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Jiucheng He, Haydee E. P. Bazan; Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor-α on Corneal Myofibroblast Apoptosis. Invest. Ophthalmol. Vis. Sci. 2006;47(3):883-891. doi: 10.1167/iovs.05-0581.
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purpose. Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-α in corneal myofibroblast apoptosis was explored.
methods. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm2). Mouse anti-α-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-α receptors 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-α (20 ng/mL), and TNF-α+cPAF, 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.
results. Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF-α receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF-α for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, demonstrating a synergistic action between PAF and TNF-α. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF.
conclusions. Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF-α suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.
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