of Histology and Embryology, Ege University School of Medicine, Izmir, and 3Department of Biochemistry, Dumlupinar University Evliya Celebi Training and Research Hospital, Kutahya, Turkey
Abstract Aim: Endometriosis is a common disease in women of reproductive age, and many different treatments have been developed, although none has provided a cure. In this study, the efficacy of losartan, an angiotensin II type 1 receptor blocker and an antiangiogenic and anti-inflammatory agent, on regression of experimental endometriotic implants in a rat model was investigated. Methods: Peritoneal endometriosis was surgically induced in 16 mature female Sprague–Dawley rats. The peritoneal endometriotic implant was confirmed after 28 days, and the animals were divided randomly into two groups. The control group (n = 8) was given 4 mL/day tap water by oral gavage, and the losartan group (n = 8) was given 20 mg/kg per day losartan p.o. We compared endometriotic implant size, extent and severity of adhesion, as well as plasma and peritoneal lavage fluid cytokine levels including vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α, plasma inflammatory factor pentraxin-3 (PTX-3) and C-reactive protein (CRP) between the treatment groups. Results: Mean surface endometriotic area, histological score of implants, adhesion formation, plasma VEGF, TNF, PTX-3 and CRP levels were significantly lower in the losartan group compared with control (P < 0.05). Furthermore, the peritoneal VEGF level was lower in the losartan group than in the control group (P < 0.001), but peritoneal TNF-α was similar in both groups (P > 0.05). Conclusion: Losartan suppressed the implant surface area of experimental endometriosis in rats and reduced the levels of plasma VEGF, TNF-α, PTX-3 and CRP. Key words: angiogenic factors, angiotensin II receptor blocker, endometriosis, losartan, vascular endothelial growth factor.
Introduction Endometriosis is characterized by the presence of endometrial glands and stroma at extrauterine locations.1 It can cause dysmenorrhea, dyspareunia,
chronic pelvic pain and subfertility,2 and is a common gynecologic disorder, occurring in 10–15% of women of reproductive age.3 The pathogenesis of endometriosis growth and development remains unclear, although diverse etiologies have been proposed,
Received: March 31 2014. Accepted: July 31 2014. Reprint request to: Dr Bulent Cakmak, Department of Obstetrics and Gynecology, Faculty of Medicine, Gaziosmanpasa University, Sevki Erek Yerleskesi, Fisunoglu Street, No: 68, Postal code: 60100, Tokat, Turkey. Email: [email protected] The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/EHQQya.
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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including immune system effects,4 genetic predisposition,5 changes in the peritoneum and retrograde menstruation6 related to complex mechanisms that involve estrogen and progesterone activity,7 apoptosis,7,8 inflammation9 and angiogenesis.10 Sufficient angiogenesis has an important role in the normal function of endometrial tissue in the uterine cavity.11 Induction of angiogenesis is also critical to the development of adequate vascularization for the intense blood vessel network found in endometriotic lesions12,13 and plays a critical role in the progression and regression of the condition.14 Vascular endothelial growth factor (VEGF) is the major angiogenic factor mediating this process, and endometriotic lesions with high proliferative activity are accompanied by higher local angiogenic activity and higher levels of VEGF in the serum and peritoneal fluid.15 Various antiangiogenic agents including growth factor inhibitors, statins, cyclooxygenase-2 (COX-2) inhibitors, peroxisome proliferator-activated receptors (PPAR) agonists, progestins and dopamine agonists have been used in experimental studies to induce regression of endometriotic lesions by targeting their blood supply to reduce microvessel density and decrease VEGF expression.16 Angiotensin II (AngII) is involved in cardiovascular circulation and is the most effective peptide of the renin–angiotensin system (RAS). AngII acts in different tissues via two different receptors, the AngII type 1 and type 2 receptors (AngII R1 and AngII R2, respectively).17 AngII stimulates the synthesis of VEGF via AngII R1 and induces angiogenesis, cellular proliferation and inflammation.18 AngII R1, under control of the local RAS and reproductive hormones, is present in both the human and rat uterus19,20 and mediates AngII in endometrial stromal cells.21 Angiotensin II R1 blockers, such as losartan, block the biological activities of AngII and are used widely to treat hypertensive disorders without serious sideeffects. Several experimental studies have reported that AngII R1 blockers have strong antiangiogenic activity and can inhibit the growth of pancreatic, breast and endometrial cancers.22–24 This indicates that losartan has antiproliferative effects on cancer cells via antiangiogenic activity mediated by suppressed VEGF expression and vascularization in the tumor.23 In the present study, we hypothesized that losartan acts as an AngII R1 selective blocker and may have therapeutic benefit in endometriosis. We investigated the effect of losartan on experimental endometriotic lesions in a rat model by histopathologic examination
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and analysis of the angiogenic factors VEGF, tumor necrosis factor (TNF)-α and plasma inflammatory factor pentraxin-3 (PTX-3).
Materials and Methods Animals In this study, we used 16 mature female Sprague– Dawley albino rats at 12 weeks of age, weighing 200– 220 g. Animals were fed ad libitum and housed in pairs in steel cages in a temperature-controlled environment (22 ± 2°C) with a 12-h light/dark cycle. The experimental procedures were approved by the Committee for Animal Research of Gaziosmanpasa University. All animal studies strictly conformed to the animal experiment guidelines of the Committee for Human Care at our institution. Experimental protocol The abdomen was opened through a 5-cm midline incision, and the left uterine horn was ligated at both the uterotubal junction and the cervical end prior to removal. The segmental horn was immersed in sterile saline solution, and the endometrium exposed by bisection of the anti-mesenteric axis to prepare 5 mm × 5 mm × 1-mm sections. These explants were sutured to the left anterolateral endometrial peritoneal surface by 7-0 polypropylene (Prolene; Ethicon). Abdominal layers were closed anatomically using 4-0 polyglactin 910 (Vicryl; Ethicon). After 4 weeks, the endometriotic explants were surgically visualized. All subjects had confirmed peritoneal endometriotic foci documented and measured according the formula A × B × C × 0.52 mm3 (A, length; B, width; C, height of implants). After the second operation, all rats were allowed a 3-day resting period. The rats were then randomly divided into two groups (n = 8 per group). The control group was given 4 mL/ day tap water by oral gavage via orogastric tubes, and the rats in the losartan group were given 20 mg/kg per day losartan p.o. Losartan was prepared from a tablet containing 50 mg losartan (Cozaar; MSD) that was crushed and suspended in tap water to yield a concentration of 10 mg/mL. The drug solution, calibrated to the weight of each rat, was suspended in 4 mL tap water. Four weeks after the start of treatment, a third laparotomy was performed, and the rats were killed by ketamine anesthesia. In the third laparotomy, the length and width of implants was measured macroscopically according to the lesion margin, and the
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Regression of endometriosis with losartan
volume calculated again. We also obtained an aspirate by peritoneal lavage with 1 mL isotonic solution. The extent and severity of adhesions at the operation site of endometriotic implants were evaluated using an established scoring system25 as follows: 0, no adhesions; 1, 25% of traumatized area; 2, 50% of traumatized area; and 3, total involvement. Fractional scores were given for the extent of adhesions between the above grades. The severity of the adhesions was measured as follows: 0, no resistance to separation; 0.5, some resistance (moderate force was required); and 1, sharp dissection needed.
