The role of estrogen receptors in fibrosis: in search of therapeutics

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dc.contributor.advisorSmith, Carineen_ZA
dc.contributor.advisorOllewagen, Traceyen_ZA
dc.contributor.authorAlberts, Jannekeen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.en_ZA
dc.date.accessioned2024-02-05T15:38:13Zen_ZA
dc.date.accessioned2024-04-27T00:01:26Zen_ZA
dc.date.available2024-02-05T15:38:13Zen_ZA
dc.date.available2024-04-27T00:01:26Zen_ZA
dc.date.issued2024-02en_ZA
dc.descriptionThesis (MMed)--Stellenbosch University, 2024.en_ZA
dc.description.abstractENGLISH ABSTRACT: Background: Autoimmune rheumatic diseases, affecting 5-8% of people globally, exhibit a 2:1 female prevalence bias, influencing severity and prognosis. Chronic inflammation leads to immune complex formation, which causes vessel occlusion, and resulting in fibrosis, contributing to cardiovascular diseases, particularly myocarditis. Fibrosis - driven by activated fibroblasts - results from extracellular matrix dysregulation, causing tissue scarring and organ dysfunction. Therefore, investigating antifibrotic interventions is crucial for global healthcare. 17β-estradiol (E2), the predominant female hormone, has the potential to modulate fibrosis, but literature in this niche is contradictory. Zebrafish have been identified as an ideal model for studying sex bias in autoimmune rheumatic-related myocarditis, exploring the role of E2 in chronic inflammation and fibrosis. Methods: To elucidate underlying mechanisms, a multidisciplinary approach was imperative. Employing diverse model systems, the thesis incorporated two pilot studies. The initial pilot study utilised male human dermal fibroblasts (HDFs) in vitro to assess the impact of E2 on cell function linked to fibrotic outcome. Cell viability and fibronectin production were assessed after 24 hours (h) E2- exspoure relative to that of untreated cells. Subsequently, a second pilot study evaluated behavioural outcomes in larval zebrafish, in response to exposure of a range of E2 dosages for 60 h, informing the selection of an appropriate, potentially beneficial, E2 concentration for the main experiment. A significant innovation was the establishment of a novel larval zebrafish myocarditis model, by means of microinjection with a sclerosing agent (bleomycin). Using this model, the effect of added E2 and modulation of estrogen receptor (ER) signalling on fibrosis was determined by staining for collagen (Masson-Goldner Trichrome) and vimentin (immunofluorescence staining). Results: Results generated in HDFs elucidated that treatment with E2 did not significantly alter the fibroblast viability or fibronectin production. Microinjected zebrafish larvae exhibited lower basal activity levels than controls (p<O.05). Placebo and E2 + Tamoxifen (TAM)-treated groups showed decreased activity (p<O.05). Post-hoc testing revealed significant vimentin fluorescence differences post-microinjection (p<O.0001) independent of whether a sclerosing agent or phosphate-buffered saline (PBS) was injected. Both PBS and bleomycin microinjected larvae had more collagen deposition when compared to the noninjected control (p<O.0001). E2 mitigated both the fibroblast infiltration and collagen deposition effects (p<O.0001), despite bleomycin microinjection. However, treatment with ER antagonists (TAM, 4-[2-Phenyl-5,7-is(trifluoromethyl)pyrazolo[1,5-a]-pyrimidin-3-yl]phenol (PHTPP)) negated the effect of E2, leading to significant increases in vimentin expression (p<O.01 — p<O.0001) and collagen deposition compared to E2 exposure alone (p<O.0001). Conclusion: Male (HDF) cells did not exhibit a robust response to exogenous E2, likely due to insufficient ER expression for the context in which they were used. For the purposes of the current investigation, larval zebrafish models proved more suitable as research tool. The observed in vivo effects of E2 on collagen deposition and fibroblast abundance in chronic myocarditis in zebrafish larvae highlight the beneficial role of E2 in the context of cardiac fibrotic outcome. Moreover, our model did not incorporate autoimmune components, however, it serves as a foundational framework for research within the context of autoimmune rheumatic diseases.