Gene Expression of Matrix and Cell Adhesion Molecules in PAF–Treated Corneal Myofibroblasts

Abstract

Abstract: : Purpose:Recent studies from our laboratory demonstrated that the inflammatory mediator platelet–activating factor (PAF) induces, by a receptor–mediated mechanism, the expression of selective matrix metalloproteinases (MMPs) in corneal myofibroblasts. (Ottino et al, IOVS, in press) Since myofibroblasts are key components of contraction of collagen and wound healing, in this study we compared the gene–expression profiles of matrix and adhesion molecules of rabbit corneal keratocytes and myofibroblasts and the effect of PAF on the expression of these genes in myofibroblasts. Methods:A human extracellular matrix and adhesion molecules gene array containing 96 genes (Superarray) was used. Corneal keratocytes were isolated from rabbit corneas. Myofibroblasts were obtained from corneal fibroblast subcultured at low cell density in DMEM–F12 with 2%–5% PPHS. The cells were identified by morphology and a–smooth muscle actin expression. Myofibroblasts were serum–starved overnight and treated with or without 100 nM PAF for 4 h and RNA was isolated. Changes in gene expression of more than three fold were considered significant. Real–time PCR was employed to confirm the cDNA array results and to identify the fold increases of gene expression. Results:cDNA expression array analysis comparing keratocytes with myofibroblasts showed that the cell adhesion molecule MICA and the MMPs –10,– 13 and –24 were down–regulated in myofibroblasts, while the extracellular protein caveolin–1 and the integrin alpha 3 were up–regulated. Stimulation of myofibroblasts with PAF induced up–regulation of the cell adhesion molecule contactin –1, and the integrin beta 1 (ITGB1), the extracellular matrix proteins collagen type 1 and fibrinogen beta, the MMPs –11 and –20, in addition to those already described. Gene expression of ITGB1 increased 3.5 fold at 8 hours as assayed by real–time. PCR. Conclusions:Changes in the expression of cell–matrix adhesion molecules between keratocytes and myofibroblasts could reveal differences in function between these two cell phenotypes. The up–regulation of specific extracellular matrix and cell adhesion genes, such as ITGB1, suggests a role of the inflammatory mediator PAF in cell–matrix adhesion.

Keywords: cornea: stroma and keratocytes • extracellular matrix • gene microarray