European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 167–171
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effects of repeated propranolol administration in a rat model of surgically induced endometriosis Ozlem Uzunlar a,*, Sebnem Ozyer a, Yaprak Engin-Ustun a, Ozlem Moraloglu a, H. Cavidan Gulerman a, Muzaffer Caydere b, Senem M. Keskin a, Leyla Mollamahmutoglu a a b
Department of Obstetrics and Gynecology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey Department of Pathology, Ankara Education and Research Hospital, Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 April 2014 Received in revised form 30 August 2014 Accepted 9 September 2014
Objectives: To determine whether propranolol has an inhibitory effect on the angiogenesis of endometriosis in an experimental rat model or not. Study design: This was an experimental animal model study. Twenty-four female Wistar albino rats (200–250 g) were used to create a model for surgical induction of endometriosis. Two rats died during the surgeries. The rats were randomly divided into treatment (n = 11) and control groups (n = 10), which were treated with daily intraperitoneal propranolol (10 mg/kg) and saline (2 mL), respectively. Study duration was 8 weeks. The volumes and histopathological findings of the implants, and immunochemistry for vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-2, and MMP-9 were evaluated. Results: Viable endometriotic implants were created in all animals. In the propranolol-treated group, the mean implant volume significantly decreased after treatment (142.5 vs. 32.1 mm3, respectively; p = 0.008), while the mean implant volume significantly increased in the control group (141.0 vs. 174.2 mm3, respectively; p = 0.009). There were also significant reductions in VEGF immunoreactivity scores and both stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores in the propranololtreated group compared with the control group (p < 0.005 for all scores). Conclusions: Propranolol may suppress endometrial tissue by its antiangiogenic activity through inhibitory actions on VEGF, MMP-2, and MMP-9. Therefore, propranolol is a promising candidate drug for effective treatment of patients with endometriosis, which needs to be confirmed with further studies. ß 2014 Published by Elsevier Ireland Ltd.
Keywords: Endometriosis Propranolol Angiogenesis
Introduction Endometriosis is an estrogen-dependent disease affecting 8% to 10% of females of reproductive age. It is diagnosed in 71% to 87% of females with chronic pelvic pain and in 30% of women with infertility [1]. It is defined as adherence and growth of the functional layer of the endometrium outside the uterine cavity. In endometriotic lesions, the development of new blood vessels from pre-existing ones is necessary to supply oxygen and essential nutrients [2], a process that is coordinated by a sequence of humoral and cellular interactions [3]. Vascular endothelial growth factor (VEGF) is of primary importance as a mediator of angiogenesis in endometriosis in addition to its potent endothelial
* Corresponding author. Tel.: +90 532 747 0999; fax: +90 312 311 0298. E-mail address: [email protected] (O. Uzunlar). http://dx.doi.org/10.1016/j.ejogrb.2014.09.025 0301-2115/ß 2014 Published by Elsevier Ireland Ltd.
cell-mitogen-, morphogen-, and vascular permeability-inducing activities [4,5]. Furthermore, metalloproteinases (MMPs) influence the outcome of inflammatory reactions, angiogenesis, and tissue remodeling through regulation of extracellular matrix turnover [6,7]. Hormonal therapies, such as dienogest and danazol, widely used to reduce functional endometrial tissue by causing atrophy, thus inducing regression of the symptoms of endometriosis [8,11]. However, treatment with these hormonal agents is associated with adverse events that limit their use, such as estrogen-withdrawal symptoms (e.g. headache, hot flushes, vaginal dryness, decreased libido, and bone demineralization), androgenic effects, adverse effects on lipids, arterial thrombosis, liver dysfunction, increased glucocorticoid activity, and a high incidence of abnormal menstrual bleeding [10,11]. Therefore, there is a need for alternative treatments that are both effective and safe against endometriosis.
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O. Uzunlar et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 167–171
Propranolol is a non-selective beta-blocker that demonstrates equal affinity for adrenoreceptors and therefore acts on multiple tissues. Propranolol has been found to be potent and safe for treatment of hemangiomas by inhibition of angiogenesis [12]. Beta-blockers also decrease the expression of VEGF, thus preventing angiogenesis [12,13]. Based on these data, we hypothesized that propranolol inhibits angiogenesis, which plays a critical role in the pathogenesis of endometriosis. Thus, the aim of this study was to investigate whether propranolol has an inhibitory effect on the angiogenesis of endometriosis in an experimental rat model. For this aim, we evaluated VEGF, MMP2 and MMP9 immunoreactivity in endometriosis lesions.
