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Effects of hyperprolactinemia treatment with the dopamine agonist quinagolide on endometriotic lesions in patients with endometriosis-associated hyperprolactinemia
Effects of hyperprolactinemia treatment with the dopamine agonist quinagolide on endometriotic lesions in patients with endometriosis-associated hyperprolactinemia Raul G omez, Ph.D.,a Antonio Abad, M.D.,b Francisco Delgado, Ph.D.,a Silvia Tamarit, M.D.,b Carlos Sim on, M.D.,a and Antonio Pellicer, M.D.a,c a
Instituto Universitario Instituto Valenciano de Infertilidad, Valencia University School of Medicine and (Fundacion) Investigaci on Clınico Valencia; b Hospital Universitario Dr. Peset; and c Hospital Universitario La Fe, Valencia, Spain
Objective: To assess whether dopamine receptor 2 agonists reduced the size of peritoneal lesions in women with endometriosis and elucidate whether affectation of vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2)–dependent angiogenesis was mediating the observed effects. Design: Proof-of-concept study. Setting: University hospital and a university-affiliated private IVF research center. Patient(s): Hyperprolactinemic patients (n ¼ 9) with endometriosis requiring a first surgical intervention (L1) and benefiting from a second-look laparoscopy (L2) were evaluated. Intervention(s): During L1, four to six peritoneal red lesions were identified. One-half of the lesions were removed and the remaining one-half were labeled with silk knot sutures. After L1, quinagolide was administered in a titrated manner (25–75 mg/d) for 18–20 weeks. During L2, the remaining lesions were surgically excised. Main Outcome Measure(s): Both L1 and L2 were video recorded to compare the effects of quinagolide treatment on lesion size. Lesions removed at L1 and L2 were compared by means of: 1) histologic analysis; 2) immunohistochemical quantitative analysis of angiogenesis; and 3) quantitative fluorescence polymerase chain reaction array analysis of 84 chemokines and pro-/antiangiogenic molecules. Result(s): Quinagolide induced a 69.5% reduction in the size of the lesions, with 35% vanishing completely. Histologic analysis showed tissue degeneration, which was supported by down-regulation of VEGF/VEGFR2, three proangiogenic cytokines (CCL2, RUNX1, and AGGF1) and plasminogen activator inhibitor (PAI) 1, a potent inhibitor of fibrinolysis in the L2 lesions. Conclusion(s): By interfering with angiogenesis, enhancing fibrinolysis, and reducing inflammation, quinagolide reduces or eliminates peritoneal endometriotic lesions in women with endometriosis. (Fertil Steril 2011;95:882–8. 2011 by American Society for Reproductive Medicine.) Key Words: Endometriosis, endometrium, dopamine, dopamine agonists, quinagolide, angiogenesis, dopamine receptor 2, VEGF, VEGF receptor 2
Current treatment of endometriosis consists of surgical removal of diseased tissue and/or hormonal manipulation to create a hypoestrogenic milieu (GnRH agonist, aromatase inhibitors) (1). Unfortunately, the success of this treatment is often limited, and recurrence of the disease is very common (1). Therefore, new approaches to its treatment are necessary (2, 3).
Received May 2, 2010; revised September 10, 2010; accepted October 13, 2010; published online November 5, 2010. R.G. has nothing to disclose. A.A. has nothing to disclose. F.D. has nothing to disclose. S.T. has nothing to disclose. C.S. has conducted clinical research sponsored by Ferring. A.P. has conducted clinical research sponsored by Organon, Schering-Plough, Serono, Merck Serono, and Ferring Pharmaceuticals. The first two authors contributed equally to this work. Clinicaltrials.gov identifier: NCT 00625950. Supported by grant SAF2007–65334 from the Spanish Government, a Lilly Foundation Grant for Research in Clinical Medicine, and Ferring Pharmaceuticals, Copenhagen, Denmark. mez, Ph.D., Instituto Valenciano de Infertilidad, Reprint requests: Raul Go Plaza de la Policıa Local, 3, 46015, Valencia, Spain (E-mail: apellicer@ ivi.es).
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Analysis of endometriotic lesions has demonstrated that a prerequisite for their formation and maintenance is the development of an adequate blood supply (4). The regulation of the ‘‘endometriotic vasculature’’ involves canonic pathways of angiogenesis, including vascular endothelial growth factor (VEGF) acting through the VEGF receptor 2 (VEGFR2) (5). Several antiangiogenic agents that target this pathway have been successfully used in animal models of endometriosis (6). However, these agents can induce severe side effects (7), preventing their use for treating endometriosis in young and otherwise healthy women. We theorized that dopamine receptor 2 agonists (Drd2-A) might be an alternative to commercial antiangiogenic agents. In animal models, Drd2-A inhibit pathologic angiogenesis in tumors (8) by inactivating VEGFR2 signaling (9). However, this type of medication has an acceptable safety profile and does not interfere with the normal establishment and progression of pregnancy (10, 11). We previously demonstrated in a well established experimental endometriosis model that cabergoline prevented the formation of typical endometriosis lesions by inhibiting angiogenesis (12). These results encouraged us to perform a pilot study to evaluate the efficacy of Drd2-A in the treatment of peritoneal endometriosis in humans.
Fertility and Sterility Vol. 95, No. 3, March 1, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.10.024
MATERIALS AND METHODS Human Subjects and Study Design The study was designed as a proof-of-concept pilot study and approved by the ETHICS COmmittee of Hospital Universitario Dr. Peset. We were requested to involve only patients newly diagnosed with hyperprolactinemia (PRL >30 ng/mL), who were simultaneously suffering from severe endometriosis in whom a first surgical intervention (L1) was indicated, but the likelihood and benefit of a second-look laparoscopy (L2) was high owing to the risk of adhesion formation. Informed consents from prospective subjects (n ¼ 10) were obtained before their inclusion in the study. Additional inclusion criteria were: 1) open fallopian tubes; 2) patients not on any other treatment for endometriosis or family planning before or after L1; 3) body mass index <22 kg/m2; and 4) at least four red endometriotic lesions located in the culde-sac and situated R2 cm away from all other lesions, as diagnosed during L1. These red lesions were labeled index lesions. L1 and L2 were performed by two experienced surgeons using modern laparoscopic equipment, and were recorded by video. During L1, four to six peritoneal red (index) lesions were identified before surgery was initiated. One-half of these index lesions were surgically removed and stored as described below. The remaining onehalf were marked with a nonabsorbable silk sutures at a distance of 1 cm. All nonindex lesions were subsequently removed and discarded. One week after L1, oral administration of quinagolide (Norprolac; Ferring Pharmaceuticals, Madrid, Spain) was titrated, starting at 25 mg/d for the first 15 days, followed by 50 mg/d during the next 15 days and 75 mg/d for the remaining treatment period of 18–20 weeks, to avoid intolerance to the medication. During the 4-month treatment period (i.e., between L1 and L2), patients were monitored on a monthly basis to assess the severity of the side effects of Drd2-A, which include dizziness, nausea, and vomiting (13). During these visits, blood was drawn for subsequent enzyme-linked immunosorbent assay measurement of serum PRL, a marker of the level of Drd2 activity, to monitor compliance with taking the medication. When treatment had terminated, L2 was performed and video recorded. During surgery, the silk sutures were removed and the index lesions that had been untouched in L1 were surgically excised. If significant adhesion formation was detected, lysis of adhesion was performed. The index lesions removed at L1 and L2 were fixed in formalin (75% of each lesion tissue) for histologic analysis and immunohistochemical quantification of vascularization, vessel immaturity, Drd2 expression, cell proliferation, VEGFR2 expression, and VEGFR2 activation. The remaining portion of each lesion tissue (25%) was homogenized in Trizol and cryopreserved at 80 C for subsequent superarray quantitative fluorescence polymerase chain reaction analysis (QF-PCR). In addition, during both L1 and L2, endometrial tissue was obtained to confirm the proliferative phase of the menstrual cycle by histologic methods.
Macroscopic Analysis All laparoscopies were recorded with a Karl Storz (Munich, Germany) video camera. Video recordings were displayed to find illustrative frames of a blunt metal probe with black/gray cap of known size, in perpendicular position in relation to the lesion. Eligible frames were captured and opened with ImagePro Plus version 6.03 (Media Cybernetics, Bethesda, MD) software for image processing. The size of the black/gray cap served as a reference for measuring at L2 the changes in size induced by quinagolide on the area of the lesions left untouched during L1.
