Abstract
Purpose: :
Corneal transplantation is the most common tissue transplantation worldwide, and donor corneas are in short supply. Corneal endothelial cells (EC) maintain corneal transparency and EC loss during storage is a major reason for discarding donor corneas. The goal of this study was to develop a gene therapy approach to enhance EC survival during storage.
Methods: :
Gene transfer with anti-apoptotic molecules p35 (baculoviral) or bcl-xL (mammalian) to EC of human corneas was accomplished with the lentiviral vector pHAGE-CMV-MCS-IZsGreen (3 x 10^5 IU/ml). These corneas were compared to untreated controls and to corneas being transduced with IZsGreen only or with bcl-xL at a high titer (1.2 x 10^8 IU/ml). EC loss was studied under typical eye bank storage conditions, hypothermic (Optisol GS, 4oC) or organ culture (37oC, n=22). Changes in EC density, cell morphology and phases of cell death were assessed by phase contrast microscopy during long-term storage. Apoptosis was evaluated by TUNEL staining and confocal laser scanning microscopy.
Results: :
Increased expression of either anti-apoptotic molecule, p35 or bcl-xL, enhanced survival of EC. During hypothermic (11 weeks) as well as organ culture storage (5 weeks) corneas expressing p35 or bcl-xL demonstrated significantly higher cell counts (p<0.01) and improved physiological morphology (p<0.01). Nuclear fragmentation, loss of EC monolayer integrity or massive EC loss occurred at later timepoints. In addition, apoptosis could be determined as the underlying mechanism for EC loss during storage. Expression of p35 or bcl-xL resulted in less apoptosis.
Conclusions: :
Protection of EC by expression of the proteins p35 or bcl-xL is an effective method to increase EC survival during long-term storage of donor corneas. Translation of this technique could decrease donor tissue loss in eye banking without the need to increase donor figures, reduce graft failure after transplantation and is of specific interest as to precut corneas and DSAEK/DMEK procedures.
Keywords: gene transfer/gene therapy • cornea: endothelium • apoptosis/cell death