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Increased immunoreactivity to SLIT/ROBO1 and its correlation with severity of dysmenorrhea in adenomyosis

2010; Elsevier BV; Volume: 95; Issue: 3 Linguagem: Inglês

10.1016/j.fertnstert.2010.09.027

1556-5653

Jichan Nie, Xishi Liu, Yu Zheng, Jian-Guo Geng, Sun‐Wei Guo,

Uterine Myomas and Treatments

Compared with normal endometrium, SLIT expression was statistically significantly higher in ectopic endometrium from women with adenomyosis, while roundabout 1 (ROBO1) immunoreactivity and microvessel density (MVD) level were statistically significantly higher in both eutopic and ectopic endometrium than normal endometrium. Both SLIT immunoreactivity in ectopic endometrium and MVD in eutopic endometrium were positively correlated with the severity of dysmenorrhea and found to be significant predicators for dysmenorrhea severity in women with adenomyosis. Compared with normal endometrium, SLIT expression was statistically significantly higher in ectopic endometrium from women with adenomyosis, while roundabout 1 (ROBO1) immunoreactivity and microvessel density (MVD) level were statistically significantly higher in both eutopic and ectopic endometrium than normal endometrium. Both SLIT immunoreactivity in ectopic endometrium and MVD in eutopic endometrium were positively correlated with the severity of dysmenorrhea and found to be significant predicators for dysmenorrhea severity in women with adenomyosis. Dysmenorrhea is one of top complaints from women with symptomatic adenomyosis, a disease characterized by the benign invasion of endometrial tissues into the myometrium and a diffuse enlargement of the uterus (1Bergeron C. Amant F. Ferenczy A. Pathology and physiopathology of adenomyosis.Best Pract Res Clin Obstet Gynaecol. 2006; 20: 511-521Crossref PubMed Scopus (270) Google Scholar, 2Peric H. Fraser I.S. The symptomatology of adenomyosis.Best Pract Res Clin Obstet Gynaecol. 2006; 20: 547-555Crossref PubMed Scopus (113) Google Scholar). The molecular mechanisms underlying adenomyosis-associated dysmenorrhea are poorly understood. Thus, there is a need for the identification of key targets involved in adenomyosis-related dysmenorrhea. We have recently reported that decreased immunoreactivity to progesterone receptor isoform B (PR-B) and increased immunoreactivity to nuclear factor κB (NF-κB), oxytocin receptor (OTR), and transient receptor potential vanilloid type 1 (TRPV1) were correlated with the severity of dysmenorrhea in women with adenomyosis (3Nie J. Lu Y. Liu X. Guo S.W. Immunoreactivity of progesterone receptor isoform B, nuclear factor κB, and IκBα in adenomyosis.Fertil Steril. 2009; 92: 886-889Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 4Nie J. Liu X. Guo S.W. Immunoreactivity of oxytocin receptor and transient receptor potential vanilloid type 1 and its correlation with dysmenorrhea in adenomyosis.Am J Obstet Gynecol. 2010; 202 (346.e1–8)Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), suggesting that these proteins may be involved in adenomyosis-associated dysmenorrhea. Myometrial infiltration depth is also reported to play a role (5Cirpan T. Yeniel O. Ulukus M. Ozbal A. Gundem G. Ozsener S. et al.Clinical symptoms and histopathological findings in subjects with adenomyosis uteri.Clin Exp Obstet Gynecol. 2008; 35: 48-53PubMed Google Scholar). Although myometrial infiltration of endometrial cells involves many factors, angiogenesis is of critical importance. As in endometriosis, ectopic endometrium requires a nutritional supply and thus neovascularization to maintain proliferation and to infiltrate into myometrium (6Groothuis P.G. Nap A.W. Winterhager E. Grummer R. Vascular development in endometriosis.Angiogenesis. 2005; 8: 147-156Crossref PubMed Scopus (160) Google Scholar). Indeed, vascularization is found to be markedly increased in adenomyosis (7Ota H. Igarashi S. Tanaka T. Morphometric evaluation of stromal vascularization in the endometrium in adenomyosis.Hum Reprod. 