Morphologic Studies Tissue samples of active endometriotic lesions were evaluated for histologic hallmarks, including the presence and appearance of glands and stroma, by microscopic analysis of hematoxylin and eosin–stained sections from L1 and L2.
Glostrup, Denmak), Drd2, VEGFR2 (Santa Cruz, Santa Cruz, CA), and VEGFR2Y951, VEGFR2Y1054, and VEGFR2Y1075 (Cell Signaling, Danvers, MA) for each determination. Slides incubated overnight with the primary antibodies were revealed by using the Dako Kit (Dakocytomation). For immunofluorescence purposes, directly labeled primary antibodies or tyramide signal–amplification fluorescein (TSA; Perkin Elmer;) instead of 3,30 -diaminobenzidine was used.
Quantification of Immunohistochemistry For each immunohistochemical parameter evaluated, three noncontiguous slides were photographed in four random high-power (40) fields with a Nikon Eclipse E800 (Nikon, NY) camera and were analyzed using Image-Pro Plus 6.3 analysis software to outline, highlight, and quantify stained areas.
mRNA Analysis by PCR Superarray of Genes Involved in Inflammation and Angiogenesis To evaluate how inflammatory and angiogenic pathways were modulated by quinagolide, we used a commercially available superarray (PAHS-072E-4; SAB Biosciences, Frederick, MD) containing 84 cytokines, quimoquines, growth factors, and inhibitors of angiogenesis. The cDNAs from L1 and L2 index lesions were amplified using the ABI Prism 7900 (Perkin Elmer) with universal QF-PCR conditions. The final results, expressed as n-fold differences normalized to five constitutive housekeeping genes were determined by the 2DDCt method using specific software form SAB Bioscience.
Statistical Analysis Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS, Chicago, IL). Data were expressed as mean SD. A Wilcoxon matched pairs test was used to determine differences in lesion size and PRL levels. A Mann-Whitney test was used for quantitative analysis of immunohistochemistry. Significance was defined to be P<.05.
RESULTS Compliance with Taking Medication and Side Effects Lowered PRL levels 1 month after initiation of quinagolide treatment and subsequent maintenance below baseline levels (<20 ng/mL) provided evidence that patients were taking the medication as prescribed (Supplemental Fig. 1, available online). No major complications were detected except for one patient who discontinued her participation in the study owing to vomiting when the dose of quinagolide was increased to 75 mg/kg/d.
Macroscopic Appearance and Surface Area of Endometriotic Lesions All index endometriotic lesions removed during L1 and those left untouched and recovered during L2 had a red appearance. In two of the nine patients, all index endometriotic lesions were absent during L2 (Figs. 1A–1D), suggesting that quinagolide induced regression of peritoneal endometriotic lesions. Indeed, in three patients some lesions vanished and those that persisted decreased in size (Figs. 1E and 1F). In the remaining four patients, despite all of the index lesions persisting at L2, they had all shrunk in size. Of a total of 23 red index lesions labelled at L1, eight vanished and only 15 were recovered at L2, of which one was larger, one unchanged, and the remaining 13 smaller in size than in L1 (Fig. 2A). Assuming that vanished lesions represent a 100% decrease in size, quinagolide reduced the surface area of index lesions by 69.5% (Fig. 2B)
Immunohistochemistry/Immunofluorescence of L1 and L2 Lesions
Histologic Appearance
Standard immunohistochemistry was performed using specific primary antibodies to CD31 (Abcam, Cambridge, UK), Ki-67 (Dakocytomation,
Index lesions surgically removed during L1 exhibited histologic characteristics typical of peritoneal endometriosis: Glandular tissue
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FIGURE 1 The effect of quinagolide treatment on lesion size in three different patients (A-B, C-D, and E-F). Images in the first column show index lesions left untouched during first laparoscopy (L1). Images in the right column show the appearance of the same index lesions during second-look laparoscopy (L2) after quinagolide administration. The corresponding index lesions or the place where lesions were expected to be found during L2 have also been circled. White arrows point to original L1 lesions which had vanished at L2. Black arrow points to a lesion originally labelled during L1 and whose size has been reduced. Paired images A-B and C-D show two cases in which all index endometriotic samples disappeared after quinagolide treatment. Paired images E-F show a case in which lesions had either vanished or been reduced in size after quinagolide treatment. Blue arrowhead in F points to a silk suture marking an index lesion left untouched during L1.
G omez. Effects of quinagolide on endometriotic lesions. Fertil Steril 2011.
was often circular, with a hollow center with glandular secretions, and the stroma was highly organized with clearly visible fibroblasts and noncellular stroma filling the intercellular space. After quinagolide administration, the typical highly organized gland/stroma pattern had disappeared, with the matrix of the stroma becoming looser and less cellular, thus indicating that a process of tissue regression had been initiated (Figs. 3A–3D).
3J). Signals obtained for VEGFR2-Tyr1054 and VEGFR2Tyr1212 were extremely faint and were not quantified. The reduction in phsosphorylation of VEGFR2-Tyr 951 paralleled that observed for VEGFR2 (almost 50%). Therefore, it is likely that the decreased VEGFR2 phosphorylation was due to reduced VEGFR2 expression.
PCR Array in Endometriotic Lesions Immunohistochemical Evaluation Comparison of L1 and L2 lesions showed no difference in vascular density (Figs. 3E and 3F), quantity of immature vessels, or cell proliferation (data not shown). Drd2 staining was found to be spotty and up-regulated by quinagolide treatment (Figs. 3G and 3H). Surprisingly, Drd2 staining was absent in vessel structures. The density of VEGFR2 was reduced by twofold in L2 lesions (Figs. 3I and
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To assess the pattern of angiogenic and inflammatory pathways modulated by Drd2 activation, we performed a PCR superarray with 16 (L1 ¼ 9; L2 ¼ 7) pooled (per patient) endometriotic cDNA index lesion samples. Statistical analyses showed significant (P<.05) up-regulation of Serpine 1, CCL2, VEGF, AGGF1, and RUNX1 and down-regulation of CXCL10 genes (Table 1) by quinagolide.
Effects of quinagolide on endometriotic lesions
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FIGURE 2 Image-Pro Plus software size measurements of the same endometriotic lesion during the first (L1) and second (L2; after 4 months of quinagolide therapy) laparoscopies. Lesion size area is expressed in mm2. (A) Each of the lesions analyzed during the study period is represented by a case (‘‘serie’’) number. Note that all but two endometriotic lesions undergo a reduction in size or disappear (value 0) after quinagolide treatment. A Wilcoxon paired test was used for statistical comparison: *P< .05. (B) Hatched bar represents mean SD surface area of index lesions left untouched and video recorded during L1. Bricked bar represents mean SD surface area of the same (paired) index lesions after 4 months of quinagolide treatment (L2). Note an overall 69.5% decrease in lesion size after quinagolide treatment. A Mann Whitney test was used for statistical comparison, *P< .05.
A
100
Lesion size before and after quinagolide treatment
Individual lesion area in mm2
90 80 70 60
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50 40 30 20 10 0 L1
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L2
Serie1 Serie2 Serie3 Serie4 Serie5 Serie6 Serie7 Serie8 Serie9 Serie10 Serie11 Serie12 Serie13 Serie14 Serie15 Serie16 Serie17 Serie18 Serie19 Serie20 Serie21 Serie22 Serie23
lesion size before and after quinagolide treatment
Mean lesion area in mm2
50
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G omez. Effects of quinagolide on endometriotic lesions. Fertil Steril 2011.
