Purpose: Contraction of the scar tissue during corneal wound healing changes the shape of the cornea and corneal refraction. In a previous study, it was found that corneal epithelial cells secrete the factor that stimulates collagen gel contraction by keratocytes in vitro. The purpose of the present study was to purify and identify the contraction-stimulating factor derived from corneal epithelial cells.
Methods: The cultured medium of rabbit corneal epithelial cells was collected and used as an epithelial cell-conditioned medium (ECCM). Subcultured rabbit keratocytes were embedded in a collagen gel, and collagen gel contraction was investigated. The contraction-stimulating factor in the ECCM was purified through acetone precipitation, affinity chromatography (heparin Sepharose), gel filtration, and reversed-phase chromatography. The amino acid sequence of a contraction-stimulating factor was analyzed.
Results: Collagen gel contraction by keratocytes was enhanced by the addition of ECCM in a dose-dependent manner. The amino acids sequence of the contraction-stimulating factor was homologous to a 32-kDa glycoprotein, a secreted protein that is acidic and rich in cysteine (SPARC). Western blot analysis confirmed that SPARC was contained in the ECCM. Collagen gel contraction by keratocytes was enhanced by the addition of purified SPARC in a dose-dependent manner. SPARC was found in the basal layer of the migrating epithelium and activated keratocytes adjacent to the wound 3 days and 1 week after perforating injury in rabbit corneas.
Conclusions: Epithelial cells secrete SPARC, which modulates the contraction of scar tissue in the corneal stroma.