Materials and methods The study was conducted in the Animal Research Center of Ankara Education and Research Hospital with the approval of the Experimental Animals Ethics Committee of the same institution. All procedures were performed in compliance with the international guidelines for the care and use of experimental animals. Twenty-four female, non-pregnant Wistar albino rats weighing 200 to 250 g were used to create a model for the experimental induction of endometriosis. The rats were caged individually in a controlled environment with a 12-h light/dark cycle and were fed ad libitum. The animals were sexually mature and demonstrated normal estrous cycle changes in uterine histology (data not shown). All rats were observed for several days to ascertain their health before the operation. Unlike human females, rats do not menstruate, nor do they spontaneously develop endometriosis. Therefore, endometriosis was surgically induced in rats by transplanting an autologous fragment of endometrial tissue onto the inner surface of the abdominal wall as proposed by Vernon and Wilson [14] with modifications by Lebovic et al. [15]. The rats were anesthetized by intramuscular administration of 40 mg/kg of ketamine hydrochloric acid (Ketalar; Eczacıbasi Warner-Lambert, Levent, Istanbul, Turkey) and 10 mg/kg of xylazine hydrochloric acid (Rompun; Bayer, Sisli, Istanbul, Turkey). They were immobilized on a standard rat surgery board [16]. Before surgery, the abdominal skin was shaved, and antisepsis was obtained by 10% povidone iodine solution. A 4- to 5-cm ventral vertical incision 2 cm over the pubis was made to expose the reproductive organs using a sterile technique. The operation was limited to 25 min for each rat to limit the effect of room air on tissue drying. The abdominal incision was closed in a continuous interlocking manner with 2-0 silk sutures. All rats underwent three consecutive laparotomies. The first operation involved creation of the rat model of endometriosis. Vaginal smears were performed on a daily basis, and only rats exhibiting regular 4- to 5-day estrous cycles were used. Surgery was performed under aseptic conditions as described above. When a rat was in estrous, a distal segment 1 cm in length was resected from the right uterine horn and placed in warm sterile phosphate-buffered saline (PBS). Without removing the myometrium, a 5- 5-mm piece of this fragment was implanted onto the inner surface of the right side of the abdominal wall close to an artery with the serosal surface apposed and sutured with 4-0 vicryl (polyglactin 910, Medico Huaian Co., Ltd Jiangsu, China) sutures at two edges. Yilmaz et al., secured with non absorbable 4-0 polypropylenesutures at 2 edges and demonstrated successful, viable endometrial implants in their experimental rat model [21]. Attachment of the implant at 2 edges would be less traumatic and it would be less effected by the course of the endometriosis model. Because the endometriosis is also a chronic inflammatory disease.
Before closure of the abdominal wall, 2 mL of saline were administered into the abdominal cavity to prevent drying and to minimize adhesion formation, and the incision was closed in a continuous interlocking manner with 2-0 silk sutures. After this operation, all rats were observed for 4 weeks without medication. Two rats died during this period. The remaining 22 rats underwent a second laparotomy to examine the endometrial implants for size and viability. One of the investigators measured the endometrial implants in three dimensions ((length width height in mm) using a caliper as described by Demirel et al. [31]. The volumes of the implants were calculated using the ellipsoid formula: V (mm3) = 0.52 A B C, where A, B, and C indicate width, length, and height, respectively. Tissues were photographed using a digital camera, and all measurements were recorded. The rats were randomly divided into treatment (propranolol) and control groups. The rats in the treatment group (n = 11) were given 10 mg/kg of intraperitoneal propranolol daily (Dideral 40mg tablet; Sanofi Aventis, France). The drug was dissolved in distilled water and given in a volume of 2 mL. The drug dose was determined based on a previous experimental rat study related to propranolol [17,18]. The rats in the control group (n = 11) received daily intraperitoneal injections of 2 mL of saline as a vehicle solution. The injections were applied at approximately the same time each day for 3 weeks. One rat in the control group died the day after the second laparotomy; thus, the final number of rats in the treatment group was 11 and that in the control group was 10. At the end of the 3 weeks, the rats were euthanized by ketamine anesthesia, and the third laparotomy was performed. The sizes of the endometrial implants were measured again with the same caliper method by the same investigator, who was blinded to the groups. The implants were photographed, then excised and fixed in 10% formaldehyde buffer for histopathologic and immunohistochemical examination. The formalin-fixed endometriotic foci were embedded in paraffin blocks, sectioned at 5-mm thickness, and stained with hematoxylin and eosin. All tissues were examined by a blinded observer using a 40 objective lens on a light microscope (Olympus BX51, Tokyo, Japan). The histologic diagnosis of endometriosis was based on the morphologic diagnosis of endometrial glandular tissue and stroma of the endometrial type, with epithelial lining and luminal formation. Semiquantitative analysis of endometriotic explants was performed according to a scoring system as follows: 3, well-preserved epithelial layer; 2, moderately preserved epithelium with a leukocytic infiltrate; 1, poorly preserved epithelium (occasional epithelial cells only); and 0, no epithelium [19,20]. Immunochemistry A Leica polymer detection kit was used (DS9800; Leica, Newcastle, United Kingdom) on a Leica BOND-MAX automated IHC ISH system for MMP-2, MMP-9, and VEGF. Kidney, colon, breast cancer, and ovarian cancer tissues were used as positive controls for MMP-2 and MMP-9; corpus luteum, small intestine, and ovary tissues were used as positive controls for VEGF. Paraffin-embedded tissues were sectioned at 3-mm thickness, deparaffinized in the fully automated systems, boiled in EDTA for 20 min, and incubated with MMP-2 antibody (MMP2-507; Leica Bond, Newcastle, United Kingdom) or MMP-9 antibody (MMP9439; Leica Bond, Newcastle, United Kingdom) at a 1:40 dilution or in VEGF (C-1) (sc-7269; Santa Cruz Biotechnology, Inc. Dallas, Texas 75220 U.S.A) at a 1:100 dilution for 20 min. For secondary elements, a polymer detection kit was used (DS9800; Leica Bond, Newcastle, United Kingdom). The sections were dehydrated and
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Fig. 1. ((a) and (b)) After the implantation of myometrium, well-vascularized, cystic, viable endometriotic implants were observed in all cases during the second operation (a). In the treatment group, endometriotic implant regressed after 3 weeks of propronalol treatment (b).
