We recently reported that Slit/Roundabout (ROBO) 1 pathway may be a constituent biomarker for recurrence of endometriosis, likely through promoting angiogenesis. In this study, we sought to determine as whether Slit2 overexpression can facilitate angiogenesis, increase lesion size, and induce hyperalgesia in mice with induced endometriosis. We used 30 Slit2 transgenic (S) and 29 wild-type (W) mice and cross-transplanted endometrial fragments from S to W (group SW) and vice versa (group WS), and also within the S and W (groups SS and WW, respectively), into the peritoneal cavity, inducing endometriosis. We also performed a sham surgery within both S and W mice (groups Sm and Wm, respectively). The size of the ectopic implants, microvessel density (MVD) and immunoreactivity to ROBO1, and vascular endothelial cell growth factor (VEGF) in ectopic and eutopic endometrium, along with hotplate and tail-flick tests in all mice, were then evaluated. We found that the induction of endometriosis resulted in generalized hyperalgesia, which was unaffected by Slit2 overexpression. Slit2 overexpression did increase the lesion size significantly and correlated positively with the MVD in ectopic and eutopic endometrium. Slit2 expression levels appear to correlate with the MVD, but not with VEGF immunoreactivity, in ectopic endometrium. Consequently, we conclude that Slit2 may play an important role in angiogenesis in endometriosis. The increased angiogenesis, as measured by MVD, but not VEGF immunoreactivity, likely resulted in increased lesion size in induced endometriosis. Thus, SLIT2/ROBO1 pathway may be a potential therapeutic target for treating endometriosis.
TaylorRNLebovicDIMuellerMD.Angiogenic factors in endometriosis. Ann N Y Acad Sci. 2002;955:89–100; discussion 118, 396-406.
6.
TaylorRNYuJTorresPBMechanistic and therapeutic implications of angiogenesis in endometriosis. Reprod Sci. 2009;16(2):140–146.
7.
BeckerCMD'AmatoRJ. Angiogenesis and antiangiogenic therapy in endometriosis. Microvasc Res. 2007;74(2-3):121–130.
8.
FerreroSRagniNRemorgidaV. Antiangiogenic therapies in endometriosis. Br J Pharmacol. 2006;149(2):133–135.
9.
Van LangendoncktADonnezJDefrereSDunselmanGAGroothuisPG. Antiangiogenic and vascular-disrupting agents in endometriosis: pitfalls and promises. Mol Hum Reprod. 2008;14(5):259–268.
10.
McLarenJPrenticeACharnock-JonesDSSmithSK. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod. 1996;11(1):220–223.
11.
DeguchiMIshikoOSumiTYoshidaHYamamotoKOgitaS. Expression of angiogenic factors in extrapelvic endometriosis. Oncol Rep. 2001;8(6):1317–1319.
12.
HullMLCharnock-JonesDSChanCLAntiangiogenic agents are effective inhibitors of endometriosis. J Clin Endocrinol Metab. 2003;88(6):2889–2899.
13.
NapAWGriffioenAWDunselmanGAAntiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab. 2004;89(3):1089–1095.
14.
LaschkeMWSchwenderCScheuerCVollmarBMengerMD. Epigallocatechin-3-gallate inhibits estrogen-induced activation of endometrial cells in vitro and causes regression of endometriotic lesions in vivo. Hum Reprod. 2008;23(10):2308–2318.
15.
LaschkeMWElitzschAVollmarBVajkoczyPMengerMD. Combined inhibition of vascular endothelial growth factor (VEGF), fibroblast growth factor and platelet-derived growth factor, but not inhibition of VEGF alone, effectively suppresses angiogenesis and vessel maturation in endometriotic lesions. Hum Reprod. 2006;21(1):262–268.
16.
McBrideWG. (1961). Thalidomide and congenital abnormalities. Lancet. 278, 1358.
17.
D'AmatoRJLoughnanMSFlynnEFolkmanJ. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A. 1994;91(9):4082–4085.
18.
KlauberNRohanRMFlynnED'AmatoRJ. Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470. Nat Med. 1997;3(4):443–446.
19.
BroseKBlandKSWangKHSlit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell. 1999;96(6):795–806.
20.
WangKHBroseKArnottDBiochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell. 1999;96(6):771–784.
21.
