The human tissue-engineered cornea as a model to study the impact of altering the PI3K/Akt signal transduction pathway on corneal wound healing

Abstract

Purpose : Upon injury to the cornea, the composition of the extracellular matrix (ECM) rapidly changes to promote wound healing through its interactions with integrins. We hypothesize that ECM remodelling occurring during corneal wound healing causes the activation of very specific signal transduction mediators that favor faster closure of the wound. Our goal is to proceed to the pharmacological inhibition and/or activation of the PI3K/Akt mediators Akt and CREB using the human tissue-engineered cornea (hTECs) as a model.

Methods : hTECs produced by the self-assembly approach were wounded with a 8-mm diameter biopsy punch and deposited on another reconstructed human corneal stroma to allow wound closure on a natural ECM. Total RNAs and proteins were prepared from the epithelial cells of wounded and unwounded areas and their gene expression pattern was determined by microarrays. The wounded tissues were then incubated with or without C646 (a CREB inhibitor) or with or without SC79 (an AKT activator). DMSO (the vehicle) was used alone as a negative control. Closure of the wounds was monitored over a period of 5 days to determine whether Akt activation and CREB inhibition will alter wound closure.

Results : Analysis of the microarray data indicates that important alterations in the expression of a few mediators is occurring primarily in the PI3K/Akt pathway in response to the ECM remodeling taking place during hTECs wound healing. Pharmacological inhibition of CREB with C646 considerably accelerated wound closure compared to controls whereas complete wound closure was observed when hTECs were grown in the presence of both SC-79 and C646.

Conclusions : We demonstrated for the first time that corneal wound healing can be monitored in vitro using as a model hTECs that are very close to the native tissue. Most of all, our results validated both the Akt and CREB genes as potential targets on which we may influence to alter the wound healing dynamic of the cornea. This will certainly lead to progress in the clinical field of corneal blindness.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.