Date of this Version
2011 The Association for Research in Vision and Ophthalmology, Inc.
PURPOSE. Intracellular reactive oxygen species have been reported to associate with growth factor and integrin signalings in promoting cell adhesion in many cell types. This study is to explore if exogenous H2O2 at low levels can be beneficial to cell adhesion, migration, and wound healing.
METHODS. Primary rabbit corneal epithelial cells treated with 0–70 M H2O2 were tested for viability by MTT assay, adhesion by centrifugation assay, focal contacts of vinculin and F-actin by immunofluorescence, activated Src(pY416), EGF receptor (pY845), vinculin(pY1065), FAK(pY397), and FAK(pY576) by immunoblotting. Cell migration was examined with 0–50 M H2O2 using the scratch wound technique. Corneal wound healing of ex vivo pig model and in vivo mouse model was examined using H2O2 with and without antioxidant N-acetylcysteine (NAC).
RESULTS. Compared with the untreated control, H2O2 at 10–50 M stimulated cell viability and facilitated adhesion and migration with clear induction of vinculin-rich focal adhesions and F-actin– containing stress fibers by increasing activated Src, FAK(pY576), and vinculin(pY1065). H2O2 also increased phosphorylation of EGFR(Y845) parallel to that of activated Src, but both were eliminated by NAC and PP1 (Src inhibitor). Finally, H2O2 induced faster wound healing in cornea both in vitro and in vivo, but the healing was diminished by NAC.
CONCLUSIONS. These findings suggest that H2O2 at low levels promotes cell adhesion, migration, and wound healing in cornea cells or tissue, and the interaction of H2O2 with Src plays a major role.
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