DISCUSSION Recently we showed for the first time that Drd2-A prevented ovarian hyperstimulaton syndrome in women (14, 15) by blocking VEGFR2–mediated vascular permeability (16). Herein we show for the first Fertility and Sterility
time that peritoneal endometriotic lesions in humans decrease in size or disappear when exposed to prolonged treatment with the Drd2-A quinagolide. Histologic evaluation suggested that quinagolide induced tissue degeneration of endometriotic lesions. In areas
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FIGURE 3 (A–D) Histologic analysis of index lesions obtained during first (L1) and second (L2) laparoscopies. Intermediate-power (20) representative images of index red lesions obtained during L1 (A) and L2 (B). Morphological analysis confirms glandular (purple) presence of endometrium surrounded by dried blood (redish color) typical of red lesions. High-power magnification of L1 untreated (C) and L2 quinagolide-treated (D) index red lesions. Endometriotic lesions in L1 show a typical high cellular stroma and glands well defined and surrounded by peritoneal tissue. In L2 lesions, a lax stroma is observed with a nonprominent glandular epithelium suggesting an atropic or degenerative status of tissue (B). (E–L) Representative paired images of immunohistochemical analysis in index endometritic lesions removed before (L1; left column) and left behind and recovered after (L2; middle column) quinagolide treatment.. Vascularizaton (vessel density) was assayed by immunostaining against CD31 antigen (brown color) in blood vessels (E, F). Repesentative images (G, H) of dopamine receptor 2 (Drd2; brown staining). Note the significant increase in brown staining after quinagolide treatment in H vs. G. Broad expression of vascular endothelial growth factor receptor 2 (VEGFR2) (I, J) staining (brown staining) was detected in both untreated and treated lesions. A slight decrease in VEGFR2 levels is observed in treated vs. untreated lesion. An antibody recognizing VEGFR2 specifically phosphorylated at tyrosine site 951 reveals faint antigen expression (brown color) before (K) and after (L) quinagolide treatment. The right column shows corresponding quantitative analyses of the stained areas in the row pair. Positive-stained area for each immunohistochemical parameter was outlined by setting a background noise level automatically with Image-Pro Plus. Area of interest was subsequently highlighted, quantified, compared with the total tissue, and expressed as percentage stained area. Results represent the ratio of stained/total area multiplied by 100. A Mann-Whitney test was used for comparisions, *P< .05.
G omez. Effects of quinagolide on endometriotic lesions. Fertil Steril 2011.
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TABLE 1 Statistical analysis using SAB Bioscience software to identify genes significantly deregulated by quinagolide in cDNA from L1 and L2 index endometriotic samples after quantitative fluorescence polymerase chain reaction amplification using superarray (PAHS-072E-4) plates.
Symbol SERPINE1 AGGF1 CCL2 VEGF CCL10 RUNX1 CXCL12 BTG1 FST RHOB FGFBP1 FGF1 SERPINF1 STAB
P value vs. control
Fold regulation vs. control
.03254a .0376a .04389a .04428a .04770a .04901a .08206 .086207 .10483 .12902 .15187 .165346 .183201 .19273
19.8491 3.1954 6.1199 5.8199 6.7693 4.3878 3.3952 3.3776 3.0136 1.7303 3.0283 2.5955 1.9339 5.4869
Note: The table shows a list of the 14 genes most likely to be deregulated among the 84 angiogenic and inflammatory citokines included in the kit. Gene candidates are sorted according to their P values (middle column). A lower P value indicates a higher probability that the deregulaton observed is not due to random variation. The right column shows the down-regulation (negative values) and upregulation (positive values) of genes in L2 (quinagolide-treated index lesion) vs L1 (index lesions not treated with quinagolide). a P< .05. G omez. Effects of quinagolide on endometriotic lesions. Fertil Steril 2011.
where the lesions had disappeared, the normal peritoneal tissue organisation had been reestablished. Endometriosis is a progressive disease (17) which deteriorates significantly over a short period of time in R50% of patients (18). In the absence of medical intervention, mature endometriotic lesions such as those identified in the present study are expected to increase or maintain their size over a 6-month period (18). Therefore, medication-naive endometriotic lesions removed during L1 are comparable to hypothetically placebo-exposed lesions removed during L2. Given that this was a proof-of-concept study, the nine patients eventually included were sufficient for achieving its goal and will serve as groundwork for a larger clinical trial. Based on findings in animal models (9, 12, 16), we expected that quinagolide would up-regulate Drd2 in endothelial cells, leading to decreased VEGFR2 phosphorylation in an autocrine fashion, thereby reducing vessel proliferation and density. Indeed, quinagolide increased Drd2 expression and decreased VEGF/VEGFR2 expression, but it did not reduce vessel density in L2 lesions. We do not rule out that, by taking place in vanished/regressed tissue, the antiangiogenic actions exerted by Drd2-A might have been masked. In fact a very recent report on the successful treatment of rectal endometriosis (19) supports our hypothesis that Drd2-A manifests its antiangiogenic effect by acting via VEGFR2. We speculate that the effects of quinagolide on ectopic lesion size may be mediated by different molecular mechanisms from those suggested by our heterologous nude mice model (12), who lack an Fertility and Sterility
immune system and where human endometrial vessels are replaced by host endothelial cells. Endometriotic lesions contain mature vessels not expected to be susceptible to the antiproliferative effects of antiangiogenic therapy, so additional effects must be exerted by quinagolide to induce shrinking and vanishing of peritoneal lesions. Recent studies suggest that even mature endometriotic vessels are somehow different to those present in the eutopic endometrium, and thus can be selectively disrupted by targeting aberrantly expressed tissue factor in their endothelium (20). Because endometriotic vessels lacked Drd2, a hypothetical selective disruption of pathologic mature vessels by quinagolide would be mediated by paracrine mechanisms. Most immune cell subtypes express Drd2 (21), and among these, macrophages are known to express large amounts of VEGF (6). Drd2 activation decreases VEGF production (22) and VEGF-mediated angiogenesis (8), and the ability of macrophages to reduce angiogenesis when activated by dopamine has recently been described (23). Therefore, we suggest that the paracrine cross-talk between Drd2 and the VEGF/VEGFR2 system and the additional effects exerted by quinagolide in the present study may be mediated by macrophages. In this regard, Drd2 activation in macrophages is known to increase their phagocytic activity (24). In addition, our PCR arrays revealed that PAI-1, a potent inhibitor of fibrinolysis, was the most down-regulated gene in L2 lesions. Therefore, we hypothesize that, by promoting selective fibronolytic activity, macrophages destabilize the peritoneal lesions scaffolding when activated by quinagolide. The composition of peritoneal fluid (25) and endometriotic lesions (26) suggests that inflammatory pathways involving macrophages are highly up-regulated during endometriosis. A role for inflammatory angiogenesis is supported by our finding that the cytokine CXCL10, a potent antiangiogenic regulator (27), was up-regulated by quinagolide. In addition, we also found that three proangiogenic cytokines (CCL2, RUNX1 and AGGF1 (28–30) and VEGF (25), also a potent chemoattractor (31)) were down-regulated in L2 lesions. This body of evidence suggests that a major mechanism of action of quinagolide consists of reverting a proangiogenic environment associated with untreated lesions into an antiangiogenic/inflammatory environment by affecting the inflammatory process. Because their different mechanism of action, Drd2-A are not expected to interfere with the menstrual cycle, offering an important advantage over medication such as GnRH agonists and aromatase inhibitors, which induce osteoporosis and hot flashes by creating a hypoestrogenic milieu (32, 33), in the treatment of women who are infertile due to the disease. Nevertheless, in vitro experiments showed that cabergoline blocks P4 production by granulose cells, suggesting that Drd2-A might exert an aromatase inhibitory action (34). Moreover, PRL deprivation may affect reproductive functions by inhibiting intermittent GnRH (35). Thus, by affecting the hypothalamic-pituitary axis, Drd2-A might influence endometriotic lesions by mechanisms other than the ones suggested in the present study. Despite the good safety profile of Drd2-A, as has been demonstrated in women treated for prolactinomas (36), potential side effects should be reduced by using alternative routes of administration, such as vaginal delivery of the drug. In the future, a combination of drugs can be the treatment of choice in endometriosis patients. Quinagolide is a nonergot-derived Drd2-A which lacks an effect on the serotonin (5-hydroxytryptamine [5-HT]) receptor subtype 2b at relevant concentrations. This may be clinically important, because stimulation of 5-HT2b receptors in cardiac valve tissue can lead to proliferation of fibroblasts (37). Drd2-A are also
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differentiated by their pharmacokinetic profile; quinagolide has a much shorter half-life (17 hours) than Cb2 (63–69 hours) (38), thus minimizing exposure during organogenesis in women who may wish to become pregnant. In summary, this is the first study in humans that shows unequivocally that the Drd2-A quinagolide reverses the size of endometriotic
lesions, causing some to completely disappear. The long-term side effect profile of dopamine agonists is favorable over the antisteroidogenic agents. Before adding this very promising type of medication to the armamentarium for the treatment of endometriosis, larger clinical trials need to be conducted to confirm our results and investigate in more depth its mechanism of action.