Histopathologic examination of endometriosis foci For histopathologic studies, all animals were anesthetized by an i.p. injection of 40 mg/kg ketamine and 4 mg/kg xylazine, then perfused with 200 mL 4% formaldehyde in 0.1 mol phosphate-buffer saline (PBS). The implants were then excised and fixed in 10% formalin, embedded in paraffin blocks, sectioned at 5-μm thickness, stained with hematoxylin–eosin and examined under a light microscope. The persistence of epithelial cells in endometrial explants was semiquantitatively evaluated as follows: 3, wellpreserved epithelial layer; 2, moderately preserved epithelium with leukocyte infiltrate; 1, poorly preserved epithelium (occasional epithelial cells only); and 0, no epithelium. This evaluation was based on a previous rat endometriosis study.25 These measurements and evaluations were made by one operator blinded to the study. Biochemical assays Measurement of plasma and peritoneal fluids cytokine levels For cytokine measurements, blood and peritoneal fluids were centrifuged at 1200 g for 10 min at room
temperature and stored at −20°C until assay. Cytokine levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits for VEGF (Invitrogen), TNF-α (Uscn Life Science) and C-reactive protein (CRP; Uscn Life Science). Samples from each animal were assayed in duplicate according to the manufacturer’s guide. Evaluation of plasma PTX-3 levels Plasma PTX-3 levels were measured in each 100-μL sample by ELISA at 450 nm using a PTX-3 kit (Uscn Life Science). PTX-3 levels were determined in duplicate according to the manufacturer’s guidelines. The detection range for the PTX-3 assay was 0.078–5 ng/mL.
Statistical analysis Data analyses were performed using SPSS version 15.0 for Windows. Parametric variables were compared using Student’s t-test. Non-parametric variables were compared by the Mann–Whitney U-test. Results are shown as mean ± standard error of the mean. P < 0.05 was considered statistically significant, and P < 0.001 was accepted as highly statistically significant.
Results Histopathology of endometriotic implants Endometriotic implant sizes, scores and adhesion formation In the second laparotomy, the endometriotic implant surface area was similar in all animals before treatment (Table 1). After 28 days of treatment, the endometriotic implant surface area decreased significantly in the losartan group compared with the control (P < 0.05). An example of endometriosis implant on the
Table 1 Histopathologic comparison of the endometriotic implants Histopathologic features of implants Mean surface area of implants (mm2) Before medication After medication Mean volume of implants (mm3) Before medication After medication Histopathologic score of implants Extent of adhesion Severity of adhesion
Control
Losartan (20 mg/kg)
P
12.6 ± 4.3 16.2 ± 5.4
15.9 ± 6.1 8.5 ± 4.9
0.563 0.035
36.8 ± 8.1 41.2 ± 6.3 2.32 ± 0.5 2.28 ± 0.18 0.85 ± 0.09
41.7 ± 7.2 22.6 ± 4.9 1.25 ± 0.65 1.14 ± 0.34 0.42 ± 0.17
0.27 0.043 0.008 0.005 0.036
Data are expressed as means ± standard error of the mean.
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Table 2 Comparison of plasma cytokine levels between groups
3mm
3mm
Figure 1 An endometriosis implant is shown on the peritoneal surface of the abdominal wall. (a) Control group. (b) Losartan-treated group. The size of ectopic endometriotic implants regressed significantly with losartan treatment.
Figure 2 Histological sections from the control and losartan groups (original magnification, ×100). Arrow indicates the epithelium of the endometriotic implant. (a) Histological section from a control rat demonstrating well-preserved viable epithelium of the endometriotic implant (score = 3). (b) Histological section from a losartan-treated rat showing marked atrophy and regression of the epithelium of the endometriotic implant (score = 1).
peritoneal surface of the abdominal wall is shown in Figure 1. After treatment, the histopathologic score of endometriotic implants was lower in the losartan group compared with control (P < 0.01). Histological sections from the control and losartan groups are shown in Figure 2. Furthermore, the extent and severity of adhesion formation was significantly lower in the losartan group compared with the control (P < 0.05).
Biochemical assays Plasma and peritoneal fluid cytokine levels The plasma levels of VEGF and TNF-α were significantly lower in the losartan group compared with control after treatment (Table 2). Additionally, the inflammatory markers CRP and PTX-3 were decreased
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Plasma cytokine levels
Control
Losartan (20 mg/kg)
P
VEGF (pg/mL) TNF-α (pg/mL) PTX-3 (ng/mL) CRP (mg/dL)
195.7 ± 10.8 79.2 ± 2.8 4.8 ± 0.3 0.75 ± 0.16
137.5 ± 6.6 50.7 ± 5.9 3.2 ± 0.5 0.54 ± 0.11
0.032 0.013 0.025 0.044
Data are expressed as means ± standard error of the mean. CRP, C-reactive protein; PTX-3, pentraxin-3; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor.
Figure 3 Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α levels in the peritoneal fluid of the control and losartan-treated groups. Data are expressed as mean ± standard error of the mean.
in the losartan treatment group compared with the post-treatment control group. Peritoneal lavage VEGF levels were significantly different between the losartan (25.8 ± 1.7 pg/mL) and control groups (56.7 ± 3.5 pg/mL) after treatment. However, the TNF-α level in peritoneal lavage was similar in the two groups after treatment (losartan, 33.5 ± 2.2 pg/mL; control, 36.8 ± 3.2 pg/mL), as shown in Figure 3.
