Complement activation contributes to subretinal fibrosis through the induction of epithelial-to-mesenchymal transition (EMT) in retinal pigment epithelial cells

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• dc.contributor.author Llorián-Salvador, María
• dc.contributor.author Byrne, Eimear M.
• dc.contributor.author Szczepan, Manon
• dc.contributor.author Little, Karis
• dc.contributor.author Chen, Mei
• dc.contributor.author Xu, Heping
• dc.date.accessioned 2022-11-03T07:10:18Z
• dc.date.available 2022-11-03T07:10:18Z
• dc.date.issued 2022
• dc.description.abstract Background: We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved. Methods: Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10-100 ng/mL) or TGF-β2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-β1, TGF-β2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis. Results: Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II+ cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-β2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-β1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I+ lesions only mildly reduced. Conclusions: Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a-C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis.
• dc.format.mimetype application/pdf
• dc.identifier.citation Llorián-Salvador M, Byrne EM, Szczepan M, Little K, Chen M, Xu H. Complement activation contributes to subretinal fibrosis through the induction of epithelial-to-mesenchymal transition (EMT) in retinal pigment epithelial cells. J Neuroinflammation. 2022 Jul 14;19(1):182. DOI: 10.1186/s12974-022-02546-3
• dc.identifier.doi http://dx.doi.org/10.1186/s12974-022-02546-3
• dc.identifier.issn 1742-2094
• dc.identifier.uri http://hdl.handle.net/10230/54663
• dc.language.iso eng
• dc.publisher BioMed Central
• dc.relation.ispartof J Neuroinflammation. 2022 Jul 14;19(1):182
• dc.rights © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword Age-related macular degeneration
• dc.subject.keyword C3a
• dc.subject.keyword C5a
• dc.subject.keyword Complement system
• dc.subject.keyword Epithelial-to-mesenchymal transition
• dc.subject.keyword Inflammation
• dc.subject.keyword Macular fibrosis
• dc.subject.keyword Retinal pigment epithelial cell
• dc.subject.keyword Subretinal fibrosis
• dc.title Complement activation contributes to subretinal fibrosis through the induction of epithelial-to-mesenchymal transition (EMT) in retinal pigment epithelial cells
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion