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J Tissue Eng Regen Med. 2019 Jun 17. doi: 10.1002/term.2912. [Epub ahead of print]

Contribution of the WNK1 kinase to corneal wound healing using the tissue-engineered human cornea as an in vitro model.

Desjardins P1,2,3,4, Couture C1,2,3,4, Germain L1,2,3,4, Guérin SL1,3.

Author information

1
CUO-Recherche, Médecine Régénératrice - Centre de recherche du CHU de Québec, Université Laval, Québec, Canada.
2
Centre de Recherche en Organogénèse expérimentale de l'Université Laval/LOEX, Québec, Canada.
3
Département d'Ophtalmologie, Université Laval, Québec, QC, Canada.
4
Département de Chirurgie, Faculté de médecine, Université Laval, Québec, QC, Canada.

Abstract

Damage to the corneal epithelium triggers important changes in the extracellular matrix (ECM) to which basal human corneal epithelial cells (hCECs) attach. These changes are perceived by integrin receptors that activate different intracellular signaling pathways, ultimately leading to re-epithelialization of the injured epithelium. In this study, we investigated the impact of pharmacological inhibition of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and the tissue-engineered human cornea (hTECs) as an in vitro 3D model. RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The impact of WNK1 inhibition was evaluated on the wounded hTECs as well as on hCECs monolayers using a scratch wound assay. Gene profiling and protein kinase arrays revealed that expression and activity of several mediators from the integrin-dependent signaling pathways were altered in response to the ECM changes occurring during corneal wound healing. Phosphorylation of the WNK1 kinase turned out to be the most striking activation event going on during this process. The inhibition of WNK1 by WNK463 reduced the rate of corneal wound closure in both the hTEC and hCECs grown in monolayer compared to their respective negative controls. WNK463 also reduced phosphorylation of the WNK1 downstream targets SPAK/OSR1 in wounded hTECs. These in vitro results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and identified WNK1 as a kinase important to ensure proper wound healing of the cornea.

KEYWORDS:

Tissue-engineering; WNK1 kinase; biomaterial; cornea; signal transduction pathway; wound healing

PMID:
31207112
DOI:
10.1002/term.2912

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