Discussion This study demonstrated that losartan, a selective AngII R1 blocker, could be used experimentally to treat endometriosis by shrinking peritoneal surgical endometriosis implants and reducing adhesion formation and VEGF levels in plasma and peritoneal lavage fluid. There are several treatment options for endometriosis. Gonadotropin-releasing hormone (GnRH) analogs have demonstrated efficacy in experimental studies
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Regression of endometriosis with losartan
and are used in humans as a conventional treatment, and immunomodulators and aromatase inhibitors are newer treatment options that are gaining popularity.26–31 Several medications used in endometriosis treatment decrease vasculogenesis by reducing angiogenesis and decreasing VEGF expression and secretion.16 GnRH analogs used in the treatment of endometriosis cause a hypoestrogenic effect that inhibits the growth of endometriotic foci. Additionally, other important mechanisms of action have been reported in humans and in human endometriotic cell culture studies including reduction of anti-inflammatory interleukin (IL)-1, transforming growth factor (TGF)-β1 and VEGF.26,27 An experimental model of endometriosis in rats treated with the GnRH analog leuprolide showed a significant decrease in endometriotic implant surface area and VEGF in the peritoneal fluid.28 In our study, plasma and peritoneal lavage fluid VEGF levels were decreased significantly in the losartantreated group. Additionally, plasma TNF-α, PTX-3 and CRP levels were also decreased significantly, although there was no significant change in TNF-α levels in peritoneal lavage fluid. Ceyhan et al.29 reported that the aromatase inhibitor letrozole suppressed endometriotic foci by providing locally reduced levels of estrogen and that immunomodulators were effective in treatment of endometriosis in a surgically created endometriosis rat model. In that study, letrozole treatment decreased the endometriosis implant area and VEGF immunoreactivity, but there was no change in peritoneal VEGF levels. Furthermore, peritoneal endometrial implants regressed following treatment with the immunomodulators infliximab and etanercept, with no change in VEGF level or immunoreactivity. Uygur et al.30,31 also reported that the immunomodulator leflunomide decreased endometriotic foci in two studies investigating the efficacy of leflunomide for endometriotic implant regression. In those studies, the surface areas and weights of endometriotic implants decreased significantly with leflunomide treatment, but angiogenesis and inflammatory reactions were not evaluated. In our study, endometriotic implant surface area and histopathologic scores were decreased after losartan treatment. These changes were accompanied by reduced plasma and peritoneal fluid levels of VEGF and other angiogenic inflammatory markers, with the exception of TNF-α. Additionally, the extent and severity of adhesions were lower in the losartan group. Many studies using experimental animal models have examined the efficacy of PPAR-γ agonists for
endometriosis treatment.32–34 In previous studies, expression of PPAR in endometrial and endometriotic cells was reported, and PPAR receptor activation inhibited cell proliferation and VEGF expression.35,36 Using an experimental rat model, Aytan et al.33 found that the PPAR-γ agonist rosiglitazone caused a significant regression in peritoneal endometriotic implant length, width, height and spherical volume. The effect of losartan on PPAR-γ activity in tissue from the female genital tissue has not yet been studied. However, in studies of mouse hepatic injury and human macrophages in vitro, the efficacy of partial PPAR-γ activation on reduced TNF-α, IL-8 and COX-2 expression was reported.37,38 Based on our study, the reduction of the endometriotic foci and decreased plasma concentrations of inflammatory factors such as TNF-α, PTX-3 and CRP in the group treated with losartan highly suggest that in addition to its AngII R1 blocker effect losartan may also have a partial PPAR-γ activation effect. In the present study, we demonstrated that losartan suppressed endometriotic implants by acting as an AngII R1 inhibitor with antiangiogenic activity. Many other AngII R1 blockers are used as antihypertensive agents, such as telmisartan. An experimental mouse model study investigating telmisartan efficacy for endometriosis found that telmisartan was more effective than the PPAR-γ agonist pioglitazone39 and that telmisartan acted as an AngII R1 inhibitor and PPAR-γ activator. Nenicu et al.39 noted that telmisartan induced upregulation of PPAR-γ and downregulation of AngII R1 proteins in endometriosis-like lesions, which was associated with decreased density of CD31-positive microvessels, reduced immune cell content, and a lower number of Ki67-positive proliferating cells. Furthermore, that study demonstrated that telmisartan inhibited the expression of several angiogenic and inflammatory genes, which was similar to the approach we used in this study to assess the effect of losartan. There are several limitations that should be noted in this study. The first is that side-effects of losartan were not evaluated in the normal endometrial tissue found in the remaining uterine horn. The second is that we did not determine whether losartan affected ovarian functioning by following the progression of the regular estrous cycle and/or estrogen levels. The third is that we only used a single standard losartan dose rather than evaluating a dose-dependent effect of this drug on endometriotic implant regression. In conclusion, the findings of this study indicate that in an experimental endometriosis model, losartan
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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caused significant regression of endometriotic implant size and resulted in lower VEGF levels in the peritoneal fluid, along with decreased plasma TNF-α, PTX-3 and CRP. This is the first study to determine that losartan could be beneficial in endometriosis treatment.
Disclosure None of the authors has anything to disclose.
References 1. Galle PC. Clinical presentation and diagnosis of endometriosis. Obstet Gynecol Clin North Am 1989; 16: 29–42. 2. Farquhar CM. Extracts from ‘Clinical Evidence’ Endometriosis. BMJ 2000; 320: 1449–1452. 3. Ranney B. Endometriosis: Pathogenesis, symptoms, and findings. Clin Obstet Gynecol 1980; 23: 865–874. 4. Berkkanoglu M, Arici A. Immunology and endometriosis. Am J Reprod Immunol 2003; 50: 48–59. 5. Stefansson H, Geirsson RT, Steinthorsdottir V et al. Genetic factors contribute to the risk of developing endometriosis. Hum Reprod 2002; 17: 555–559. 6. Valle RF. Endometriosis: Current concepts and therapy. Int J Gynaecol Obstet 2002; 78: 107–119. 7. Reis FM, Petraglia F, Taylor RN. Endometriosis: Hormone regulation and clinical consequences of chemotaxis and apoptosis. Hum Reprod Update 2013; 19: 406–418. 8. Taniguchi F, Kaponis A, Izawa M et al. Apoptosis and endometriosis. Front Biosci (Elite Ed) 2011; 3: 648–662. 9. Lousse JC, Van Langendonckt A, Defrere S, Ramos RG, Colette S, Donnez J. Peritoneal endometriosis is an inflammatory disease. Front Biosci (Elite Ed) 2012; 4: 23–40. 10. Laschke MW, Giebels C, Menger MD. Vasculogenesis: A new piece of the endometriosis puzzle. Hum Reprod Update 2011; 17: 628–636. 11. Girling JE, Rogers PAW. Recent advances in endometrial angiogenesis research. Angiogenesis 2005; 8: 89–99. 12. Nisolle M, Casanas Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993; 59: 681–684. 13. Taylor RN, Lebovic DI, Mueller MD. Angiogenic factors in endometriosis. Ann N Y Acad Sci 2002; 955: 89–100. 14. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13: 1686–1690. 15. Bourlev V, Volkov N, Pavlovitch S, Lets N, Larsson A, Olovsson M. The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction 2006; 132: 501–509. 16. Laschke MW, Menger MD. Anti-angiogenic treatment strategies for the therapy of endometriosis. Hum Reprod Update 2012; 18: 682–702. 17. Neri Serneri GG, Boddi M, Coppo M et al. Evidence for the existence of a functional cardiac renin–angiotensin system in humans. Circulation 1996; 94: 1886–1893.