REFERENCES 1. Ozkan S, Arici A. Advances in treatment options of endometriosis. Gynecol Obstet Invest 2009;67: 81–91. 2. Mihalyi A, Simsa P, Mutinda KC, Meuleman C, Mwenda JM. d’Hooghe TM. Emerging drugs in endometriosis. Expert Opin Emerg Drugs 2006;11: 503–24. 3. Bruner-Tran KL, Osteen KG, Duleba AJ. Simvastatin protects against the development of endometriosis in a nude mouse model. J Clin Endocrinol Metab 2009;94:2489–94. 4. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, et al. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996;81:3112–8. 5. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, M€ uller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. 6. Nap AW, Griffioen AW, Dunselman GA, Bouma–ter Steege JC, Thijssen VL, Evers JL, et al. Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 2004;89:1089–95. 7. Eremina V, Jefferson JA, Kowalewska J, Hochster H, Haas M, Weisstuch J, et al. VEGF inhibition and renal thrombotic microangiopathy. N Engl J Med 2008; 358:1129–36. 8. Basu S, Sarkar C, Chakroborty D, Nagy J, Mitra RB, Dasgupta PS, et al. Ablation of peripheral dopaminergic nerves stimulates malignant tumor growth by inducing vascular permeability factor/vascular endothelial growth factor–mediated angiogenesis. Cancer Res 2004;64:5551–5. 9. Basu S, Nagy JA, Pal S, Vasile E, Eckelhoefer IA, Bliss VS. The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor. Nat Med 2001;7:569–74. 10. Robert E, Musatti L, Piscitelli G, Ferrari CI. Pregnancy outcome after treatment with the ergot derivative, cabergoline. Reprod Toxicol 1996;10:333–7. 11. Ricci E, Parazzini F, Motta T, Ferrari CI, Colao A, Clavenna A, et al. Pregnancy outcome after cabergoline treatment in early weeks of gestation. Reprod Toxicol 2002;16:791–3. 12. Novella-Maestre E, Carda C, Noguera I, Ruiz-Saurı A, Garcia-Velasco JA, Simon C, et al. Dopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis. Hum Reprod 2009; 24:1025–35. 13. Vance ML, Cragun JR, Reimnitz C, Chang RJ, Rashef E, Blackwell RE. CV 205-502 treatment of hyperprolactinemia. J Clin Endocrinol Metab 1989; 8:336–9.
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14. Alvarez C, Martı-Bonmatı L, Novella-Maestre E, Sanz R, Gomez R, Fernandez-Sanchez M, et al. Dopamine agonist cabergoline reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted reproduction. J Clin Endocrinol Metab 2007;92:2931–7. 15. Busso C, Fernandez-Sanchez M, Garcıa-Velasco JA, Landeras J, Ballesteros A, Mu~noz E, et al. The nonergot derived dopamine agonist quinagolide in prevention of early ovarian hyperstimulation syndrome in IVF patients: a randomized, double-blind, placebocontrolled trial. Hum Reprod 2010;25:995–1004. 16. Gomez R, Gonzalez-Izquierdo M, Zimmermann RC, Novella-Maestre E, Alonso-Muriel I, SanchezCriado J, et al. Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)–mediated vascular hyperpermeability without altering VEGF receptor 2–dependent luteal angiogenesis in a rat ovarian hyperstimulation model. Endocrinology 2006;147:5400–11. 17. Hoshiai H, Ishikawa M, Sawatari Y, Noda K, Fukaya T. Laparoscopic evaluation of the onset and progression of endometriosis. Am J Obstet Gynecol 1993;169:714–9. 18. Thomas EJ, Cooke ID. Impact of gestrinone on the course of asymptomatic endometriosis. Br Med J 1987;294:272–4. 19. Kurt M, Ibis M, Karaman K, Bostanci EB, Temucin T. Rectal endometriosis and prolactinoma. Hum Reprod 2010;25:1588–9. 20. Krikun G, Hu Z, Osteen K, Bruner-Tran KL, Schatz F, Taylor HS, et al. The immunoconjugate ‘‘icon’’ targets aberrantly expressed endothelial tissue factor causing regression of endometriosis. Am J Pathol 2010;176:1050–6. 21. McKenna F, McLaughlin PJ, Lewis BJ, Sibbring GC, Cummerson JA, Bowen-Jones D, et al. Dopamine receptor expression on human T- and B-lymphocytes, monocytes, neutrophils, eosinophils and NK cells: a flow cytometric study. J Neuroinmunol 2002; 132:34–40. 22. Cristina C, Dıaz-Torga G, Baldi A, Gongora A, Rubinstein M, Low MJ, et al. Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice. Endocrinology 2005;146:2952–62. 23. Chakroborty D, Chowdhury UR, Sarkar C, Baral R, Dasgupta PS, Basu S. Dopamine regulates endothelial progenitor cell mobilization from mouse bone marrow in tumor vascularization. J Clin Invest 2008;118:1380–9. 24. Sookhai S, Wang JH, McCourt M, O’Connell D, Redmond HP. Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism. Surgery 1999;126:314–22. 25. Oral E, Olive DL, Arici A. The peritoneal environment in endometriosis. Hum Reprod Update 1996; 2:385–98.
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26. Kyama CM, Mihalyi A, Simsa P, Falconer H, Fulop V, Mwenda J, et al. Role of cytokines in the endometrialperitoneal cross-talk and development of endometriosis. Front Biosci 2009;1:444–54. 27. Yoshino O, Osuga Y, Koga K, Hirota Y, Tsutsumi O, Yano T, et al. Concentrations of interferon-gamma– induced protein-10 (IP-10), an antiangiogenic substance, are decreased in peritoneal fluid of women with advanced endometriosis. Am J Reprod Immunol 2003;50:60–5. 28. Ulukus M, Ulukus EC, Tavmergen Goker EN, Tavmergen E, Zheng W, Arici A. Expression of interleukin-8 and monocyte chemotactic protein 1 in women with endometriosis. Fertil Steril 2009;91: 687–93. 29. Konno R, Fujiwara H, Netsu S, Odagiri K, Shimane M, Nomura H, et al. Gene expression profiling of the rat endometriosis model. Am J Reprod Immunol 2007;58:330–43. 30. Tian XL, Kadaba R, You SA, Liu M, Timur AA, Yang L, et al. Identification of an angiogenic factor that when mutated causes susceptibility to KlippelTrenaunay syndrome. Nature 2004;427:640–5. 31. Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, et al. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 1990;172: 1535–45. 32. Gregoriou O, Konidaris S, Vitoratos N, Papadias C, Papoulias I, Chryssicopoulos A. Gonadotropinreleasing hormone analogue plus hormone replacement therapy for the treatment of endometriosis: a randomized controlled trial. Int J Fertil Womens Med 1997;42:406–11. 33. Walch K, Unfried G, Huber J, Kurz C, van Trotsenburg M, Pernicka E. Implanon versus medroxyprogesterone acetate: effects on pain scores in patients with symptomatic endometriosis—a pilot study. Contraception 2009;79:29–34. 34. Negishi H, Furuta I, Ushigoe K, Fujimoto S, Koide SS. In vitro production of progesterone and estradiol by rat granulosa cells regulated by cabergoline and prolactin. Endocr Res 1998;24:195–203. 35. Sauder SE, Frager M, Case GD, Kelch RP, Marshall JC. Abnormal patterns of pulsatile luteinizing hormone secretion in women with hyperprolactinemia and amenorrhea: responses to bromocriptine. J Clin Endocrinol Metab 1984;59:941–8. 36. Bevan JS, Webster J, Burke CW, Scanlon MF. Dopamine agonists and pituitary tumor shrinkage. Endocr Rev 1992;13:220–40. 37. Roth BL. Drugs and valvular heart disease. N Engl J Med 2007;356:6–7. 38. Colao A, di Sarno A, Pivonello R, di Somma C, Lombardi G. Dopamine receptor agonists for treating prolactinomas. Expert Opin Investig Drugs 2002;11: 787–800.
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SUPPLEMENTAL FIGURE 1 Prolactin levels as measured by enzyme-linked immunoassay in blood serum obtained from our nine parients at different time points during the quinagolide treatment. Patients started daily quinagolide administration 1 week after surgery (QX) and continued for 4 months. Pre-QX ¼ blood samples taken with first laparoscopy just before starting the quinagolide treatment. Post-Qx 1 month ¼ blood samples taken during the first monthly visit after 1 month of quinagolide administration. Post-QX 3 months ¼ blood samples taken during the third monthly visit after 3 months of quinagolide administration. Prolactin levels were found to be <20 ng/mL in all cases after 1 month of quinagolide treatment and continued below that baseline during treatment. Wilcoxon paired test was for statiscal comparision: ** P< .01 compared with Pre-QX.
