J Korean Ophthalmol Soc.  2003 Sep;44(9):2128-2136.

The Apoptosis Induced by Ceramide in the Endothelial Cell

• Lee SY
• Kim TI
• Tchag H

Abstract

PURPOSE
To evaluate the effect of variable ceramides on the apoptosis of corneal endothelial cell and then, if ceramide induce the apoptosis in endothelial cells, via which pathway apoptosis occur. METHODS: Corneal endothelial cells were isolated from fresh rabbit cornea and cultured. Cultured corneal endothelial cells were exposed to 10, 20, 40 and 80 micro M of ceramide type II, VI and phytoceramide type II, VI. And then, apoptosis was evaluated with Hoechst staining and flow cytometric analysis with Annexin V for evaluation of apoptotic response. Corneal endothelial cells were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor(IETD-CHO(R)) and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 20 M of 4 types of ceramide. 12 hours later, LDH assay was done. Cytochrome c immunostaining was done after exposure to 4 types of ceramide.
RESULTS
Shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation were observed on Hoechst staining. In flow cytometric analysis, early apoptotic responses were identified. Apoptotic response increased significantly at the concentration of 10M and more 12 hours later. CPP32-like protease inhibitor, caspase-8, 9 inhibitor reduced the LDH activity. Apoptotic endothelial cells induced by ceramide were stained with cytochrome c antibody.
CONCLUSIONS
Ceramide induced apoptosis in cultured corneal endothelial cells. This apoptosis developed via caspase and mitochondrial pathway.

Keyword

Apoptosis; Caspase; Ceramide; Corneal endothelial cell; Mitochondrial pathway; Phytoceramide

MeSH Terms

Annexin A5
Apoptosis*
Caspase 8
Caspase 9
Ceramides
Cornea
Cytochromes c
Cytoplasm
Endothelial Cells*
Protease Inhibitors
Annexin A5
Caspase 8
Caspase 9
Ceramides
Cytochromes c
Protease Inhibitors