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Effect of antiangiogenic treatment on peritoneal endometriosis-associated nerve fibers
Effect of antiangiogenic treatment on peritoneal endometriosis-associated nerve fibers María Vila-Vives, M.D.,b,c Edurne Novella-Maestre, Ph.D.,a,b Sonia Herraiz, Ph.D.,b Jose Carmen Carda, Ph.D., M.D.,d Amparo Ruiz-Sauri, Ph.D., M.D.,d and Antonio Pellicer, Ph.D., M.D.b,c,e a tica, Hospital Universitario y Polite cnico La Fe; b Grupo de Investigacio n de Medicina Reproductiva, Unidad de Gene cnico La Fe; n Sanitario La Fe; c Area de Salud de la Mujer, Hospital Universitario y Polite Instituto de Investigacio d Departamento de Patología, Facultad de Medicina y Odontología, Universidad de Valencia; and e Instituto de Infertilidad de Valencia, Universidad de Valencia (IVI-IU), Valencia, Spain
Objective: To investigate the effect of antiangiogenic treatment on experimental endometriotic lesion nerve fibers. Design: Heterologous mouse model of endometriosis. Setting: University Institute IVI, University Hospital La Fe. Animal(s): Ovariectomized nude mice (n ¼ 16) receiving human endometrial fragments from oocyte donors (n ¼ 4). Intervention(s): Endometrium fragments stuck in the peritoneum of 5-week-old female nude mice treated with vehicle (n ¼ 8) and antiangiogenic agent cabergoline (n ¼ 8; Cb2, 0.05 mg/kg/day) for 14 days. Main Outcome Measure(s): Immunofluorescence analysis of von-Willebrand factor (vWF) and vascular smooth muscle cells (aSMA) for evaluating the number of immature blood vessels (IBV) and microvascular density (MVD); immunochemical analysis of proteingene product 9.5 (PGP 9.5) to assess nerve fibers density (NFD), and blue toluidine staining to confirm presence of mast cells and macrophages in endometriotic lesions. Result(s): All the results were quantified by morphometric techniques. The IBV, NFD, and number of macrophages and mast cells were statistically significantly decreased in the Cb2-treated group when compared with controls. Conclusion(s): Antiangiogenic treatment statistically significantly diminishes new blood vessel formation after macrophage, mast cell, and nerve fiber reduction, providing a rationale to test antiangiogenic agents as a novel therapeutic approach to severe pelvic pain associated with human peritoneal endometriosis. (Fertil SterilÒ 2012;98:1209–17. Use your smartphone Ó2012 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Angiogenesis, dopamine agonists, endometriosis, nerve fibers, vascular and connect to the endothelial growth factor Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/novella-maestree-antiangiogenic-treatment-peritonealendometriosis-nerve-fibers/
E
ndometriosis, an estrogendependent inflammatory disease characterized by the presence of endometrial tissue outside the uterus, predominantly affects women of reproductive age. The real incidence of endometriosis is unknown, but it is estimated that this pathology is present in
more than 15% of reproductive age women and in up to 50% of women with pelvic pain (1). Pain, in the form of dysmenorrhea, dyspareunia, or dyschezia, is the most common symptom in patients with endometriosis and may be due to nociceptive, inflammatory, or neuropathic mechanisms.
Received January 25, 2012; revised July 8, 2012; accepted July 11, 2012; published online August 21, 2012. E.N.-M. has nothing to disclose. S.H. has nothing to disclose. J.M.V.-V. has nothing to disclose. C.C. has nothing to disclose. A.R.-S. has nothing to disclose. A.P. has nothing to disclose. Reprint requests: Edurne Novella-Maestre, Ph.D., Laboratorio de FIV, Torre B planta 1, Hospital Unicnico La Fe, Bulevar Sur s/n, 46026, Valencia, Spain (E-mail: edurnenovella@ versitario y Polite yahoo.es). Fertility and Sterility® Vol. 98, No. 5, November 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1103 VOL. 98 NO. 5 / NOVEMBER 2012
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Surgical resection of endometriotic lesions provides temporary relief in terms of reduced chronic pelvic pain and severe dysmenorrhea (2), but the recurrence rate at 2 years after surgery is as high as 75% of cases. Thus, medical treatment is employed at any stage of the disease in many patients worldwide. The presence of nerve fibers in the peritoneal endometriotic lesions (3–8) and eutopic endometrium of women with endometriosis (9, 10) has received much attention recently. Nerve fibers in peritoneal lesions are located in or near endometriotic stromal cells and are colocalized with 1209
ORIGINAL ARTICLE: ENDOMETRIOSIS immature blood vessels (5, 6). These findings suggest that human peritoneal endometriotic lesions are innervated by nerve fibers that may interact with each other and with many active molecules released by endometriotic lesions to cause pain and local tenderness (11). Studies have shown that nerve fiber density (NFD) is significantly greater in the peritoneal lesions of patients with painful endometriosis than in patients with asymptomatic disease, which suggests a direct association between pain and NFD (3, 12). Some current treatments for endometriosis, such as combinations of oral contraceptives and progestogens, significantly decrease NFD in the endometrium and myometrium in women with endometriosis (13, 14). Steroids act via a different route and have no effect on vascular endothelial growth factor (VEGF) expression, so they cannot be employed in all patients. Thus, the search for new routes in the medical treatment of endometriosis must continue. Different proteins and growth factors have been described as regulators of nerve fibers growth. Vascular endothelial growth factor, produced by macrophages that are elevated in the peritoneal fluid and endometriotic lesions of women with endometriosis, has been reported to act as a neurotrophic factor, stimulating nerve-fiber growth (15). These findings suggest that VEGF may also play a role in increasing NFD in peritoneal endometriotic lesions (8). The role of VEGF in endometriosis-related angiogenesis is well established (16, 17). Expression of VEGF is increased in active red lesions (18, 19) and deep infiltrating endometriosis (20), which are characterized by an active angiogenic process and a rich vascular net formed by immature blood vessels. In previous studies, we targeted VEGF in an experimental endometriosis model that examined the dopamine/dopamine receptor 2 (DRD2) pathway, whose activation is involved in the regulation of angiogenic events mediated by VEGF/ VEGF receptor 2 (KDR) signaling (19, 21). By targeting the VEGF system, we were able to inhibit the angiogenic process in endometriotic lesions and reduce the number of immature blood vessels (21). This concept has also been successfully addressed in humans (22). Moreover, it is wellknown that neovascularization plays an important role during neurogenesis (5). Based on this background, we hypothesized that the antiangiogenic properties that dopamine agonists exert on endometriotic lesions targeting VEGF could also diminish NFD through the inhibition of new blood vessel development in endometriotic lesions.
