CA2-HYDROXYCHOLESTEROL IN HUMAN AORTIC SMOOTH-MUSCLE CELLS( CHANNEL BLOCKERS VERAPAMIL AND NIFEDIPINE INHIBIT APOPTOSIS INDUCED BY 25)

We have characterized the death of human aortic smooth muscle cells in duced by 25-hydroxycholesterol, an oxidation product of cholesterol. C hromatin condensation characteristic of apoptosis was observed by enzy matic (TUNEL) staining of chromatin, and by electron microscopy. Fourt een percent of cells treated with 5 mu g/ml of 25-hydroxycholesterol f or 24 h displayed chromatin degradation as determined by positive TUNE L staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas tile cytokines alone were without effect. After 48 h, 40% of the cells trea ted with 5 mu g/ml of 25-hydroxycholesterol were TUNEL positive, and 2 1% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by ag-arose gel e lectrophoresis. Furthermore, activation of the ICE-like protease caspa se 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the cu lture medium inhibited apoptosis by more than 70% and reduced cytotoxi city, while removal of Ca2+ from culture medium reduced apoptosis by 4 2%. Within a few minutes after-addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3 -0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addit ion of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cy tokines can increase the cytotoxicity of lipid oxidation products.