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

Mikko P.S. Ares, M. Isabella Pörn-Ares, Johan Thyberg, Lisa Juntti-Berggren, Per Olof Berggren, Ulf Diczfalusy, Bengt Kallin, Ingemar Björkhem, Sten Orrenius, Jan Nilsson

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81 Scopus citations

Abstract

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 μg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNFα (10 ng/ml) and IFNγ (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 μg/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 TNFα and IFNγ enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.

Original languageEnglish (US)
Pages (from-to)2049-2061
Number of pages13
JournalJournal of Lipid Research
Volume38
Issue number10
StatePublished - Oct 1 1997

Keywords

  • Ca
  • Caspase 3
  • Chromatin condensation
  • CPP32
  • DNA fragmentation
  • Oxysterol

ASJC Scopus subject areas

  • Endocrinology

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    Ares, M. P. S., Pörn-Ares, M. I., Thyberg, J., Juntti-Berggren, L., Berggren, P. O., Diczfalusy, U., Kallin, B., Björkhem, I., Orrenius, S., & Nilsson, J. (1997). Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells. Journal of Lipid Research, 38(10), 2049-2061.