The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis

The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research note The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis Fiona L Cousins, Johanna K Farley, Rebecca Kerrigan, Shayanti Mukherjee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-45953/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2020 Read the published version in BMC Research Notes → Version 2 posted 5 You are reading this latest preprint version Show more versions Abstract Objective: Endometriosis is a common and painful condition characterized by the formation of endometrial lesions within the peritoneal cavity. Previous studies have suggested a role for hedgehog signalling in the pathogenesis of endometriosis. We investigated the role of hedgehog signalling in the establishment of endometriosis lesions using 5E1, a hedgehog ligand neutralising antibody, and a mouse model of endometriosis. To mimic the initiation of by endometriosis by retrograde menstruation, which is believed to occur in humans, donor mice underwent an artificial menstruation protocol. Fragments of menstrual endometrium were injected into the peritoneal cavity of estrogen primed recipients. Recipients received twice weekly injections of 5E1 or an isotype matched control antibody for three weeks. Lesions were collected and analysed for markers of epithelium, proliferation and apoptosis by immunofluorescence microscopy. Results: Treatment with 5E1, reduced the number of lesions found on the mesentery. No significant changes were found in the size of lesions, abundance of endometrial epithelial cells, proliferation or apoptosis. Obstetrics & Gynecology Endometrium endometriosis hedgehog signalling mouse model Figures Figure 1 Figure 2 Introduction Endometriosis is a complex disorder of unknown aetiology, defined by the growth of endometrial fragments outside the uterine cavity. Endometriosis is thought to occur via the retrograde menstruation of endometrial fragments into the peritoneal cavity which then persist and form lesions on intra-peritoneal organs[1]. Retrograde menstruation occurs in 90% of menstruating females, yet only 10% go on to develop endometriosis, indicating that additional factors are involved[2]. Published data highlights that eutopic endometrial stromal cells from women with endometriosis exhibit increased adherence and proliferation in vitro [3,4]. It is likely that cell signalling and cell growth pathways of eutopic endometrial cells are altered in women with endometriosis. Current non-surgical treatments for endometriosis are largely oral contraceptives, to limit retrograde menstruation and growth of established lesions, or analgesics for pain. Non-hormonal therapeutics are desperately needed and targeting cell signalling pathways may reveal new avenues for drug design. Hedgehog signalling is a developmental pathway that is activated in some endometrial cancers[5] and the endometrium of women with endometriosis[6]. Sonic hedgehog ( SHH ) and its downstream signalling transcription factor GLI1 are upregulated in the eutopic endometrium of women with endometriosis in comparison to healthy controls[6]. Hedgehog signalling genes Shh, Gli1, Stil1 and Jag2 are also upregulated in the ectopic lesions of a mouse model of endometriosis featuring enhanced lesion formation[7]. Hedgehog signalling regulates cell proliferation, differentiation and stromal fibroblast maintenance in the endometrium via the FAK/ERK1/2 and PI3K/Akt signalling pathways[8]. Both of these pathways have been implicated in the pathogenesis of endometriosis[9], with prolonged phosphorylation of ERK in endometrial epithelial[10] and stromal cells[11] and endometriomas[12] and increased phosphorylated Akt in ovarian endometriomas[13]. HOXA10 is a developmental transcription factor that has been linked to endometriosis, and, like hedgehog signalling, is involved in progesterone induced endometrial differentiation[14]. This evidence suggests that hedgehog signalling contributes to the pathogenesis of endometriosis. Thus, hedgehog signalling may have value as a diagnostic marker for endometriosis and as a therapeutic target. Highly specific antibodies are increasingly being used in therapeutic applications[15]. The 5E1 monoclonal antibody recognises mammalian hedgehog ligands and prevents hedgehog signal transduction via the Patched receptor. 5E1 is commonly used as tool to block hedgehog signalling in mouse-based in vivo experiments[16,17]. In the current study we investigated the potential of hedgehog blocking antibodies as a therapy for endometriosis by using 5E1 in a mouse model that closely mimics the human condition. Materials And Methods Animal ethics All animals were held in the Monash Medical Centre Animal Facility and housed on a 12-hour light/day cycle with access to normal chow and water ab libitum . Approval for all procedures described was obtained from Monash Medical Centre Animal Ethics Committee A. Mouse model of endometriosis C57BL/6J background mice 8-12 weeks age underwent the mouse model of endometriosis as published previously[18]. Briefly, donor mice were ovariectomised, then given sub-cutaneous injections of β-estradiol (1µg/ml) on days 7-9. A progesterone secreting pellet was placed sub-cutaneously from days 13-19 (500ng/day) in combination with β-estradiol sub-cutaneous injections from days 13-15 (50ng/ml). On day 15 the endometrium was artificial decidualised by injecting 20µl of oil into the uterine lumen. Progesterone support was removed on day 19 and 4 hours later the menstrual-like endometrium was removed from the outer myometrium, minced into fragments approximately 1mm 3 and injected into ovariectomised estrogen primed (β-estradiol secreting pellet, 100ng/day days 7-19) recipients (approximately 20 fragments, 200mg of