Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer-A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts.
Invest Ophthalmol Vis Sci
; 60(8): 3170-3177, 2019 07 01.
Article
in En
| MEDLINE
| ID: mdl-31335954
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 â¼45% 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 â¼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an â¼25% 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.
Full text:
1
Database:
MEDLINE
Main subject:
RNA, Messenger
/
Endothelium, Corneal
/
Gene Expression Regulation
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Corneal Transplantation
/
Gene Transfer Techniques
/
Zonula Occludens-1 Protein
Limits:
Humans
Language:
En
Year:
2019
Type:
Article