The endothelium is the monolayer of cells that maintains corneal clarity. Corneal endothelial cell density decreases with age and cell loss can be accelerated by age-related diseases, and by ocular surgery or laser procedures. Maintenance of corneal clarity requires an intact endothelial monolayer, and transparency can be lost when cell density is reduced. Currently, visual loss caused by endothelial dysfunction can only be restored by corneal transplantation. Decreased cell density over time indicates that the rate of cell division does not keep pace with the rate of cell loss. The long-term goal of this project is to develop therapies to increase endothelial cell density. The proposed approach is to use molecular biology methods to transiently stimulate proliferation. Studies from this laboratory indicate that, in vivo, corneal endothelial cells are arrested in G1-phase (the part of the cell cycle that prepares cells for DNA synthesis) and are actively maintained in this non-replicative state. G1- phase arrest results from the activity of specific inhibitors that prevent induction of S-phase genes by the transcription factor, E2F. The hypothesis for these studies is that proliferation can be induced in corneal endothelial cells either by bypassing the point of G1-phase arrest and inducing the next downstream event in the cell cycle, or by decreasing the cellular concentration of G1-phase inhibitors.
Two specific aims are proposed: 1) Stimulate proliferation and increase cell density in human corneal endothelium by overexpressing the transcription factor, E2F-2, and 2) Stimulate proliferation and increase cell density in human corneal endothelium by decreasing expression of the cell cycle inhibitor, p27kipl, using antisense technology.
Both aims use methods that should induce a limited number of division cycles in a limited number of cells. This is based on the hypothesis that, to increase cell density to a level that will support endothelial function, only limited proliferation is necessary and desirable. For both aims, studies will be conducted in ex vivo human donor corneas. A rabbit model will also be used to permit evaluation at several levels of biological complexity, i.e., in tissue culture; in ex vivo corneal culture; and in treated ex vivo corneas transplanted back into a rabbit host. Immunocytochemistry (ICC), Western blots, and RT-PCR will detect alterations in E2F-2 and p27kip1 protein and mRNA expression. Proliferation will be detected by ICC, Western blot and RT-PCR for S- phase and cell cycle markers, and by flow cytometric analysis of DNA content. Cell density will be calculated by image analysis. Electron microscopy (TEM) and ICC for markers of plasma membrane polarity will evaluate endothelial morphology and ultrastructure following cDNA or antisense treatments. Endothelial function in treated, transplanted rabbit corneas will be tested in vivo by ultrasonic pachymetry measurements of corneal thickness. Apoptosis will be detected by annexin V assay and by gel assay of DNA fragments. TEM will detect multiple cell layers, providing evidence for cell transformation upon cDNA or antisense treatment.
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