Ceramide-Induced Apoptosis in Rabbit Corneal Fibroblasts

Abstract

To evaluate the effect of various ceramides on the apoptosis of corneal fibroblasts and to determine the pathway on which they act. Corneal fibroblasts isolated and cultured from New Zealand white rabbits were exposed to various concentrations of ceramide types II and VI and phytoceramide types II and VI, and their apoptotic response was evaluated using an LDH assay and Hoechst and Annexin V staining. Corneal fibroblasts were preincubated with various concentrations of the CPP32-like protease inhibitor Z-VAD-FMK, the caspase-8 inhibitor IETD-CHO, and the caspase-9 inhibitor Z-LEHD-FMK before treatment with ceramide, and apoptotic response was assayed by LDH assay. In addition, cells treated with ceramide or phytoceramide were stained with an antibody to cytochrome c. At concentrations of 20 microM and higher, all 4 ceramides increased fibroblast apoptotic response significantly after 12 hours. Hoechst staining showed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation after ceramide exposure, and Annexin V staining showed small vesicles around the cell membrane. The CPP32-like protease inhibitor reduced the apoptotic response to all 4 ceramides. The specific caspase-8 inhibitor reduced the apoptotic response to ceramide type VI and phytoceramide types II and VI, whereas the specific caspase-9 inhibitor significantly reduced the apoptotic response to phytoceramide types II and VI. Following exposure to ceramides, corneal fibroblasts stained positively with antibody to cytochrome c. Ceramide induced apoptosis in cultured corneal fibroblasts. This apoptosis involved the caspase cascade and the mitochondrial pathway.