Inhibition of the rapamycin-insensitive mTORC1 /4E-BP1 axis attenuates TGF-β1-induced fibrotic response in human Tenon's fibroblasts

Jiayu Zou; Binrong Wu; Yan Tao; Zuimeng Liu; Huanyu Zhao; Pin Wang; Yuanbo Liang; Jia Qu; Shaodan Zhang

doi:10.1016/j.exer.2024.109927

Inhibition of the rapamycin-insensitive mTORC1 /4E-BP1 axis attenuates TGF-β1-induced fibrotic response in human Tenon's fibroblasts

Exp Eye Res. 2024 May 13:244:109927. doi: 10.1016/j.exer.2024.109927. Online ahead of print.

Authors

Jiayu Zou¹, Binrong Wu¹, Yan Tao¹, Zuimeng Liu¹, Huanyu Zhao¹, Pin Wang¹, Yuanbo Liang², Jia Qu³, Shaodan Zhang⁴

Affiliations

¹ The Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China.
² The Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China; National Clinical Research Center for Ocular Diseases, Wenzhou, China; Glaucoma Research Institute, Wenzhou Medical University, Wenzhou, China.
³ The Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China; National Clinical Research Center for Ocular Diseases, Wenzhou, China. Electronic address: jia.qu@eye.ac.cn.
⁴ The Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China; National Clinical Research Center for Ocular Diseases, Wenzhou, China; Glaucoma Research Institute, Wenzhou Medical University, Wenzhou, China. Electronic address: shaodan_zhang@eye.ac.cn.

PMID: 38750784
DOI: 10.1016/j.exer.2024.109927

Abstract

Subconjunctival fibrosis is the major cause of failure in both conventional and modern minimally invasive glaucoma surgeries (MIGSs) with subconjunctival filtration. The search for safe and effective anti-fibrotic agents is critical for improving long-term surgical outcomes. In this study, we investigated the effect of inhibiting the rapamycin-insensitive mTORC1/4E-BP1 axis on the transforming growth factor-beta 1(TGF-β1)-induced fibrotic responses in human Tenon's fibroblasts (HTFs), as well as in a rat model of glaucoma filtration surgery (GFS). Primary cultured HTFs were treated with 3 ng/mL TGF-β1 for 24 h, followed by treatment with 10 μM CZ415 for additional 24 h. Rapamycin (10 μM) was utilized as a control for mTORC1/4E-BP1 signaling insensitivity. The expression levels of fibrosis-associated molecules were measured using quantitative real-time PCR, Western blotting, and immunofluorescence analysis. Cell migration was assessed through the scratch wound assay. Additionally, a rat model of GFS was employed to evaluate the anti-fibrotic effect of CZ415 in vivo. Our findings indicated that both rapamycin and CZ415 treatment significantly reduced the TGF-β1-induced cell proliferation, migration, and the expression of pro-fibrotic factors in HTFs. CZ415 also more effectively inhibited TGF-β1-mediated collagen synthesis in HTFs compared to rapamycin. Activation of mTORC1/4E-BP signaling following TGF-β1 exposure was highly suppressed by CZ415 but was only modestly inhibited by rapamycin. Furthermore, CZ415 was found to decrease subconjunctival collagen deposition in rats post GFS. Our results suggest that rapamycin-insensitive mTORC1/4E-BP1 signaling plays a critical role in TGF-β₁-driven collagen synthesis in HTFs. This study demonstrated that inhibition of the mTORC1/4E-BP1 axis offers superior anti-fibrotic efficacy compared to rapamycin and represents a promising target for improving the success rate of both traditional and modern GFSs.

Keywords: CZ415; Collagen; Fibrosis; Glaucoma; mTORC1/4E-BP1.