mounted with hydrogen peroxide for 10 min, post polymer for 6 min, polymer for 6 min, DAB for 6 min, and hematoxylin for 5 min. A value of histologic score was derived from the sum of the percentages of positively stained epithelial cells multiplied by the weighted intensity of staining: histological score SPi(I + 1), where I represents staining intensity (0 = no expression, 1 = mild, 2 = moderate, and 3 = intense) and Pi is the percentage of stained cells for each intensity [21]. Statistical analysis was performed by IBM SPSS Statistics version 21. Data were given as median (min–max). Mann Whitney U test was used to compare the differences between treatment and control groups, and Wilcoxon signed-rank test was used to compare the pre- and post-treatment differences within groups. p Value less than 0.05 was considered as statistically significant. Results Two rats died after the first operation, and one rat died after the second operation. The remaining 21 rats were followed up daily in terms of general well being, weight, and the presence of any complications. There were no complications associated with the incision area, and the treatment had no adverse effects on the general wellness and estrous cycles of the rats. The constitution of the implants with respect to the appearance and implant surface area was examined at the second operation, before the treatment. Well-vascularized, cystic, viable endometriotic implants were created in 21 animals (Fig. 1). The morphological characteristics of the endometriotic lesions were similar in both groups 4 weeks after the implantation. The pre-treatment volumes of the endometrial implants in the propranolol and control groups were not significantly different (141.0 vs. 142.5 mm3, respectively; p = 0.94). In the propranololtreated group, the mean implant volume decreased significantly after treatment (142.5 vs. 32.1 mm3 before and after treatment, respectively; p = 0.008), while the mean implant volume increased significantly 3 weeks after the second operation (141.0 vs. 174.2 mm3, respectively; p = 0.009). The post-treatment volumes were also significantly different between the control and propranolol-treated groups (174.0 vs. 32.1 mm3, respectively; p = 0.001) (Table 1, Fig. 2).
At the end of the treatment period, the mean score of the histopathological examination was lower in the treatment group than in the control group (2 vs. 3, respectively; p = 0.048). There was also a significant reduction in VEGF immunoreactivity scores and both the stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores in the propranolol-treated group compared with the control group (p < 0.005 for all scores) (Table 2, Fig. 3). Comments This is the first controlled study to evaluate the effect of propranolol in a rat model of surgically induced endometriosis. We evaluated the change in volume of endometriotic implant areas along with histopathology and immunochemistry scores induced by propranolol treatment compared with a control group treated with saline as the vehicle solution. Our findings showed that propranolol significantly decreased the size of the endometriotic lesions, and immunoreactivity for VEGF, MMP-2, and MMP-9. Current therapeutic agents used to treat endometriosis have limiting side effects. For example, progestins (especially depot preparations), which have been used to treat the pain associated with endometriosis, delay the resumption of ovulation and increase glucocorticoid activity [8–11]. Dienogest, an effective, usually well-tolerated therapeutic agent for the long-term treatment of endometriosis, is generally highly selective for the progesterone receptor and displays strong progestational effects with few hypo-estrogenic effects and moderate anti-gonadotrophic effects, but no androgenic, glucocorticoid, or mineralocorticoid activity. However, the efficacy of dienogest was sustained during long-term treatment for more than 1 year, and it was associated with a high incidence of abnormal menstrual bleeding patterns [11]. Therefore, there is still a need to establish both safe and effective therapeutic options for use in the treatment of endometriosis. Antiangiogenic agents are known to be effective inhibitors of endometriosis [22]. We hypothesized that propranolol, which has been shown to be effective against infantile hemangiomas, has inhibitory actions on endometriotic lesions and can be used in the treatment of endometriosis. The effect of propranolol on infantile hemangiomas was detected coincidentally in patients treated with
Table 1 The pre-treatment and the post-treatment implant volumes in the study groups. Implant volume (mm3)
Control (saline-injected) group (n = 10)
Treatment (propranolol-treated) group (n = 11)
pa
Pre-treatment Post-treatment pb
141 (19–230) 174.2 (20.1–230.6) 0.009
142.5 (21.7–209.1) 32.1 (3.3–181.2) 0.008
0.944 0.001
Data are given as mean (minimum–maximum [range]). a Mann Whitney U test. b Wilcoxon signed-rank test.
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Fig. 2. The mean implant volumes before and 3 weeks after treatment in the control and propranolol-treated groups. There was a statistically significant increase and decrease in implant volumes of the control and treatment groups (p = 0.009 and p = 0.008, respectively). The implant volumes were also significantly different between the control and treatment groups after 3 weeks of treatment (p = 0.001).
propranolol for obstructive hypertrophic cardiomyopathy with coexisting nasal hemangioma [12,23,24]. Endometriotic lesions are known to stimulate angiogenesis and neovascularization [25]. A dynamic process involving humoral and cellular interactions plays a role in the formation of new vessels [3]. The stimulation of angiogenic growth factors causes deterioration of the stabilization of mature blood vessel walls, which triggers separation of the mural cells and deconstruction of the extracellular matrix. This constitutes the primordial step of the formation of new vessels. Based on the inhibitory effect of propranolol on hemangiomas, we tested its effect on endometrial implants. In our experimental model, well-vascularized, cystic, viable endometriotic implants were obtained in 21 animals. In rats treated with propranolol for 3 weeks, the volume of implants decreased significantly (4–5-fold), while the volume of implants in the control group increased significantly. The histopathological examination score, which was based on the morphologic diagnosis of endometrial glandular tissue and stroma, was also lower in the treatment group than in the control group. We suggest that propranolol has a suppressive effect on endometriotic implants by inhibiting angiogenesis. To test this hypothesis, we also evaluated the immunoreactivity of VEGF, MMP-2, and MMP-9 in samples obtained from endometriotic implants. VEGF, the most potent angiogenic protein identified to date, has been detected in the epithelium of endometriotic implants and in the peritoneal fluid of patients with endometriosis [5]. It is not only an important mediator of angiogenesis but also a potent endothelial cell-mitogen-, morphogen-, vascular permeability-inducing agent [26,27]. VEGF is produced by endometriotic lesions. Activated peritoneal macrophages have also been suggested to contribute to the secretion and
release of VEGF [26]. Chen et al. [13] showed that serum VEGF levels were higher in patients with infantile hemangioma and decreased after 1 month of oral propranolol treatment. Similar results, although less pronounced, were found after 3 months of treatment. Lesion volume and serum VEGF levels were significantly correlated [13]. Similarly, in our experimental model, we found that the VEGF immunoreactivity score was significantly lower in endometrial tissue of propranolol-treated rats than in control rats. In addition to VEGF, MMPs have been suggested to play a role in the angiogenesis of endometriosis [28]. MMPs are a family of zincand calcium-dependent endoproteases involved in the degradation of the extracellular matrix and basement membrane. MMPs influence the outcome of inflammatory reactions, angiogenesis, and tissue remodeling through regulation of extracellular matrix turnover [6]. Endometrial cells transmigrate into the surrounding interstitium, resulting in the formation of capillary branching [29]. The endothelial cells proliferate behind the migrating endothelium, continuously increasing the length and diameter of newly developing blood vessels. Adherence of pericytes, smooth muscle cells, and extracellular matrix compounds provides the stabilization of the new vessel wall [30]. In addition, higher MMP-9 and lower tissue inhibitor of MMP-1 immunostaining in ectopic and eutopic endometrium [29] have been reported. We found that both the stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores were significantly lower in the propranolol-treated group than in the control group. The limitations of the study worth noting are lack of immunohistochemical data to analyze microvascular density in the lesions using CD31 plus quantification of angiogenesis relative to lesion size. We have also not used intra-vital imaging methods to measure blood flow of endometrial tissue. Additionally, we did not evaluate the presence of beta-adrenergic receptors in the blood vessels of the lesions. Although these limitations prevents us from reaching a definitive conclusion, the present study presents initial immunohistochemical data on the inhibitory function of propranolol on endometrial tissue, which need to be confirmed by further studies. In conclusion, our results suggest that propranolol may suppress the endometrial tissue by its antiangiogenic activity through inhibitory actions on VEGF, MMP-2, and MMP-9. Based on our findings in this experimental animal study with auto grafts, further clinical and immunohistochemical studies should be implemented to draw a final conclusion on the inhibitory action of propranolol on endometrial tissue and to evaluate the efficacy, safety, and potential use of propranolol in patients with endometriosis. The administration of propranolol had previously described for different conditions [13,32] and different vias (oral, intraperitoneal) of administration were described. Intraperitoneal propranolol was demonstrated as an effective route [32]. Since the effect was faster, we chose intraperitoneal route for propranolol administration.
Table 2 Comparison of the post-treatment histopathological and immunoreactivity scores between the study groups. Scoring
Control (saline-injected) group (n = 10)
Treatment (propranolol-treated) group (n = 11)
Histopatological score Immunoreactivity scores VEGF H score MMP-2 epithelium H score MMP-2 stroma H score MMP-9 epithelium H score MMP-9 stroma H score
3 (2–3)
2 (1–3)
6 (4–15) 2 (1–9) 2 (1–6) 4 (1–9) 3.5 (1–6)
2 0 0 0 1
(1–6) (0–2) (0–2) (0–2) (0–4)
Data are given as mean (minimum–maximum [range]). MMP: metalloproteinase, VEGF: vascular endothelial growth factor. a Mann–Whitney U test.
pa 0.048
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effects of repeated propranolol administration in a rat model of surgically induced endometriosis Ozlem Uzunlar a,*, Sebnem Ozyer a, Yaprak Engin-Ustun a, Ozlem Moraloglu a, H. Cavidan Gulerman a, Muzaffer Caydere b, Senem M. Keskin a, Leyla Mollamahmutoglu a a b
Department of Obstetrics and Gynecology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey Department of Pathology, Ankara Education and Research Hospital, Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 April 2014 Received in revised form 30 August 2014 Accepted 9 September 2014
Objectives: To determine whether propranolol has an inhibitory effect on the angiogenesis of endometriosis in an experimental rat model or not. Study design: This was an experimental animal model study. Twenty-four female Wistar albino rats (200–250 g) were used to create a model for surgical induction of endometriosis. Two rats died during the surgeries. The rats were randomly divided into treatment (n = 11) and control groups (n = 10), which were treated with daily intraperitoneal propranolol (10 mg/kg) and saline (2 mL), respectively. Study duration was 8 weeks. The volumes and histopathological findings of the implants, and immunochemistry for vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-2, and MMP-9 were evaluated. Results: Viable endometriotic implants were created in all animals. In the propranolol-treated group, the mean implant volume significantly decreased after treatment (142.5 vs. 32.1 mm3, respectively; p = 0.008), while the mean implant volume significantly increased in the control group (141.0 vs. 174.2 mm3, respectively; p = 0.009). There were also significant reductions in VEGF immunoreactivity scores and both stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores in the propranololtreated group compared with the control group (p < 0.005 for all scores). Conclusions: Propranolol may suppress endometrial tissue by its antiangiogenic activity through inhibitory actions on VEGF, MMP-2, and MMP-9. Therefore, propranolol is a promising candidate drug for effective treatment of patients with endometriosis, which needs to be confirmed with further studies. ß 2014 Published by Elsevier Ireland Ltd.
Keywords: Endometriosis Propranolol Angiogenesis
Introduction Endometriosis is an estrogen-dependent disease affecting 8% to 10% of females of reproductive age. It is diagnosed in 71% to 87% of females with chronic pelvic pain and in 30% of women with infertility [1]. It is defined as adherence and growth of the functional layer of the endometrium outside the uterine cavity. In endometriotic lesions, the development of new blood vessels from pre-existing ones is necessary to supply oxygen and essential nutrients [2], a process that is coordinated by a sequence of humoral and cellular interactions [3]. Vascular endothelial growth factor (VEGF) is of primary importance as a mediator of angiogenesis in endometriosis in addition to its potent endothelial
* Corresponding author. Tel.: +90 532 747 0999; fax: +90 312 311 0298. E-mail address: [email protected] (O. Uzunlar). http://dx.doi.org/10.1016/j.ejogrb.2014.09.025 0301-2115/ß 2014 Published by Elsevier Ireland Ltd.
cell-mitogen-, morphogen-, and vascular permeability-inducing activities [4,5]. Furthermore, metalloproteinases (MMPs) influence the outcome of inflammatory reactions, angiogenesis, and tissue remodeling through regulation of extracellular matrix turnover [6,7]. Hormonal therapies, such as dienogest and danazol, widely used to reduce functional endometrial tissue by causing atrophy, thus inducing regression of the symptoms of endometriosis [8,11]. However, treatment with these hormonal agents is associated with adverse events that limit their use, such as estrogen-withdrawal symptoms (e.g. headache, hot flushes, vaginal dryness, decreased libido, and bone demineralization), androgenic effects, adverse effects on lipids, arterial thrombosis, liver dysfunction, increased glucocorticoid activity, and a high incidence of abnormal menstrual bleeding [10,11]. Therefore, there is a need for alternative treatments that are both effective and safe against endometriosis.
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O. Uzunlar et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 167–171
Propranolol is a non-selective beta-blocker that demonstrates equal affinity for adrenoreceptors and therefore acts on multiple tissues. Propranolol has been found to be potent and safe for treatment of hemangiomas by inhibition of angiogenesis [12]. Beta-blockers also decrease the expression of VEGF, thus preventing angiogenesis [12,13]. Based on these data, we hypothesized that propranolol inhibits angiogenesis, which plays a critical role in the pathogenesis of endometriosis. Thus, the aim of this study was to investigate whether propranolol has an inhibitory effect on the angiogenesis of endometriosis in an experimental rat model. For this aim, we evaluated VEGF, MMP2 and MMP9 immunoreactivity in endometriosis lesions.