WuWWongKChenJDirectional guidance of neuronal migration in the olfactory system by the protein Slit. Nature. 1999;400(6742):331–336.
HuminieckiLGornMSuchtingSPoulsomRBicknellR. 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(4):547–552.
24.
WangBXiaoYDingBBInduction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell. 2003;4(1):19–29.
25.
DunawayCMHwangYLindsleyCWCooperative signaling between Slit2 and Ephrin-A1 regulates a balance between angiogenesis and angiostasis. Mol Cell Biol. 2011;31(3):404–416.
26.
BedellVMYeoSY, Park KW, et al. roundabout4 is essential for angiogenesis in vivo. Proc Natl Acad Sci U S A. 2005;102(18):6373–6378.
27.
GronroosMSalmiTAVuentoMHMass screening for endometrial cancer directed in risk groups of patients with diabetes and patients with hypertension. Cancer. 1993;71(4):1279–1282.
28.
WangLJZhaoYHanBTargeting Slit-Roundabout signaling inhibits tumor angiogenesis in chemical-induced squamous cell carcinogenesis. Cancer Sci. 2008;99(3):510–517.
29.
ShenFLiuXGengJGGuoSW. Increased immunoreactivity to SLIT/ROBO1 in ovarian endometriomas: a likely constituent biomarker for recurrence. Am J Pathol. 2009;175(2):479–488.
30.
SuchtingSBicknellREichmannA. Neuronal clues to vascular guidance. Exp Cell Res. 2006;312(5):668–675.
31.
MaiKTTeoIAl MoghrabiHMargineanECVeinotJP. 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(5):493–499.
32.
PlumpASErskineLSabatierCSlit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron. 2002;33(2):219–232.
33.
YangXMHanHXSuiFDaiYMChenMGengJG. Slit-Robo signaling mediates lymphangiogenesis and promotes tumor lymphatic metastasis. Biochem Biophys Res Commun. 2010;396(2):571–577.
34.
ZhouWJGengZHChiSSlit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis. Cell Res. 2011;21(4):609–626.
35.
Council NR. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academies Press; 1996.
36.
BeckerCMSampsonDARupnickMAEndostatin inhibits the growth of endometriotic lesions but does not affect fertility. Fertil Steril. 2005;84 (suppl 2):1144–1155.
37.
BerkleyKJDmitrievaNCurtisKSPapkaRE. Innervation of ectopic endometrium in a rat model of endometriosis. Proc Natl Acad Sci U S A. 2004;101(30):11094–11098.
38.
CummingsAMMetcalfJL. Induction of endometriosis in mice: a new model sensitive to estrogen. Reprod Toxicol. 1995;9(3):233–238.
39.
CasonAMSamuelsenCLBerkleyKJ. Estrous changes in vaginal nociception in a rat model of endometriosis. Horm Behav. 2003;44(2):123–131.
40.
MatsuzakiSCanisMDarchaCDechelottePJPoulyJLMageG. Effects of a protein kinase C inhibitor on the initial development of ectopic implants in a syngeneic mouse model of endometriosis. Fertil Steril. 2008;89(1):206–211.
41.
LuYNieJLiuXZhengYGuoSW. Trichostatin A, a histone deacetylase inhibitor, reduces lesion growth and hyperalgesia in experimentally induced endometriosis in mice. Hum Reprod. 2010;25(4):1014–1025.
42.
BannonAWMalmbergAB.Models of nociception: hot-plate, tail-flick, and formalin tests in rodents. Curr Protoc Neurosci. 2007;Chapter 8:Unit 8.9.
43.
SompuramSRKodelaVZhangKA novel quality control slide for quantitative immunohistochemistry testing. J Histochem Cytochem. 2002;50(11):1425–1434.
44.
Wang-TilzYTilzCWangBTilzGPStefanH. Influence of lamotrigine and topiramate on MDR1 expression in difficult-to-treat temporal lobe epilepsy. Epilepsia. 2006;47(2):233–239.
45.
WeidnerNSempleJPWelchWRFolkmanJ. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. N Engl J Med. 1991;324(1):1–8.
46.
MaSLiuXGengJGGuoSW.Increased SLIT immunoreactivity as a biomarker for recurrence in endometrial carcinoma. Am J Obstet Gynecol. 2010;202(1):68.e1–e68.e11.
47.
TukeyJW. Exploratory Data Analysis. Reading, MA: Addison-Wesley; 1977.
