Abstract
Purpose
To investigate sphingolipid expression profile in alkali‐burned cornea and subsequently evaluate effects of the selected species on corneal epithelial wound healing.
Methods
All experiments were performed using porcine cornea organ culture or porcine corneal epithelial cells (PCEC). For sphingolipid profiling samples were spiked with internal standards, extracted by Bligh&Dyer method and analyzed using liquid chromatography‐mass spectrometry. PCEC viability/proliferation and migration were assessed using MTT and Boyden chamber assays, respectively. Epithelial wound healing in cornea organ culture was followed by fluorescein staining. Molecular mechanisms underlying the action of selected sphingolipids were studied using iTRAQ‐based quantitative proteomics.
Results
Sphingolipid profiling of alkali‐burned corneas revealed early decrease in sphingosine 1‐phosphate and lysosphingomyelin with concomitant increase of ceramide 1‐phosphate. We tested 7 lipids from aforesaid subclasses for their ability to affect proliferation/viability and migration of PCEC. 2 lipids: A, B improved viability/proliferation of alkali‐injured PCEC, induced PCEC chemotaxis as well as promoted re‐epithelialization after alkali burn in cornea organ culture. Another lipid: C potently improved viability/proliferation of healthy PCEC in a wide range of concentrations. In response to A/B/C treatments PCEC displayed differential expression of 114/375/47 proteins, majority with binding and catalytic activity functions. Proteins ↑ upon A/B were involved mainly in proliferation, adhesion and glycolysis, while upon C ‐ in maintenance of corneal epithelial homeostasis.
Conclusions
We identified 2 sphingolipids that facilitate cornea re‐epithelialization by promoting proliferation and migration of corneal epithelial cells and another that promotes healthy corneal epithelial cell phenotype.