MATERIALS AND METHODS This study was approved by our institutional review board, and informed consent was obtained from patients before the collection of biopsy samples. Similarly, all the procedures employing animals were performed according to European Directive 86/609/CEE and NIH Guidelines for the Care and Use of Laboratory Animals.
Heterologous Mice Model of Endometriosis Sixteen 5-week-old ovariectomized female nude mice (Hsd: athymic Nude-nu; Harlan Iberica S.L.) were used to develop the endometriosis model, as previously described elsewhere 1210
(19). Sixty-day-release sterile capsules containing 18 mg of 17b-estradiol (E2) (Innovative Research of America) were placed subcutaneously in the neck of each animal. Four days later, fresh human endometrial biopsy samples (employing a Pipelle cannula) were obtained at ovum pickup from oocyte donors (n ¼ 4, age range: 18 to 34 years) with normal menstrual cycles and no history of endometriosis. Informed consent was obtained before the human endometrial collection. Although names were kept confidential, patient age, cycle stage, and medication history were made available. The biopsy samples were placed in a prewarmed, sterile phosphate-buffered saline (PBS) solution at pH 7.4 and transported to the laboratory, where the specimens were cut into pieces of approximately 2 3 mm. A part of each biopsy sample was fixed in 4% buffered formaldehyde and embedded in paraffin for histologic confirmation of the proliferative phase by use of established criteria (23) and to analyze the presence or absence of nerve fibers by immunohistochemical analysis. The remaining tissue was fixed in the peritoneum of each mouse (four human endometrial fragments per mouse) using an n-butyl-ester cyanoacrylate adhesive (3M Animal Care). Human endometrial samples from each individual donor were used in two animals of each experimental group, and four implants were introduced per animal. Three weeks after establishment of experimental lesions, the animals were divided into two experimental groups. Vehicle solution (1:6 alcohol in a sterile water mixture) was used to dilute Cb2 (Pfizer Laboratories), and this solution was administered daily and orally by gavage at doses of 0.00 mg/kg (control, n ¼ 8) and 0.01 mg/kg (n ¼ 8) for 14 days. Two weeks after Cb2 treatment, animals were killed, and the endometriotic implants were examined under a binocular microscope.
Histologic Evaluation Lesions were fixed in 4% neutral buffered formalin overnight at 4 C before being routinely embedded in paraffin wax and cut into 4-mm serial sections. Four to five noncontiguous sections from each specimen were stained with hematoxylin and eosin (Sigma) and examined microscopically for the presence of the histologic hallmarks (glands and stroma) of endometriosis. Only active lesions, the ones that presented both glandular and stromal elements, were included in this study. Lesions that did not show both elements, presenting an atrophic epithelium surrounded by fibrotic tissue instead of stroma, were considered inactive lesions and discarded. A morphologic study was performed by blue toluidine staining to study the mast cell and macrophage localization in endometriotic lesions.
Immunofluorescence Staining and Laser Scanning Confocal Microscopy A double-fluorescent was applied as previously described elsewhere (20). Human and murine endothelial cells were identified by use of a polyclonal rabbit anti-von Willebrand factor (vWF, Dako Corp.) conjugated with Zenon Alexa Fluor-647 (Molecular Probes), used according to the manufacturer's instructions. Rabbit anti-mouse immunoglobulins (Dako Corp.) were used as the negative control. Human VOL. 98 NO. 5 / NOVEMBER 2012
Fertility and Sterility® and murine pericytes were detected using a monoclonal mouse anti-human a smooth muscle actin fluorescein isothiocyanate conjugated (aSMA-FITC; Sigma) antibody. An isotype-matched mouse IgG (Dako Corp.) was used as a negative control. Due to the revascularization process and the native graft vessels disappearance, antibodies employed in this study detected both human and murine blood vessels, in order to be able to analyze the Cb2 effect. Fluorescently stained sections were mounted and counterstained with DAPI (Molecular Probes). The blood vessels that expressed only vWF (vWFþ/aSMA), present in endothelial cells, were considered new or immature (IBV), and those that expressed both vWF and aSMA (vWFþ/aSMAþ) were classified as old or mature. Imaging was performed with a TCS-SP2 True confocal laser scanning microscope (Leica Microsystems). A morphometric study was done to analyze double immunofluorescent staining.
Immunohistochemistry The PGP 9.5 expression was studied by employing a rabbit anti-PGP 9.5 antibody (Dako Corp.) at the 1:100 dilution. Mouse and human central nervous system were used as positive controls. Sections were deparaffinized and rehydrated through graded ethanol, rinsed in distilled water, and treated with 0.3% H2O2 and 10% normal horse serum to block endogenous peroxidase and nonspecific binding, respectively. Antigen retrieval was performed by pressure cooker boiling for 3 minutes in 10 mmol/L of citrate buffer (pH 6.0). For the secondary antibody incubation, a biotin/streptavidin (LSAB method; DakoCytomation) reaction was used according to the manufacturer's instructions. Sections were incubated in biotinylated secondary antibody in PBS for 30 minutes at room temperature followed by horseradish peroxidasestreptavidin solution incubation for 30 minutes at room temperature. The detection was performed with 3,3diaminobenzidine incubation for 3 minutes. For each tissue section, a serial section was employed as a negative control. Immunostaining was analyzed via morphometric study.