Materials and methods The study was conducted in the Animal Research Center of Ankara Education and Research Hospital with the approval of the Experimental Animals Ethics Committee of the same institution. All procedures were performed in compliance with the international guidelines for the care and use of experimental animals. Twenty-four female, non-pregnant Wistar albino rats weighing 200 to 250 g were used to create a model for the experimental induction of endometriosis. The rats were caged individually in a controlled environment with a 12-h light/dark cycle and were fed ad libitum. The animals were sexually mature and demonstrated normal estrous cycle changes in uterine histology (data not shown). All rats were observed for several days to ascertain their health before the operation. Unlike human females, rats do not menstruate, nor do they spontaneously develop endometriosis. Therefore, endometriosis was surgically induced in rats by transplanting an autologous fragment of endometrial tissue onto the inner surface of the abdominal wall as proposed by Vernon and Wilson [14] with modifications by Lebovic et al. [15]. The rats were anesthetized by intramuscular administration of 40 mg/kg of ketamine hydrochloric acid (Ketalar; Eczacıbasi Warner-Lambert, Levent, Istanbul, Turkey) and 10 mg/kg of xylazine hydrochloric acid (Rompun; Bayer, Sisli, Istanbul, Turkey). They were immobilized on a standard rat surgery board [16]. Before surgery, the abdominal skin was shaved, and antisepsis was obtained by 10% povidone iodine solution. A 4- to 5-cm ventral vertical incision 2 cm over the pubis was made to expose the reproductive organs using a sterile technique. The operation was limited to 25 min for each rat to limit the effect of room air on tissue drying. The abdominal incision was closed in a continuous interlocking manner with 2-0 silk sutures. All rats underwent three consecutive laparotomies. The first operation involved creation of the rat model of endometriosis. Vaginal smears were performed on a daily basis, and only rats exhibiting regular 4- to 5-day estrous cycles were used. Surgery was performed under aseptic conditions as described above. When a rat was in estrous, a distal segment 1 cm in length was resected from the right uterine horn and placed in warm sterile phosphate-buffered saline (PBS). Without removing the myometrium, a 5- 5-mm piece of this fragment was implanted onto the inner surface of the right side of the abdominal wall close to an artery with the serosal surface apposed and sutured with 4-0 vicryl (polyglactin 910, Medico Huaian Co., Ltd Jiangsu, China) sutures at two edges. Yilmaz et al., secured with non absorbable 4-0 polypropylenesutures at 2 edges and demonstrated successful, viable endometrial implants in their experimental rat model [21]. Attachment of the implant at 2 edges would be less traumatic and it would be less effected by the course of the endometriosis model. Because the endometriosis is also a chronic inflammatory disease.
Before closure of the abdominal wall, 2 mL of saline were administered into the abdominal cavity to prevent drying and to minimize adhesion formation, and the incision was closed in a continuous interlocking manner with 2-0 silk sutures. After this operation, all rats were observed for 4 weeks without medication. Two rats died during this period. The remaining 22 rats underwent a second laparotomy to examine the endometrial implants for size and viability. One of the investigators measured the endometrial implants in three dimensions ((length width height in mm) using a caliper as described by Demirel et al. [31]. The volumes of the implants were calculated using the ellipsoid formula: V (mm3) = 0.52 A B C, where A, B, and C indicate width, length, and height, respectively. Tissues were photographed using a digital camera, and all measurements were recorded. The rats were randomly divided into treatment (propranolol) and control groups. The rats in the treatment group (n = 11) were given 10 mg/kg of intraperitoneal propranolol daily (Dideral 40mg tablet; Sanofi Aventis, France). The drug was dissolved in distilled water and given in a volume of 2 mL. The drug dose was determined based on a previous experimental rat study related to propranolol [17,18]. The rats in the control group (n = 11) received daily intraperitoneal injections of 2 mL of saline as a vehicle solution. The injections were applied at approximately the same time each day for 3 weeks. One rat in the control group died the day after the second laparotomy; thus, the final number of rats in the treatment group was 11 and that in the control group was 10. At the end of the 3 weeks, the rats were euthanized by ketamine anesthesia, and the third laparotomy was performed. The sizes of the endometrial implants were measured again with the same caliper method by the same investigator, who was blinded to the groups. The implants were photographed, then excised and fixed in 10% formaldehyde buffer for histopathologic and immunohistochemical examination. The formalin-fixed endometriotic foci were embedded in paraffin blocks, sectioned at 5-mm thickness, and stained with hematoxylin and eosin. All tissues were examined by a blinded observer using a 40 objective lens on a light microscope (Olympus BX51, Tokyo, Japan). The histologic diagnosis of endometriosis was based on the morphologic diagnosis of endometrial glandular tissue and stroma of the endometrial type, with epithelial lining and luminal formation. Semiquantitative analysis of endometriotic explants was performed according to a scoring system as follows: 3, well-preserved epithelial layer; 2, moderately preserved epithelium with a leukocytic infiltrate; 1, poorly preserved epithelium (occasional epithelial cells only); and 0, no epithelium [19,20]. Immunochemistry A Leica polymer detection kit was used (DS9800; Leica, Newcastle, United Kingdom) on a Leica BOND-MAX automated IHC ISH system for MMP-2, MMP-9, and VEGF. Kidney, colon, breast cancer, and ovarian cancer tissues were used as positive controls for MMP-2 and MMP-9; corpus luteum, small intestine, and ovary tissues were used as positive controls for VEGF. Paraffin-embedded tissues were sectioned at 3-mm thickness, deparaffinized in the fully automated systems, boiled in EDTA for 20 min, and incubated with MMP-2 antibody (MMP2-507; Leica Bond, Newcastle, United Kingdom) or MMP-9 antibody (MMP9439; Leica Bond, Newcastle, United Kingdom) at a 1:40 dilution or in VEGF (C-1) (sc-7269; Santa Cruz Biotechnology, Inc. Dallas, Texas 75220 U.S.A) at a 1:100 dilution for 20 min. For secondary elements, a polymer detection kit was used (DS9800; Leica Bond, Newcastle, United Kingdom). The sections were dehydrated and
O. Uzunlar et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 182 (2014) 167–171
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Fig. 1. ((a) and (b)) After the implantation of myometrium, well-vascularized, cystic, viable endometriotic implants were observed in all cases during the second operation (a). In the treatment group, endometriotic implant regressed after 3 weeks of propronalol treatment (b).
mounted with hydrogen peroxide for 10 min, post polymer for 6 min, polymer for 6 min, DAB for 6 min, and hematoxylin for 5 min. A value of histologic score was derived from the sum of the percentages of positively stained epithelial cells multiplied by the weighted intensity of staining: histological score SPi(I + 1), where I represents staining intensity (0 = no expression, 1 = mild, 2 = moderate, and 3 = intense) and Pi is the percentage of stained cells for each intensity [21]. Statistical analysis was performed by IBM SPSS Statistics version 21. Data were given as median (min–max). Mann Whitney U test was used to compare the differences between treatment and control groups, and Wilcoxon signed-rank test was used to compare the pre- and post-treatment differences within groups. p Value less than 0.05 was considered as statistically significant. Results Two rats died after the first operation, and one rat died after the second operation. The remaining 21 rats were followed up daily in terms of general well being, weight, and the presence of any complications. There were no complications associated with the incision area, and the treatment had no adverse effects on the general wellness and estrous cycles of the rats. The constitution of the implants with respect to the appearance and implant surface area was examined at the second operation, before the treatment. Well-vascularized, cystic, viable endometriotic implants were created in 21 animals (Fig. 1). The morphological characteristics of the endometriotic lesions were similar in both groups 4 weeks after the implantation. The pre-treatment volumes of the endometrial implants in the propranolol and control groups were not significantly different (141.0 vs. 142.5 mm3, respectively; p = 0.94). In the propranololtreated group, the mean implant volume decreased significantly after treatment (142.5 vs. 32.1 mm3 before and after treatment, respectively; p = 0.008), while the mean implant volume increased significantly 3 weeks after the second operation (141.0 vs. 174.2 mm3, respectively; p = 0.009). The post-treatment volumes were also significantly different between the control and propranolol-treated groups (174.0 vs. 32.1 mm3, respectively; p = 0.001) (Table 1, Fig. 2).
At the end of the treatment period, the mean score of the histopathological examination was lower in the treatment group than in the control group (2 vs. 3, respectively; p = 0.048). There was also a significant reduction in VEGF immunoreactivity scores and both the stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores in the propranolol-treated group compared with the control group (p < 0.005 for all scores) (Table 2, Fig. 3). Comments This is the first controlled study to evaluate the effect of propranolol in a rat model of surgically induced endometriosis. We evaluated the change in volume of endometriotic implant areas along with histopathology and immunochemistry scores induced by propranolol treatment compared with a control group treated with saline as the vehicle solution. Our findings showed that propranolol significantly decreased the size of the endometriotic lesions, and immunoreactivity for VEGF, MMP-2, and MMP-9. Current therapeutic agents used to treat endometriosis have limiting side effects. For example, progestins (especially depot preparations), which have been used to treat the pain associated with endometriosis, delay the resumption of ovulation and increase glucocorticoid activity [8–11]. Dienogest, an effective, usually well-tolerated therapeutic agent for the long-term treatment of endometriosis, is generally highly selective for the progesterone receptor and displays strong progestational effects with few hypo-estrogenic effects and moderate anti-gonadotrophic effects, but no androgenic, glucocorticoid, or mineralocorticoid activity. However, the efficacy of dienogest was sustained during long-term treatment for more than 1 year, and it was associated with a high incidence of abnormal menstrual bleeding patterns [11]. Therefore, there is still a need to establish both safe and effective therapeutic options for use in the treatment of endometriosis. Antiangiogenic agents are known to be effective inhibitors of endometriosis [22]. We hypothesized that propranolol, which has been shown to be effective against infantile hemangiomas, has inhibitory actions on endometriotic lesions and can be used in the treatment of endometriosis. The effect of propranolol on infantile hemangiomas was detected coincidentally in patients treated with
Table 1 The pre-treatment and the post-treatment implant volumes in the study groups. Implant volume (mm3)
Control (saline-injected) group (n = 10)
Treatment (propranolol-treated) group (n = 11)
pa
Pre-treatment Post-treatment pb
141 (19–230) 174.2 (20.1–230.6) 0.009
142.5 (21.7–209.1) 32.1 (3.3–181.2) 0.008
0.944 0.001
Data are given as mean (minimum–maximum [range]). a Mann Whitney U test. b Wilcoxon signed-rank test.
