Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells

• Main Authors: M P Ares, M I Pörn-Ares, J Thyberg, L Juntti-Berggren, P O Berggren, U Diczfalusy, B Kallin, I Björkhem, S Orrenius, J Nilsson
• Format: Article
• Language: English
• Published: Elsevier 1997-10-01
• Series: Journal of Lipid Research
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520371352

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30﹪, whereas the cytokines alone were without effect. After 48 h, 40﹪ of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21﹪ of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70﹪ and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42﹪. 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. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.