Morphometric Analysis To quantify the immunohistochemical and immunofluorescence studies, five images randomly selected from each section were captured by an Olympus BH2-UMA microscope connected to a JVC/TK-1270 video camera and a computerdigitized plate (Olympus). The images were analyzed by Image-Pro Plus 5.1 software (Media Cybernetics) to calculate the stromal microscopic area of each lesion (expressed as mm2) and the positive immunostained cells of each sample. Macrophages and mast cells were calculated and expressed as the total number of cells per mm2. The IBV was calculated and expressed as the total number of immature blood vessels per square millimeter. Microvascular density (MVD) was calculated and expressed as the total number of mature and immature blood vessels per square millimeter. The presence of nerve fibers in experimental-endometriosis lesions was investigated by use of the PGP 9.5 labeling index, where the total antigen expression per mm2 of stoma (nerve fibers density, NFD) was quantified by a morphometric analysis. VOL. 98 NO. 5 / NOVEMBER 2012
Statistical Analysis Statistical analyses were performed using GraphPad Instat V3.0 (GraphPad Software). A Kruskal-Wallis analysis was employed for the overall analysis of the data to determine if they followed a normal distribution. Nonparametric MannWhitney and Dunn's multiple comparisons test were used to compare individual means. Numerical data were expressed as mean standard deviation (SD). P< .05 was considered statistically significant.
RESULTS Macroscopic and Histologic Findings The animals were killed and their peritoneal cavities opened. The four human endometrium implants transplanted onto the peritoneal mouse wall before Cb2 treatment were macroscopically observed in all animals (100%), as in previous studies (19, 21) (Fig. 1A). The histologic study confirmed the endometrium fragments of human origin with well-defined glands and a cellular stroma (see Fig. 1B). The control mice lesions presented plenty of areas with blood vessels, macrophages (see Fig. 1C), and mast cells (see Fig. 1D). However, the macrophages and mast cell areas were reduced in the Cb2-treated mice lesions (see Fig. 1E and F). After histologic study combined with morphometric techniques, we found that the number of macrophages per lesion of the stromal area was statistically significantly (P< .05) decreased in the lesions from Cb2-treated mice (57.1 37.1) as compared with the lesions from control mice (93.5 56.0) (Fig. 1G). The number of total inactivated and activated mast cells per lesion stromal area was statistically significantly (P< .05) decreased when Cb2 was administrated (1.5 0.7, 1.1 0.6, 0.4 0.3) as compared with the control group (8.0 1.2, 4.2 1.5, 3.9 1.8) (see Fig. 1H).
Vascularization of Experimental Endometriotic Lesions Microvascular density (MVD) and newly formed or immature blood vessels (IBV) were studied by a double immunostaining and laser scanning confocal microscopy analysis (Fig. 2). Human and murine pericytes were observed in mature blood vessels (vWFþ/aSMAþ) from experimental endometriosis lesions; however, they were absent in new blood vessels (vWFþ/aSMA). The IBV statistically significantly decreased (P< .001) in Cb2-treated lesions (0.8 0.3) (see Fig. 2A and C) in comparison with control mice lesions (6.9 2.1) (see Fig. 2B and C). No statistically significant differences were found among the groups for MVD (P¼ .1391) (see Fig. 2C).
Nerve Fibers Density in Experimental Endometriotic Lesions We found that PGP 9.5 staining was absent in all of the five noncontiguous sections of each of the four human endometrial samples employed to develop the heterologous mouse model of endometriosis before implantation in the mouse peritoneum (Fig. 3A). Three weeks after implantation and 1211
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FIGURE 1
Macroscopic evaluation of human endometrial implants on the peritoneal wall of mice. (A) Experimental lesions show a well-developed vascular net and adhesions that resemble human endometriotic lesions. (B) Histologic study shows implants present a cellular stroma surrounding the glandular areas attached to the peritoneal murine tissue (stain: hematoxylin and eosin; magnification: 100). (C–D) Control mice lesions present a cellular stroma with rich areas of blood vessels and full areas of (C) hemosiderin-macrophage pigments (stain: hematoxylin and eosin; magnification: 200) and (D) mast cells (stain: blue toluidine; magnification: 200). (E–F) Deprived areas of (E) macrophages (stain: hematoxylin and eosin; magnification: 200) and (F) mast cells (stain: blue toluidine; magnification: 200) are observed in the lesion from Cb2-treated mice when compared with control mice. (G–H) Morphometric study of the samples reveals a statistically significant decrease (P<.05) of (G) macrophages and (H) total inactivated and activated mast cells in the lesions from Cb2-treated mice when compared with the control group. Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
2 weeks after treatment, PGP 9.5 was expressed in all the lesions (see Fig. 3A–3D) and healthy peritoneum (see Fig. 3F) from the two experimental groups. In the control1212
mice lesions, nerve fibers were located in the stroma, close to the endometriotic glands and blood vessels. A lower level of PGP 9.5 staining was observed in the stroma of the VOL. 98 NO. 5 / NOVEMBER 2012
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FIGURE 2
Vascularization analysis in controls and the Cb2-treated group. (A) Lesions from the control group presented a higher prevalence of newly formed blood vessels areas (red: þvWF(red)/aSMA) with respect to mature blood vessels areas (green: þvWF/þaSMA), indicating an active angiogenic process in untreated mice lesions. (B) In the lesions from Cb2-treated mice, the reversal of this process was observed with predominant mature blood vessel areas over the newly formed ones, indicating an inhibition of the angiogenic process. (C) When morphometric analysis was performed, a decreasing trend was observed for microvascular density (MVD) in the Cb2-treated groups, and immature blood vessels (IBV) were statistically significantly decreased (*P<.001) in Cb2-treated lesions when compared with controls. Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
Cb2-treated animals (see Fig. 3D and E) as compared with the control lesions (see Fig. 3B and C). When the NFD was calculated, it was statistically significantly (P< .05) lower in the mice treated with Cb2 (0.4 0.1) than in the control animals (1.3 1.2) (see Fig. 3G).
