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

Research output: Contribution to journalArticle

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

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Nifedipine
Verapamil
Smooth Muscle Myocytes
Muscle
Cells
In Situ Nick-End Labeling
Chromatin
Apoptosis
Cytotoxicity
Culture Media
Condensation
Cytokines
Oxidation
Staining and Labeling
Caspase 1
Cell membranes
Electrophoresis
Ion Channels
Agar Gel Electrophoresis
Caspase 3

Keywords

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

ASJC Scopus subject areas

  • Endocrinology

Cite this

Ares, M. P. S., Pörn-Ares, M. I., Thyberg, J., Juntti-Berggren, L., Berggren, P. O., Diczfalusy, U., ... 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.

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells. / Ares, Mikko P S; Pörn-Ares, M. Isabella; Thyberg, Johan; Juntti-Berggren, Lisa; Berggren, Per Olof; Diczfalusy, Ulf; Kallin, Bengt; Björkhem, Ingemar; Orrenius, Sten; Nilsson, Jan.

In: Journal of Lipid Research, Vol. 38, No. 10, 01.10.1997, p. 2049-2061.

Research output: Contribution to journalArticle

Ares, MPS, Pörn-Ares, MI, Thyberg, J, Juntti-Berggren, L, Berggren, PO, 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, vol. 38, no. 10, pp. 2049-2061.
Ares MPS, Pörn-Ares MI, Thyberg J, Juntti-Berggren L, Berggren PO, Diczfalusy U et al. Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells. Journal of Lipid Research. 1997 Oct 1;38(10):2049-2061.
Ares, Mikko P S ; Pörn-Ares, M. Isabella ; Thyberg, Johan ; Juntti-Berggren, Lisa ; Berggren, Per Olof ; Diczfalusy, Ulf ; Kallin, Bengt ; Björkhem, Ingemar ; Orrenius, Sten ; Nilsson, Jan. / Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells. In: Journal of Lipid Research. 1997 ; Vol. 38, No. 10. pp. 2049-2061.
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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.",
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AU - Juntti-Berggren, Lisa

AU - Berggren, Per Olof

AU - Diczfalusy, Ulf

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AU - Orrenius, Sten

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N2 - 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.

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