Sodium orthovanadate enhances corneal epithelial wound healing through increased Src-family kinase activity

Abstract

Purpose : Corneal diseases is one of the leading global causes of blindness, and the only definitive treatments available are surgical. Therapeutic approaches that enhance corneal epithelial wound healing can minimize the risk of corneal scarring and blindness. Our previous study showed delayed wound healing in murine derived corneal epithelial cells due to decreased levels of Src-family kinases (SFK). Here, we demonstrate enhanced wound healing in primary human corneal epithelial cells using a competitive inhibitor for protein phosphotyrosyl phosphatases, sodium orthovanadate. Using an in vitro scratch assay, live imaging and western blot analysis were used to calculate cell migration rates and associated protein levels.

Methods : For our in vitro scratch assay, monolayers of primary human corneal epithelial cells (HCEC) were established and scratched with a sharkstooth comb. Cells were incubated with either 0.5uM or 1.0uM of sodium orthovanadate or control vehicle an hour prior to wounding. Scratched areas were imaged with a live imaging system every hour for 24 hours, and images were subsequently analyzed with Image J software. A parallel set of cells was established for protein analysis, and lysates were collected at 0, 4, 16, and 24 hours after wounding and subjected to western blot analysis.

Results : Sodium orthovanadate enhanced wound healing in HCEC at both concentrations compared to control that was statistically significant. There was no difference, however, between the two concentrations. Faster gap healing rates correlated with higher levels of activated SFK and AKT.

Conclusions : Diminished corneal epithelial wound healing was previously observed in murine derived corneal epithelial cells associated with decreased levels of activated SFK. Treatment with dasatinib, a tyrosine kinase inhibitor, also recapitulated the murine corneal epithelial findings in HCEC, where lower levels of activated SFK correlated with delayed wound healing. Results from this study demonstrate enhanced wound healing in HCEC through phosphatase inhibition leading to elevated levels of activated SFK and AKT. This opens up the potential for future therapeutic development for treating corneal epithelial wounds. Further studies to delineate the downstream effectors of SFK in regards to wound healing should be conducted.

This is a 2020 ARVO Annual Meeting abstract.