DISCUSSION Our study reveals that antiangiogenic agents such dopamine agonist are able to diminish the NFD present in experimental endometriosis lesions. Simultaneously, immature blood vessels formation also diminished employing Cb2. This finding further supports the use of dopamine agonists in the medical treatment of endometriosis. Dopamine agonists have been shown to play an active role in reducing endometriosis implants in humans (22). Herein, we demonstrate that they can reduce the presence of nerve fibers, which have been directly related to the presence of chronic pelvic pain and dysmenorrhea (3, 4). In the model employed, revascularization started 5 to 8 days after implantation and involved the disappearance of native graft vessels, concurrent with the invasion of the interface and stroma by murine vessels (24). This could explain the similarity among groups in terms of MVD. Ours is the first study to show nerve fibers in a mouse model of endometriosis. We initially tested the human endometrial tissue employed in the heterologous mouse model of VOL. 98 NO. 5 / NOVEMBER 2012
endometriosis, and no nerve fibers were seen. This reveals that nerve fibers were of a mouse origin and grew after establishing implants with appropriate vessel development. When dopamine agonists were administered, NFD and IBV significantly decreased, thus confirming our initial hypothesis that antiangiogenic agents diminish NFD as a result of the inhibition of new blood vessel development in endometriotic lesions. Nerves are located in close proximity to blood vessels and coordinate to form a proper network. Neurovascular coordination requires a sharing of the major signaling pathways involved in pathfinding, growth, migration, and differentiation. Nerve growth factor (NGF) belongs to the neurotrophin family involved in neuronal proliferation, survival, and pathfinding. Besides their usual functions in neuronal cells, it has been suggested that they play other roles in non-neuronal tissues, especially blood vessels. Studies have described NGF as activating endothelial cell proliferation and migration in vitro and inducing angiogenesis in in vivo assays (25) through cross-talk with VEGF signaling (26, 27). Neural and vascular cells reciprocally affect their own growth by paracrine mechanisms and use common signals (28, 29). Additionally, NGF promotes angiogenesis (30), and, mutually, vascular signaling affects neuronal precursor cells (31–34). In addition to its well-known role in angiogenesis, VEGF promotes neurogenesis and neuroprotection by mediating both proliferation and chemoattraction (35, 36) under normal or ischemic conditions (37). 1213
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FIGURE 3
Nerve fibers analysis with controls and the Cb2-treated group. (A) Protein gene product (PGP 9.5) staining was absent in human endometrial samples before implantation in the mouse peritoneum (magnification: 200). (B–E) PGP 9.5 was fully expressed in all the lesions (B, D: magnification 100; and C, E: magnification 200) and healthy mouse peritoneum (F: magnification 200). However, Cb2-treated lesions show poor PGP9.5 stained areas (D, E: magnification 100 and 200, respectively). The nerve fibers density (NFD) study showed a statistically significantly lower (*P<.05) NFD in Cb2-treated lesions when compared with controls (G). Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
Endometriosis is a chronic inflammatory disease. Inflammation is associated with the activation of resident macrophages, mast cells, and fibroblasts, all of which produce large quantities of proangiogenic factors (38, 39). Mast cell– derived modulators of the microvascular function include histamine, heparin, VEGF, nitric oxide, cytokines, chemokines, proteases, and lipid mediators (36–49). Some of these mediators have been shown to contribute to inflammation-induced permeability responses in a variety of animal models. Mast cell-specific tryptases and chymases 1214
promote vascular permeability via indirect and perhaps direct mechanisms (50–56). Our findings also support a beneficial effect of Cb2, astreated animals showed reduced amounts of macrophages and mast cells. Interleukin-1, a principal macrophagederived and major proinflammatory cytokine, plays an important role in the inflammatory cascade associated with endometriosis and in propagating endometriotic implants through a proinflammatory stimulus, synthesis of chemokines, growth, and angiogenic factors (57–60). Increased VOL. 98 NO. 5 / NOVEMBER 2012
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FIGURE 4
Dopamine neurotransmitter binds to dopamine receptor-2, located on the endothelial cell surface, and promotes the VEGFR-2 endocytosis, preventing VEGF-VEGFR-2 from binding. The receptor phosphorylation is blocked, and the angiogenesis process is inhibited. Consequently, there is blood vessel regression and a neuroendocrine and immune disequilibrium in the endometriotic tissue. Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
concentrations of this cytokine were found in ectopic endometrial implants of women with endometriosis (61). The fact that T cells synthesize and secrete VEGF has implications for both the physiology and pathophysiology of inflammation, and it has been shown that activated T cells can serve as vehicles for neurotrophic factors (62, 63). The homing of T cells to sites of inflammation provides a mobile source of VEGF that can be controlled by the degree of Tcell activation. Moreover, hypoxia associated with tissue damage and also with endometriosis process would lead to the production angiogenic factors such as VEGF, which push T cells visiting the site into a proinflammatory TH1 mode and amplify inflammation (62). Our study has shown that Cb2 decreases IBV and NFD in an experimental mouse model for endometriosis. A statistically significant decrease in macrophages and mast cells in treated animals through the interruption of the pathway leading to inflammation seems to be the major finding that could explain these results (Fig. 4). These results open the possibility of using dopamine agonists in the treatment of endometriosis-associated pelvic pain.