tissue in 200µl of PBS). Lesions were allowed to develop over 21 days (days 19-40) before collection on day 40. During this 21-day period mice were randomly allocated to one of two groups and treated twice weekly with either 250µg of anti-SHH antibody 5E1 (n=15, DSHB) or an isotype matched control antibody (n=18, BioXCell, MOPC-21 IgG1) in 200µl of sterile PBS. The 5E1 dose administered was based on previous reports that used this antibody to block hedgehog signalling in mouse in vivo tumour models[19]. Lesion collection and fixation Recipient mice were euthanized using rising concentrations of carbon dioxide and cervical dislocation. Upon dissection all body cavities were photographed, the number of lesions found recorded and their size measured. Lesions were carefully removed from peritoneal organs and immersed in 4% w/v paraformaldehyde in PBS overnight at 4°C and cryoprotected in 30% w/v sucrose in PBS overnight at 4°C. Tissues were frozen in optimal cutting temperature medium and cryo-sectioned at 8µm thick. Immunofluorescence analysis Unless otherwise stated all sections underwent the following staining protocol. Sections were permeabilised in 0.2% Triton X-100 in PBS for 15 minutes, blocked in DAKO blocking solution for 1 hour and then stained for hedgehog activation, (GLI1 Rabbit monoclonal Thermo MA5-32553 10µg/ml), epithelial (EpCAM-PE rat anti mouse eBioscience 12-5791-81 2µg/ml), proliferation (Ki67-EF450 rat anti mouse eBioscience 50-5698-80 2µg/ml) and apoptosis (Caspase 3 Rabbit polyclonal R&D AF835 5µg/ml) markers for 1 hour at room temperature in 1% bovine serum albumin in PBS. For unconjugated primary antibodies, sections were incubated with secondary antibodies for 1 hour at room temperature (Donkey anti rabbit Alexafluor 568 LifeTech A10042 4µg/ml). Nuclei were counterstained with 5µg/ml Hoescht 33258 in PBS for 3 mins. Images were captured on an Olympus FV1200 confocal microscope using a 20x objective lens and adjusted for brightness and contrast in a linear manner using FIJI software[20]. Analysis of lesions Histological analysis Haematoxylin and eosin staining was performed as per standard protocols on the 15 th slide of each lesion (2 sections/slide x 8µm x 15 slides= 240µm deep). Lesion cross-sectional area was measured using FIIJ, using the trace outline and measure area tools. Prior to the analysis of epithelial, proliferation and apoptosis markers, slides were blinded to reduce any bias during analysis. Markers were counted manually in FIJI. Total cell nuclei were calculated by counting the number of nuclei using the “threshold”, “watershed” and “analyse particles” functions. A minimum of 6 lesions were analysed per treatment group. A minimum of 3 fields of view per section were imaged and the average percentage of positive cell types in the total cell population was calculated for each lesion. Results were then unblinded and each lesion was then plotted as a single data point. Statistical analysis Group sizes were determined using G*Power software. For lesion number, we assumed an observed effect size of 0.5, alpha of 0.05 and Power of 0.8, the total sample size required was 26. For changes in lesion characteristics we assumed an effect size of 0.65, alpha of 0.05 and Power of 0.8, the total sample size required was 13. All statistical analyses were performed in Graphpad Prism 8.0. Raw data was subjected to D’Agostino-Pearson normality testing prior to statistical analyses. Non-parametric analysis was performed when one of the sets of data failed to pass normality testing. All lesion analyses were subjected to unpaired, two-tailed Mann-Whitney testing. Significance was accepted where P≤0.05 and data represented graphically as individual data points and median. Results And Discussion The location and incidence of lesions Lesions were found at various locations in the isotype control and anti-SHH treated group (Figure 1A). The occurrence of lesions, indicative of endometriosis, was 100% in the isotype control treated group and 80% in anti-SHH treated group (p=0.08) (Figure 1B). In line with other reports using this model, irrespective of treatment, most lesions were detected on the mesentery (83% in the isotype control group, 67% in the 5E1 group)[18]. Lesions were also found on the body wall, on the external surface of the uterus and on the digestive tract (includes stomach, small intestine, large intestine) (Figure 1C). There was a significant decrease in the number of lesions per recipient detected on the mesentery after anti-SHH treatment compared to isotype control (p<0.01) (Figure 1D). This decrease was not observed for other locations but these lesions were infrequent and the dataset was small (Figure 1D). Given the decrease in the number of mesenteric lesions we assessed their lesion area independent of other lesions but found that this sub-group did not display any difference in area (p=0.0932, data not shown). Gli1 expression as an indicator of Hedgehog activation Gli1 was examined by immunostaining to detect hedgehog activation in collected lesions. Gli1 was readily detected in mouse brain, a known site of hedgehog signalling[21]. Very weak to no signal was detected in isotype and anti-SHH lesions from the endometriosis model (Figure 2A, representative of 3 animals). This suggests that endometriotic lesions do not have the high levels of hedgehog activation found in some tumours[5,22–24]. GLI1 expression is increased in the endometrium of women with endometriosis in comparison to healthy controls[6], and in ovarian endometriomas[25] but expression in human lesions at other sites is yet to be assessed. Our mouse model does not address ovarian endometriomas due to recipients being ovariectomised, therefore we cannot comment on whether the ovarian location would result in differing GLI1 expression. Furthermore, the