606
18. Shi RZ, Wang JC, Huang SH, Wang XJ, Li QP. Angiotensin II induces vascular endothelial growth factor synthesis in mesenchymal stem cells. Exp Cell Res 2009; 315: 10–15. 19. Saridogan E, Djahanbakhch O, Puddefoot JR et al. Type 1 angiotensin II receptors in human endometrium. Mol Hum Rep 1996; 2: 659–664. 20. Pawlikowski M, Melen-Mucha G, Mucha S. The involvement of the renin-angiotensin system in the regulation of cell proliferation in the rat endometrium. Cell Mol Life Sci 1999; 55: 506–510. 21. Herr D, Bekes I, Wulff C. Local renin-angiotensin system in the reproductive system. Front Endocrinol 2013; 4: 150. 22. Noguchi R, Yoshiji H, Ikenaka Y et al. Synergistic inhibitory effect of gemcitabine and angiotensin type-1 receptor blocker, losartan, on murine pancreatic tumor growth via antiangiogenic activities. Oncol Rep 2009; 22: 355–360. 23. Chen X, Meng Q, Zhao Y et al. Angiotensin II type 1 receptor antagonists inhibit cell proliferation and angiogenesis in breast cancer. Cancer Lett 2013; 328: 318–324. 24. Choi CH, Park YA, Choi JJ et al. Angiotensin II type I receptor and miR-155 in endometrial cancers: Synergistic antiproliferative effects of anti-miR-155 and losartan on endometrial cancer cells. Gynecol Oncol 2012; 126: 124–131. 25. Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A. Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999; 72: 135–141. 26. Küpker W, Schultze-Mosgau A, Diedrich K. Paracrine changes in the peritoneal environment of women with endometriosis. Hum Reprod Update 1998; 4: 719–723. 27. Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C, Baraneao RI. Effect of GnRH analogues on apoptosis and release of interleukin-1b and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 2003; 18: 1767–1771. 28. Oktem M, Esinler I, Eroglu D, Haberal N, Bayraktar N, Zeyneloglu HB. High-dose atorvastatin causes regression of endometriotic implants: A rat model. Hum Reprod 2007; 22: 1474–1480. 29. Ceyhan ST, Onguru O, Fidan U et al. Comparison of aromatase inhibitor (letrozole) and immunomodulators (infliximab and etanercept) on the regression of endometriotic implants in a rat model. Eur J Obstet Gynecol Reprod Biol 2011; 154: 100–104. 30. Uydur D, Aytan H, Zergeroglu S, Batioglu S. Leflunomide -an immonumodulator- induces regression of endometrial explants in a rat model of endometriosis. J Soc Gynecol Invest 2006; 13: 378–383. 31. Aytan H, Caglar P, Uydur D, Zergeroglu S, Batioglu S. Effect of the immunomodulator leflunomide on the induction of endometriosis in an experimental rat model. Fertil Steril 2007; 87: 698–701. 32. Demirturk F, Aytan H, Caliskan AC, Aytan P, Koseoglu DR. Effect of peroxisome proliferator-activated receptor-gamma agonist rosiglitazone on the induction of endometriosis in an experimental rat model. J Soc Gynecol Investig 2006; 13: 58–62. 33. Aytan H, Caliskan AC, Demirturk F, Aytan P, Koseoglu DR. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone reduces the size of experimental endometriosis in the rat model. Aust N Z J Obstet Gynaecol 2007; 47: 321– 325.
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34. Lebovic DI, Mwenda JM, Chai DC, Santi A, Xu X, D’Hooghe T. Peroxisome proliferator-activated receptor-(gamma) receptor ligand partially prevents the development of endometrial explants in baboons: A prospective, randomized, placebo-controlled study. Endocrinology 2010; 151: 1846–1852. 35. Ohama Y, Harada T, Iwabe T, Taniguchi F, Takenaka Y, Terakawa N. Peroxisome proliferator-activated receptorgamma ligand reduced tumor necrosis factor-alpha-induced interleukin-8 production and growth in endometriotic stromal cells. Fertil Steril 2008; 89: 311–317. 36. Peeters LL, Vigne JL, Tee MK, Zhao D, Waite LL, Taylor RN. PPAR gamma represses VEGF expression in human endometrial cells: Implications for uterine angiogenesis. Angiogenesis 2005; 8: 373–379.
37. Koh EJ, Yoon SJ, Lee SM. Losartan protects liver against ischaemia/reperfusion injury through PPAR-γ activation and receptor for advanced glycation end-products downregulation. Br J Pharmacol 2013; 169: 1404–1416. 38. An J, Nakajima T, Kuba K, Kimura A. Losartan inhibits LPSinduced inflammatory signaling through a PPARγ-dependent mechanism in human THP-1 macrophages. Hypertens Res 2010; 33: 831–835. 39. Nenicu A, Korbel C, Gu Y, Menger MD, Laschke MW. Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor-γ by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions. Hum Reprod 2014; 29: 1011–1024.