1998; 13: 715-719Crossref PubMed Scopus (56) Google Scholar, 8Schindl M. Birner P. Obermair A. Kiesel L. Wenzl R. Increased microvessel density in adenomyosis uteri.Fertil Steril. 2001; 75: 131-135Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Although in endometriosis the involvement of vascular endothelial cell growth factor (VEGF) and other angiogenic mediators has long been recognized (9McLaren J. Prentice A. Charnock-Jones D.S. Smith S.K. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis.Hum Reprod. 1996; 11: 220-223Crossref PubMed Scopus (328) Google Scholar, 10Donnez J. Smoes P. Gillerot S. Casanas-Roux F. Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis.Hum Reprod. 1998; 13: 1686-1690Crossref PubMed Scopus (470) Google Scholar, 11Deguchi M. Ishiko O. Sumi T. Yoshida H. Yamamoto K. Ogita S. Expression of angiogenic factors in extrapelvic endometriosis.Oncol Rep. 2001; 8: 1317-1319PubMed Google Scholar, 12Taylor R.N. Lebovic D.I. Mueller M.D. Angiogenic factors in endometriosis.Ann NY Acad Sci. 2002; 955 (118, 396–406): 89-100Crossref PubMed Scopus (264) Google Scholar), we have shown recently that SLIT immunoreactivity is increased in endometriosis and its elevation may be a constitutive biomarker for recurrence of endometriosis (13Shen F. Liu X. Geng J.G. Guo S.W. Increased immunoreactivity to SLIT/ROBO1 in ovarian endometriomas: a likely constituent biomarker for recurrence.Am J Pathol. 2009; 175: 479-488Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). SLIT is a secretory glycoprotein consisting of three members, SLIT1–3, originally found to be secreted repellents in axon guidance and neuronal migration and shown to be endogenously available inhibitors of leukocyte chemotaxis (14Wu J.Y. Feng L. Park H.T. Havlioglu N. Wen L. Tang H. et al.The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors.Nature. 2001; 410: 948-952Crossref PubMed Scopus (363) Google Scholar). The receptor for SLIT is the transmembrane protein roundabout (ROBO), which currently consists of four members: ROBO1–4 (15Huminiecki L. Gorn M. Suchting S. Poulsom R. Bicknell R. Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis.Genomics. 2002; 79: 547-552Crossref PubMed Scopus (296) Google Scholar). SLIT can attract vascular endothelial cells in vitro and promote tumor-induced angiogenesis in a xenograft model of human malignant melanoma (16Wang B. Xiao Y. Ding B.B. Zhang N. Yuan X. Gui L. et al.Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity.Cancer Cell. 2003; 4: 19-29Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Consistent with this finding, SLIT-ROBO4 is reported to function as a chemoattractant to recruit vascular endothelial cells to sites for vasculogenesis (17Bedell V.M. Yeo S.Y. Park K.W. Chung J. Seth P. Shivalingappa V. et al.Roundabout4 is essential for angiogenesis in vivo.Proc Natl Acad Sci USA. 2005; 102: 6373-6378Crossref PubMed Scopus (187) Google Scholar, 18Gronroos M. Salmi T.A. Vuento M.H. Jalava E.A. Tyrkko J.E. Maatela J.I. et al.Mass screening for endometrial cancer directed in risk groups of patients with diabetes and patients with hypertension.Cancer. 1993; 71: 1279-1282Crossref PubMed Scopus (54) Google Scholar). Increased SLIT expression was associated with higher tumor angiogenesis, as measured by microvessel density (MVD) (19Wang L.J. Zhao Y. Han B. Ma Y.G. Zhang J. Yang D.M. et al.Targeting Slit-roundabout signaling inhibits tumor angiogenesis in chemical-induced squamous cell carcinogenesis.Cancer Sci. 2008; 99: 510-517Crossref PubMed Scopus (69) Google Scholar). We hypothesized that SLIT/ROBO1 may be involved in adenomyosis, and their expression levels may correlate with the severity of dysmenorrhea. Hence, we investigated the expression and localization of SLIT, ROBO1, and CD34, which is considered to be a marker for MVD (20Mai K.T. Teo I. Al Moghrabi H. Marginean E.C. Veinot J.P. Calretinin and CD34 immunoreactivity of the endometrial stroma in normal endometrium and change of the immunoreactivity in dysfunctional uterine bleeding with evidence of 'disordered endometrial stroma'.Pathology. 