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Thomas Armin Fuchsluger, Daniel Thieme, Friedrich E Kruse, Siddharth Mahajan; Corneal endothelium can be protected against cell death by anti-Bax and anti-Bak siRNA. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4438. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Apoptotic cell death has been implicated in corneal endothelial cell (EC) loss during storage and after transplantation. Strategies to prevent apoptosis depend on the efficient targeting of apoptosis pathway proteins. In this study, we describe a siRNA-based approach for silencing pro-apoptotic proteins which in turn confers protection to corneal EC against apoptosis.
Xtreme gene siRNA transfection reagent was used to transfect HCEC-12 cells (70% confluency) and donor corneal endothelium with anti-Bax and/or anti-Bak siRNA (20nM, 40nM, 100nM; Santa Cruz, Heidelberg, Germany). The transfection efficiency was determined using fluorophore-conjugated control siRNA and flow cytometry. Following transfection with test siRNAs, presence of Bax and Bak proteins was analyzed at different time intervals by western blotting of cell lysates. Metabolic EC activity was measured by CCK-8 assay. In donor corneas, endohelium was peeled and for western blot analysis of Bax and Bak expression. Apoptosis was respectively studied by caspase-3 activity assay (following apoptosis induction with etoposide).
Transfection efficiency in HCEC-12 cells was highest in the 100nM siRNA groups (70±8%). The reduction of both Bax (65±12%) and Bak expression (50± 12%) was highly significant, compared to respective controls (p<0.005). Caspase-3 activity could be reduced by 52±6% (anti-Bax, p<0.05) / 46±8 (anti-Bak, p<0.05), compared to control-siRNA. Interestingly, there was no significant further reduction in EC treated with both anti-Bax+anti-Bak. In donor corneas’ endothelium siRNA transfection resulted in 22±8 % reduction of Bax expression and in a 24±9 % reduction of Bak expression (p<0.05, each, Actin-normalized). Here, caspase-3 activity was reduced by 19±6% (anti-Bax, p<0.05) / 21±8% (anti-Bak, p<0.05), respectively compared to Etoposide controls.
In this study we successfully demonstrated a transient reduction in pro-apoptosis proteins Bax and Bak by non-viral siRNA transfer both in HCEC-12 cells and in peeled donor corneal endothelium. We further showed that silencing of pro-apoptotic proteins confers protection against apoptosis in cells and experimental corneal tissue. This could open a new translational strategy to protect corneal EC.
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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