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doi:10.1002/jog.12711
J. Obstet. Gynaecol. Res. Vol. 41, No. 3: 489 495, March 2015
Regression of experimental endometriotic implants in a rat model with the angiotensin II receptor blocker losartan Bulent Cakmak1, Turker Cavusoglu2, Utku Ates2, Ayfer Meral3, Mehmet Can Nacar1 and Oytun Erbas¸4 Departments of 1Obstetrics and Gynecology, and 4Physiology, School of Medicine, Gaziosmanpasa University, Tokat, 2 Department of Histology and Embryology, Ege University School of Medicine, Izmir, and 3Department of Biochemistry, Dumlupinar University Evliya Celebi Training and Research Hospital, Kutahya, Turkey
Abstract Aim: Endometriosis is a common disease in women of reproductive age, and many different treatments have been developed, although none has provided a cure. In this study, the efficacy of losartan, an angiotensin II type 1 receptor blocker and an antiangiogenic and anti-inflammatory agent, on regression of experimental endometriotic implants in a rat model was investigated. Methods: Peritoneal endometriosis was surgically induced in 16 mature female Sprague–Dawley rats. The peritoneal endometriotic implant was confirmed after 28 days, and the animals were divided randomly into two groups. The control group (n = 8) was given 4 mL/day tap water by oral gavage, and the losartan group (n = 8) was given 20 mg/kg per day losartan p.o. We compared endometriotic implant size, extent and severity of adhesion, as well as plasma and peritoneal lavage fluid cytokine levels including vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α, plasma inflammatory factor pentraxin-3 (PTX-3) and C-reactive protein (CRP) between the treatment groups. Results: Mean surface endometriotic area, histological score of implants, adhesion formation, plasma VEGF, TNF, PTX-3 and CRP levels were significantly lower in the losartan group compared with control (P < 0.05). Furthermore, the peritoneal VEGF level was lower in the losartan group than in the control group (P < 0.001), but peritoneal TNF-α was similar in both groups (P > 0.05). Conclusion: Losartan suppressed the implant surface area of experimental endometriosis in rats and reduced the levels of plasma VEGF, TNF-α, PTX-3 and CRP. Key words: angiogenic factors, angiotensin II receptor blocker, endometriosis, losartan, vascular endothelial growth factor.
Introduction Endometriosis is characterized by the presence of endometrial glands and stroma at extrauterine locations.1 It can cause dysmenorrhea, dyspareunia,
chronic pelvic pain and subfertility,2 and is a common gynecologic disorder, occurring in 10–15% of women of reproductive age.3 The pathogenesis of endometriosis growth and development remains unclear, although diverse etiologies have been proposed,
Received: March 31 2014. Accepted: July 31 2014. Reprint request to: Dr Bulent Cakmak, Department of Obstetrics and Gynecology, Faculty of Medicine, Gaziosmanpasa University, Sevki Erek Yerleskesi, Fisunoglu Street, No: 68, Postal code: 60100, Tokat, Turkey. Email: [email protected] The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/EHQQya.
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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including immune system effects,4 genetic predisposition,5 changes in the peritoneum and retrograde menstruation6 related to complex mechanisms that involve estrogen and progesterone activity,7 apoptosis,7,8 inflammation9 and angiogenesis.10 Sufficient angiogenesis has an important role in the normal function of endometrial tissue in the uterine cavity.11 Induction of angiogenesis is also critical to the development of adequate vascularization for the intense blood vessel network found in endometriotic lesions12,13 and plays a critical role in the progression and regression of the condition.14 Vascular endothelial growth factor (VEGF) is the major angiogenic factor mediating this process, and endometriotic lesions with high proliferative activity are accompanied by higher local angiogenic activity and higher levels of VEGF in the serum and peritoneal fluid.15 Various antiangiogenic agents including growth factor inhibitors, statins, cyclooxygenase-2 (COX-2) inhibitors, peroxisome proliferator-activated receptors (PPAR) agonists, progestins and dopamine agonists have been used in experimental studies to induce regression of endometriotic lesions by targeting their blood supply to reduce microvessel density and decrease VEGF expression.16 Angiotensin II (AngII) is involved in cardiovascular circulation and is the most effective peptide of the renin–angiotensin system (RAS). AngII acts in different tissues via two different receptors, the AngII type 1 and type 2 receptors (AngII R1 and AngII R2, respectively).17 AngII stimulates the synthesis of VEGF via AngII R1 and induces angiogenesis, cellular proliferation and inflammation.18 AngII R1, under control of the local RAS and reproductive hormones, is present in both the human and rat uterus19,20 and mediates AngII in endometrial stromal cells.21 Angiotensin II R1 blockers, such as losartan, block the biological activities of AngII and are used widely to treat hypertensive disorders without serious sideeffects. Several experimental studies have reported that AngII R1 blockers have strong antiangiogenic activity and can inhibit the growth of pancreatic, breast and endometrial cancers.22–24 This indicates that losartan has antiproliferative effects on cancer cells via antiangiogenic activity mediated by suppressed VEGF expression and vascularization in the tumor.23 In the present study, we hypothesized that losartan acts as an AngII R1 selective blocker and may have therapeutic benefit in endometriosis. We investigated the effect of losartan on experimental endometriotic lesions in a rat model by histopathologic examination
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and analysis of the angiogenic factors VEGF, tumor necrosis factor (TNF)-α and plasma inflammatory factor pentraxin-3 (PTX-3).
