Abstract
Abstract: :
Purpose: The observation that corneal transplant failure is accompanied by apoptosis of resident graft endothelial cells has led to the hypothesis that preventing death of the endothelial cells within the graft could be a powerful way of averting corneal transplant failure. We chose 4 anti–apoptosis genes (Bcl–2, Bcl–xL, survivin and p35), which will be tested for their ability to inhibit death in corneal endothelial cells. Methods: Four anti–apoptotic genes were cloned into a retroviral plasmid vector. Retroviruses were used to infect the corneal endothelial cells (CEC). Apoptosis of endothelial cells was induced by etoposide, hydrogen peroxide (H2O2) and a combination of two cytokines, interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα). Apoptosis was detected by annexin V and Propidium Iodide staining and flow cytometry analysis and by the presence of active caspase 3. Results: We show that Bcl–xL, Bcl–2 and p35 all inhibited caspase 3 cleavage. Apoptosis of Bcl–2, survivin and p35 overexpressing cells was not significantly different from the empty vector control, suggesting that these genes alone are not capable of preventing apoptosis in these cells. A significant protective effect was observed with Bcl–xL when overexpressing cells were treated with etoposide (p=0.0162). Conclusions: Out of 4 candidate anti–apoptotic genes, we found that Bcl–xL worked best at protecting corneal endothelial cells from apotosis
Keywords: apoptosis/cell death • cornea: endothelium • gene transfer/gene therapy