The Matricellular Protein SPARC Is Constitutively Expressed by Limbal Fibroblasts and Inhibits Intercellular Adhesion of Corneal Epithelial Cells in vitro

Abstract

Abstract: : Purpose:To identify and characterize proteins preferentially secreted by limbal fibroblasts in vitro. Methods:Human donor limbal and corneal fibroblasts were explant–cultured in DMEM (15% serum) until confluence, and condition in serum free medium. The supernatant from each cell was condensed and subjected to proteomic analysis. The predominant protein identified (SPARC) was further examined by real–time PCR, western blot analysis and immunohistochemistry of limbal tissue. The effects of mouse recombinant SPARC (10 µg/ml) on cell adhesion and morphology was observed using an immortalized human epithelial cell line (HCEC) cultured in EpiLife^® medium supplemented with 0 to 1 mM Ca²⁺. Results:Proteomic analysis of supernatants revealed greater levels of SPARC, TIMP 2, TIMP 2 precursor, vimentin and alpha 2 pro–collagen secreted by limbal fibroblasts compared with corneal fibroblasts. Western blot analysis and real–time PCR confirmed the higher levels of SPARC in cultured limbal fibroblasts. Anti–SPARC staining was strongest in the subepithelial region of the limbus in immunohistology. Recombinant SPARC (10 µg/ml) significantly inhibited intercellular adhesion of HCEC in serum–free EpiLife^® medium with 1 mM Ca²⁺ for up to 48 hours (p<0.05). Conclusions: SPARC secreted constitutively by limbal fibroblasts may regulate cell to cell interaction in the basal limbal epithelium.

Keywords: cornea: basic science • proteomics • cornea: stroma and keratocytes