Materials and Methods Animals In this study, we used 16 mature female Sprague– Dawley albino rats at 12 weeks of age, weighing 200– 220 g. Animals were fed ad libitum and housed in pairs in steel cages in a temperature-controlled environment (22 ± 2°C) with a 12-h light/dark cycle. The experimental procedures were approved by the Committee for Animal Research of Gaziosmanpasa University. All animal studies strictly conformed to the animal experiment guidelines of the Committee for Human Care at our institution. Experimental protocol The abdomen was opened through a 5-cm midline incision, and the left uterine horn was ligated at both the uterotubal junction and the cervical end prior to removal. The segmental horn was immersed in sterile saline solution, and the endometrium exposed by bisection of the anti-mesenteric axis to prepare 5 mm × 5 mm × 1-mm sections. These explants were sutured to the left anterolateral endometrial peritoneal surface by 7-0 polypropylene (Prolene; Ethicon). Abdominal layers were closed anatomically using 4-0 polyglactin 910 (Vicryl; Ethicon). After 4 weeks, the endometriotic explants were surgically visualized. All subjects had confirmed peritoneal endometriotic foci documented and measured according the formula A × B × C × 0.52 mm3 (A, length; B, width; C, height of implants). After the second operation, all rats were allowed a 3-day resting period. The rats were then randomly divided into two groups (n = 8 per group). The control group was given 4 mL/ day tap water by oral gavage via orogastric tubes, and the rats in the losartan group were given 20 mg/kg per day losartan p.o. Losartan was prepared from a tablet containing 50 mg losartan (Cozaar; MSD) that was crushed and suspended in tap water to yield a concentration of 10 mg/mL. The drug solution, calibrated to the weight of each rat, was suspended in 4 mL tap water. Four weeks after the start of treatment, a third laparotomy was performed, and the rats were killed by ketamine anesthesia. In the third laparotomy, the length and width of implants was measured macroscopically according to the lesion margin, and the
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volume calculated again. We also obtained an aspirate by peritoneal lavage with 1 mL isotonic solution. The extent and severity of adhesions at the operation site of endometriotic implants were evaluated using an established scoring system25 as follows: 0, no adhesions; 1, 25% of traumatized area; 2, 50% of traumatized area; and 3, total involvement. Fractional scores were given for the extent of adhesions between the above grades. The severity of the adhesions was measured as follows: 0, no resistance to separation; 0.5, some resistance (moderate force was required); and 1, sharp dissection needed.
Histopathologic examination of endometriosis foci For histopathologic studies, all animals were anesthetized by an i.p. injection of 40 mg/kg ketamine and 4 mg/kg xylazine, then perfused with 200 mL 4% formaldehyde in 0.1 mol phosphate-buffer saline (PBS). The implants were then excised and fixed in 10% formalin, embedded in paraffin blocks, sectioned at 5-μm thickness, stained with hematoxylin–eosin and examined under a light microscope. The persistence of epithelial cells in endometrial explants was semiquantitatively evaluated as follows: 3, wellpreserved epithelial layer; 2, moderately preserved epithelium with leukocyte infiltrate; 1, poorly preserved epithelium (occasional epithelial cells only); and 0, no epithelium. This evaluation was based on a previous rat endometriosis study.25 These measurements and evaluations were made by one operator blinded to the study. Biochemical assays Measurement of plasma and peritoneal fluids cytokine levels For cytokine measurements, blood and peritoneal fluids were centrifuged at 1200 g for 10 min at room
temperature and stored at −20°C until assay. Cytokine levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits for VEGF (Invitrogen), TNF-α (Uscn Life Science) and C-reactive protein (CRP; Uscn Life Science). Samples from each animal were assayed in duplicate according to the manufacturer’s guide. Evaluation of plasma PTX-3 levels Plasma PTX-3 levels were measured in each 100-μL sample by ELISA at 450 nm using a PTX-3 kit (Uscn Life Science). PTX-3 levels were determined in duplicate according to the manufacturer’s guidelines. The detection range for the PTX-3 assay was 0.078–5 ng/mL.
Statistical analysis Data analyses were performed using SPSS version 15.0 for Windows. Parametric variables were compared using Student’s t-test. Non-parametric variables were compared by the Mann–Whitney U-test. Results are shown as mean ± standard error of the mean. P < 0.05 was considered statistically significant, and P < 0.001 was accepted as highly statistically significant.
Results Histopathology of endometriotic implants Endometriotic implant sizes, scores and adhesion formation In the second laparotomy, the endometriotic implant surface area was similar in all animals before treatment (Table 1). After 28 days of treatment, the endometriotic implant surface area decreased significantly in the losartan group compared with the control (P < 0.05). An example of endometriosis implant on the
Table 1 Histopathologic comparison of the endometriotic implants Histopathologic features of implants Mean surface area of implants (mm2) Before medication After medication Mean volume of implants (mm3) Before medication After medication Histopathologic score of implants Extent of adhesion Severity of adhesion
Control
Losartan (20 mg/kg)
P
12.6 ± 4.3 16.2 ± 5.4
15.9 ± 6.1 8.5 ± 4.9
0.563 0.035
36.8 ± 8.1 41.2 ± 6.3 2.32 ± 0.5 2.28 ± 0.18 0.85 ± 0.09
41.7 ± 7.2 22.6 ± 4.9 1.25 ± 0.65 1.14 ± 0.34 0.42 ± 0.17
0.27 0.043 0.008 0.005 0.036
Data are expressed as means ± standard error of the mean.
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Table 2 Comparison of plasma cytokine levels between groups
3mm
3mm
Figure 1 An endometriosis implant is shown on the peritoneal surface of the abdominal wall. (a) Control group. (b) Losartan-treated group. The size of ectopic endometriotic implants regressed significantly with losartan treatment.
Figure 2 Histological sections from the control and losartan groups (original magnification, ×100). Arrow indicates the epithelium of the endometriotic implant. (a) Histological section from a control rat demonstrating well-preserved viable epithelium of the endometriotic implant (score = 3). (b) Histological section from a losartan-treated rat showing marked atrophy and regression of the epithelium of the endometriotic implant (score = 1).
peritoneal surface of the abdominal wall is shown in Figure 1. After treatment, the histopathologic score of endometriotic implants was lower in the losartan group compared with control (P < 0.01). Histological sections from the control and losartan groups are shown in Figure 2. Furthermore, the extent and severity of adhesion formation was significantly lower in the losartan group compared with the control (P < 0.05).
Biochemical assays Plasma and peritoneal fluid cytokine levels The plasma levels of VEGF and TNF-α were significantly lower in the losartan group compared with control after treatment (Table 2). Additionally, the inflammatory markers CRP and PTX-3 were decreased
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Plasma cytokine levels
Control
Losartan (20 mg/kg)
P
VEGF (pg/mL) TNF-α (pg/mL) PTX-3 (ng/mL) CRP (mg/dL)
195.7 ± 10.8 79.2 ± 2.8 4.8 ± 0.3 0.75 ± 0.16
137.5 ± 6.6 50.7 ± 5.9 3.2 ± 0.5 0.54 ± 0.11
0.032 0.013 0.025 0.044
Data are expressed as means ± standard error of the mean. CRP, C-reactive protein; PTX-3, pentraxin-3; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor.
Figure 3 Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α levels in the peritoneal fluid of the control and losartan-treated groups. Data are expressed as mean ± standard error of the mean.
in the losartan treatment group compared with the post-treatment control group. Peritoneal lavage VEGF levels were significantly different between the losartan (25.8 ± 1.7 pg/mL) and control groups (56.7 ± 3.5 pg/mL) after treatment. However, the TNF-α level in peritoneal lavage was similar in the two groups after treatment (losartan, 33.5 ± 2.2 pg/mL; control, 36.8 ± 3.2 pg/mL), as shown in Figure 3.
