Mitochondrial miRNA494-3p in extracellular vesicles participates in cellular interplay of iPS-Derived human retinal pigment epithelium with macrophages. (July 2021)

• Record Type: Journal Article
• Authors: Mukai, Atsushi; Otsuki, Yohei; Ito, Eiko; Fujita, Tomoko; Ueno, Morio; Maeda, Tadao; Kinoshita, Shigeru; Sotozono, Chie; Hamuro, Junji
• Is Part Of: Experimental eye research. Volume 208(2021)
• Publication Date: 2021-07
• Subjects: iPS derived RPE -- Macrophage -- EV miRNA -- Innate cross talk -- AMD; hRPE induced pluripotent stem cell (iPS)-induced human retinal pigment epithelium -- mpRPE mouse primary RPE -- EV extracellular vesicle -- CD63+ EV EV-associated CD63+ proteins -- miR microRNAs -- Mps macrophages -- hMps human macrophages -- THP-hMps phorbol 12-myristate 13-acetate induced differentiated human macrophages from THP-1 cells -- VEGF vascular endothelial growth factor -- PEDF pigment epithelium-derived factor -- MCP-1 monocyte chemotactic protein -- FBS fetal bovine serum -- DMEM Dulbecco's modified Eagle's medium -- AMD age related macular degeneration -- Elisa enzyme linked immunosorbent assay -- CS culture supernatant -- Sup supernatant -- FACS fluorescence activated cell sorting -- LPS lipopolysaccharide -- UC ultracentrifugation -- NTA nano-particle tracking assay -- TLR4 toll like receptor 4 -- PMA phorbol 12-myristate 13-acetate -- ZO-1 zonula occludin-1; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2021.108621
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: To explore new molecular targets for therapy in human model systems by discerning the role of extracellular vesicle (EV) microRNAs (miRs) secreted by human retinal pigment epithelium (hRPE) cells and their cellular interplay with macrophages (Mps). Human Mps differentiated from THP-1 cells stimulated by phorbol myristate acetate were co-cultured with induced pluripotent stem cell-derived differentiated hRPE (iPS-hRPE) cells in Transwell® system separated by 0.40 μm or 0.03 μm filters. EV-associated CD63 + proteins (CD63 + EV) were detected by western blotting, and secreted EVs were analyzed by Nanosight tracking. The miR profiles of the secreted EVs were determined using 3D-gene human microRNA chips (Toray Industries, Inc.). Levels of CD63 + EV were increased in co-cultures concomitantly with the increased production of EV particles (50–150 nm). The increased production of EVs was associated with higher production of MCP-1, IL-6, IL-8 from hRPE cells, and VEGF and repressed production of TNF-α from Mps and pigment epithelium-derived factor (PEDF) from RPE cells. Ultracentrifugation of semi-purified EVs increased the secretion of these pro-inflammatory cytokines and EV particles from hRPE cells, but this effect was eliminated in transwells equipped with 0.03 μm filters, whereas no repression of PEDF and TNF-α secretion occurred. 3D-gene miR analysis revealed a selective increase in secretion of miR494-3p in EVs from iPS-hRPE cells during the interplay with Mps. The miRs in EVs secreted by hRPE cells may have a critical role in the vicious inflammatory cycle, whereas repression of TNF-α and PEDF require cell-to-cell contact that is independent of EVs or exosomes. MiR494-3p may be a candidate molecular target of diagnosis and therapy for age-related macular degeneration. Highlights: CD63 + EV was increased in co-cultures of iPS derived hRPE cells with macrophages from THP-1. The increase of EVs was associated with the production of MCP-1, IL-6, IL-8, and VEGF from hRPE. EV-miRNAs aggravate inflammatory cycles, while TNF-α and PEDF was repressed independent of EV. MiRNA494-3p was found in EV secreted by hRPE through the interplay with macrophages.