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Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, Greenberg DA. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest 2003;111:1843–51. Sokolov DI, Solodovnikova NG, Pavlov OV, Niauri DA, Volkov NN, Sel'kov SA. Study of cytokine profile and angiogenic potential of peritoneal fluid in patients with external genital endometriosis. Bull Exp Biol Med 2005;140:541–4. Huang HF, Hong LH, Tan Y, Sheng JZ. Matrix metal-loproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis. Fertil Steril 2004;81:1235–9. Drake TS, O'Brien WF, Ramwell PW, Metz SA. Peritoneal fluid throm- boxane B2 and 6-keto-prostaglandin F1 alpha in endometriosis. Am J Obstet Gynecol 1981;140:401–4. Moon Y, Leung P, Ho Y. Prostaglandin F in human endometriotic tissue. Obset Gynecol 1981;141:344. Lin YJ, Lai MD, Lei HY, Wing LY. Neutrophils and macrophages promote angiogenesis in the early stage of endometriosis in a mouse model. Endocrinology 2006;147:1278–86. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994;55:410–22. Halme J, Becker S, Hammond MG, Raj MH, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333–7. Ryan IP, Tseng JF, Schriock ED, Khorram O, Landers DV, Taylor RN. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil Steril 1995;63:929–32. Witz CA, Montoya IA, Dey TD, Schenken RS. Characterization of lymphocyte subpopulations and T cell activation in endometriosis. Am J Reprod Immunol 1994;32:173–9. Jones RK, Bulmer JN, Searle RF. Immunohistochemical characterization of stromal leukocytes in ovarian endometriosis: comparison of eutopic and ectopic endometrium with normal endometrium. Fertil Steril 1996;66:81–9. Akoum A, Lemay A, Maheux R. Estradiol and interleu- kin-1beta exert a synergistic stimulatory effect on the expression of the chemokine regulated upon activation, normal T cell expressed, and secreted in endometriotic cells. J Clin Endocrinol Metab 2002;87:5785–92. Khorram O, Taylor RN, Ryan IP, Schall TJ, Landers DV. Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol 1993;169:1545–9. Kubes P, Granger DN. Leukocyte-endothelial cell interactions evoked by mast cells. Cardiovasc Res 1996;32:699–708. Ryan JJ, Fernando JF. Mast cell modulation of the immune response. Curr Allergy Asthma Rep 2009;9:353–9. Klesnikoff J, Galli SJ. New developments in mast cell biology. Nat Immunol 2008;9:1215–23. Strbian D, Karjalainen-Lindsberg ML, Tatlisumak T, Lindsberg PJ. Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation. J Cereb Blood Flow Metab 2006;26:605–12. Lee KS, Kim SR, Min KH, Lee KY, Choe YH, Park SY, et al. Mast cells can mediate vascular permeability through regulation of the PI3K-HIF-1alphaVEGF axis. Am J Respir Crit Care Med 2008;178:787–97. Iuvone T, Den Bossche RV, D'Acquisto F, Carnuccio R, Herman AG. Evidence that mast cell degranulation, histamine and tumor necrosis factor release occur in LPS-induced plasma leakage in rat skin. Br J Pharmacol 1999;128: 700–4. Miller HR, Pemberton AD. Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology 2002;105:375–90. Akoum A, Lemay A, Brunet C, Hebert J. Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture. Groupe D'Investigation en Gynecologie. Am J Obstet Gynecol 1995;172:594–600. Akoum A, Lemay A, Paradis I, Rheault N, Maheux R. Secretion of interleukin6 by human endometriotic cells and regulation by proinflammatory cytokines and sex steroids. Hum Reprod 1996;11:2269–75. Akoum A, Lawson C, McColl S, Villeneuve M. Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol Hum Reprod 2001;7:859–66.
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Objective: To investigate the effect of antiangiogenic treatment on experimental endometriotic lesion nerve fibers. Design: Heterologous mouse model of endometriosis. Setting: University Institute IVI, University Hospital La Fe. Animal(s): Ovariectomized nude mice (n ¼ 16) receiving human endometrial fragments from oocyte donors (n ¼ 4). Intervention(s): Endometrium fragments stuck in the peritoneum of 5-week-old female nude mice treated with vehicle (n ¼ 8) and antiangiogenic agent cabergoline (n ¼ 8; Cb2, 0.05 mg/kg/day) for 14 days. Main Outcome Measure(s): Immunofluorescence analysis of von-Willebrand factor (vWF) and vascular smooth muscle cells (aSMA) for evaluating the number of immature blood vessels (IBV) and microvascular density (MVD); immunochemical analysis of proteingene product 9.5 (PGP 9.5) to assess nerve fibers density (NFD), and blue toluidine staining to confirm presence of mast cells and macrophages in endometriotic lesions. Result(s): All the results were quantified by morphometric techniques. The IBV, NFD, and number of macrophages and mast cells were statistically significantly decreased in the Cb2-treated group when compared with controls. Conclusion(s): Antiangiogenic treatment statistically significantly diminishes new blood vessel formation after macrophage, mast cell, and nerve fiber reduction, providing a rationale to test antiangiogenic agents as a novel therapeutic approach to severe pelvic pain associated with human peritoneal endometriosis. (Fertil SterilÒ 2012;98:1209–17. Use your smartphone Ó2012 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Angiogenesis, dopamine agonists, endometriosis, nerve fibers, vascular and connect to the endothelial growth factor Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/novella-maestree-antiangiogenic-treatment-peritonealendometriosis-nerve-fibers/
E
ndometriosis, an estrogendependent inflammatory disease characterized by the presence of endometrial tissue outside the uterus, predominantly affects women of reproductive age. The real incidence of endometriosis is unknown, but it is estimated that this pathology is present in
more than 15% of reproductive age women and in up to 50% of women with pelvic pain (1). Pain, in the form of dysmenorrhea, dyspareunia, or dyschezia, is the most common symptom in patients with endometriosis and may be due to nociceptive, inflammatory, or neuropathic mechanisms.