human study used a polyclonal antibody, whereas the antibody used in our mouse studies was a monoclonal (clone JF09-08) which may account for discrepancy in results. Lesion characteristics Lesion establishment and survival relies on cross-talk between epithelial cells and the surrounding stroma[26]. Lesions were investigated for expression of the epithelial cell marker EpCAM, proliferation marker Ki67 and apoptosis marker Caspase 3 (Figure 2B). Variable expression of EpCAM was identified in the isotype control group. Fifty-eight percent of isotype lesions (7 of 12) had detectable EpCAM expression ranging from 0.17-26% positive cells in the total population. In the anti-SHH group, only 37.5% of lesions had detectable expression of EpCAM (3 of 8 lesions), only 1 lesion had a detectable level over 1% positive in the total cell population. Despite no significant decrease in EpCAM expression, the observed downward trend is in line with another report where a hedgehog inhibitor decreased endometrial epithelial cell proliferation in vitro [27]. No significant differences were identified between Isotype control and Anti-SHH groups for Ki67 and Caspase 3 (Figure 2B), which suggests 5E1 has no direct effect on overall proliferation or apoptosis of endometriotic lesions. Conclusions Differences in hedgehog pathway activation have been detected in women with endometriosis and in mouse models of endometriosis[6,7,28]. This study aimed to see whether the hedgehog neutralising antibody 5E1 would prevent lesion establishment and progression in a mouse model of endometriosis. Significantly fewer lesions were found on the mesentery of animals treated twice weekly with 5E1. However, no significant differences were observed in the size and morphology of the lesions that formed. These data suggest that hedgehog signalling is not integral to lesion progression, but may play a role in the survival of menstrual fragments in the peritoneal cavity and lesion initiation. Limitations No effect of anti-SHH treatment was observed at other sites within the peritoneal cavity, however the number of lesions obtained from these sites was small (n=4-6 lesions) meaning it is not possible to draw clear conclusions. It is difficult to determine the mechanism by which mesenteric lesions are reduced from the data obtained. List Of Abbreviations 5E1 Sonic hedgehog neutralising monoclonal antibody, clone 5E1 EpCAM Epithelial cell adhesion molecule SHH Sonic hedgehog Declarations Ethics: All mouse procedures were performed under approval MMCA2016-57 from Monash Medical Centre Animal Ethics Committee A. Consent for Publication: Not Applicable. Availability of data and materials: All relevant data generated in this study is presented in the manuscript. Competing interests: The authors declare they have no competing interests. Funding: This study was supported by the Victorian Government’s Operational Infrastructure fund and a Ferring Pharmaceuticals Innovation grant awarded to JA Deane, CE Gargett and FL Cousins. Ferring Pharmaceuticals had no input into design, collection or interpretation of data. Author’s contributions: FLC, CEG and JAD designed the study. FLC, CEG and JAD obtained funding. FLC, SM, SD and JAD collected data. FLC, JKF, RK and JAD analysed data. FLC and JAD wrote the manuscript. All authors read and approved the final version of the manuscript. Acknowledgements: We would like to acknowledge the support of the Monash Health Translational Precinct’s Research Platforms Monash Micro-Imaging and Monash Histology and animal technicians for animal husbandry and care. References Sampson JA. Metastatic or Embolic Endometriosis, due to the Menstrual Dissemination of Endometrial Tissue into the Venous Circulation. Am J Pathol. 1927 Mar;3(2):93-110.43. Giudice LC, Kao LC. Endometriosis. Lancet. 2004 Nov;364(9447):1789–99. Beste MT, Pfaffle-Doyle N, Prentice EA, Morris SN, Lauffenburger DA, Isaacson KB, et al. Molecular Network Analysis of Endometriosis Reveals a Role for c-Jun-Regulated Macrophage Activation. Sci Transl Med. 2014 Feb 5;6(222). Klemmt PAB, Carver JG, Koninckx P, McVeigh EJ, Mardon HJ. 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Supplementary Files COUSINSAuthorChecklistFull.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2020 Read the published version in BMC Research Notes → Version 2 posted Editorial decision: Accept 18 Sep, 2020 Editor assigned by journal 11 Sep, 2020 Reviewers invited by journal 11 Sep, 2020 Submission checks completed at journal 10 Sep, 2020 Editor invited by journal 10 Sep, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-45953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research note","associatedPublications":[],"authors":[{"id":2272601,"identity":"45bd2c34-322a-4f50-9b6e-60aa03f5aa0b","order_by":0,"name":"Fiona L Cousins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIie3QPUvEMBjA8ScU2iVn14TqfYZKwbH9KgmB3nKD4NLBoSDERdwF8TPcdHMkUJdA1w4i59LpBsc7cDA9RDhM8dxE8p9CeH7kBcDn+4NFV19LVANUCkD9QLDeI+YQsjeA5CEkCprVBvJp2mr5un145retblZQveR1ZFInCcLZ6Q2ILO34dTZZ9vyuK2cpmAtR47mTFAE+IxgUX3RIJmip7cLuIMkEgJtgS+j7QNpHSbf3lrTmk8TrUZLsTlFckkltiZrvSA5k7JSwTI5TkdGOywQ3OrNvKQkzjIWkP3eSWDd0XeXTo/app5tLfTL8GHmrWBHHYuH+5qFvF2AAXI7Pj1T8Wvh8Pt9/7QNfMmBYS1VwiAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7064-8596","institution":"Hudson Institute of Medical Research","correspondingAuthor":true,"prefix":"","firstName":"Fiona","middleName":"L","lastName":"Cousins","suffix":""},{"id":2272602,"identity":"deb3496d-c97f-4d97-ab59-6275122c5131","order_by":1,"name":"Johanna K Farley","email":"","orcid":"","institution":"Hudson Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Johanna","middleName":"K","lastName":"Farley","suffix":""},{"id":2272603,"identity":"06ab1456-1032-4a7b-af3b-c072e265fd1e","order_by":2,"name":"Rebecca