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Instituto Universitario Instituto Valenciano de Infertilidad, Valencia University School of Medicine and (Fundacion) Investigaci on Clınico Valencia; b Hospital Universitario Dr. Peset; and c Hospital Universitario La Fe, Valencia, Spain
Objective: To assess whether dopamine receptor 2 agonists reduced the size of peritoneal lesions in women with endometriosis and elucidate whether affectation of vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2)–dependent angiogenesis was mediating the observed effects. Design: Proof-of-concept study. Setting: University hospital and a university-affiliated private IVF research center. Patient(s): Hyperprolactinemic patients (n ¼ 9) with endometriosis requiring a first surgical intervention (L1) and benefiting from a second-look laparoscopy (L2) were evaluated. Intervention(s): During L1, four to six peritoneal red lesions were identified. One-half of the lesions were removed and the remaining one-half were labeled with silk knot sutures. After L1, quinagolide was administered in a titrated manner (25–75 mg/d) for 18–20 weeks. During L2, the remaining lesions were surgically excised. Main Outcome Measure(s): Both L1 and L2 were video recorded to compare the effects of quinagolide treatment on lesion size. Lesions removed at L1 and L2 were compared by means of: 1) histologic analysis; 2) immunohistochemical quantitative analysis of angiogenesis; and 3) quantitative fluorescence polymerase chain reaction array analysis of 84 chemokines and pro-/antiangiogenic molecules. Result(s): Quinagolide induced a 69.5% reduction in the size of the lesions, with 35% vanishing completely. Histologic analysis showed tissue degeneration, which was supported by down-regulation of VEGF/VEGFR2, three proangiogenic cytokines (CCL2, RUNX1, and AGGF1) and plasminogen activator inhibitor (PAI) 1, a potent inhibitor of fibrinolysis in the L2 lesions. Conclusion(s): By interfering with angiogenesis, enhancing fibrinolysis, and reducing inflammation, quinagolide reduces or eliminates peritoneal endometriotic lesions in women with endometriosis. (Fertil Steril 2011;95:882–8. 2011 by American Society for Reproductive Medicine.) Key Words: Endometriosis, endometrium, dopamine, dopamine agonists, quinagolide, angiogenesis, dopamine receptor 2, VEGF, VEGF receptor 2
Current treatment of endometriosis consists of surgical removal of diseased tissue and/or hormonal manipulation to create a hypoestrogenic milieu (GnRH agonist, aromatase inhibitors) (1). Unfortunately, the success of this treatment is often limited, and recurrence of the disease is very common (1). Therefore, new approaches to its treatment are necessary (2, 3).
Received May 2, 2010; revised September 10, 2010; accepted October 13, 2010; published online November 5, 2010. R.G. has nothing to disclose. A.A. has nothing to disclose. F.D. has nothing to disclose. S.T. has nothing to disclose. C.S. has conducted clinical research sponsored by Ferring. A.P. has conducted clinical research sponsored by Organon, Schering-Plough, Serono, Merck Serono, and Ferring Pharmaceuticals. The first two authors contributed equally to this work. Clinicaltrials.gov identifier: NCT 00625950. Supported by grant SAF2007–65334 from the Spanish Government, a Lilly Foundation Grant for Research in Clinical Medicine, and Ferring Pharmaceuticals, Copenhagen, Denmark. mez, Ph.D., Instituto Valenciano de Infertilidad, Reprint requests: Raul Go Plaza de la Policıa Local, 3, 46015, Valencia, Spain (E-mail: apellicer@ ivi.es).
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Analysis of endometriotic lesions has demonstrated that a prerequisite for their formation and maintenance is the development of an adequate blood supply (4). The regulation of the ‘‘endometriotic vasculature’’ involves canonic pathways of angiogenesis, including vascular endothelial growth factor (VEGF) acting through the VEGF receptor 2 (VEGFR2) (5). Several antiangiogenic agents that target this pathway have been successfully used in animal models of endometriosis (6). However, these agents can induce severe side effects (7), preventing their use for treating endometriosis in young and otherwise healthy women. We theorized that dopamine receptor 2 agonists (Drd2-A) might be an alternative to commercial antiangiogenic agents. In animal models, Drd2-A inhibit pathologic angiogenesis in tumors (8) by inactivating VEGFR2 signaling (9). However, this type of medication has an acceptable safety profile and does not interfere with the normal establishment and progression of pregnancy (10, 11). We previously demonstrated in a well established experimental endometriosis model that cabergoline prevented the formation of typical endometriosis lesions by inhibiting angiogenesis (12). These results encouraged us to perform a pilot study to evaluate the efficacy of Drd2-A in the treatment of peritoneal endometriosis in humans.
Fertility and Sterility Vol. 95, No. 3, March 1, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.10.024
MATERIALS AND METHODS Human Subjects and Study Design The study was designed as a proof-of-concept pilot study and approved by the ETHICS COmmittee of Hospital Universitario Dr. Peset. We were requested to involve only patients newly diagnosed with hyperprolactinemia (PRL >30 ng/mL), who were simultaneously suffering from severe endometriosis in whom a first surgical intervention (L1) was indicated, but the likelihood and benefit of a second-look laparoscopy (L2) was high owing to the risk of adhesion formation. Informed consents from prospective subjects (n ¼ 10) were obtained before their inclusion in the study. Additional inclusion criteria were: 1) open fallopian tubes; 2) patients not on any other treatment for endometriosis or family planning before or after L1; 3) body mass index <22 kg/m2; and 4) at least four red endometriotic lesions located in the culde-sac and situated R2 cm away from all other lesions, as diagnosed during L1. These red lesions were labeled index lesions. L1 and L2 were performed by two experienced surgeons using modern laparoscopic equipment, and were recorded by video. During L1, four to six peritoneal red (index) lesions were identified before surgery was initiated. One-half of these index lesions were surgically removed and stored as described below. The remaining onehalf were marked with a nonabsorbable silk sutures at a distance of 1 cm. All nonindex lesions were subsequently removed and discarded. One week after L1, oral administration of quinagolide (Norprolac; Ferring Pharmaceuticals, Madrid, Spain) was titrated, starting at 25 mg/d for the first 15 days, followed by 50 mg/d during the next 15 days and 75 mg/d for the remaining treatment period of 18–20 weeks, to avoid intolerance to the medication. During the 4-month treatment period (i.e., between L1 and L2), patients were monitored on a monthly basis to assess the severity of the side effects of Drd2-A, which include dizziness, nausea, and vomiting (13). During these visits, blood was drawn for subsequent enzyme-linked immunosorbent assay measurement of serum PRL, a marker of the level of Drd2 activity, to monitor compliance with taking the medication. When treatment had terminated, L2 was performed and video recorded. During surgery, the silk sutures were removed and the index lesions that had been untouched in L1 were surgically excised. If significant adhesion formation was detected, lysis of adhesion was performed. The index lesions removed at L1 and L2 were fixed in formalin (75% of each lesion tissue) for histologic analysis and immunohistochemical quantification of vascularization, vessel immaturity, Drd2 expression, cell proliferation, VEGFR2 expression, and VEGFR2 activation. The remaining portion of each lesion tissue (25%) was homogenized in Trizol and cryopreserved at 80 C for subsequent superarray quantitative fluorescence polymerase chain reaction analysis (QF-PCR). In addition, during both L1 and L2, endometrial tissue was obtained to confirm the proliferative phase of the menstrual cycle by histologic methods.
Macroscopic Analysis All laparoscopies were recorded with a Karl Storz (Munich, Germany) video camera. Video recordings were displayed to find illustrative frames of a blunt metal probe with black/gray cap of known size, in perpendicular position in relation to the lesion. Eligible frames were captured and opened with ImagePro Plus version 6.03 (Media Cybernetics, Bethesda, MD) software for image processing. The size of the black/gray cap served as a reference for measuring at L2 the changes in size induced by quinagolide on the area of the lesions left untouched during L1.
