Caspase Activation in Human Retinal Pigment Epithelial Cells (ARPE–19) After Exposure to 7–Ketocholesterol

Abstract

Purpose: : To determine if caspase pathways are activated in human retinal pigment epithelial (ARPE–19) cells after exposure to 7–ketocholesterol (7kCh).

Methods: : : ARPE–19 cells were plated in 6 well tissue culture plates. Cells were treated with 40 µg/ml of 7 kCh (Sigma–Aldrich, St. Louis, MO) , an oxidized cholesterol, dissolved in dimethyl sulfoxide (DMSO) and incubated for 2, 6 and 24 hours. Activities of caspase 3, 8 and 9 were detected using Fluorochrome Inhibitor of Caspases (FLICA) kits (Immunochemistry Technologies LLC, Bloomington, MN) and compared with non–treated cultures. Caspase 12 activity was analyzed with Western blotting. Some cultures were treated with 20 µM z–VAD–fmk, a pan–caspase inhibitor, 1 hour before and during exposure to 7 kCh and caspase 3 activity was measured after that.

Results: : Caspases 3, 8 and 12 were activated in ARPE–19 cells after exposure to 40 µg/ml of 7–ketocholesterol. Activation of caspase 3 was blocked in cultures treated with z–VAD–fmk. Caspase 9 was not activated in cell cultures treated with 7kCh.

Conclusions: : The apoptosis induced by 7kCh in ARPE–19 cells uses the extrinsic FAS and FAS–ligand (caspase 8) and endoplasmic reticulum stress (caspase 12) pathways. The mitochondrial caspase pathway (caspase 9) was not activated. Our results suggest that oxysterols such as 7kCh may cause loss of cell viability via caspase dependent apoptosis and be oxidative stressors leading to damage of the RPE cell layer.

Keywords: retinal pigment epithelium • nutritional factors • retinal culture