Kerrigan","email":"","orcid":"","institution":"Hudson Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Kerrigan","suffix":""},{"id":2272604,"identity":"cbf1606a-5faf-4bd9-9494-54d11dad8b1a","order_by":3,"name":"Shayanti Mukherjee","email":"","orcid":"","institution":"Hudson Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Shayanti","middleName":"","lastName":"Mukherjee","suffix":""},{"id":2272605,"identity":"f3394314-ad1d-4b26-8ae2-c68b49129392","order_by":4,"name":"Saeedeh Darzi","email":"","orcid":"","institution":"Hudson Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Saeedeh","middleName":"","lastName":"Darzi","suffix":""},{"id":2272606,"identity":"dc7fc24b-a1bd-4eff-983f-50b428cb9b12","order_by":5,"name":"Caroline E Gargett","email":"","orcid":"","institution":"Hudson Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"E","lastName":"Gargett","suffix":""},{"id":2272607,"identity":"4f3f70f6-7673-41f3-acda-add4956089a9","order_by":6,"name":"James A Deane","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACxgYwxQxmPwCzJYCYh0gtzAZEaYECsBY2CaK0MLefTnz4g8Fajn9287GqGxV35OVnNzA+eNuGx2E9uZuNeRjSjSXuHEu7nXPmmeGGOweYDefi09KQu02ageFwYsONHLPbuW2HGTdIJLBJ8+LT0v92+88fDIfr59/I/1YM1GI/f0YC+2+8WmbkbgN69nCCwY0cNmagFqB1CWzM+LW83SzNY5BuuPFGmrF0zpnDyRtuJDZLzjmHW4thf+7Gjz8qrOXlbiQ//JxTcdh2/ozkgx/elOHR0gAiDVBtbsCtHgjk8cqOglEwCkbBKAABAJHXVcpqgKTjAAAAAElFTkSuQmCC","orcid":"","institution":"Monash University Department of Obstetrics and Gynaecology","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"A","lastName":"Deane","suffix":""}],"badges":[],"createdAt":"2020-07-20 10:53:06","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-45953/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-45953/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-020-05299-5","type":"published","date":"2020-09-25T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2398575,"identity":"724346b1-9389-4dfa-a9d9-1b25a96eac4f","added_by":"auto","created_at":"2020-09-14 17:40:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88426,"visible":true,"origin":"","legend":"Endometriosis incidence and lesion location following treatment with SHH antibody. A) Lesions (dashed circles) were identified on different organs within the body cavity, including the body wall, mesentery, uterus and intestine. Scale bars 3mm. B) Endometriosis incidence was calculated as the percentage of mice with detectable lesions at three weeks, isotype control n=18, Anti-SHH n=15. Data displayed as mean±SEM. C) The percentage of mice with lesions at different sites. D) The number of lesions/recipient at each site. Isotype v anti-SHH for each site was tested using an unpaired, two-tailed Mann-Whitney test. *** p\u003c0.001. Pink line denotes median. ","description":"","filename":"CousinsBMCFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45953/v2/CousinsBMCFigure1.jpg"},{"id":2398576,"identity":"fd98a0ac-e4be-438e-8dfa-3db833bdad56","added_by":"auto","created_at":"2020-09-14 17:40:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158896,"visible":true,"origin":"","legend":"Expression of SHH pathway and cell identity markers did not change with anti-SHH treatment. A) Gli1 expression was detected in the mouse brain but was barely detected in lesions from either group. Inset rabbit IgG1 isotype control. B) Dual immunofluorescence for EpCAM and Ki67, insert isotype control. C) Immunofluorescence for Caspase 3, insert isotype control. B+C quantification; EpCAM, Ki67 and Caspase 3 expressed as a percentage of total cell population, isotype control (black circles) and Anti-Shh (white circles). Data analysed by an unpaired, two-tailed Mann-Whitney test. 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Endometriosis is thought to occur via the retrograde menstruation of endometrial fragments into the peritoneal cavity which then persist and form lesions on intra-peritoneal organs[1]. Retrograde menstruation occurs in 90% of menstruating females, yet only 10% go on to develop endometriosis, indicating that additional factors are involved[2]. Published data highlights that eutopic endometrial stromal cells from women with endometriosis exhibit increased adherence and proliferation \u003cem\u003ein\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e[3,4]. It is likely that cell signalling and cell growth pathways of eutopic endometrial cells are altered in women with endometriosis. Current non-surgical treatments for endometriosis are largely oral contraceptives, to limit retrograde menstruation and growth of established lesions, or analgesics for pain. Non-hormonal therapeutics are desperately needed and targeting cell signalling pathways may reveal new avenues for drug design.\u003c/p\u003e\n\u003cp\u003eHedgehog signalling is a developmental pathway that is activated in some endometrial cancers[5] and the endometrium of women with endometriosis[6]. Sonic hedgehog (\u003cem\u003eSHH\u003c/em\u003e) and its downstream signalling transcription factor \u003cem\u003eGLI1 \u003c/em\u003eare upregulated in the eutopic endometrium of women with endometriosis in comparison to healthy controls[6]. Hedgehog signalling genes \u003cem\u003eShh, Gli1, Stil1\u003c/em\u003e and \u003cem\u003eJag2\u003c/em\u003e are also upregulated in the ectopic lesions of a mouse model of endometriosis featuring enhanced lesion formation[7]. Hedgehog signalling regulates cell proliferation, differentiation and stromal fibroblast maintenance in the endometrium via the FAK/ERK1/2 and PI3K/Akt signalling pathways[8]. Both of these pathways have been implicated in the pathogenesis of endometriosis[9], with prolonged phosphorylation of ERK in endometrial epithelial[10] and stromal cells[11] and endometriomas[12] and increased phosphorylated Akt in ovarian endometriomas[13]. HOXA10 is a developmental transcription factor that has been linked to endometriosis, and, like hedgehog signalling, is involved in progesterone induced endometrial differentiation[14]. This evidence suggests that hedgehog signalling contributes to the pathogenesis of endometriosis. Thus, hedgehog signalling may have value as a diagnostic marker for endometriosis and as a therapeutic target.