Morphologic Studies Tissue samples of active endometriotic lesions were evaluated for histologic hallmarks, including the presence and appearance of glands and stroma, by microscopic analysis of hematoxylin and eosin–stained sections from L1 and L2.
Glostrup, Denmak), Drd2, VEGFR2 (Santa Cruz, Santa Cruz, CA), and VEGFR2Y951, VEGFR2Y1054, and VEGFR2Y1075 (Cell Signaling, Danvers, MA) for each determination. Slides incubated overnight with the primary antibodies were revealed by using the Dako Kit (Dakocytomation). For immunofluorescence purposes, directly labeled primary antibodies or tyramide signal–amplification fluorescein (TSA; Perkin Elmer;) instead of 3,30 -diaminobenzidine was used.
Quantification of Immunohistochemistry For each immunohistochemical parameter evaluated, three noncontiguous slides were photographed in four random high-power (40) fields with a Nikon Eclipse E800 (Nikon, NY) camera and were analyzed using Image-Pro Plus 6.3 analysis software to outline, highlight, and quantify stained areas.
mRNA Analysis by PCR Superarray of Genes Involved in Inflammation and Angiogenesis To evaluate how inflammatory and angiogenic pathways were modulated by quinagolide, we used a commercially available superarray (PAHS-072E-4; SAB Biosciences, Frederick, MD) containing 84 cytokines, quimoquines, growth factors, and inhibitors of angiogenesis. The cDNAs from L1 and L2 index lesions were amplified using the ABI Prism 7900 (Perkin Elmer) with universal QF-PCR conditions. The final results, expressed as n-fold differences normalized to five constitutive housekeeping genes were determined by the 2DDCt method using specific software form SAB Bioscience.
Statistical Analysis Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS, Chicago, IL). Data were expressed as mean SD. A Wilcoxon matched pairs test was used to determine differences in lesion size and PRL levels. A Mann-Whitney test was used for quantitative analysis of immunohistochemistry. Significance was defined to be P<.05.
RESULTS Compliance with Taking Medication and Side Effects Lowered PRL levels 1 month after initiation of quinagolide treatment and subsequent maintenance below baseline levels (<20 ng/mL) provided evidence that patients were taking the medication as prescribed (Supplemental Fig. 1, available online). No major complications were detected except for one patient who discontinued her participation in the study owing to vomiting when the dose of quinagolide was increased to 75 mg/kg/d.
Macroscopic Appearance and Surface Area of Endometriotic Lesions All index endometriotic lesions removed during L1 and those left untouched and recovered during L2 had a red appearance. In two of the nine patients, all index endometriotic lesions were absent during L2 (Figs. 1A–1D), suggesting that quinagolide induced regression of peritoneal endometriotic lesions. Indeed, in three patients some lesions vanished and those that persisted decreased in size (Figs. 1E and 1F). In the remaining four patients, despite all of the index lesions persisting at L2, they had all shrunk in size. Of a total of 23 red index lesions labelled at L1, eight vanished and only 15 were recovered at L2, of which one was larger, one unchanged, and the remaining 13 smaller in size than in L1 (Fig. 2A). Assuming that vanished lesions represent a 100% decrease in size, quinagolide reduced the surface area of index lesions by 69.5% (Fig. 2B)
Immunohistochemistry/Immunofluorescence of L1 and L2 Lesions
Histologic Appearance
Standard immunohistochemistry was performed using specific primary antibodies to CD31 (Abcam, Cambridge, UK), Ki-67 (Dakocytomation,
Index lesions surgically removed during L1 exhibited histologic characteristics typical of peritoneal endometriosis: Glandular tissue
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FIGURE 1 The effect of quinagolide treatment on lesion size in three different patients (A-B, C-D, and E-F). Images in the first column show index lesions left untouched during first laparoscopy (L1). Images in the right column show the appearance of the same index lesions during second-look laparoscopy (L2) after quinagolide administration. The corresponding index lesions or the place where lesions were expected to be found during L2 have also been circled. White arrows point to original L1 lesions which had vanished at L2. Black arrow points to a lesion originally labelled during L1 and whose size has been reduced. Paired images A-B and C-D show two cases in which all index endometriotic samples disappeared after quinagolide treatment. Paired images E-F show a case in which lesions had either vanished or been reduced in size after quinagolide treatment. Blue arrowhead in F points to a silk suture marking an index lesion left untouched during L1.
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was often circular, with a hollow center with glandular secretions, and the stroma was highly organized with clearly visible fibroblasts and noncellular stroma filling the intercellular space. After quinagolide administration, the typical highly organized gland/stroma pattern had disappeared, with the matrix of the stroma becoming looser and less cellular, thus indicating that a process of tissue regression had been initiated (Figs. 3A–3D).
3J). Signals obtained for VEGFR2-Tyr1054 and VEGFR2Tyr1212 were extremely faint and were not quantified. The reduction in phsosphorylation of VEGFR2-Tyr 951 paralleled that observed for VEGFR2 (almost 50%). Therefore, it is likely that the decreased VEGFR2 phosphorylation was due to reduced VEGFR2 expression.
PCR Array in Endometriotic Lesions Immunohistochemical Evaluation Comparison of L1 and L2 lesions showed no difference in vascular density (Figs. 3E and 3F), quantity of immature vessels, or cell proliferation (data not shown). Drd2 staining was found to be spotty and up-regulated by quinagolide treatment (Figs. 3G and 3H). Surprisingly, Drd2 staining was absent in vessel structures. The density of VEGFR2 was reduced by twofold in L2 lesions (Figs. 3I and
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To assess the pattern of angiogenic and inflammatory pathways modulated by Drd2 activation, we performed a PCR superarray with 16 (L1 ¼ 9; L2 ¼ 7) pooled (per patient) endometriotic cDNA index lesion samples. Statistical analyses showed significant (P<.05) up-regulation of Serpine 1, CCL2, VEGF, AGGF1, and RUNX1 and down-regulation of CXCL10 genes (Table 1) by quinagolide.
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FIGURE 2 Image-Pro Plus software size measurements of the same endometriotic lesion during the first (L1) and second (L2; after 4 months of quinagolide therapy) laparoscopies. Lesion size area is expressed in mm2. (A) Each of the lesions analyzed during the study period is represented by a case (‘‘serie’’) number. Note that all but two endometriotic lesions undergo a reduction in size or disappear (value 0) after quinagolide treatment. A Wilcoxon paired test was used for statistical comparison: *P< .05. (B) Hatched bar represents mean SD surface area of index lesions left untouched and video recorded during L1. Bricked bar represents mean SD surface area of the same (paired) index lesions after 4 months of quinagolide treatment (L2). Note an overall 69.5% decrease in lesion size after quinagolide treatment. A Mann Whitney test was used for statistical comparison, *P< .05.
A
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Lesion size before and after quinagolide treatment
Individual lesion area in mm2
90 80 70 60
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B
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Serie1 Serie2 Serie3 Serie4 Serie5 Serie6 Serie7 Serie8 Serie9 Serie10 Serie11 Serie12 Serie13 Serie14 Serie15 Serie16 Serie17 Serie18 Serie19 Serie20 Serie21 Serie22 Serie23
lesion size before and after quinagolide treatment
Mean lesion area in mm2
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10
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DISCUSSION Recently we showed for the first time that Drd2-A prevented ovarian hyperstimulaton syndrome in women (14, 15) by blocking VEGFR2–mediated vascular permeability (16). Herein we show for the first Fertility and Sterility
time that peritoneal endometriotic lesions in humans decrease in size or disappear when exposed to prolonged treatment with the Drd2-A quinagolide. Histologic evaluation suggested that quinagolide induced tissue degeneration of endometriotic lesions. In areas
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FIGURE 3 (A–D) Histologic analysis of index lesions obtained during first (L1) and second (L2) laparoscopies. Intermediate-power (20) representative images of index red lesions obtained during L1 (A) and L2 (B). Morphological analysis confirms glandular (purple) presence of endometrium surrounded by dried blood (redish color) typical of red lesions. High-power magnification of L1 untreated (C) and L2 quinagolide-treated (D) index red lesions. Endometriotic lesions in L1 show a typical high cellular stroma and glands well defined and surrounded by peritoneal tissue. In L2 lesions, a lax stroma is observed with a nonprominent glandular epithelium suggesting an atropic or degenerative status of tissue (B). (E–L) Representative paired images of immunohistochemical analysis in index endometritic lesions removed before (L1; left column) and left behind and recovered after (L2; middle column) quinagolide treatment.. Vascularizaton (vessel density) was assayed by immunostaining against CD31 antigen (brown color) in blood vessels (E, F). Repesentative images (G, H) of dopamine receptor 2 (Drd2; brown staining). Note the significant increase in brown staining after quinagolide treatment in H vs. G. Broad expression of vascular endothelial growth factor receptor 2 (VEGFR2) (I, J) staining (brown staining) was detected in both untreated and treated lesions. A slight decrease in VEGFR2 levels is observed in treated vs. untreated lesion. An antibody recognizing VEGFR2 specifically phosphorylated at tyrosine site 951 reveals faint antigen expression (brown color) before (K) and after (L) quinagolide treatment. The right column shows corresponding quantitative analyses of the stained areas in the row pair. Positive-stained area for each immunohistochemical parameter was outlined by setting a background noise level automatically with Image-Pro Plus. Area of interest was subsequently highlighted, quantified, compared with the total tissue, and expressed as percentage stained area. Results represent the ratio of stained/total area multiplied by 100. A Mann-Whitney test was used for comparisions, *P< .05.
