Abstract
Abstract: :
Purpose: Stromal keratocytes as well as myofibroblasts express the platelet activating factor (PAF) receptor, while fibroblasts do not (He and Bazan, ARVO, 2003). Because of the importance of myofibroblasts during corneal wound healing, we investigated the effect of the lipid cytokine PAF in these cells. We also identified the localization of the PAF receptor in myofibroblasts Methods: Keratocytes isolated from rabbit and pig corneas were plated either in DMEM/F12 to maintain the keratocyte phenotype, or in DMEM/F12 containing 10% FBS to transform the keratocytes to fibroblasts. Myofibroblasts were obtained from subcultured fibroblasts plating at a low density (1000 cells/ml). PAF–receptor immunofluorescence was performed with both rabbit anti–PAF receptor (N terminus) antibody and goat anti–PAF receptor (C terminus) antibodies. Mouse anti–vimentin and anti–α–SM antibodies were used to identify the cell phenotype. Apoptosis was assayed by TUNEL staining and DNA ladder. DAPI was used for nuclear staining. Results: . PAF receptor in the porcine corneal myofibroblasts localized in the plasma membrane and nuclear membrane as identified by immunofluorescence with a rabbit anti–human PAF receptor (N terminus) antibody. PAF receptor distribution on the nuclear membrane was confirmed by double staining with an anti–nuclear membrane antibody TUNEL–staining showed that addition of cPAF (300nM) to the medium of 7–day–cultured rabbit keratocytes for 6 hours induced more than 60% of the cells into apoptosis, while no positive staining was found in myofibroblasts cultures from 6 to 24 hours. When incubated with cPAF for 48 to 72 hours about 20–30% myofibroblasts were apoptotic. Pre–incubation with a PAF antagonist blocked PAF–induced apoptosis in both keratocytes and myofibroblasts. Those results were further confirmed by DNA– ladder Conclusions: Although both keratocytes and myofibroblasts express the PAF receptor, the induction of apoptosis in these cells differs in intensity and time, suggesting that different signal mechanisms are activated in these two cell types. In addition, the presence of a nuclear PAF receptor in myofibroblasts suggest that PAF, which is synthesized after injury and retained in the cells, could activate through this receptor the expression of specific genes, such as selective MMPs (Axelrod et al ARVO 2003) whose over– induction could contribute to the formation of scar tissue.
Keywords: cornea: stroma and keratocytes • receptors • cytokines/chemokines