Advanced Glycation End Product and its Receptor RAGE promote the in-vitro wound healing of HCE corneal epithelial cells

Abstract

Purpose : Corneal epithelium wound healing, following severe ocular injuries (such as chemical burns), is of primary importance to recover a normal visual acuity. The aim of this work is to decipher the involvement of a ligand/receptor couple called AGE/RAGE in such phenomenon, by using human corneal epithelial cell line (HCE) and in-vitro scratch assay.

Methods : HCE (Human Corneal Epithelial) cell line were cultured under standard conditions (5% CO2, humidified air, 37°C) in DMEM-F12+GlutaMAX^TM-I (Gibco) supplemented with 10% fetal bovine serum and additives (insulin, cholera toxin, antibiotics, EGF and DMSO). Cells were seeded 24h prior to scratch assay done with a 200µl pipette tip. Then, cells were treated (or not for control condition) every 24 hours (h) and for 48 h with Advanced Glycation End Product (AGE) at various concentrations (ranging from 1 to 200 µg/ml of cell media). Photography's were taken every 12h and residual wound areas were measured using imageJ software. Following that, cells were used to study the "AGE" influence on proliferation and migration by using 5-Bromo-2’deoxy-uridine labelling and migration chambers. Finally, the activation of NF-kB signalling pathway by the AGE/RAGE couple in HCE cells was tested using Luciferase NF-kB reporter assay system and by the quantification of NF-KB target genes linked to wound healing process (such as for example connexin 43). Statistical analyses for all experiments were conducted using non parametric test (Mann-Whitney U-test).

Results : A significant improvement of wound healing area was obtained after treatment by AGE at 100 µg/mL. Nevertheless, no significant difference on HCE cell migration and proliferation were demonstrated. Then, AGE treatment induces an early activation of NF-kB pathway using luciferase gene reporter system and demonstrate its ability to regulate connexin 43 gene expression during earlier stages of wound healing.

Conclusions : Our results, obtained using HCE cell line demonstrate the implications of AGE/RAGE on this phenomenon. This work will be followed by a global study of AGE/RAGE target genes in corneal epithelium using RNAseq. Furthermore, it paves the way for the future use of in-vivo RAGE-/- mice model (available in the lab) to clarify this pathway importance in corneal wound healing.

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