2008; 40: 493-499Abstract Full Text PDF PubMed Scopus (19) Google Scholar), in women with adenomyosis and in control endometrium. Based on the menstrual phase in which the tissue samples were harvested, we selected 50 women with diffuse adenomyosis (excluding endometriosis as determined by ultrasound, surgical observation, and histologic analysis) admitted to the Shanghai Obstetrics and Gynecology Hospital of Fudan University from 2004 to 2005. Their archived, formalin-fixed, paraffin-embedded ectopic and homologous eutopic endometrial tissue blocks were retrieved. For the controls, we also collected paraffin-embedded endometrial tissue samples from 18 women with surgically diagnosed benign ovarian cysts, none of whom had endometriosis, adenomyosis, or myoma. In both study and control samples, precisely half were in the proliferative or the secretory phases, respectively, and none had a history of hormone therapy or intrauterine device use for ≥6 months before the surgery or tissue collection. For all 50 patients, information was collected on age at surgery, uterus size, severity of dysmenorrhea (none, mild, moderate, or severe), duration of dysmenorrhea, and gravidity, as previously reported elsewhere (3Nie J. Lu Y. Liu X. Guo S.W. Immunoreactivity of progesterone receptor isoform B, nuclear factor κB, and IκBα in adenomyosis.Fertil Steril. 2009; 92: 886-889Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This study was approved by the institutional ethics review board of Shanghai Obstetrics and Gynecology Hospital. Serial 4-μm sections were obtained from each block to confirm the pathologic diagnosis and to stain for pan-SLIT, ROBO1, and CD34. Routine deparaffinization and rehydration procedures were performed, followed by routine immunohistochemical analysis (13Shen F. Liu X. Geng J.G. Guo S.W. Increased immunoreactivity to SLIT/ROBO1 in ovarian endometriomas: a likely constituent biomarker for recurrence.Am J Pathol. 2009; 175: 479-488Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Antibodies to ROBO1, pan-SLIT, and the preimmune IgG were prepared and characterized as reported previously elsewhere (16Wang B. Xiao Y. Ding B.B. Zhang N. Yuan X. Gui L. et al.Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity.Cancer Cell. 2003; 4: 19-29Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Mouse monoclonal anti-CD34 (M-0117) was purchased from Changdao Bio-Reagent Company (Shanghai, China). The concentration used for the pan-SLIT and ROBO1 antibody was 5 μg/mL for both; the concentration for CD34 was 1 μg/mL. The scoring of the immunoreactivity to SLIT and ROBO1 performed by use of digital image analysis with Image Pro-Plus 6.0 (Media Cybernetics, Bethesda, MD) as described elsewhere (21Wang-Tilz Y. Tilz C. Wang B. Tilz G.P. Stefan H. Influence of lamotrigine and topiramate on MDR1 expression in difficult-to-treat temporal lobe epilepsy.Epilepsia. 2006; 47: 233-239Crossref PubMed Scopus (70) Google Scholar); the evaluator had no prior knowledge of the clinicopathologic information. A series of 10 random images were taken for each immunostained parameter to obtain a mean value. Staining was defined via color intensity, and the scoring was performed as reported previously (3Nie J. Lu Y. Liu X. Guo S.W. Immunoreactivity of progesterone receptor isoform B, nuclear factor κB, and IκBα in adenomyosis.Fertil Steril. 2009; 92: 886-889Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 4Nie J. Liu X. Guo S.W. Immunoreactivity of oxytocin receptor and transient receptor potential vanilloid type 1 and its correlation with dysmenorrhea in adenomyosis.Am J Obstet Gynecol. 