Purchase this article with an account.
Thomas Armin Fuchsluger, Daniel Thieme, Friedrich E Kruse, Siddharth Mahajan; Silencing of pro-apoptotic proteins as anti-apoptotic therapy for corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2164.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Apoptotic cell death has been identified as a major contributor in corneal endothelial cell (EC) loss both during corneal storage and after transplantation. Prevention of apoptosis has been suggested as a strategy to protect EC. To achieve this aim efficient targeting of apoptosis pathway proteins is required. In this proof-of-principle study, siRNA-mediated silencing of pro-apoptotic proteins was used to achieve protection of EC against apoptotic cell death.
EC were transfected in vitro and ex vivo with anti-Bax and anti-Bak siRNAs at a concentration of 1000nM. EC were studied at different time intervals for the presence of Bax and Bak proteins by western blotting. Metabolic activity was analyzed after transfection by CCK-8 assay. Induction of apoptosis was performed using etoposide (25µg/mL, 21 hours) and staurosporine (250nM, 6 hours) following siRNA transfection. Apoptosis was analyzed with caspase-3 activity assay and by analysis of downstream apoptosis marker proteins (cleaved Lamin A, cleaved PARP) by western blotting. Fluorescence intensity (Ex 360 nm, Em 450 nm) was measured using Fluoroskan Ascent FL fluorometer and luminometer for microplates (Thermo Scientific, Waltham, MA). Both untreated EC and those treated with a control siRNA served as controls.
Silencing of pro-apoptotic proteins Bax and Bak was achieved both in vitro and in vivo. Caspase-3 activity could be significantly reduced after both etoposide (52±3%) and staurosporine (38±6%) by combined Bax+Bak siRNA treatments relative to control-siRNA or untransfected EC. Downstream apoptosis marker proteins (cleaved Lamin A, cleaved PARP) were significantly reduced (83±6%, 79±4%) in Bax+Bak transfected EC compared to controls. In corneal donor tissue, Bax and Bak could be reduced by 30±5%. CCK-8 assay data did not show a significant effect of siRNA treatment on EC viability.
In this study, the proof-of-principle of successful transient reduction in pro-apoptosis proteins Bax and Bak and in downstream apoptosis markers by non-viral siRNA transfer was demonstrated in EC both in vitro and in vivo. The treatment did not result in a significant loss in metabolic activity. Therefore, silencing of pro-apoptotic proteins could be a functional approach to protect EC against apoptosis. These findings could be of high relevance in both corneal cultivation, donor graft cultivation and posterior lamellar graft preparation.
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