Received January 25, 2012; revised July 8, 2012; accepted July 11, 2012; published online August 21, 2012. E.N.-M. has nothing to disclose. S.H. has nothing to disclose. J.M.V.-V. has nothing to disclose. C.C. has nothing to disclose. A.R.-S. has nothing to disclose. A.P. has nothing to disclose. Reprint requests: Edurne Novella-Maestre, Ph.D., Laboratorio de FIV, Torre B planta 1, Hospital Unicnico La Fe, Bulevar Sur s/n, 46026, Valencia, Spain (E-mail: edurnenovella@ versitario y Polite yahoo.es). Fertility and Sterility® Vol. 98, No. 5, November 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1103 VOL. 98 NO. 5 / NOVEMBER 2012
discussion forum for this article now.*
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Surgical resection of endometriotic lesions provides temporary relief in terms of reduced chronic pelvic pain and severe dysmenorrhea (2), but the recurrence rate at 2 years after surgery is as high as 75% of cases. Thus, medical treatment is employed at any stage of the disease in many patients worldwide. The presence of nerve fibers in the peritoneal endometriotic lesions (3–8) and eutopic endometrium of women with endometriosis (9, 10) has received much attention recently. Nerve fibers in peritoneal lesions are located in or near endometriotic stromal cells and are colocalized with 1209
ORIGINAL ARTICLE: ENDOMETRIOSIS immature blood vessels (5, 6). These findings suggest that human peritoneal endometriotic lesions are innervated by nerve fibers that may interact with each other and with many active molecules released by endometriotic lesions to cause pain and local tenderness (11). Studies have shown that nerve fiber density (NFD) is significantly greater in the peritoneal lesions of patients with painful endometriosis than in patients with asymptomatic disease, which suggests a direct association between pain and NFD (3, 12). Some current treatments for endometriosis, such as combinations of oral contraceptives and progestogens, significantly decrease NFD in the endometrium and myometrium in women with endometriosis (13, 14). Steroids act via a different route and have no effect on vascular endothelial growth factor (VEGF) expression, so they cannot be employed in all patients. Thus, the search for new routes in the medical treatment of endometriosis must continue. Different proteins and growth factors have been described as regulators of nerve fibers growth. Vascular endothelial growth factor, produced by macrophages that are elevated in the peritoneal fluid and endometriotic lesions of women with endometriosis, has been reported to act as a neurotrophic factor, stimulating nerve-fiber growth (15). These findings suggest that VEGF may also play a role in increasing NFD in peritoneal endometriotic lesions (8). The role of VEGF in endometriosis-related angiogenesis is well established (16, 17). Expression of VEGF is increased in active red lesions (18, 19) and deep infiltrating endometriosis (20), which are characterized by an active angiogenic process and a rich vascular net formed by immature blood vessels. In previous studies, we targeted VEGF in an experimental endometriosis model that examined the dopamine/dopamine receptor 2 (DRD2) pathway, whose activation is involved in the regulation of angiogenic events mediated by VEGF/ VEGF receptor 2 (KDR) signaling (19, 21). By targeting the VEGF system, we were able to inhibit the angiogenic process in endometriotic lesions and reduce the number of immature blood vessels (21). This concept has also been successfully addressed in humans (22). Moreover, it is wellknown that neovascularization plays an important role during neurogenesis (5). Based on this background, we hypothesized that the antiangiogenic properties that dopamine agonists exert on endometriotic lesions targeting VEGF could also diminish NFD through the inhibition of new blood vessel development in endometriotic lesions.
MATERIALS AND METHODS This study was approved by our institutional review board, and informed consent was obtained from patients before the collection of biopsy samples. Similarly, all the procedures employing animals were performed according to European Directive 86/609/CEE and NIH Guidelines for the Care and Use of Laboratory Animals.
Heterologous Mice Model of Endometriosis Sixteen 5-week-old ovariectomized female nude mice (Hsd: athymic Nude-nu; Harlan Iberica S.L.) were used to develop the endometriosis model, as previously described elsewhere 1210
(19). Sixty-day-release sterile capsules containing 18 mg of 17b-estradiol (E2) (Innovative Research of America) were placed subcutaneously in the neck of each animal. Four days later, fresh human endometrial biopsy samples (employing a Pipelle cannula) were obtained at ovum pickup from oocyte donors (n ¼ 4, age range: 18 to 34 years) with normal menstrual cycles and no history of endometriosis. Informed consent was obtained before the human endometrial collection. Although names were kept confidential, patient age, cycle stage, and medication history were made available. The biopsy samples were placed in a prewarmed, sterile phosphate-buffered saline (PBS) solution at pH 7.4 and transported to the laboratory, where the specimens were cut into pieces of approximately 2 3 mm. A part of each biopsy sample was fixed in 4% buffered formaldehyde and embedded in paraffin for histologic confirmation of the proliferative phase by use of established criteria (23) and to analyze the presence or absence of nerve fibers by immunohistochemical analysis. The remaining tissue was fixed in the peritoneum of each mouse (four human endometrial fragments per mouse) using an n-butyl-ester cyanoacrylate adhesive (3M Animal Care). Human endometrial samples from each individual donor were used in two animals of each experimental group, and four implants were introduced per animal. Three weeks after establishment of experimental lesions, the animals were divided into two experimental groups. Vehicle solution (1:6 alcohol in a sterile water mixture) was used to dilute Cb2 (Pfizer Laboratories), and this solution was administered daily and orally by gavage at doses of 0.00 mg/kg (control, n ¼ 8) and 0.01 mg/kg (n ¼ 8) for 14 days. Two weeks after Cb2 treatment, animals were killed, and the endometriotic implants were examined under a binocular microscope.
Histologic Evaluation Lesions were fixed in 4% neutral buffered formalin overnight at 4 C before being routinely embedded in paraffin wax and cut into 4-mm serial sections. Four to five noncontiguous sections from each specimen were stained with hematoxylin and eosin (Sigma) and examined microscopically for the presence of the histologic hallmarks (glands and stroma) of endometriosis. Only active lesions, the ones that presented both glandular and stromal elements, were included in this study. Lesions that did not show both elements, presenting an atrophic epithelium surrounded by fibrotic tissue instead of stroma, were considered inactive lesions and discarded. A morphologic study was performed by blue toluidine staining to study the mast cell and macrophage localization in endometriotic lesions.
Immunofluorescence Staining and Laser Scanning Confocal Microscopy A double-fluorescent was applied as previously described elsewhere (20). Human and murine endothelial cells were identified by use of a polyclonal rabbit anti-von Willebrand factor (vWF, Dako Corp.) conjugated with Zenon Alexa Fluor-647 (Molecular Probes), used according to the manufacturer's instructions. Rabbit anti-mouse immunoglobulins (Dako Corp.) were used as the negative control. Human VOL. 98 NO. 5 / NOVEMBER 2012
Fertility and Sterility® and murine pericytes were detected using a monoclonal mouse anti-human a smooth muscle actin fluorescein isothiocyanate conjugated (aSMA-FITC; Sigma) antibody. An isotype-matched mouse IgG (Dako Corp.) was used as a negative control. Due to the revascularization process and the native graft vessels disappearance, antibodies employed in this study detected both human and murine blood vessels, in order to be able to analyze the Cb2 effect. Fluorescently stained sections were mounted and counterstained with DAPI (Molecular Probes). The blood vessels that expressed only vWF (vWFþ/aSMA), present in endothelial cells, were considered new or immature (IBV), and those that expressed both vWF and aSMA (vWFþ/aSMAþ) were classified as old or mature. Imaging was performed with a TCS-SP2 True confocal laser scanning microscope (Leica Microsystems). A morphometric study was done to analyze double immunofluorescent staining.
