Purchase this article with an account.
Genta Nakayama, Naoki Okumura, Takeshi Oshima, Emi Ueda, Kyoko Watanabe, Theofilos Tourtas, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Noriko Koizumi; Feasibility of mTOR inhibitor for the treatment of Fuchs endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3878.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We previously reported that the activation of transforming growth factor-β (TGF-β) signaling plays an important role in the death of corneal endothelial cells in Fuchs endothelial corneal dystrophy (FECD) (Okumura et al., Sci Rep. 2017). We screened the FDA-approved drug library by cell-based screening system, and found that the mammalian target of rapamycin (mTOR) inhibitor suppressed the apoptosis of corneal endothelial cells derived from patients with FECD. In this study, we investigated the feasibility of mTOR inhibitors as a potential drug for treating FECD.
Corneal endothelial cells obtained from FECD patients were cultured and immortalized to produce an FECD cell model (iFECD), and were then treated with TGF-β2 (10 ng/ml) to induce cell death. The effect of mTOR inhibitors (rapamycin, everolimus, and temsirolimus) on cell death was evaluated by phase contrast microscopy. The inhibitory effect on apoptosis was examined by western blotting, Caspase-Glo®3/7 Assay, and flow cytometry. The effect of mTOR knockdown by siRNA on cell death was also evaluated.
Phase contrast microscopy showed that TGF-β2 induced iFECD cell death. On the other hand, rapamycin, everolimus, and temsirolimus suppressed TGF-β2-mediated cell death. Flow cytometry showed that Annexin V positive apoptotic cells were significantly decreased by rapamycin in comparison to the TGF-β2-treated iFECD (8.1±0.8% and 15.3±1.4%, respectively) (p<0.01). Western blotting showed that TGF-β2 induced cleavages of caspase 3 and poly (ADP-ribose) polymerase (PARP), yet mTOR inhibitors suppressed the cleavage of those apoptotic proteins. Caspase-Glo®3/7 Assay showed that mTOR inhibitors significantly decreased the caspase 3/7 activity in comparison to the TGF-β2-treated iFECD (p<0.01). As with the mTOR inhibitors, flow cytometry showed that Annexin V positive apoptotic cells were significantly decreased in mTOR siRNA in comparison to in the control (p<0.01). Western blotting showed that TGF-β2-mediated cleavages of caspase 3 and PARP were suppressed by mTOR siRNA.
The findings of this study showed that mTOR inhibitors suppressed TGF-β-signaling mediated apoptosis in FECD cell models, and that mTOR signaling pathway might be the potential druggable target for the treatment of FECD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only