\u003c/p\u003e\n\u003cp\u003eHighly specific antibodies are increasingly being used in therapeutic applications[15]. The 5E1 monoclonal antibody recognises mammalian hedgehog ligands and prevents hedgehog signal transduction via the Patched receptor. 5E1 is commonly used as tool to block hedgehog signalling in mouse-based \u003cem\u003ein vivo\u003c/em\u003e experiments[16,17]. In the current study we investigated the potential of hedgehog blocking antibodies as a therapy for endometriosis by using 5E1 in a mouse model that closely mimics the human condition.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003eAnimal ethics\u003c/h2\u003e\n\u003cp\u003eAll animals were held in the Monash Medical Centre Animal Facility and housed on a 12-hour light/day cycle with access to normal chow and water \u003cem\u003eab libitum\u003c/em\u003e. Approval for all procedures described was obtained from Monash Medical Centre Animal Ethics Committee A.\u003c/p\u003e\n\u003ch2\u003eMouse model of endometriosis\u003c/h2\u003e\n\u003cp\u003eC57BL/6J background mice 8-12 weeks age underwent the mouse model of endometriosis as published previously[18]. Briefly, donor mice were ovariectomised, then given sub-cutaneous injections of \u0026beta;-estradiol (1\u0026micro;g/ml) on days 7-9. A progesterone secreting pellet was placed sub-cutaneously from days 13-19 (500ng/day) in combination with \u0026beta;-estradiol sub-cutaneous injections from days 13-15 (50ng/ml). On day 15 the endometrium was artificial decidualised by injecting 20\u0026micro;l of oil into the uterine lumen. Progesterone support was removed on day 19 and 4 hours later the menstrual-like endometrium was removed from the outer myometrium, minced into fragments approximately 1mm\u003csup\u003e3\u003c/sup\u003e and injected into ovariectomised estrogen primed (\u0026beta;-estradiol secreting pellet, 100ng/day days 7-19) recipients (approximately 20 fragments, 200mg of tissue in 200\u0026micro;l of PBS). Lesions were allowed to develop over 21 days (days 19-40) before collection on day 40. During this 21-day period mice were randomly allocated to one of two groups and treated twice weekly with either 250\u0026micro;g of anti-SHH antibody 5E1 (n=15, DSHB) or an isotype matched control antibody (n=18, BioXCell, MOPC-21 IgG1) in 200\u0026micro;l of sterile PBS. The 5E1 dose administered was based on previous reports that used this antibody to block hedgehog signalling in mouse \u003cem\u003ein vivo\u003c/em\u003e tumour models[19].\u003c/p\u003e\n\u003ch2\u003eLesion collection and fixation\u003c/h2\u003e\n\u003cp\u003eRecipient mice were euthanized using rising concentrations of carbon dioxide and cervical dislocation. Upon dissection all body cavities were photographed, the number of lesions found recorded and their size measured. Lesions were carefully removed from peritoneal organs and immersed in 4% w/v paraformaldehyde in PBS overnight at 4\u0026deg;C and cryoprotected in 30% w/v sucrose in PBS overnight at 4\u0026deg;C. Tissues were frozen in optimal cutting temperature medium and cryo-sectioned at 8\u0026micro;m thick.\u003c/p\u003e\n\u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e\n\u003cp\u003eUnless otherwise stated all sections underwent the following staining protocol. Sections were permeabilised in 0.2% Triton X-100 in PBS for 15 minutes, blocked in DAKO blocking solution for 1 hour and then stained for hedgehog activation, (GLI1 Rabbit monoclonal Thermo MA5-32553 10\u0026micro;g/ml), epithelial (EpCAM-PE rat anti mouse eBioscience 12-5791-81 2\u0026micro;g/ml), proliferation (Ki67-EF450 rat anti mouse eBioscience 50-5698-80 2\u0026micro;g/ml) and apoptosis (Caspase 3 Rabbit polyclonal R\u0026amp;D AF835 5\u0026micro;g/ml) markers for 1 hour at room temperature in 1% bovine serum albumin in PBS. For unconjugated primary antibodies, sections were incubated with secondary antibodies for 1 hour at room temperature (Donkey anti rabbit Alexafluor 568 LifeTech A10042 4\u0026micro;g/ml). Nuclei were counterstained with 5\u0026micro;g/ml Hoescht 33258 in PBS for 3 mins. Images were captured on an Olympus FV1200 confocal microscope using a 20x objective lens and adjusted for brightness and contrast in a linear manner using FIJI software[20].\u003c/p\u003e\n\u003ch2\u003eAnalysis of lesions\u003c/h2\u003e\n\u003ch2\u003eHistological analysis\u003c/h2\u003e\n\u003cp\u003eHaematoxylin and eosin staining was performed as per standard protocols on the 15\u003csup\u003eth\u003c/sup\u003e slide of each lesion (2 sections/slide x 8\u0026micro;m x 15 slides= 240\u0026micro;m deep). Lesion cross-sectional area was measured using FIIJ, using the trace outline and measure area tools.\u003c/p\u003e\n\u003cp\u003ePrior to the analysis of epithelial, proliferation and apoptosis markers, slides were blinded to reduce any bias during analysis. Markers were counted manually in FIJI. Total cell nuclei were calculated by counting the number of nuclei using the \u0026ldquo;threshold\u0026rdquo;, \u0026ldquo;watershed\u0026rdquo; and \u0026ldquo;analyse particles\u0026rdquo; functions. A minimum of 6 lesions were analysed per treatment group. A minimum of 3 fields of view per section were imaged and the average percentage of positive cell types in the total cell population was calculated for each lesion. Results were then unblinded and each lesion was then plotted as a single data point.