Contribution of the WNK1 kinase to corneal wound healing using a human tissue-engineered cornea as a model

Abstract

Purpose : Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix (ECM) to which the basal human corneal epithelial cells (hCECs) attach. These changes are perceived by integrins, a family of trans-membrane receptors that activate different intracellular signalling pathways, ultimately leading to re-epithelialization of the injured epithelium. Our goal is to study the impact of the pharmacological inhibition of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas (hTECs) as in vitro models.

Methods : hTECs were produced by the self-assembly approach and wounded with a 8-mm biopsy punch. Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The wounded tissues were then incubated either with various concentrations (50nM, 1uM or 10 uM) of the WNK1 inhibitor WNK463 or with the vehicle alone (DMSO; negative control) and wound healing was monitored for 6 days (n=4). The impact of WNK1 inhibition on hCECs monolayers was determined using a scratch wound assay (n=4).

Results : Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signalling pathways were altered in response to the ECM changes taking place during corneal wound healing. Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing. The pharmacological inhibition of WNK1 by WNK463 significantly reduced corneal wound closure time in our hTECs (p=1.93E-07) and hCECs monolayers (p=2.58E-05) compared to their respective negative controls (77.9% reduction and 48.9% reduction respectively).

Conclusions : These results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing. Most of all, they identified the WNK1 kinase as an important player in ensuring proper wound healing of the cornea since its inhibition considerably impedes that process.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.