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TABLE 1 Statistical analysis using SAB Bioscience software to identify genes significantly deregulated by quinagolide in cDNA from L1 and L2 index endometriotic samples after quantitative fluorescence polymerase chain reaction amplification using superarray (PAHS-072E-4) plates.
Symbol SERPINE1 AGGF1 CCL2 VEGF CCL10 RUNX1 CXCL12 BTG1 FST RHOB FGFBP1 FGF1 SERPINF1 STAB
P value vs. control
Fold regulation vs. control
.03254a .0376a .04389a .04428a .04770a .04901a .08206 .086207 .10483 .12902 .15187 .165346 .183201 .19273
19.8491 3.1954 6.1199 5.8199 6.7693 4.3878 3.3952 3.3776 3.0136 1.7303 3.0283 2.5955 1.9339 5.4869
Note: The table shows a list of the 14 genes most likely to be deregulated among the 84 angiogenic and inflammatory citokines included in the kit. Gene candidates are sorted according to their P values (middle column). A lower P value indicates a higher probability that the deregulaton observed is not due to random variation. The right column shows the down-regulation (negative values) and upregulation (positive values) of genes in L2 (quinagolide-treated index lesion) vs L1 (index lesions not treated with quinagolide). a P< .05. G omez. Effects of quinagolide on endometriotic lesions. Fertil Steril 2011.
where the lesions had disappeared, the normal peritoneal tissue organisation had been reestablished. Endometriosis is a progressive disease (17) which deteriorates significantly over a short period of time in R50% of patients (18). In the absence of medical intervention, mature endometriotic lesions such as those identified in the present study are expected to increase or maintain their size over a 6-month period (18). Therefore, medication-naive endometriotic lesions removed during L1 are comparable to hypothetically placebo-exposed lesions removed during L2. Given that this was a proof-of-concept study, the nine patients eventually included were sufficient for achieving its goal and will serve as groundwork for a larger clinical trial. Based on findings in animal models (9, 12, 16), we expected that quinagolide would up-regulate Drd2 in endothelial cells, leading to decreased VEGFR2 phosphorylation in an autocrine fashion, thereby reducing vessel proliferation and density. Indeed, quinagolide increased Drd2 expression and decreased VEGF/VEGFR2 expression, but it did not reduce vessel density in L2 lesions. We do not rule out that, by taking place in vanished/regressed tissue, the antiangiogenic actions exerted by Drd2-A might have been masked. In fact a very recent report on the successful treatment of rectal endometriosis (19) supports our hypothesis that Drd2-A manifests its antiangiogenic effect by acting via VEGFR2. We speculate that the effects of quinagolide on ectopic lesion size may be mediated by different molecular mechanisms from those suggested by our heterologous nude mice model (12), who lack an Fertility and Sterility
immune system and where human endometrial vessels are replaced by host endothelial cells. Endometriotic lesions contain mature vessels not expected to be susceptible to the antiproliferative effects of antiangiogenic therapy, so additional effects must be exerted by quinagolide to induce shrinking and vanishing of peritoneal lesions. Recent studies suggest that even mature endometriotic vessels are somehow different to those present in the eutopic endometrium, and thus can be selectively disrupted by targeting aberrantly expressed tissue factor in their endothelium (20). Because endometriotic vessels lacked Drd2, a hypothetical selective disruption of pathologic mature vessels by quinagolide would be mediated by paracrine mechanisms. Most immune cell subtypes express Drd2 (21), and among these, macrophages are known to express large amounts of VEGF (6). Drd2 activation decreases VEGF production (22) and VEGF-mediated angiogenesis (8), and the ability of macrophages to reduce angiogenesis when activated by dopamine has recently been described (23). Therefore, we suggest that the paracrine cross-talk between Drd2 and the VEGF/VEGFR2 system and the additional effects exerted by quinagolide in the present study may be mediated by macrophages. In this regard, Drd2 activation in macrophages is known to increase their phagocytic activity (24). In addition, our PCR arrays revealed that PAI-1, a potent inhibitor of fibrinolysis, was the most down-regulated gene in L2 lesions. Therefore, we hypothesize that, by promoting selective fibronolytic activity, macrophages destabilize the peritoneal lesions scaffolding when activated by quinagolide. The composition of peritoneal fluid (25) and endometriotic lesions (26) suggests that inflammatory pathways involving macrophages are highly up-regulated during endometriosis. A role for inflammatory angiogenesis is supported by our finding that the cytokine CXCL10, a potent antiangiogenic regulator (27), was up-regulated by quinagolide. In addition, we also found that three proangiogenic cytokines (CCL2, RUNX1 and AGGF1 (28–30) and VEGF (25), also a potent chemoattractor (31)) were down-regulated in L2 lesions. This body of evidence suggests that a major mechanism of action of quinagolide consists of reverting a proangiogenic environment associated with untreated lesions into an antiangiogenic/inflammatory environment by affecting the inflammatory process. Because their different mechanism of action, Drd2-A are not expected to interfere with the menstrual cycle, offering an important advantage over medication such as GnRH agonists and aromatase inhibitors, which induce osteoporosis and hot flashes by creating a hypoestrogenic milieu (32, 33), in the treatment of women who are infertile due to the disease. Nevertheless, in vitro experiments showed that cabergoline blocks P4 production by granulose cells, suggesting that Drd2-A might exert an aromatase inhibitory action (34). Moreover, PRL deprivation may affect reproductive functions by inhibiting intermittent GnRH (35). Thus, by affecting the hypothalamic-pituitary axis, Drd2-A might influence endometriotic lesions by mechanisms other than the ones suggested in the present study. Despite the good safety profile of Drd2-A, as has been demonstrated in women treated for prolactinomas (36), potential side effects should be reduced by using alternative routes of administration, such as vaginal delivery of the drug. In the future, a combination of drugs can be the treatment of choice in endometriosis patients. Quinagolide is a nonergot-derived Drd2-A which lacks an effect on the serotonin (5-hydroxytryptamine [5-HT]) receptor subtype 2b at relevant concentrations. This may be clinically important, because stimulation of 5-HT2b receptors in cardiac valve tissue can lead to proliferation of fibroblasts (37). Drd2-A are also
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differentiated by their pharmacokinetic profile; quinagolide has a much shorter half-life (17 hours) than Cb2 (63–69 hours) (38), thus minimizing exposure during organogenesis in women who may wish to become pregnant. In summary, this is the first study in humans that shows unequivocally that the Drd2-A quinagolide reverses the size of endometriotic
lesions, causing some to completely disappear. The long-term side effect profile of dopamine agonists is favorable over the antisteroidogenic agents. Before adding this very promising type of medication to the armamentarium for the treatment of endometriosis, larger clinical trials need to be conducted to confirm our results and investigate in more depth its mechanism of action.