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eGroup sizes were determined using G*Power software. For lesion number, we assumed an observed effect size of 0.5, alpha of 0.05 and Power of 0.8, the total sample size required was 26. For changes in lesion characteristics we assumed an effect size of 0.65, alpha of 0.05 and Power of 0.8, the total sample size required was 13.\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed in Graphpad Prism 8.0. Raw data was subjected to D\u0026rsquo;Agostino-Pearson normality testing prior to statistical analyses. Non-parametric analysis was performed when one of the sets of data failed to pass normality testing. All lesion analyses were subjected to unpaired, two-tailed Mann-Whitney testing. Significance was accepted where P\u0026le;0.05 and data represented graphically as individual data points and median.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003ch2\u003eThe location and incidence of lesions\u003c/h2\u003e\n\u003cp\u003eLesions were found at various locations in the isotype control and anti-SHH treated group (Figure 1A). The occurrence of lesions, indicative of endometriosis, was 100% in the isotype control treated group and 80% in anti-SHH treated group (p=0.08) (Figure 1B). In line with other reports using this model, irrespective of treatment, most lesions were detected on the mesentery (83% in the isotype control group, 67% in the 5E1 group)[18]. Lesions were also found on the body wall, on the external surface of the uterus and on the digestive tract (includes stomach, small intestine, large intestine) (Figure 1C). There was a significant decrease in the number of lesions per recipient detected on the mesentery after anti-SHH treatment compared to isotype control (p\u0026lt;0.01) (Figure 1D). This decrease was not observed for other locations but these lesions were infrequent and the dataset was small (Figure 1D). Given the decrease in the number of mesenteric lesions we assessed their lesion area independent of other lesions but found that this sub-group did not display any difference in area (p=0.0932, data not shown).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eGli1 expression as an indicator of Hedgehog activation\u003c/h2\u003e\n\u003cp\u003eGli1 was examined by immunostaining to detect hedgehog activation in collected lesions. Gli1 was readily detected in mouse brain, a known site of hedgehog signalling[21]. Very weak to no signal was detected in isotype and anti-SHH lesions from the endometriosis model (Figure 2A, representative of 3 animals). This suggests that endometriotic lesions do not have the high levels of hedgehog activation found in some tumours[5,22\u0026ndash;24]. GLI1 expression is increased in the endometrium of women with endometriosis in comparison to healthy controls[6], and in ovarian endometriomas[25] \u0026nbsp;but expression in human lesions at other sites is yet to be assessed. \u0026nbsp;Our mouse model does not address ovarian endometriomas due to recipients being ovariectomised, therefore we cannot comment on whether the ovarian location would result in differing GLI1 expression. \u0026nbsp;Furthermore, the human study used a polyclonal antibody, whereas the antibody used in our mouse studies was a monoclonal (clone JF09-08) which may account for discrepancy in results. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eLesion characteristics\u003c/h2\u003e\n\u003cp\u003eLesion establishment and survival relies on cross-talk between epithelial cells and the surrounding stroma[26]. Lesions were investigated for expression of the epithelial cell marker EpCAM, proliferation marker Ki67 and apoptosis marker Caspase 3 (Figure 2B). Variable expression of EpCAM was identified in the isotype control group. Fifty-eight percent of isotype lesions (7 of 12) had detectable EpCAM expression ranging from 0.17-26% positive cells in the total population. In the anti-SHH group, only 37.5% of lesions had detectable expression of EpCAM (3 of 8 lesions), only 1 lesion had a detectable level over 1% positive in the total cell population. Despite no significant decrease in EpCAM expression, the observed downward trend is in line with another report where a hedgehog inhibitor decreased endometrial epithelial cell proliferation \u003cem\u003ein vitro\u003c/em\u003e[27].\u003c/p\u003e\n\u003cp\u003eNo significant differences were identified between Isotype control and Anti-SHH groups for Ki67 and Caspase 3 (Figure 2B), which suggests 5E1 has no direct effect on overall proliferation or apoptosis of endometriotic lesions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDifferences in hedgehog pathway activation have been detected in women with endometriosis and in mouse models of endometriosis[6,7,28]. This study aimed to see whether the hedgehog neutralising antibody 5E1 would prevent lesion establishment and progression in a mouse model of endometriosis. Significantly fewer lesions were found on the mesentery of animals treated twice weekly with 5E1. However, no significant differences were observed in the size and morphology of the lesions that formed. These data suggest that hedgehog signalling is not integral to lesion progression, but may play a role in the survival of menstrual fragments in the peritoneal cavity and lesion initiation.\u003c/p\u003e\n\u003ch2\u003eLimitations\u003c/h2\u003e\n\u003cp\u003eNo effect of anti-SHH treatment was observed at other sites within the peritoneal cavity, however the number of lesions obtained from these sites was small (n=4-6 lesions) meaning it is not possible to draw clear conclusions. It is difficult to determine the mechanism by which mesenteric lesions are reduced from the data obtained.