Abstract
Purpose :
Bleb failure by fibrotic scarring remains a major problem of glaucoma filtration surgery. Dysfunctional PI3K/mTOR signaling has recently been recognized as a driver of aberrant proliferative responses in kidney, pulmonary and cardiac fibrosis. In this study, we investigated the anti-fibrotic effect of rapamycin, an mTOR inhibitor, on the TGF-b2 induced proliferation of human tenon fibroblasts (HTFs).
Methods :
Human tenon tissue was obtained from adult strabismus patients during surgery. Primary cell culture for the HTFs was performed. After 24 hour incubation with 2ng/mL TGF-β2, the cells were treated with rapamycin 1nM for 48 hours. Cell viability and proliferation were measured with MTT assay. Cell migration was assessed by scratch wound assay. Flow cytometry of AnnexinV/propidium iodide (PI) staining was performed to measure the cell apoptosis. Expression of proliferative gene MKi67, pro-apoptotic gene caspase 3, and autophagy related gene LC3B were analyzed by real-time PCR.
Results :
TGF-β2 significantly promoted the proliferation (138% of controls, p<0.01) and migration of HTFs, as well as the mRNA expression of MKi67. Rapamycin effectively attenuate the TGF-β2 induced cell proliferation (80.8% of TGF-β2gourp）, and migration. The up-regulated MKi67 expression in TGF-β2 treated HTFs was also distinctively inhibited by rapamycin. Cell apoptosis, as assessed by both flow cytometry and caspase 3 mRNA expression, was not observed in all groups. As an autophagy inducer, rapamycin significantly up-regulated the mRNA expression of LC3B, an important autophagy marker, in the HTF cells with and without TGF-β2.
Conclusions :
Rapamycin could effectively prevent the TGF-β2 induced proliferation and migration of HTF cells without affecting the cell survival. The involvement of mTOR and the underlying mechanism of autophagy need further investigation. Rapamycin may become a potential anti-fibrotic agent that may facilitate the therapeutic effect of glaucoma filtering surgery.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.