Establishment of a drug screening system for Fuchs endothelial corneal dystrophy

Abstract

Purpose : We previously reported that transforming growth factor-β (TGF-β) signaling plays an important role in the pathophysiology of Fuchs endothelial corneal dystrophy (FECD) by inducing the endoplasmic reticulum (ER) stress (Okumura et al., Sci Rep. 2017). The purpose of this study was to establish a drug screening system by using a cellular model of FECD in which caspase activity induced by TGF-β was evaluated.

Methods : After obtaining patient consent, corneal endothelial cells (CECs) were isolated from the Descemet’s membrane including corneal endothelium obtained from FECD patients who underwent Descemet's membrane endothelial keratoplasty. The CECs were cultured and then immortalized using both SV40 and hTERT to produce an iFECD cell model. The iFECD cells were seeded in a 96-well plate and treated with TGF-β2 (10 ng/mL) to induce cell death. The activation of caspase 3/7 was evaluated by Caspase-Glo^® 3/7 Assay (Promega Corp. Madison, WI) after 24 hours. The feasibility of the drug screening system was evaluated by indicators including coefficient of variation (CV) level (unevenness index), signal-to-background (S/B) ratio (signal-strength-ratio pre and post reaction), signal-to-noise (S/N) ratio (ratio of the size of the signal for the unevenness of the base), and Z'-factor (aim of the precision pro-assay). Finally, 765 FDA-approved drugs were evaluated via this drug screening system.

Results : Phase-contrast microscopy showed that iFECD cells were damaged after 24 hours of TGF-β treatment. Consistently, Caspase-Glo^® 3/7 Assay demonstrated that the percentage of caspase 3/7 activity in the TGF-β treatment group increased 224.6±5.1% more than in the non-treated control group (p<0.01). The percentage of CV level was 5.9% in the control group and 5.1% in the TGF-β treatment group (targeted value of CV: <10%). The S/B ratio was 2.25 (targeted value: 2<), the Z'-factor was 0.58 (targeted value: 0.5<), and the S/N ratio was 21.21. By screening the FDA-approved drug library, we found the potent drugs which suppress caspase 3/7 activity in iFECD.

Conclusions : The findings of this study show that we have established a reliable cell-based drug screening system via the use of a cellular model derived from FECD patients. Though further evaluation of screened drugs is necessary, this screening system might be beneficial for drug development for FECD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.