Discussion This study demonstrated that losartan, a selective AngII R1 blocker, could be used experimentally to treat endometriosis by shrinking peritoneal surgical endometriosis implants and reducing adhesion formation and VEGF levels in plasma and peritoneal lavage fluid. There are several treatment options for endometriosis. Gonadotropin-releasing hormone (GnRH) analogs have demonstrated efficacy in experimental studies
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and are used in humans as a conventional treatment, and immunomodulators and aromatase inhibitors are newer treatment options that are gaining popularity.26–31 Several medications used in endometriosis treatment decrease vasculogenesis by reducing angiogenesis and decreasing VEGF expression and secretion.16 GnRH analogs used in the treatment of endometriosis cause a hypoestrogenic effect that inhibits the growth of endometriotic foci. Additionally, other important mechanisms of action have been reported in humans and in human endometriotic cell culture studies including reduction of anti-inflammatory interleukin (IL)-1, transforming growth factor (TGF)-β1 and VEGF.26,27 An experimental model of endometriosis in rats treated with the GnRH analog leuprolide showed a significant decrease in endometriotic implant surface area and VEGF in the peritoneal fluid.28 In our study, plasma and peritoneal lavage fluid VEGF levels were decreased significantly in the losartantreated group. Additionally, plasma TNF-α, PTX-3 and CRP levels were also decreased significantly, although there was no significant change in TNF-α levels in peritoneal lavage fluid. Ceyhan et al.29 reported that the aromatase inhibitor letrozole suppressed endometriotic foci by providing locally reduced levels of estrogen and that immunomodulators were effective in treatment of endometriosis in a surgically created endometriosis rat model. In that study, letrozole treatment decreased the endometriosis implant area and VEGF immunoreactivity, but there was no change in peritoneal VEGF levels. Furthermore, peritoneal endometrial implants regressed following treatment with the immunomodulators infliximab and etanercept, with no change in VEGF level or immunoreactivity. Uygur et al.30,31 also reported that the immunomodulator leflunomide decreased endometriotic foci in two studies investigating the efficacy of leflunomide for endometriotic implant regression. In those studies, the surface areas and weights of endometriotic implants decreased significantly with leflunomide treatment, but angiogenesis and inflammatory reactions were not evaluated. In our study, endometriotic implant surface area and histopathologic scores were decreased after losartan treatment. These changes were accompanied by reduced plasma and peritoneal fluid levels of VEGF and other angiogenic inflammatory markers, with the exception of TNF-α. Additionally, the extent and severity of adhesions were lower in the losartan group. Many studies using experimental animal models have examined the efficacy of PPAR-γ agonists for
endometriosis treatment.32–34 In previous studies, expression of PPAR in endometrial and endometriotic cells was reported, and PPAR receptor activation inhibited cell proliferation and VEGF expression.35,36 Using an experimental rat model, Aytan et al.33 found that the PPAR-γ agonist rosiglitazone caused a significant regression in peritoneal endometriotic implant length, width, height and spherical volume. The effect of losartan on PPAR-γ activity in tissue from the female genital tissue has not yet been studied. However, in studies of mouse hepatic injury and human macrophages in vitro, the efficacy of partial PPAR-γ activation on reduced TNF-α, IL-8 and COX-2 expression was reported.37,38 Based on our study, the reduction of the endometriotic foci and decreased plasma concentrations of inflammatory factors such as TNF-α, PTX-3 and CRP in the group treated with losartan highly suggest that in addition to its AngII R1 blocker effect losartan may also have a partial PPAR-γ activation effect. In the present study, we demonstrated that losartan suppressed endometriotic implants by acting as an AngII R1 inhibitor with antiangiogenic activity. Many other AngII R1 blockers are used as antihypertensive agents, such as telmisartan. An experimental mouse model study investigating telmisartan efficacy for endometriosis found that telmisartan was more effective than the PPAR-γ agonist pioglitazone39 and that telmisartan acted as an AngII R1 inhibitor and PPAR-γ activator. Nenicu et al.39 noted that telmisartan induced upregulation of PPAR-γ and downregulation of AngII R1 proteins in endometriosis-like lesions, which was associated with decreased density of CD31-positive microvessels, reduced immune cell content, and a lower number of Ki67-positive proliferating cells. Furthermore, that study demonstrated that telmisartan inhibited the expression of several angiogenic and inflammatory genes, which was similar to the approach we used in this study to assess the effect of losartan. There are several limitations that should be noted in this study. The first is that side-effects of losartan were not evaluated in the normal endometrial tissue found in the remaining uterine horn. The second is that we did not determine whether losartan affected ovarian functioning by following the progression of the regular estrous cycle and/or estrogen levels. The third is that we only used a single standard losartan dose rather than evaluating a dose-dependent effect of this drug on endometriotic implant regression. In conclusion, the findings of this study indicate that in an experimental endometriosis model, losartan
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caused significant regression of endometriotic implant size and resulted in lower VEGF levels in the peritoneal fluid, along with decreased plasma TNF-α, PTX-3 and CRP. This is the first study to determine that losartan could be beneficial in endometriosis treatment.
Disclosure None of the authors has anything to disclose.
