Proteomic analysis of soluble factors secreted by limbal fibroblasts

Purpose: To identify soluble factors selectively secreted by limbal fibroblasts as possible regulators of limbal basal epithelium. Methods: Limbal, corneal, and conjunctival fibroblasts were first expanded in vitro in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, and then maintained in serum-free medium for two weeks. Proteomic analysis of culture supernatants was done to compare differences in secreted matricellular proteins. Real time PCR and western blots were done to confirm the expression of secreted protein acid and rich in cysteine (SPARC), a protein found in abundance in extracellular proteins secreted by limbal fibroblasts. Immunohistochemistry of SPARC was done in human limbal tissue to show the spatial distribution of the protein. An adhesion assay was designed to demonstrate the effects of SPARC on an SV40 immortalized human corneal epithelial cell line (HCEC). Results: Proteomic analysis revealed several proteins selectively secreted by limbal fibroblasts. The particular spots were identified as SPARC, vimentin, serine protease, collagen alpha 2 precursor, tissue inhibitor of metalloproteinase 2 (TIMP-2), and 5,10-methlenetetrahdrofolate reductase (FADH2). The expression of SPARC was confirmed by western blot analysis, and mRNA expression was significantly higher in limbal fibroblasts compared to central corneal fibroblasts when analyzed by real time PCR. Immunohistochemistry revealed higher distribution of SPARC in the subepithelial stroma of the limbus compared to the central cornea. The addition of 10 μg/ml murine SPARC in HCEC significantly reduced cell spreading at three h. Conclusions: The matricellular protein SPARC is preferentially secreted by limbal fibroblasts, and may modulate intercellular adhesion of basal limbal epithelial cells.