REFERENCES 1. Ozkan S, Arici A. Advances in treatment options of endometriosis. Gynecol Obstet Invest 2009;67: 81–91. 2. Mihalyi A, Simsa P, Mutinda KC, Meuleman C, Mwenda JM. d’Hooghe TM. Emerging drugs in endometriosis. Expert Opin Emerg Drugs 2006;11: 503–24. 3. Bruner-Tran KL, Osteen KG, Duleba AJ. Simvastatin protects against the development of endometriosis in a nude mouse model. J Clin Endocrinol Metab 2009;94:2489–94. 4. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, et al. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996;81:3112–8. 5. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, M€ uller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. 6. Nap AW, Griffioen AW, Dunselman GA, Bouma–ter Steege JC, Thijssen VL, Evers JL, et al. Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 2004;89:1089–95. 7. Eremina V, Jefferson JA, Kowalewska J, Hochster H, Haas M, Weisstuch J, et al. VEGF inhibition and renal thrombotic microangiopathy. N Engl J Med 2008; 358:1129–36. 8. Basu S, Sarkar C, Chakroborty D, Nagy J, Mitra RB, Dasgupta PS, et al. Ablation of peripheral dopaminergic nerves stimulates malignant tumor growth by inducing vascular permeability factor/vascular endothelial growth factor–mediated angiogenesis. Cancer Res 2004;64:5551–5. 9. Basu S, Nagy JA, Pal S, Vasile E, Eckelhoefer IA, Bliss VS. The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor. Nat Med 2001;7:569–74. 10. Robert E, Musatti L, Piscitelli G, Ferrari CI. Pregnancy outcome after treatment with the ergot derivative, cabergoline. Reprod Toxicol 1996;10:333–7. 11. Ricci E, Parazzini F, Motta T, Ferrari CI, Colao A, Clavenna A, et al. Pregnancy outcome after cabergoline treatment in early weeks of gestation. Reprod Toxicol 2002;16:791–3. 12. Novella-Maestre E, Carda C, Noguera I, Ruiz-Saurı A, Garcia-Velasco JA, Simon C, et al. Dopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis. Hum Reprod 2009; 24:1025–35. 13. Vance ML, Cragun JR, Reimnitz C, Chang RJ, Rashef E, Blackwell RE. CV 205-502 treatment of hyperprolactinemia. J Clin Endocrinol Metab 1989; 8:336–9.
888
Gomez et al.
14. Alvarez C, Martı-Bonmatı L, Novella-Maestre E, Sanz R, Gomez R, Fernandez-Sanchez M, et al. Dopamine agonist cabergoline reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted reproduction. J Clin Endocrinol Metab 2007;92:2931–7. 15. Busso C, Fernandez-Sanchez M, Garcıa-Velasco JA, Landeras J, Ballesteros A, Mu~noz E, et al. The nonergot derived dopamine agonist quinagolide in prevention of early ovarian hyperstimulation syndrome in IVF patients: a randomized, double-blind, placebocontrolled trial. Hum Reprod 2010;25:995–1004. 16. Gomez R, Gonzalez-Izquierdo M, Zimmermann RC, Novella-Maestre E, Alonso-Muriel I, SanchezCriado J, et al. Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)–mediated vascular hyperpermeability without altering VEGF receptor 2–dependent luteal angiogenesis in a rat ovarian hyperstimulation model. Endocrinology 2006;147:5400–11. 17. Hoshiai H, Ishikawa M, Sawatari Y, Noda K, Fukaya T. Laparoscopic evaluation of the onset and progression of endometriosis. Am J Obstet Gynecol 1993;169:714–9. 18. Thomas EJ, Cooke ID. Impact of gestrinone on the course of asymptomatic endometriosis. Br Med J 1987;294:272–4. 19. Kurt M, Ibis M, Karaman K, Bostanci EB, Temucin T. Rectal endometriosis and prolactinoma. Hum Reprod 2010;25:1588–9. 20. Krikun G, Hu Z, Osteen K, Bruner-Tran KL, Schatz F, Taylor HS, et al. The immunoconjugate ‘‘icon’’ targets aberrantly expressed endothelial tissue factor causing regression of endometriosis. Am J Pathol 2010;176:1050–6. 21. McKenna F, McLaughlin PJ, Lewis BJ, Sibbring GC, Cummerson JA, Bowen-Jones D, et al. Dopamine receptor expression on human T- and B-lymphocytes, monocytes, neutrophils, eosinophils and NK cells: a flow cytometric study. J Neuroinmunol 2002; 132:34–40. 22. Cristina C, Dıaz-Torga G, Baldi A, Gongora A, Rubinstein M, Low MJ, et al. Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice. Endocrinology 2005;146:2952–62. 23. Chakroborty D, Chowdhury UR, Sarkar C, Baral R, Dasgupta PS, Basu S. Dopamine regulates endothelial progenitor cell mobilization from mouse bone marrow in tumor vascularization. J Clin Invest 2008;118:1380–9. 24. Sookhai S, Wang JH, McCourt M, O’Connell D, Redmond HP. Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism. Surgery 1999;126:314–22. 25. Oral E, Olive DL, Arici A. The peritoneal environment in endometriosis. Hum Reprod Update 1996; 2:385–98.
Effects of quinagolide on endometriotic lesions
26. Kyama CM, Mihalyi A, Simsa P, Falconer H, Fulop V, Mwenda J, et al. Role of cytokines in the endometrialperitoneal cross-talk and development of endometriosis. Front Biosci 2009;1:444–54. 27. Yoshino O, Osuga Y, Koga K, Hirota Y, Tsutsumi O, Yano T, et al. Concentrations of interferon-gamma– induced protein-10 (IP-10), an antiangiogenic substance, are decreased in peritoneal fluid of women with advanced endometriosis. Am J Reprod Immunol 2003;50:60–5. 28. Ulukus M, Ulukus EC, Tavmergen Goker EN, Tavmergen E, Zheng W, Arici A. Expression of interleukin-8 and monocyte chemotactic protein 1 in women with endometriosis. Fertil Steril 2009;91: 687–93. 29. Konno R, Fujiwara H, Netsu S, Odagiri K, Shimane M, Nomura H, et al. Gene expression profiling of the rat endometriosis model. Am J Reprod Immunol 2007;58:330–43. 30. Tian XL, Kadaba R, You SA, Liu M, Timur AA, Yang L, et al. Identification of an angiogenic factor that when mutated causes susceptibility to KlippelTrenaunay syndrome. Nature 2004;427:640–5. 31. Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, et al. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 1990;172: 1535–45. 32. Gregoriou O, Konidaris S, Vitoratos N, Papadias C, Papoulias I, Chryssicopoulos A. Gonadotropinreleasing hormone analogue plus hormone replacement therapy for the treatment of endometriosis: a randomized controlled trial. Int J Fertil Womens Med 1997;42:406–11. 33. Walch K, Unfried G, Huber J, Kurz C, van Trotsenburg M, Pernicka E. Implanon versus medroxyprogesterone acetate: effects on pain scores in patients with symptomatic endometriosis—a pilot study. Contraception 2009;79:29–34. 34. Negishi H, Furuta I, Ushigoe K, Fujimoto S, Koide SS. In vitro production of progesterone and estradiol by rat granulosa cells regulated by cabergoline and prolactin. Endocr Res 1998;24:195–203. 35. Sauder SE, Frager M, Case GD, Kelch RP, Marshall JC. Abnormal patterns of pulsatile luteinizing hormone secretion in women with hyperprolactinemia and amenorrhea: responses to bromocriptine. J Clin Endocrinol Metab 1984;59:941–8. 36. Bevan JS, Webster J, Burke CW, Scanlon MF. Dopamine agonists and pituitary tumor shrinkage. Endocr Rev 1992;13:220–40. 37. Roth BL. Drugs and valvular heart disease. N Engl J Med 2007;356:6–7. 38. Colao A, di Sarno A, Pivonello R, di Somma C, Lombardi G. Dopamine receptor agonists for treating prolactinomas. Expert Opin Investig Drugs 2002;11: 787–800.
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SUPPLEMENTAL FIGURE 1 Prolactin levels as measured by enzyme-linked immunoassay in blood serum obtained from our nine parients at different time points during the quinagolide treatment. Patients started daily quinagolide administration 1 week after surgery (QX) and continued for 4 months. Pre-QX ¼ blood samples taken with first laparoscopy just before starting the quinagolide treatment. Post-Qx 1 month ¼ blood samples taken during the first monthly visit after 1 month of quinagolide administration. Post-QX 3 months ¼ blood samples taken during the third monthly visit after 3 months of quinagolide administration. Prolactin levels were found to be <20 ng/mL in all cases after 1 month of quinagolide treatment and continued below that baseline during treatment. Wilcoxon paired test was for statiscal comparision: ** P< .01 compared with Pre-QX.
G omez. Effects of quinagolide on endometriotic lesions. Fertil Steril 2011.
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