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J.C. McAlister, N.C. Joyce, A.J. Thrasher, R.R. Ali, D.F. Larkin; Increase in Corneal Endothelial Cell Density in Human Organ Culture following E2F2 Gene Delivery . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4334.
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Purpose: One of the main reasons for corneal graft failure is low donor corneal endothelial cell density, which results in an increased risk in primary and late secondary graft failure. The aim of this study is to develop a gene-based approach to increase the cell density in donor tissue prior to transplantation. We assessed changes in human corneal endothelial cell density and morphology following adenovirus-mediated transfer of a gene encoding human E2F2. E2F2 belongs to a family of transcription factors that are critical regulators of the cell division cycle. Methods: Human corneas were exposed for 2hrs to (i) recombinant adenoviral vector, AdU, bearing a GFP cDNA driven by a CMV promoter , (ii) AdUE bearing an identical GFP construct plus E2F2 cDNA driven by a CMV promoter, or (iii) mock-infection, and then maintained in ex vivo culture. Corneal endothelial cell density was assessed prior to and following infection for up to 3 weeks. Morphology was also assessed using haematoxylin-eosin stained wax sections. In order to assess the level of contact inhibition following treatment, the corneal endothelium was immunostained for ZO-1 Results: Human E2F2 (AdUE) over-expression resulted in a significant increase in cell density after one (paired T test p<0.005) and two (paired T test p<0.01) weeks of culture. AdU and mock infection had no such effect at each time point examined. GFP expression was largely extinguished by the second week in the AdUE-treated group whereas in the AdU treated group, GFP expression was undiminished. One week after E2F2 gene delivery there was intracellular ZO-1 immunostaining over large areas of the corneal endothelium consistent with cell proliferation. At two weeks ZO-1 staining was mostly localised to cell junctions indicating a high level of contact-inhibition. Consistently high and stable GFP expression as well as classical ZO-1 staining was maintained in the AdU-treated group. Analysis of H+E stained wax sections suggested that an increase in cell density occurred throughout the AdUE-treated cornea in comparison to controls. Discussion: An increase in corneal endothelial cell density in human donor corneas is possible by over-expression of the transcription factor E2F2.
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