Stimulation of quiescent corneal endothelial cells by direct delivery of the SV40 large T-antigen protein

Abstract

Purpose. To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells. Methods. Liposome-mediated delivery of proteins into bovine corneal endothelial cells (BCEC) was utilized in this study. Initially, beta-galactosidase was used as a marker protein for cell delivery and cells were assayed colorimetrically for beta-galactosidase activity. Subsequently, SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry 24 hours after liposome-protein treatment. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the SV40 large T-antigen was detected by standard immunohistochemical methods. Results. Beta-galactosidase or SV40 large T antigen were introduced into BCECs using liposome transfer methods. The transfer efficiency of beta-galactosidase was > 30% of the cells. SV40 large T antigen was successfully introduced and was localized to the nuclei of BCECs. The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation. Co-labeling confirmed that only cells containing SV40 large T antigen were positive for de novo DNA synthesis. Conclusions. This study demonstrates that proteins can be inserted directly into corneal endothelial cells. In the case of the SV40 large T-antigen, the protein localized to the nucleus and maintained its bioactivity by inducing DNA synthesis. This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.