Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor-α on Corneal Myofibroblast Apoptosis

METHODS. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm). Mouse anti-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNFreceptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF(20 ng/mL), and TNFcPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope.