Cellular Interplay Through Extracellular Vesicle miR-184 Alleviates Corneal Endothelium Degeneration

Tomoko Yamashita; Kazuko Asada; Morio Ueno; Nao Hiramoto; Tomoko Fujita; Munetoyo Toda; Chie Sotozono; Shigeru Kinoshita; Junji Hamuro

doi:10.1016/j.xops.2022.100212

Cellular Interplay Through Extracellular Vesicle miR-184 Alleviates Corneal Endothelium Degeneration

Ophthalmol Sci. 2022 Aug 18;2(4):100212. doi: 10.1016/j.xops.2022.100212. eCollection 2022 Dec.

Authors

Tomoko Yamashita¹, Kazuko Asada², Morio Ueno², Nao Hiramoto², Tomoko Fujita², Munetoyo Toda¹, Chie Sotozono², Shigeru Kinoshita¹, Junji Hamuro²

Affiliations

¹ Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
² Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Abstract

Objective: The objective of the study was to reveal the presence of cellular interplay through extracellular vesicle (EV) microRNAs (miRs), to dampen the vicious cycle to degenerate human corneal endothelium (HCE) tissues.

Design: Prospective, comparative, observational study.

Methods: The miR levels in neonate-derived corneal tissues, in the aqueous humor (AqH) of bullous keratoplasty and cataract patients, as well as in the culture supernatant (CS) and EV of cultured human corneal endothelial cells (hCECs), were determined using 3D-Gene human miR chips and then validated using the real-time polymerase chain reaction. The extracellularly released miRs were profiled after the forced downregulation of cellular miR-34a, either by an miR-34a inhibitor or exposure to H₂O₂. The senescence-associated secretory phenotypes and mitochondrial membrane potential (MMP) were assessed to determine the functional features of the released miRs.

Main outcome measures: Identification of functional miRs attenuating HCE degeneration.

Results: The miRs in AqH were classified into 2 groups: expression in 1 group was significantly reduced in neonate-derived tissues, whereas that in the other group remained almost constant, independent of aging. The miR-34a and -29 families were typical in the former group, whereas miR-184 and -24-3p were typical in the latter. Additionally, a larger amount of the latter miRs was detected in AqH compared with those of the former miRs. There was also a greater abundance of miR-184 and -24-3p in hCECs, EV, and CS in fully mature CD44^-/dull hCEC, leading to sufficient clinical tissue regenerative capacity in cell injection therapy. The repression of cellular miR-34a, either due to miR-34a inhibitors or exposure to oxidative stress, unexpectedly resulted in the elevated release of miR-184 and -24-3p. Secretions of VEGF, interleukin 6, monocyte chemotactic protein-1, and MMP were all repressed in both mature CD44^-/dull and degenerated CD44⁺⁺⁺ hCEC, transfected with an miR-184 mimic.

Conclusions: The elevated release of miR-184 into AqH may constitute cellular interplay that prevents the aggravation of HCE degeneration induced by oxidative stress, thereby sustaining tissue homeostasis in HCE.

Keywords: AQP-1, aquaporin 1; AqH, aqueous humor; CS, culture supernatant; Corneal endothelium degeneration; ECD, endothelial cell density; ER, endoplasmic reticulum; EV, extracellular vesicle; Extracellular vesicle; HCE, human corneal endothelium; IL-6, interleukin 6; MCP-1, monocyte chemotactic protein-1; MMP, mitochondrial membrane potential; MiR-184; Mitochondria metabolic homeostasis; Oxidative stress; SASP, senescence-associated secretory phenotype; SLC4A11, solute carrier family 4 member 11; SP, subpopulation; hCEC, cultured human corneal endothelial cell; miR, microRNA.