Low Concentration of H2O2 Promoted–Cell Adhesion in Cultured Rabbit Corneal Epithelial Cells

Abstract

Purpose: : Fibronectin–integrin system plays an essential role in cornea epithelial cell adhesion. Intracellular ROS have been reported to associate with integrin signaling in promoting cell adhesion in some cell types. The purpose of this study is to investigate the effect of exogenous low dose of H₂O₂ on rabbit corneal epithelial cell adhesion.

Methods: : Primary rabbit corneal epithelial cells were grown to confluence in SHEM medium with 5% FBS. The cells were gradually serum starved by first culturing in 0.5% FBS overnight and then serum–free medium for 30 min before use. MTT assay was used to test the viability of cells treated with various concentrations of H₂O₂ (0, 1, 5, 10, 20, 50, 60, and 70 µM) for 2 hours. The same dosages of H₂O₂ were tested for cell adhesion to fibronectin–coated plates by adding H₂O₂ with and without mannitol, a free radical scavenger, in the cell suspension before plating. After centrifugation, the adhered cells were counted and photographed at 30 and 60 min. The vinculin–containing focal contacts of cells treated with low–dose–H₂O₂ were determined by immunofluorescence.

Results: : H₂O₂ at 5–50 µM did not reduce the viability of rabbit corneal epithelial cells compared with non–treated cells, but concentrations higher than 50 µM were harmful. However, H₂O₂ at the range of 5–50 µM enhanced cell adhesion and increased cell growth in a dose–dependent manner in comparison with the control (no H₂O₂). The enhanced adhesion and growth were suppressed in the presence of mannitol. Immunofluorescence assay confirmed that H₂O₂ at these low concentrations facilitated the formation of vinculin–containing focal adhesion complexes, which were not present in the non–treated cells.

Conclusions: : These findings suggest that exogenous H₂O₂ at low level can promote cultured rabbit corneal cell adhesion to fibronectin. This is the first report of oxidation–associated cell adhesion in corneal epithelial cells.

Keywords: cornea: epithelium • cell adhesions/cell junctions • cornea: basic science