Abstract
Purpose :
Subconjunctival fibrosis is the main cause of surgery failure for both conventional trabeculectomy and tube-shunt implantation, and modern subconjunctival minimal invasive glaucoma surgeries. We investigate the effect of rapamycin-insensitive mTORC1/4E-BP1 inhibition on the transforming growth factor-beta 1(TGF-β1)-induced fibrotic responses of human Tenon’s fibroblasts (HTFs), as well as in a rat model of glaucoma filtration surgery (GFS)
Methods :
Primary cultured HTFs were obtained from healthy adults during stabismus surgery. HTFs were incubated in the medium containing 3ng/mL TGF-β1, and subsequently treated with 10μM CZ415, a pan-mTOR inhibitor which demonstrated obvious mTORC1/4E-BP1 inhibition, for another 24 hours. Rapamycin was used as mTORC1/4E-BP1 signaling-insensitive control. Expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col-I), and fibronectin were evaluated by quantitative real-time PCR, Western blot and immunofluorescence analysis. Scratch-Wound assay was performed to determine the effect of CZ415 on the migration of HTFs. Inhibitory effects on mTORC1/4E-BP1 axis by CZ415 and rapamycin were also investigated. A rat model of glaucoma filtering surgery was established to further assess the anti-fibrotic effect of CZ415 in vivo.
Results :
Both rapamycin and CZ415 treatment obviously attenuated the TGF-β1-induced cell proliferation, migration and expression of profibrotic factors of HTFs. TGF-β1-mediated collagen synthesis of HTFs was significantly inhibited by CZ415 compared with rapamycin. Significant activation of mTORC1/4E-BP signaling was observed after TGF-β1 exposure, which was greatly inhibited by CZ415 instead of rapamycin. In vivo results also indicated that CZ415 effectively alleviated subconjunctival collagen deposition in rats after glaucoma filtering surgery.
Conclusions :
Rapamycin-insensitive mTORC1 signaling via 4E-BP1 is crucial for the TGF-β1-stimulated collagen synthesis in HTFs. Inhibition of mTORC1/4E-BP1 axis shows superior anti-fibrotic efficacy than rapamycin, and may become a promising target for improving the success of both conventional and modern GFSs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.