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Masakazu Sato, Naoki Okumura, Makiko Nakahara, Takahiko Sato, Koji Kitazawa, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Noriko Koizumi; The role of TCF4 in the pathophysiology of Fuchs endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4435. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that the expression level of the TCF4 gene is increased in the corneal endothelium of Fuchs corneal endothelial dystrophy (FECD) patients (Hayashi et al. ARVO 2017). We also reported that activation of transforming growth factor-β (TGF-β) signaling plays an important role in the pathophysiology of FECD via the induction of endoplasmic reticulum stress (Okumura et al., Sci Rep. 2017). The purpose of this present study was to investigate the effect of TCF4 in the pathophysiology of FECD using a cellular model established from patients with FECD.
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. TCF4 was knocked out using CRISPR/Cas9 in iFECD (TCF4-/- iFECD). iFECD and TCF4-/- iFECD were treated with TGF-β2 (10 ng/mL) to evaluated the effect of TCF4 on cell death and extracellular matrix (ECM) production.Apoptosis was assessed by flow cytometry using Annexin V staining. Activation of caspase-3 and poly (ADP-ribose) polymerase apoptosis-related proteins (PARP) were evaluated by western blotting. Snail1 and ZEB1 (epithelial-mesenchymal transition related proteins), and fibronectin were also evaluated.
Phase-contrast microscopy imaging showed that TGF-β2 induced iFECD cell death, however, the suppression of cell death was observed in TCF4-/- iFECD. Flow cytometry showed that Annexin V positive apoptotic cells were significantly increased from 9.6±0.9% to 35.8±2.7% by TGF-β2 in iFECD (p<0.01). In contrast, Annexin V positive cells were not increased by TGF-β2 in TCF4-/- iFECD (from 6.2±0.2% to 7.5±0.4%). Western blotting showed that the apoptosis-related proteins caspase-3 and PARP were cleaved (activated) by TGF-β2 in iFECD, and that this activation was suppressed in TCF4-/- iFECD. The expression level of Snail1 was decreased in TCF4-/- iFECD in comparison to iFECD, whereas ZEB1 was not altered. The expression level of fibronectin was lower in TCF4-/- iFECD than in iFECD.
Our findings suggest that TCF4 might be responsible for cell death and the upregulation of ECM production in the corneal endothelium of FECD patients.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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