Kampik, D;
Basche, M;
Georgiadis, A;
Luhmann, UFO;
Larkin, DF;
Smith, AJ;
Ali, RR;
(2019)
Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer-A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts.
Investigative Ophthalmology & Visual Science
, 60
(8)
pp. 3170-3177.
10.1167/iovs.18-26260.
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Abstract
PURPOSE: Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer. METHODS: HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment. RESULTS: LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD. CONCLUSIONS: ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.
| Type: | Article |
|---|---|
| Title: | Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer-A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1167/iovs.18-26260 |
| Publisher version: | http://dx.doi.org/10.1167/iovs.18-26260 |
| Language: | English |
| Additional information: | © 2019 The Authors. This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/). |
| Keywords: | corneal endothelial cells, ZO-1/ZONAB, gene therapy, cell proliferation |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10078955 |
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