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Fig. 2. The mean implant volumes before and 3 weeks after treatment in the control and propranolol-treated groups. There was a statistically significant increase and decrease in implant volumes of the control and treatment groups (p = 0.009 and p = 0.008, respectively). The implant volumes were also significantly different between the control and treatment groups after 3 weeks of treatment (p = 0.001).
propranolol for obstructive hypertrophic cardiomyopathy with coexisting nasal hemangioma [12,23,24]. Endometriotic lesions are known to stimulate angiogenesis and neovascularization [25]. A dynamic process involving humoral and cellular interactions plays a role in the formation of new vessels [3]. The stimulation of angiogenic growth factors causes deterioration of the stabilization of mature blood vessel walls, which triggers separation of the mural cells and deconstruction of the extracellular matrix. This constitutes the primordial step of the formation of new vessels. Based on the inhibitory effect of propranolol on hemangiomas, we tested its effect on endometrial implants. In our experimental model, well-vascularized, cystic, viable endometriotic implants were obtained in 21 animals. In rats treated with propranolol for 3 weeks, the volume of implants decreased significantly (4–5-fold), while the volume of implants in the control group increased significantly. The histopathological examination score, which was based on the morphologic diagnosis of endometrial glandular tissue and stroma, was also lower in the treatment group than in the control group. We suggest that propranolol has a suppressive effect on endometriotic implants by inhibiting angiogenesis. To test this hypothesis, we also evaluated the immunoreactivity of VEGF, MMP-2, and MMP-9 in samples obtained from endometriotic implants. VEGF, the most potent angiogenic protein identified to date, has been detected in the epithelium of endometriotic implants and in the peritoneal fluid of patients with endometriosis [5]. It is not only an important mediator of angiogenesis but also a potent endothelial cell-mitogen-, morphogen-, vascular permeability-inducing agent [26,27]. VEGF is produced by endometriotic lesions. Activated peritoneal macrophages have also been suggested to contribute to the secretion and
release of VEGF [26]. Chen et al. [13] showed that serum VEGF levels were higher in patients with infantile hemangioma and decreased after 1 month of oral propranolol treatment. Similar results, although less pronounced, were found after 3 months of treatment. Lesion volume and serum VEGF levels were significantly correlated [13]. Similarly, in our experimental model, we found that the VEGF immunoreactivity score was significantly lower in endometrial tissue of propranolol-treated rats than in control rats. In addition to VEGF, MMPs have been suggested to play a role in the angiogenesis of endometriosis [28]. MMPs are a family of zincand calcium-dependent endoproteases involved in the degradation of the extracellular matrix and basement membrane. MMPs influence the outcome of inflammatory reactions, angiogenesis, and tissue remodeling through regulation of extracellular matrix turnover [6]. Endometrial cells transmigrate into the surrounding interstitium, resulting in the formation of capillary branching [29]. The endothelial cells proliferate behind the migrating endothelium, continuously increasing the length and diameter of newly developing blood vessels. Adherence of pericytes, smooth muscle cells, and extracellular matrix compounds provides the stabilization of the new vessel wall [30]. In addition, higher MMP-9 and lower tissue inhibitor of MMP-1 immunostaining in ectopic and eutopic endometrium [29] have been reported. We found that both the stroma and epithelium MMP-2 and MMP-9 immunoreactivity scores were significantly lower in the propranolol-treated group than in the control group. The limitations of the study worth noting are lack of immunohistochemical data to analyze microvascular density in the lesions using CD31 plus quantification of angiogenesis relative to lesion size. We have also not used intra-vital imaging methods to measure blood flow of endometrial tissue. Additionally, we did not evaluate the presence of beta-adrenergic receptors in the blood vessels of the lesions. Although these limitations prevents us from reaching a definitive conclusion, the present study presents initial immunohistochemical data on the inhibitory function of propranolol on endometrial tissue, which need to be confirmed by further studies. In conclusion, our results suggest that propranolol may suppress the endometrial tissue by its antiangiogenic activity through inhibitory actions on VEGF, MMP-2, and MMP-9. Based on our findings in this experimental animal study with auto grafts, further clinical and immunohistochemical studies should be implemented to draw a final conclusion on the inhibitory action of propranolol on endometrial tissue and to evaluate the efficacy, safety, and potential use of propranolol in patients with endometriosis. The administration of propranolol had previously described for different conditions [13,32] and different vias (oral, intraperitoneal) of administration were described. Intraperitoneal propranolol was demonstrated as an effective route [32]. Since the effect was faster, we chose intraperitoneal route for propranolol administration.
Table 2 Comparison of the post-treatment histopathological and immunoreactivity scores between the study groups. Scoring
Control (saline-injected) group (n = 10)
Treatment (propranolol-treated) group (n = 11)
Histopatological score Immunoreactivity scores VEGF H score MMP-2 epithelium H score MMP-2 stroma H score MMP-9 epithelium H score MMP-9 stroma H score
3 (2–3)
2 (1–3)
6 (4–15) 2 (1–9) 2 (1–6) 4 (1–9) 3.5 (1–6)
2 0 0 0 1
(1–6) (0–2) (0–2) (0–2) (0–4)
Data are given as mean (minimum–maximum [range]). MMP: metalloproteinase, VEGF: vascular endothelial growth factor. a Mann–Whitney U test.
pa 0.048