Immunohistochemistry The PGP 9.5 expression was studied by employing a rabbit anti-PGP 9.5 antibody (Dako Corp.) at the 1:100 dilution. Mouse and human central nervous system were used as positive controls. Sections were deparaffinized and rehydrated through graded ethanol, rinsed in distilled water, and treated with 0.3% H2O2 and 10% normal horse serum to block endogenous peroxidase and nonspecific binding, respectively. Antigen retrieval was performed by pressure cooker boiling for 3 minutes in 10 mmol/L of citrate buffer (pH 6.0). For the secondary antibody incubation, a biotin/streptavidin (LSAB method; DakoCytomation) reaction was used according to the manufacturer's instructions. Sections were incubated in biotinylated secondary antibody in PBS for 30 minutes at room temperature followed by horseradish peroxidasestreptavidin solution incubation for 30 minutes at room temperature. The detection was performed with 3,3diaminobenzidine incubation for 3 minutes. For each tissue section, a serial section was employed as a negative control. Immunostaining was analyzed via morphometric study.
Morphometric Analysis To quantify the immunohistochemical and immunofluorescence studies, five images randomly selected from each section were captured by an Olympus BH2-UMA microscope connected to a JVC/TK-1270 video camera and a computerdigitized plate (Olympus). The images were analyzed by Image-Pro Plus 5.1 software (Media Cybernetics) to calculate the stromal microscopic area of each lesion (expressed as mm2) and the positive immunostained cells of each sample. Macrophages and mast cells were calculated and expressed as the total number of cells per mm2. The IBV was calculated and expressed as the total number of immature blood vessels per square millimeter. Microvascular density (MVD) was calculated and expressed as the total number of mature and immature blood vessels per square millimeter. The presence of nerve fibers in experimental-endometriosis lesions was investigated by use of the PGP 9.5 labeling index, where the total antigen expression per mm2 of stoma (nerve fibers density, NFD) was quantified by a morphometric analysis. VOL. 98 NO. 5 / NOVEMBER 2012
Statistical Analysis Statistical analyses were performed using GraphPad Instat V3.0 (GraphPad Software). A Kruskal-Wallis analysis was employed for the overall analysis of the data to determine if they followed a normal distribution. Nonparametric MannWhitney and Dunn's multiple comparisons test were used to compare individual means. Numerical data were expressed as mean standard deviation (SD). P< .05 was considered statistically significant.
RESULTS Macroscopic and Histologic Findings The animals were killed and their peritoneal cavities opened. The four human endometrium implants transplanted onto the peritoneal mouse wall before Cb2 treatment were macroscopically observed in all animals (100%), as in previous studies (19, 21) (Fig. 1A). The histologic study confirmed the endometrium fragments of human origin with well-defined glands and a cellular stroma (see Fig. 1B). The control mice lesions presented plenty of areas with blood vessels, macrophages (see Fig. 1C), and mast cells (see Fig. 1D). However, the macrophages and mast cell areas were reduced in the Cb2-treated mice lesions (see Fig. 1E and F). After histologic study combined with morphometric techniques, we found that the number of macrophages per lesion of the stromal area was statistically significantly (P< .05) decreased in the lesions from Cb2-treated mice (57.1 37.1) as compared with the lesions from control mice (93.5 56.0) (Fig. 1G). The number of total inactivated and activated mast cells per lesion stromal area was statistically significantly (P< .05) decreased when Cb2 was administrated (1.5 0.7, 1.1 0.6, 0.4 0.3) as compared with the control group (8.0 1.2, 4.2 1.5, 3.9 1.8) (see Fig. 1H).
Vascularization of Experimental Endometriotic Lesions Microvascular density (MVD) and newly formed or immature blood vessels (IBV) were studied by a double immunostaining and laser scanning confocal microscopy analysis (Fig. 2). Human and murine pericytes were observed in mature blood vessels (vWFþ/aSMAþ) from experimental endometriosis lesions; however, they were absent in new blood vessels (vWFþ/aSMA). The IBV statistically significantly decreased (P< .001) in Cb2-treated lesions (0.8 0.3) (see Fig. 2A and C) in comparison with control mice lesions (6.9 2.1) (see Fig. 2B and C). No statistically significant differences were found among the groups for MVD (P¼ .1391) (see Fig. 2C).
Nerve Fibers Density in Experimental Endometriotic Lesions We found that PGP 9.5 staining was absent in all of the five noncontiguous sections of each of the four human endometrial samples employed to develop the heterologous mouse model of endometriosis before implantation in the mouse peritoneum (Fig. 3A). Three weeks after implantation and 1211
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FIGURE 1
Macroscopic evaluation of human endometrial implants on the peritoneal wall of mice. (A) Experimental lesions show a well-developed vascular net and adhesions that resemble human endometriotic lesions. (B) Histologic study shows implants present a cellular stroma surrounding the glandular areas attached to the peritoneal murine tissue (stain: hematoxylin and eosin; magnification: 100). (C–D) Control mice lesions present a cellular stroma with rich areas of blood vessels and full areas of (C) hemosiderin-macrophage pigments (stain: hematoxylin and eosin; magnification: 200) and (D) mast cells (stain: blue toluidine; magnification: 200). (E–F) Deprived areas of (E) macrophages (stain: hematoxylin and eosin; magnification: 200) and (F) mast cells (stain: blue toluidine; magnification: 200) are observed in the lesion from Cb2-treated mice when compared with control mice. (G–H) Morphometric study of the samples reveals a statistically significant decrease (P<.05) of (G) macrophages and (H) total inactivated and activated mast cells in the lesions from Cb2-treated mice when compared with the control group. Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
2 weeks after treatment, PGP 9.5 was expressed in all the lesions (see Fig. 3A–3D) and healthy peritoneum (see Fig. 3F) from the two experimental groups. In the control1212
mice lesions, nerve fibers were located in the stroma, close to the endometriotic glands and blood vessels. A lower level of PGP 9.5 staining was observed in the stroma of the VOL. 98 NO. 5 / NOVEMBER 2012
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FIGURE 2
Vascularization analysis in controls and the Cb2-treated group. (A) Lesions from the control group presented a higher prevalence of newly formed blood vessels areas (red: þvWF(red)/aSMA) with respect to mature blood vessels areas (green: þvWF/þaSMA), indicating an active angiogenic process in untreated mice lesions. (B) In the lesions from Cb2-treated mice, the reversal of this process was observed with predominant mature blood vessel areas over the newly formed ones, indicating an inhibition of the angiogenic process. (C) When morphometric analysis was performed, a decreasing trend was observed for microvascular density (MVD) in the Cb2-treated groups, and immature blood vessels (IBV) were statistically significantly decreased (*P<.001) in Cb2-treated lesions when compared with controls. Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
Cb2-treated animals (see Fig. 3D and E) as compared with the control lesions (see Fig. 3B and C). When the NFD was calculated, it was statistically significantly (P< .05) lower in the mice treated with Cb2 (0.4 0.1) than in the control animals (1.3 1.2) (see Fig. 3G).
