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Daniel Kampik, Mark Basche, Anastasios Georgiadis, Ulrich F. Luhmann, Alexander J. Smith, Frank Larkin, Robin R. Ali; Lentivirus Mediated Interference With the ZO-1/ZONAB Pathway Induces Cell Cycle Progression in Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6004.
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The ZO-1 protein, an integral part of tight junctions, is involved in the maintenance of G1 arrest. By cytoplasmic sequestration of transcription factor ZONAB (ZO-1-associated nucleic-acid-binding protein) it prevents ZONAB from activating genes inducing cell cycle progression. We assess the effects of lentiviral knockdown of ZO-1 or overexpression of ZONAB on corneal endothelial cells (CEC) in human corneal explants.
For ZO-1 knockdown, an HIV based lentiviral vector was constructed delivering a small hairpin RNA against ZO-1 (LNT-shZO-1). Using a similar vector backbone, ZONAB was delivered under a constitutive promoter (LNT-ZONAB). A lentiviral vector delivering green fluorescent protein gene (LNT-GFP) served as control. Human corneal specimens with normal endothelium were bisected and each half was exposed to treatment or control vector. After one week in ex vivo culture, effects were assessed by quantitative RT-PCR, Western blot and immunohistochemistry. CEC density was determined by counting nuclei on flatmounted specimens.
LNT-shZO-1 achieved a 90% knockdown of ZO-1 mRNA in ex vivo corneal endothelium. In corneal samples from donors up to 60 years of age, this led to an average increase in CEC density of 50% (p=0.016, paired t test, range 26-69%), while the cell monolayer showed intact morphology and ZO-1 staining. With a donor age >80 years, no effect on cell density was detectable. LNT-ZONAB achieved a >500-fold increase in ZONAB mRNA. This led to Ki67 upregulation as shown by immunohistochemistry, but no significant increase in cell density.
ZO-1 downregulation may be a strategy to increase CEC density by direct interference in contact inhibition and cell cycle control. Using a non-integrating lentivirus to deliver shZO-1 might lead to a temporary upregulation of CEC replication suitable for ex vivo, in situ application.
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