2010; 202 (346.e1–8)Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We assessed the MVD in the CD34 stained slides as reported previously elsewhere (13Shen F. Liu X. Geng J.G. Guo S.W. Increased immunoreactivity to SLIT/ROBO1 in ovarian endometriomas: a likely constituent biomarker for recurrence.Am J Pathol. 2009; 175: 479-488Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The microvessel count was performed on five chosen ×200 fields of the "hot plot," and the evaluator had no prior knowledge of the pathologic status of the samples under evaluation. The MVD was defined to be the mean of the vessel counts obtained in these fields. A negative control was also incorporated that used preimmune IgG instead of the primary antibody. All sections were double-inspected independently (by JCN and YZ). Discrepancies, if occurred, were resolved by consensus. The comparison of distributions of continuous variables was made using the Wilcoxon test or Kruskal-Wallis test. Spearman's correlation coefficient was used when evaluating correlations between the severity of dysmenorrhea and SLIT or ROBO1 immunoreactivity or MVD levels. To identify factors associated with the severity of dysmenorrhea, we used a semiparametric polychotomous logistic regression model and the Cox regression model (22Liu X. Guo S.W. Dysmenorrhea: risk factors in women with endometriosis.Womens Health (Lond Engl). 2008; 4: 399-411Crossref PubMed Scopus (18) Google Scholar). P<.05 was considered statistically significant. All computations were made with R 2.10.1 (23R Foundation for Statistical Computing. R: A language and environment for statistical computing. Available at: http://www.r-project.org. Accessed September 20, 2010.Google Scholar). The clinicopathologic data of the study and control groups were reported previously elsewhere (3Nie J. Lu Y. Liu X. Guo S.W. Immunoreactivity of progesterone receptor isoform B, nuclear factor κB, and IκBα in adenomyosis.Fertil Steril. 2009; 92: 886-889Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Briefly, among the 50 patients with adenomyosis, 6 (12%), 13 (26%), 17 (34%), and 14 (28%) had no, mild, moderate, or severe dysmenorrhea, respectively. The study and control groups were comparable in age, with the mean age and its standard deviation (SD) in the two groups being 43.4 years (SD = 3.9, range: 32 to 50 years) and 43.5 years (SD = 4.5, range: 30 to 51 years), respectively. The immunoreactivity of SLIT was seen mostly in glandular epithelial cells and was localized both in cytoplasm and membrane. Staining of ROBO1 was seen in both glandular epithelial and vascular endothelial cells and was localized in both the cytoplasm and the membrane of ectopic endometrium; in control and eutopic endometrium, it was seen exclusively in glandular epithelial cells. Immunostaining of CD34 was seen mostly in vascular endothelial cells in control, eutopic, and ectopic endometrium. In control and eutopic and ectopic endometrium, no statistically significant difference in SLIT, ROBO1 immunoreactivity, or MVD level was found between the proliferative and secretory phases (all P≥0.12). We found that SLIT and ROBO1 immunoreactivities in control (r = 0.83, P=1.8 × 10−5) and ectopic (r = 0.39, P=.005) endometrium were positively correlated. In addition, SLIT immunoreactivity correlated positively with the MVD level in control (r = 0.77, P=1.8 × 10−4) and eutopic (r = 0.39, P=.005) endometrium, whereas ROBO1 and MVD were positively correlated in control (r = 0.57, P=.013) and ectopic (r = 0.35, P=.012) endometrium. The SLIT expression in ectopic endometrium was statistically significantly higher than that in normal endometrium (P=.016; Fig. 1A ), as were the ROBO1 expression and MVD values in both eutopic and ectopic endometrium (all P≤.034; see Fig. 1B and C). The expression of SLIT in ectopic endometrium and the MVD level in eutopic