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003e5E1\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Sonic hedgehog neutralising monoclonal antibody, clone 5E1\u003c/p\u003e\n\u003cp\u003eEpCAM\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Epithelial cell adhesion molecule\u003c/p\u003e\n\u003cp\u003eSHH\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Sonic hedgehog\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics:\u003c/strong\u003e All mouse procedures were performed under approval MMCA2016-57 from Monash Medical Centre Animal Ethics Committee A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e Not Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All relevant data generated in this study is presented in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by the Victorian Government\u0026rsquo;s Operational Infrastructure fund and a Ferring Pharmaceuticals Innovation grant awarded to JA Deane, CE Gargett and FL Cousins. Ferring Pharmaceuticals had no input into design, collection or interpretation of data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions:\u003c/strong\u003e FLC, CEG and JAD designed the study. FLC, CEG and JAD obtained funding.\u0026nbsp; FLC, SM, SD and JAD collected data. FLC, JKF, RK and JAD analysed data.\u0026nbsp; FLC and JAD wrote the manuscript.\u0026nbsp; All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We would like to acknowledge the support of the Monash Health Translational Precinct\u0026rsquo;s Research Platforms Monash Micro-Imaging and Monash Histology and animal technicians for animal husbandry and care.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSampson JA. Metastatic or Embolic Endometriosis, due to the Menstrual Dissemination of Endometrial Tissue into the Venous Circulation. Am J Pathol. 1927 Mar;3(2):93-110.43.\u003c/li\u003e\n\u003cli\u003eGiudice LC, Kao LC. Endometriosis. Lancet. 2004 Nov;364(9447):1789\u0026ndash;99.\u003c/li\u003e\n\u003cli\u003eBeste MT, Pfaffle-Doyle N, Prentice EA, Morris SN, Lauffenburger DA, Isaacson KB, et al. Molecular Network Analysis of Endometriosis Reveals a Role for c-Jun-Regulated Macrophage Activation. Sci Transl Med. 2014 Feb 5;6(222).\u003c/li\u003e\n\u003cli\u003eKlemmt PAB, Carver JG, Koninckx P, McVeigh EJ, Mardon HJ. Endometrial cells from women with endometriosis have increased adhesion and proliferative capacity in response to extracellular matrix components: towards a mechanistic model for endometriosis progression. Hum Reprod. 2007 Dec;22(12):3139\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003eFeng Y-Z, Shiozawa T, Miyamoto T, Kashima H, Kurai M, Suzuki A, et al. Overexpression of hedgehog signaling molecules and its involvement in the proliferation of endometrial carcinoma cells. Clin Cancer Res. 2007 Mar 1;13(5):1389\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eHe Y, Guo Q, Cheng Y, Qu Y, Sun L, Kong C, et al. Abnormal activation of the sonic hedgehog signaling pathway in endometriosis and its diagnostic potency. Fertil Steril. 2018 Jul 1;110(1):128-136.e2.\u003c/li\u003e\n\u003cli\u003eHeard ME, Simmons CD, Simmen FA, Simmen RCM. Kr\u0026uuml;ppel-like factor 9 deficiency in uterine endometrial cells promotes ectopic lesion establishment associated with activated notch and hedgehog signaling in a mouse model of endometriosis. Endocrinology. 2014 Apr 29;155(4):1532\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003ePark SR, Kim SR, Park CH, Lim S, Ha SY, Hong IS, et al. Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells. Mol Ther. 2020 Feb 5;28(2):452\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eMcKinnon BD, Kocbek V, Nirgianakis K, Bersinger NA, Mueller MD. Kinase signalling pathways in endometriosis: Potential targets for non-hormonal therapeutics. Hum Reprod Update. 2016 Apr 1;22(3):382\u0026ndash;403.\u003c/li\u003e\n\u003cli\u003eYotova IY, Quan P, Leditznig N, Beer U, Wenzl R, Tschugguel W. Abnormal activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in eutopic endometrial stromal cells of patients with endometriosis. Hum Reprod. 2011;26(4):885\u0026ndash;97.\u003c/li\u003e\n\u003cli\u003eVelarde MC, Aghajanova L, Nezhat CR, Giudice LC. Increased mitogen-activated protein kinase kinase/extracellularly regulated kinase activity in human endometrial stromal fibroblasts of women with endometriosis reduces 3\u0026prime;,5\u0026prime;-cyclic adenosine 5\u0026prime;-monophosphate inhibition of cyclin D1. Endocrinology. 2009 Oct;150(10):4701\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eNg\u0026ocirc; C, Nicco C, Leconte M, Ch\u0026eacute;reau C, Arkwright S, Vacher-Lavenu MC, et al. Protein kinase inhibitors can control the progression of endometriosis in vitro and in vivo. J Pathol. 2010 Oct;222(2):148\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eCinar O, Seval Y, Uz YH, Cakmak H, Ulukus M, Kayisli UA, et al. Differential regulation of Akt phosphorylation in endometriosis. Reprod Biomed Online. 2009;19(6):864\u0026ndash;71.\u003c/li\u003e\n\u003cli\u003eZanatta A, Rocha AM, Carvalho FM, Pereira RMA, Taylor HS, Motta ELA, et al. The role of the Hoxa10/HOXA10 gene in the etiology of endometriosis and its related infertility: A review. J Assist Reprod Genet. 2010 Dec;27(12):701\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eDimitrov JD. Harnessing the Therapeutic Potential of \u0026lsquo;Rogue\u0026rsquo; Antibodies. Vol. 41, Trends in Pharmacological Sciences. Elsevier Ltd; 2020. p. 409\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eBeachy PA, Hymowitz SG, Lazarus RA, Leahy DJ, Siebold C. Interactions between Hedgehog proteins and their binding partners come into view. Vol. 24, Genes and Development. Cold Spring Harbor Laboratory Press; 2010. p. 2001\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eCucchi D, Occhione MA, Gulino A, De Smaele E. Hedgehog signaling pathway and its targets for treatment in basal cell carcinoma. Vol. 4, Journal of Experimental Pharmacology. Dove Medical Press Ltd.; 2012. p. 173\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eGreaves E, Cousins FL, Murray A, Esnal-Zufiaurre A, Fassbender A, Horne AW, et al. A Novel Mouse Model of Endometriosis Mimics Human Phenotype and Reveals Insights into the Inflammatory Contribution of Shed Endometrium. Am J Pathol. 2014 Jun;184(7):1930\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eBailey JM, Mohr AM, Hollingsworth MA. Sonic hedgehog paracrine signaling regulates metastasis and lymphangiogenesis in pancreatic cancer. Oncogene. 2009;\u003c/li\u003e\n\u003cli\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eDahmane N, S\u0026aacute;nchez P, Gitton Y, Palma V, Sun T, Beyna M, et al. The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development. 2001;128(24):5201\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eThayer SP, Di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ, Lauwers GY, et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature. 2003 Oct 23;425(6960):851\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eRuiz I Altaba A, Stecca B, S\u0026aacute;nchez P. Hedgehog-Gli signaling in brain tumors: Stem cells and paradevelopmental programs in cancer. Cancer Lett. 2004 Feb 20;204(2):145\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eWang LH, Choi Y La, Hua XY, Shin YK, Song YJ, Youn SJ, et al. Increased expression of sonic hedgehog and altered methylation of its promoter region in gastric cancer and its related lesions. Mod Pathol. 2006 May;19(5):675\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eLiu H, Zhang W, Wang L, Zhang Z, Xiong W, Zhang L, et al. GLI1 is increased in ovarian endometriosis and regulates migration, invasion and proliferation of human endometrial stromal cells in endometriosis. Ann Transl Med. 2019 Nov;7(22):663\u0026ndash;663.\u003c/li\u003e\n\u003cli\u003eHull ML, Escareno CR, Godsland JM, Doig JR, Johnson CM, Phillips SC, et al. Endometrial-peritoneal interactions during endometriotic lesion establishment. Am J Pathol. 2008 Sep;173(3):700\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eNakajima T, Iguchi T, Sato T. Hedgehog signaling plays roles in epithelial cell proliferation in neonatal mouse uterus and vagina. Cell Tissue Res. 2012 Apr 1;348(1):239\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003eHeard ME, Velarde MC, Giudice LC, Simmen FA, Simmen RCM. Kr\u0026uuml;ppel-Like Factor 13 Deficiency in Uterine Endometrial Cells Contributes to Defective Steroid Hormone Receptor Signaling but Not Lesion Establishment in a Mouse Model of Endometriosis1. Biol Reprod. 2015 Jun 1;92(6).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Endometrium, endometriosis, hedgehog signalling, mouse model","lastPublishedDoi":"10.21203/rs.3.rs-45953/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-45953/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObjective: Endometriosis is a common and painful condition characterized by the formation of endometrial lesions within the peritoneal cavity. Previous studies have suggested a role for hedgehog signalling in the pathogenesis of endometriosis. We investigated the role of hedgehog signalling in the establishment of endometriosis lesions using 5E1, a hedgehog ligand neutralising antibody, and a mouse model of endometriosis. To mimic the initiation of by endometriosis by retrograde menstruation, which is believed to occur in humans, donor mice underwent an artificial menstruation protocol. Fragments of menstrual endometrium were injected into the peritoneal cavity of estrogen primed recipients. Recipients received twice weekly injections of 5E1 or an isotype matched control antibody for three weeks. Lesions were collected and analysed for markers of epithelium, proliferation and apoptosis by immunofluorescence microscopy.\u003c/p\u003e\u003cp\u003eResults: Treatment with 5E1, reduced the number of lesions found on the mesentery. No significant changes were found in the size of lesions, abundance of endometrial epithelial cells, proliferation or apoptosis.\u003c/p\u003e","manuscriptTitle":"The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-09-14 17:40:46","doi":"10.21203/rs.3.rs-45953/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-09-18T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-09-11T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-09-11T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-10T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-10T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-08-13 21:01:21","doi":"10.21203/rs.3.rs-45953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-08-23T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-08-16T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-08-16T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewersInvited","content":"","date":"2020-08-12T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-08-10T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-08-09T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-08-09T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa922333-f236-4789-be07-313a244da407","owner":[],"postedDate":"September 14th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":294000,"name":"Obstetrics \u0026 Gynecology"}],"tags":[],"updatedAt":"2020-09-27T15:02:42+00:00","versionOfRecord":{"articleIdentity":"rs-45953","link":"https://doi.org/10.1186/s13104-020-05299-5","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2020-09-25 12:00:00","publishedOnDateReadable":"September 25th, 2020"},"versionCreatedAt":"2020-09-14 17:40:46","video":"","vorDoi":"10.1186/s13104-020-05299-5","vorDoiUrl":"https://doi.org/10.1186/s13104-020-05299-5","workflowStages":[]},"version":"v2","identity":"rs-45953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-45953","identity":"rs-45953","version":["v2"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}