DISCUSSION Our study reveals that antiangiogenic agents such dopamine agonist are able to diminish the NFD present in experimental endometriosis lesions. Simultaneously, immature blood vessels formation also diminished employing Cb2. This finding further supports the use of dopamine agonists in the medical treatment of endometriosis. Dopamine agonists have been shown to play an active role in reducing endometriosis implants in humans (22). Herein, we demonstrate that they can reduce the presence of nerve fibers, which have been directly related to the presence of chronic pelvic pain and dysmenorrhea (3, 4). In the model employed, revascularization started 5 to 8 days after implantation and involved the disappearance of native graft vessels, concurrent with the invasion of the interface and stroma by murine vessels (24). This could explain the similarity among groups in terms of MVD. Ours is the first study to show nerve fibers in a mouse model of endometriosis. We initially tested the human endometrial tissue employed in the heterologous mouse model of VOL. 98 NO. 5 / NOVEMBER 2012
endometriosis, and no nerve fibers were seen. This reveals that nerve fibers were of a mouse origin and grew after establishing implants with appropriate vessel development. When dopamine agonists were administered, NFD and IBV significantly decreased, thus confirming our initial hypothesis that antiangiogenic agents diminish NFD as a result of the inhibition of new blood vessel development in endometriotic lesions. Nerves are located in close proximity to blood vessels and coordinate to form a proper network. Neurovascular coordination requires a sharing of the major signaling pathways involved in pathfinding, growth, migration, and differentiation. Nerve growth factor (NGF) belongs to the neurotrophin family involved in neuronal proliferation, survival, and pathfinding. Besides their usual functions in neuronal cells, it has been suggested that they play other roles in non-neuronal tissues, especially blood vessels. Studies have described NGF as activating endothelial cell proliferation and migration in vitro and inducing angiogenesis in in vivo assays (25) through cross-talk with VEGF signaling (26, 27). Neural and vascular cells reciprocally affect their own growth by paracrine mechanisms and use common signals (28, 29). Additionally, NGF promotes angiogenesis (30), and, mutually, vascular signaling affects neuronal precursor cells (31–34). In addition to its well-known role in angiogenesis, VEGF promotes neurogenesis and neuroprotection by mediating both proliferation and chemoattraction (35, 36) under normal or ischemic conditions (37). 1213
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FIGURE 3
Nerve fibers analysis with controls and the Cb2-treated group. (A) Protein gene product (PGP 9.5) staining was absent in human endometrial samples before implantation in the mouse peritoneum (magnification: 200). (B–E) PGP 9.5 was fully expressed in all the lesions (B, D: magnification 100; and C, E: magnification 200) and healthy mouse peritoneum (F: magnification 200). However, Cb2-treated lesions show poor PGP9.5 stained areas (D, E: magnification 100 and 200, respectively). The nerve fibers density (NFD) study showed a statistically significantly lower (*P<.05) NFD in Cb2-treated lesions when compared with controls (G). Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
Endometriosis is a chronic inflammatory disease. Inflammation is associated with the activation of resident macrophages, mast cells, and fibroblasts, all of which produce large quantities of proangiogenic factors (38, 39). Mast cell– derived modulators of the microvascular function include histamine, heparin, VEGF, nitric oxide, cytokines, chemokines, proteases, and lipid mediators (36–49). Some of these mediators have been shown to contribute to inflammation-induced permeability responses in a variety of animal models. Mast cell-specific tryptases and chymases 1214
promote vascular permeability via indirect and perhaps direct mechanisms (50–56). Our findings also support a beneficial effect of Cb2, astreated animals showed reduced amounts of macrophages and mast cells. Interleukin-1, a principal macrophagederived and major proinflammatory cytokine, plays an important role in the inflammatory cascade associated with endometriosis and in propagating endometriotic implants through a proinflammatory stimulus, synthesis of chemokines, growth, and angiogenic factors (57–60). Increased VOL. 98 NO. 5 / NOVEMBER 2012
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FIGURE 4
Dopamine neurotransmitter binds to dopamine receptor-2, located on the endothelial cell surface, and promotes the VEGFR-2 endocytosis, preventing VEGF-VEGFR-2 from binding. The receptor phosphorylation is blocked, and the angiogenesis process is inhibited. Consequently, there is blood vessel regression and a neuroendocrine and immune disequilibrium in the endometriotic tissue. Novella-Maestre. Nerve fibers in endometriosis. Fertil Steril 2012.
concentrations of this cytokine were found in ectopic endometrial implants of women with endometriosis (61). The fact that T cells synthesize and secrete VEGF has implications for both the physiology and pathophysiology of inflammation, and it has been shown that activated T cells can serve as vehicles for neurotrophic factors (62, 63). The homing of T cells to sites of inflammation provides a mobile source of VEGF that can be controlled by the degree of Tcell activation. Moreover, hypoxia associated with tissue damage and also with endometriosis process would lead to the production angiogenic factors such as VEGF, which push T cells visiting the site into a proinflammatory TH1 mode and amplify inflammation (62). Our study has shown that Cb2 decreases IBV and NFD in an experimental mouse model for endometriosis. A statistically significant decrease in macrophages and mast cells in treated animals through the interruption of the pathway leading to inflammation seems to be the major finding that could explain these results (Fig. 4). These results open the possibility of using dopamine agonists in the treatment of endometriosis-associated pelvic pain.
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