endometrium were both statistically significantly correlated with dysmenorrhea severity (r = 0.36, P=.01 and r = 0.33, P=.019, respectively; see Fig. 1D and E). The semiparametric polychotomous logistic regression model identified the SLIT immunoreactivity level in ectopic endometrium as the only covariate that was associated with the dysmenorrhea severity; the Cox model identified uterus size (P=.042), immunoreactivity to SLIT (P=.024), and ROBO1 (P=.02) in ectopic endometrium as three covariates positively associated with dysmenorrhea severity. These data demonstrate that SLIT immunoreactivity is elevated in adenomyosis and that it correlates with the severity of dysmenorrhea in women with symptomatic adenomyosis. It has been reported that the administration of an angiogenesis inhibitor reduces the development of endometrial blood vessels, resulting in a lowered incidence of the uterine adenomyosis induced by pituitary grafting in mice, and reducing proliferation of ectopic endometrium (24Zhou Y.F. Mori T. Kudo H. Asakai R. Sassa S. Sakamoto S. Effects of angiogenesis inhibitor TNP-470 on the development of uterine adenomyosis in mice.Fertil Steril. 2003; 80: 788-794Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Yet angiogenesis may involve multiple pathways (25Casanovas O. Hicklin D.J. Bergers G. Hanahan D. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors.Cancer Cell. 2005; 8: 299-309Abstract Full Text Full Text PDF PubMed Scopus (1410) Google Scholar). Therefore, in order for the anti-angiogenic therapy to work, it may be imperative to identify all possible angiogenic molecules and pathways involved in adenomyosis. SLIT2 is reported to promote tube formation in a ROBO1- and phosphatidylinositol kinase-dependent manner, and the neutralization of Robo1 has been found to reduce MVD and tumor mass in vivo (16Wang B. Xiao Y. Ding B.B. Zhang N. Yuan X. Gui L. et al.Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity.Cancer Cell. 2003; 4: 19-29Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The difference in ROBO1 immunostaining locations between normal and ectopic endometrium observed in our study suggests that such a SLIT/ROBO1-mediated crosstalk in adenomyosis may well facilitate angiogenesis, ensuring the blood supply to growing ectopic endometrium, facilitating myometrial infiltration, and thus exacerbating dysmenorrhea. In addition, as the SLIT/ROBO system is known to play a role in neural development, increased SLIT expression may also be related to increased activity of pain mediators, though this is yet to be demonstrated. It is no coincidence that the vascular and the nervous systems share similar signaling pathways, often crosstalk, and, when deregulated, contribute to medically relevant diseases (26Carmeliet P. Blood vessels and nerves: common signals, pathways and diseases.Nat Rev Genet. 2003; 4: 710-720Crossref PubMed Scopus (442) Google Scholar). The observed close correlation of immunoreactivity levels between SLIT and ROBO1 is consistent with the structural findings (27Morlot C. Thielens N.M. Ravelli R.B. Hemrika W. Romijn R.A. Gros P. et al.Structural insights into the Slit-Robo complex.Proc Natl Acad Sci USA. 2007; 104: 14923-14928Crossref PubMed Scopus (144) Google Scholar); the similar relationship between SLIT and MVD in ectopic endometrium and between ROBO1 and MVD in control endometrium strongly supports the notion that SLIT/ROBO1 signaling is involved in angiogenesis in adenomyosis. We have found higher SLIT and ROBO1 immunoreactivity in women with adenomyosis, and that the SLIT and ROBO1 expression levels correlated closely with MVD. We also found that that SLIT immunoreactivity and MVD were positively correlated with the severity of dysmenorrhea. These results suggest that SLIT/ROBO1 may be potential therapeutic targets in treating adenomyosis-related dysmenorrhea and perhaps chronic pelvic pain as well.