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. 2025 Jan;263(1):33-46.
doi: 10.1007/s00417-024-06481-2. Epub 2024 Jul 23.

Inhibition of mTOR differently modulates planar and subepithelial fibrogenesis in human conjunctival fibroblasts

Megumi Watanabe #  1 , Yuri Tsugeno #  2 , Tatsuya Sato #  3   4 , Megumi Higashide  2 , Araya Umetsu  2 , Masato Furuhashi  3 , Hiroshi Ohguro  5
Affiliations

Inhibition of mTOR differently modulates planar and subepithelial fibrogenesis in human conjunctival fibroblasts

Megumi Watanabe et al. Graefes Arch Clin Exp Ophthalmol. 2025 Jan.

Erratum in

Abstract

Purpose: In the current investigation, the effects of the mTOR inhibitors, Rapa and Torin1 on the TGF-β2-induced conjunctival fibrogenesis were studied.

Study design: Experimental research.

Methods: 2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D).

Result: Rapa or Torin1 both significantly increased planar proliferation in the non-TGF-β2-treated 2D HconF cells, but in the TGF-β2-treated cells, this proliferation was inhibited by Rapa and enhanced by Torin1. Although Rapa or Torin1 did not affect cellular metabolism in the non-TGF-β2-treated HconF cells, mTOR inhibitors significantly decreased and increased the mitochondrial respiration and the glycolytic capacity, respectively, under conditions of TGF-β2-induced fibrogenesis. Subepithelial proliferation, as evidenced by the hardness of the 3D spheroids, was markedly down-regulated by both Rapa and Torin1 independent of TGF-β2. The mRNA expressions of several ECM molecules and their regulators fluctuated in the cases of 2D vs 3D and TGF-β2 untreated vs treated cultures.

Conclusion: The present findings indicate that mTOR inhibitors have the ability to increase and to reduce planar and subepithelial proliferation in HconF cells, depending on the inhibitor being used.

Keywords: 3D spheroid culture; Human conjunctival fibroblast; TGF-β2; mTOR inhibitor.

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Conflict of interest statement

Declarations. Research involving human participants and/or animals: All procedures performed in studies involving human participants were in accordance with the ethical standards approved by the institutional review board (IRB registration number 282–8, Sapporo Medical University Hospital, Japan,) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent to participate: Informed consent was obtained from all individual participants included in the study. Conflict of interest: All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

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