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Inleiding: Outo-immuun rumatiese siektes, wat 5-8% van die wêreldbevolking affekteer, toon 'n 2:1 oorheersende vroulike voorkomspartydigheid en beïnvloed die erns en prognose. Kroniese inflammasie kan immuunkomplekse vorm, wat bloedvaatblokkasie veroorsaak en tot fibrose lei, wat bydra tot kardiovaskulêre siektes, veral miokarditis. Fibrose, aangedryf deur geaktiveerde fibroblaste, ontstaan as gevolg van die deregulering van die ekstrasellulêre matriks, wat tot weefsellittekenvorming en orgaandisfunksie lei. Daarom is die ondersoek na antifibrotiese ingrypings van kritieke belang vir wêreldwye gesondheidsorg. 17β-estradiol (E2), die hoof vroulike hormoon, het die potensiaal om fibrose te moduleer, maar die literatuur in hierdie nis is teenstrydig. Sebravisse is geïdentifiseer as 'n ideale model vir die bestudering van geslagspartydigheid in outo-immuun rumaties-verwante miokarditis, wat die rol van E2 in chroniese inflammasie en fibrose ondersoek. Metodes: Om die onderliggende meganismes te verduidelik, was 'n multidissiplinêre benadering noodsaaklik. Deur gebruik te maak van uiteenlopende modelle, het die tesis twee lootsstudies ingesluit. Die aanvanklike lootsstudie het menslike dermale fibroblaste (HDFs) in vitro gebruik om die impak van E2 op selfunksie gekoppel aan fibrotiese uitkoms te assesseer. Sellewensvatbaarheid en fibronektienproduksie is beoordeel na 24 uur (h) E2-blootstelling relatief tot onbehandelde selle. Vervolgens het 'n tweede lootsstudie gedragsuitkomste in sebravislarwes geëvalueer, in reaksie op blootstelling van 'n reeks E2-konsentrasies vir 60 h, wat die keuse van 'n toepaslike, potensieel voordelige, E2-konsentrasie vir die hoofeksperiment ingelig het. 'n Beduidende innovering was die vestiging van 'n nuwe sebravislarwe miokarditis model, deur middel van mikro-inspuiting met 'n verhardingsmiddel (bleomisien). Met behulp van hierdie model is die effek van bygevoegde E2 en modulering van estrogeenreseptor (ER) seine op fibrose bepaal deur te kleuring vir kollageen (MassonGoldner Trichrome) en vimentien (immunofluoressensiekleuring). Resultate: Resultate wat in HDFS gegenereer is, het gewys dat behandeling met E2 nie beduidend die fibroblast lewensvatbaarheid Of fibronektienproduksie verander het nie. Mikro-ingespuite sebravislarwes het laer basale aktiwiteitvlakke as kontroles getoon (peO.05). Plasebo en E2 Tamoxifen (TAM)- behandelingsgroepe het afname in aktiwiteit getoon (p<O.05). Post-hoc toetsing het beduidende vimentienfluoresensieverskille na mikro-inspuiting aangetoon ongeag Of 'n verhardingsmiddel Of fosfaatgebufferde soutoplossing (PBS) ingespuit is. Beide PBS- en bleomisienmikro-ingespuite larwes het meer kollageenafsetting getoon in vergelyking met die oningespuite kontrole (peO.OOOI). E2 het beide die fibroblast infiltrasie en kollageenafsettingseffekte verminder (p<O.OOOI), ten spyte van mikro-inspuiting. Behandeling met ER-antagoniste (TAM, PHTPP) het egter die effek van E2 tenietgedoen, wat gelei het tot beduidende toenarnes in vimentienuitdrukking (p<0.01 - p<0.0001) en kollageenafsetting in vergelyking met blootstelling aan E2 alleen (p<0.0001). Gevolgtrekking: Manlike (HDF) selle het nie 'n robuuste reaksie op eksogeen E2 toed getoon nie, waarskynlik as gevolg van onvoldoende ER-uitdrukking vir die konteks waarin hulle gebruik is. Vir die doeleindes van die huidige ondersoek het sebravislarwemodelle meer geskik geblyk as navorsingsinstrument. Die waargenome in vivo effekte van E2 op kollageenafsetting en fibroblast- oorvloed in kroniese miokarditis in sebravislarwes beklemtoon die voordelige rol van E2 in die konteks van kardiale fibrotiese uitkoms. Boonop, ons model het nie outo-immuunkomponente ingesluit nie, maar dit dien egter as 'n fundamentele raamwerk vir navorsing binne die konteks van outo-immuun rurnatiese siektes.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxviii, 95 pages : illustrationsen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130614en_ZA
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshAutoimmune diseases -- Animal modelsen_ZA
dc.subject.lcshEstrogen -- Receptorsen_ZA
dc.subject.lcshInflammation -- Animal modelsen_ZA
dc.subject.lcshMyocarditisen_ZA
dc.subject.lcshFibrosisen_ZA
dc.titleThe role of estrogen receptors in fibrosis: in search of therapeuticsen_ZA
dc.typeThesisen_ZA
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