References 1. Galle PC. Clinical presentation and diagnosis of endometriosis. Obstet Gynecol Clin North Am 1989; 16: 29–42. 2. Farquhar CM. Extracts from ‘Clinical Evidence’ Endometriosis. BMJ 2000; 320: 1449–1452. 3. Ranney B. Endometriosis: Pathogenesis, symptoms, and findings. Clin Obstet Gynecol 1980; 23: 865–874. 4. Berkkanoglu M, Arici A. Immunology and endometriosis. Am J Reprod Immunol 2003; 50: 48–59. 5. Stefansson H, Geirsson RT, Steinthorsdottir V et al. Genetic factors contribute to the risk of developing endometriosis. Hum Reprod 2002; 17: 555–559. 6. Valle RF. Endometriosis: Current concepts and therapy. Int J Gynaecol Obstet 2002; 78: 107–119. 7. Reis FM, Petraglia F, Taylor RN. Endometriosis: Hormone regulation and clinical consequences of chemotaxis and apoptosis. Hum Reprod Update 2013; 19: 406–418. 8. Taniguchi F, Kaponis A, Izawa M et al. Apoptosis and endometriosis. Front Biosci (Elite Ed) 2011; 3: 648–662. 9. Lousse JC, Van Langendonckt A, Defrere S, Ramos RG, Colette S, Donnez J. Peritoneal endometriosis is an inflammatory disease. Front Biosci (Elite Ed) 2012; 4: 23–40. 10. Laschke MW, Giebels C, Menger MD. Vasculogenesis: A new piece of the endometriosis puzzle. Hum Reprod Update 2011; 17: 628–636. 11. Girling JE, Rogers PAW. Recent advances in endometrial angiogenesis research. Angiogenesis 2005; 8: 89–99. 12. Nisolle M, Casanas Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993; 59: 681–684. 13. Taylor RN, Lebovic DI, Mueller MD. Angiogenic factors in endometriosis. Ann N Y Acad Sci 2002; 955: 89–100. 14. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13: 1686–1690. 15. Bourlev V, Volkov N, Pavlovitch S, Lets N, Larsson A, Olovsson M. The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction 2006; 132: 501–509. 16. Laschke MW, Menger MD. Anti-angiogenic treatment strategies for the therapy of endometriosis. Hum Reprod Update 2012; 18: 682–702. 17. Neri Serneri GG, Boddi M, Coppo M et al. Evidence for the existence of a functional cardiac renin–angiotensin system in humans. Circulation 1996; 94: 1886–1893.
606
18. Shi RZ, Wang JC, Huang SH, Wang XJ, Li QP. Angiotensin II induces vascular endothelial growth factor synthesis in mesenchymal stem cells. Exp Cell Res 2009; 315: 10–15. 19. Saridogan E, Djahanbakhch O, Puddefoot JR et al. Type 1 angiotensin II receptors in human endometrium. Mol Hum Rep 1996; 2: 659–664. 20. Pawlikowski M, Melen-Mucha G, Mucha S. The involvement of the renin-angiotensin system in the regulation of cell proliferation in the rat endometrium. Cell Mol Life Sci 1999; 55: 506–510. 21. Herr D, Bekes I, Wulff C. Local renin-angiotensin system in the reproductive system. Front Endocrinol 2013; 4: 150. 22. Noguchi R, Yoshiji H, Ikenaka Y et al. Synergistic inhibitory effect of gemcitabine and angiotensin type-1 receptor blocker, losartan, on murine pancreatic tumor growth via antiangiogenic activities. Oncol Rep 2009; 22: 355–360. 23. Chen X, Meng Q, Zhao Y et al. Angiotensin II type 1 receptor antagonists inhibit cell proliferation and angiogenesis in breast cancer. Cancer Lett 2013; 328: 318–324. 24. Choi CH, Park YA, Choi JJ et al. Angiotensin II type I receptor and miR-155 in endometrial cancers: Synergistic antiproliferative effects of anti-miR-155 and losartan on endometrial cancer cells. Gynecol Oncol 2012; 126: 124–131. 25. Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A. Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999; 72: 135–141. 26. Küpker W, Schultze-Mosgau A, Diedrich K. Paracrine changes in the peritoneal environment of women with endometriosis. Hum Reprod Update 1998; 4: 719–723. 27. Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C, Baraneao RI. Effect of GnRH analogues on apoptosis and release of interleukin-1b and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 2003; 18: 1767–1771. 28. Oktem M, Esinler I, Eroglu D, Haberal N, Bayraktar N, Zeyneloglu HB. High-dose atorvastatin causes regression of endometriotic implants: A rat model. Hum Reprod 2007; 22: 1474–1480. 29. Ceyhan ST, Onguru O, Fidan U et al. Comparison of aromatase inhibitor (letrozole) and immunomodulators (infliximab and etanercept) on the regression of endometriotic implants in a rat model. Eur J Obstet Gynecol Reprod Biol 2011; 154: 100–104. 30. Uydur D, Aytan H, Zergeroglu S, Batioglu S. Leflunomide -an immonumodulator- induces regression of endometrial explants in a rat model of endometriosis. J Soc Gynecol Invest 2006; 13: 378–383. 31. Aytan H, Caglar P, Uydur D, Zergeroglu S, Batioglu S. Effect of the immunomodulator leflunomide on the induction of endometriosis in an experimental rat model. Fertil Steril 2007; 87: 698–701. 32. Demirturk F, Aytan H, Caliskan AC, Aytan P, Koseoglu DR. Effect of peroxisome proliferator-activated receptor-gamma agonist rosiglitazone on the induction of endometriosis in an experimental rat model. J Soc Gynecol Investig 2006; 13: 58–62. 33. Aytan H, Caliskan AC, Demirturk F, Aytan P, Koseoglu DR. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone reduces the size of experimental endometriosis in the rat model. Aust N Z J Obstet Gynaecol 2007; 47: 321– 325.
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34. Lebovic DI, Mwenda JM, Chai DC, Santi A, Xu X, D’Hooghe T. Peroxisome proliferator-activated receptor-(gamma) receptor ligand partially prevents the development of endometrial explants in baboons: A prospective, randomized, placebo-controlled study. Endocrinology 2010; 151: 1846–1852. 35. Ohama Y, Harada T, Iwabe T, Taniguchi F, Takenaka Y, Terakawa N. Peroxisome proliferator-activated receptorgamma ligand reduced tumor necrosis factor-alpha-induced interleukin-8 production and growth in endometriotic stromal cells. Fertil Steril 2008; 89: 311–317. 36. Peeters LL, Vigne JL, Tee MK, Zhao D, Waite LL, Taylor RN. PPAR gamma represses VEGF expression in human endometrial cells: Implications for uterine angiogenesis. Angiogenesis 2005; 8: 373–379.
37. Koh EJ, Yoon SJ, Lee SM. Losartan protects liver against ischaemia/reperfusion injury through PPAR-γ activation and receptor for advanced glycation end-products downregulation. Br J Pharmacol 2013; 169: 1404–1416. 38. An J, Nakajima T, Kuba K, Kimura A. Losartan inhibits LPSinduced inflammatory signaling through a PPARγ-dependent mechanism in human THP-1 macrophages. Hypertens Res 2010; 33: 831–835. 39. Nenicu A, Korbel C, Gu Y, Menger MD, Laschke MW. Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor-γ by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions. Hum Reprod 2014; 29: 1011–1024.
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