Increased SPARC Production During Keratocyte Fibrosis

Abstract

Abstract: : Purpose: Corneal keratocytes and their derivatives, fibroblasts and myofibroblasts, are integral to corneal wound healing. The purpose of the current study is to use an in vitro bovine keratocyte fibrosis model system to identify the secreted products of the myofibroblast. Methods: Collagenase-isolated bovine keratocytes plated at 400,000 cells per 9.5 cm₂ (high density) and 100,000 cells per 9.5 cm₂ (low density) in 1% platelet-poor horse serum were allowed to attach overnight. High density cultures were maintained as keratocytes in serum-free conditions for five days while low density cultures were cultured with either 10% fetal bovine serum or 10% FBS plus 5 ng/ml TGF-ß to induce proliferation and differentiation into fibroblasts or myofibroblasts. Phenotypes were substantiated based on morphology and reactivity to anti-α-smooth muscle actin on Western blotting. Cells were radiolabeled with ₃₅S-methionine at 100 uCi/ml from day 4 to day 5. Products secreted into the culture media were analyzed by SDS-PAGE/autoradiography. Myofibroblast media was chromatographed on Mono-Q ion exchange resin and resulting fractions were electrophoresed to visualize isolated bands in preparation for amino acid sequencing. Keratocyte, fibroblast and myofibroblast media and cell layers were subjected to Western blotting using anti-bovine SPARC antibody. Total RNA from each phenotype was treated with DNase and used to synthesize cDNAs which were amplified in TaqMan-based realtime PCR to examine the relative difference in SPARC message expression between stromal cell phenotypes. Results: A 43 kDa protein secreted into the media was identified as SPARC by amino acid sequencing of tryptic peptides and confirmed by Western blot analysis of media. Autoradiography demonstrated a 13-fold increase in SPARC synthesis from keratocytes to myofibroblasts. In contrast, realtime PCR analysis of total RNA indicated no significant difference in SPARC message expression between the three phenotypes. Conclusion: Previous studies indicate the corneal epithelium is the source of SPARC in wound healing. The results of the current study, however, show that SPARC is made by keratocytes and its synthesis increases substantially in keratocytes induced to become myofibroblasts. This increase in synthesis is likely at the translational level since no changes in SPARC mRNA were found.

Keywords: 374 cornea: stroma and keratocytes • 631 wound healing • 403 extracellular matrix