SPARC from corneal epithelial cells modulates collagen contraction by keratocytes

PURPOSE. Contraction of the scar tissue during corneal wound healing change s the shape of the cornea and corneal refraction. In a previous study, it w as found that corneal epithelial cells secrete the factor that stimulates c ollagen gel contraction by keratocytes in vitro. The purpose of the present study was to purify and identify the contraction-stimulating factor derive d from corneal epithelial cells. METHODS. The cultured medium of rabbit corneal epithelial cells was collect ed and used as an epithelial cell-conditioned medium (ECCM). Subcultured ra bbit keratocytes were embedded in a collagen gel, and collagen gel contract ion was investigated. The contraction-stimulating factor in the ECCM was pu rified through acetone precipitation, affinity chromatography (heparin Seph arose), gel filtration, and reversed-phase chromatography. The amino acid s equence of a contraction-stimulating factor was analyzed. RESULTS. Collagen gel contraction by keratocytes was enhanced by the additi on of ECCM in a dose-dependent manner. The amino acids sequence of the cont raction-stimulating factor was homologous to a 32-kDa glycoprotein, a secre ted protein that is acidic and rich in cysteine (SPARC). Western blot analy sis confirmed that SPARC was contained in the ECCM. Collagen gel contractio n by keratocytes was enhanced by the addition of purified SPARC in a dose-d ependent manner. SPARC was found in the basal layer of the migrating epithe lium and activated keratocytes adjacent to the wound 3 days and 1 week afte r perforating injury in rabbit corneas. CONCLUSIONS. Epithelial cells secrete SPARC, which modulates the contractio n of scar tissue in the corneal stroma.