The results hereby presented demonstrate clear advantages to EC expressing anti-apoptotic proteins p35 or Bcl-xL during storage, both in terms of retention of cell viability and physiologic cell morphology. Prolongation of corneal endothelial cell viability during storage by means of anti-apoptotic gene transfer is a novel finding that confirms our hypothesis that EC apoptosis is a pivotal process to loss of donor tissue quality. Furthermore, we demonstrate that gene transfer of the pan-caspase inhibitor p35 prevents apoptosis in EC more effectively than Bcl-xL during both organ (37°) and hypothermic (4°C) storage conditions. Application of this technology in eye banking could lead to an increase of donor corneas available for transplantation, and may lead to a decrease in graft failure since EC deterioration is a common denominator for all forms of corneal graft failure in humans.
Previous studies have suggested protective effects of anti-apoptotic proteins in the rat 25
, in the mouse 13
, and in murine or human corneal EC lines 13,16,26
. Interestingly, such studies have not been performed in human corneas under adapted eye bank conditions which would be crucial for application in grafted donor corneas. The findings of the present study delineate the efficacy of EC protection by anti-apoptotic gene transfer under clinical conditions both regarding the optimal type of gene and elucidating the potential impact of viral dose.
Gene transfer using a lentiviral vector is well known to lead to high gene expression in transduced cells 25,27
. Application of lentiviral vectors results in retained cell viability after infection and a rapid onset of gene expression 27,28
. Previous studies have shown that this vector can be used to successfully transduce rat, bovine, murine and human corneal endothelial cells 13,25,27
. As demonstrated in our experiments, lentiviral-mediated gene expression resulted in high IZsGreenW expression 48 hours post-infection after as few as 30 minutes. We investigated the effects of two virus titers on cell viability 29
by showing that transduction using a very high titer of Bcl-xL does not protect, but derogates, EC vitality due to increased apoptosis.
The functional relevance of EC expression of p35 or Bcl-xL is confirmed by the novel finding of prolonged cell viability and maintenance of physiological morphology under eye bank conditions. Currently, up to 30% of all donor corneas are considered unusable for corneal transplantation due to EC loss during organ preservation 4,6
, and up to 16% during hypothermic storage 11
. Our data suggest that anti-apoptotic gene transfer could increase the yield of limited donor tissue by decreasing the rate at which eye banks reject donor tissues based on EC quality.
Our findings consistently show higher EC counts and a higher percentage of physiological morphology in EC expressing p35 compared to Bcl-xL. This difference might be explained by the different ways the two proteins interact within the cell-intrinsic suicide signaling cascade. A key characteristic of the Bcl-2 protein family is that its members share sequence homology with four domains (BH1, 2, 3, 4). The BH3 domain is essential for the promotion of the death function of pro-apoptotic molecules 30
. Interestingly, the anti-apoptotic proteins Bcl-xL or Bcl-2 merely inhibit pro-apoptotic molecules of the BH3-only subfamily [such as Bad, Bid or Bim] 31
. Hence, multidomain pro-apoptotic molecules like Bax, Bak or Bok can still trigger the death program leading to apoptosis, while p35 directly inhibits a broad spectrum of caspases. Cleavage of the reactive site loop of p35 leads to translocation of its N-terminus into the active site of caspases resulting in irreversible caspase inhibition 32,33
. Our data suggest that broad suppression of caspases seems to have a higher impact on preventing EC death than inhibition of select pro-apoptotic proteins of the Bcl-2 family.
Previous studies identify apoptosis, an act of cellular “self-destruction”, as the main contributor to cell death during preservation 10-12,34
. Depending on the approach of cell death evaluation, different percentages of EC undergoing apoptosis have been reported 10,12,35
. As apoptotic cells can be entirely removed by phagocytosis within an estimated time of 30 to 60 minutes 36
, detection is only possible within a specific timeframe and results in an underestimation of overall apoptosis. In our current study, we demonstrate a significant decrease in apoptotic EC expressing anti-apoptotic genes p35 or Bcl-xL.
An important concern when applying anti-apoptotic strategies to EC is whether replication can be induced leading to local tumors in the target cell type. Thanks to the very low proliferative capacity of the corneal endothelial monolayer there are no reports of corneal EC tumors described in the literature ever. EC are not even forming a bilayer, not even in in vitro
experiments. Therefore, the chance to induce local tumors is minimal to null. Secondly, approaches targeting EC have been of two types: a) To induce proliferation of EC and b) To suppress EC death which is the strategy employed herein. Whereas by promoting proliferation there could be a viable concern of inducing certain pathologies, by suppression of EC death, tumor induction is not possible. This is confirmed by the ‘proof of principle’ experiment in the mouse model transplanting corneas with EC treated with the same viral vector construct used in this study (lenti-IZsGr-Bcl-xL). The data showed that corneal grafts with EC expressing Bcl-xL resulted in a significantly enhanced graft survival compared to controls without signs of pathological proliferation,of EC or other cell types (routine follow-up time in this model is 8 weeks) 13
. However, longer follow-up periods in this model might be necessary to further evaluate the safety of this approach.
Another important question is whether or how many viral vector particles remain on the donor tissue after washing the endothelium with PBS at the end of the transduction period. Residual viral vector could lead to transduction of other cell types in the anatomical vicinity of the corneal endothelium raising safety concerns of induction of tumorigenic processes. However, the neighboring cells to the EC in the cornea are either stromal keratocytes (with a low proliferative capacity and with no predilection for tumorogenesis) or epithelial cells (which are entirely replaced by epithelial cells of the host soon after surgery). To study potential hazards, further studies are required. In our study, we were able to detect gene expression without appreciable diminution up to 11 weeks after transduction at readily detectable levels. These data are confirmed by previous work of this group in the mouse transplantation model [13
, expression after 8 weeks readily detectable].
Given the biology of the EC as a monolayer, it is conceivable that this approach could be translated to other cell sheet transplantations. Examples could include cultured mesenchymal stem cells to enhance bone formation 37
or transplantation of tissue engineered corneal epithelial sheets 38
. Given the risk that uncontrollable cell division of cells expressing anti-apoptotic genes could result in tumor formation, the transfer of this methodology to cell types with a high proliferative capacity should be exercised with caution.
In summary, these findings provide new insights on the roles of both the mammalian anti-apoptotic protein Bcl-xL and the baculoviral protein p35 in prolonging survival of EC during preservation. To our knowledge, we have shown for the first time that cell survival of human EC can be enhanced during preservation by means of gene therapy. Moreover, application of anti-apoptotic gene transfer may be further synergized with the recent advancements in the transplantation of the corneal endothelial monolayer, which is currently being globally implemented in corneal transplantation clinics 39
. In addition, application of anti-apoptotic gene therapy in eye banks prior to transplantation could lead to an increase of available donor corneas, which could impact the availability of donor tissue for millions of people suffering from corneal blindness .