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Jennifer Young, Neil Richardson, Damien Gerard Harkin; Expression and function of Eph receptors in corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4910.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal endothelium is critically required for maintaining the optimal hydration and transparency of the cornea. Unfortunately, the corneal endothelium does not normally undergo regeneration following cell loss, and this can result in vision loss for which the only current treatment is transplant surgery. We aim to develop strategies to increase endothelial cell density in patients with endothelial dysfunction, circumventing the requirement for surgery. Previous studies indicate that some Eph receptor tyrosine kinases are expressed in corneal tissues. Since Eph receptors are known to regulate cell migration and proliferation, we have initiated a study to examine their expression and role in corneal endothelial cells (CECs). Our studies are focused on the ligand ephrin-A1, and on two of its receptors, EPHA1 and EPHA2.
Expression characteristics of ephrin-A1, EPHA1 and EPHA2 were analysed in fresh samples of human corneal endothelium, cultures of primary human CECs and in cultures of transformed human CECs (B4G12s) using immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR) techniques. EPHA1 and EPHA2 signaling activity was reduced in CECs using lithocholic acid and specific siRNAs. Scratch wound assays were then conducted to analyse migration, and quantitative RT-PCRs were performed to analyse gene expression, in cells with reduced EPHA receptor signaling.
Ephrin-A1, EPHA1 and EPHA2 proteins were expressed in fresh and cultured human CECs, and in the B4G12 cell line. While the three proteins were variably located to nuclear and perinuclear regions, they were all consistently associated with lamellipodia in migratory cells. B4G12 cells exposed to lithocholic acid migrated significantly faster in scratch wound assays, and this was correlated with decreased E-cadherin expression, and increased N-cadherin expression. Individual EPH receptor knockdown using siRNAs also resulted in a change in cadherin expression.
Our data indicates that ephrin-A1, EPHA1 and EPHA2 are expressed in human CECs, and that their inactivation can enhance cell migration. In addition, our data indicates that cell migration regulated by EPH receptor signaling may be mediated through the differential expression of cadherins. This data is supportive of a potential role for these factors in regulating the migration of endothelial cells in the adult cornea.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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