48.
InhakaRGentlemanRR. R: a language for data analysis and graphics. J comput Graph Statist. 1996;5:1923–1927.
49.
CarmelietPJainRK. Angiogenesis in cancer and other diseases. Nature. 2000;407(6801):249–257.
50.
OosterlynckDJMeulemanCSobisHVandeputteMKoninckxPR. Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil Steril. 1993;59(4):778–782.
51.
NisolleMCasanas-RouxFAnafVMineJMDonnezJ. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril. 1993;59(3):681–684.
52.
ShifrenJLTsengJFZaloudekCJOvarian 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(8):3112–3118.
53.
FascianiAD'AmbrogioGBocciGMontiMGenazzaniARArtiniPG. High concentrations of the vascular endothelial growth factor and interleukin-8 in ovarian endometriomata. Mol Hum Reprod. 2000;6(1):50–54.
54.
Di BlasioAMCentinaioGCarnitiCSomiglianaEViganoPVignaliM. Basic fibroblast growth factor messenger ribonucleic acid levels in eutopic and ectopic human endometrial stromal cells as assessed by competitive polymerase chain reaction amplification. Mol Cell Endocrinol. 1995;115(2):169–175.
55.
BeckerCMSampsonDAShortSMJavaherianKFolkmanJD'AmatoRJ. Short synthetic endostatin peptides inhibit endothelial migration in vitro and endometriosis in a mouse model. Fertil Steril. 2006;85(1);71–77.
56.
NapAWDunselmanGAGriffioenAWMayoKHEversJLGroothuisPG. Angiostatic agents prevent the development of endometriosis-like lesions in the chicken chorioallantoic membrane. Fertil Steril. 2005;83(3):793–795.
57.
Novella-MaestreECardaCNogueraIDopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis. Hum Reprod. 2009;24(5):1025–1035.
58.
BourlevVVolkovNPavlovitchSLetsNLarssonAOlovssonM. The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction. 2006;132(3):501–509.
59.
WingfieldMMacphersonAHealyDLRogersPA. Cell proliferation is increased in the endometrium of women with endometriosis. Fertil Steril. 1995;64(2):340–346.
60.
BrosensIBrosensJJBenagianoG. The eutopic endometrium in endometriosis: are the changes of clinical significance?Reprod Biomed Online. 2012;24(5):496–502.
61.
HanHXGengJG. Over-expression of Slit2 induces vessel formation and changes blood vessel permeability in mouse brain. Acta Pharmacol Sin. 2011;32(11):1327–1336.
62.
DuncanWCMcDonaldSEDickinsonREExpression of the repulsive SLIT/ROBO pathway in the human endometrium and Fallopian tube. Mol Hum Reprod. 2010;16(12):950–959.
63.
Di CarloCBonifacioMTommaselliGABifulcoGGuerraGNappiC. Metalloproteinases, vascular endothelial growth factor, and angiopoietin 1 and 2 in eutopic and ectopic endometrium. Fertil Steril. 2009;91(6):2315–2323.
64.
BerkleyKJCasonAJacobsHBradshawHWoodE. Vaginal hyperalgesia in a rat model of endometriosis. Neurosci Lett. 2001;306(3):185–188.
65.
HeWLiuXSZhangYQGuoSW.Generalized hyperalgesia in women with endometriosis and its resolution following a successful surgery. Reprod Sci. 2010;17(12):1099–1111.
66.
VierckCJ.Animal models of pain. In: McMahonSBKoltzenburgM, eds. Wall and Melzack's Textbook of Pain. 5th ed. Edinburgh: Elsevier, Churchill Livingstone; 2006:175–185.
67.
DicksonBJGilestroGF. Regulation of commissural axon pathfinding by slit and its Robo receptors. Annu Rev Cell Dev Biol. 2006;22:651–675.
68.
VercelliniPFedeleLAimiGPietropaoloGConsonniDCrosignaniPG. Association between endometriosis stage, lesion type, patient characteristics and severity of pelvic pain symptoms: a multivariate analysis of over 1000 patients. Hum Reprod. 2007;22(1):266–271.
69.
NieJLiuXZhengYGengJGGuoSW. Increased immunoreactivity to SLIT/ROBO1 and its correlation with severity of dysmenorrhea in adenomyosis. Fertil Steril. 2011;95(3):1164–1167.
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