Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells.
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.
Ares
M P
MP
King Gustav Vth Research Institute, Department of Medicine, Stockholm, Sweden.
Pörn-Ares
M I
MI
Thyberg
J
J
Juntti-Berggren
L
L
Berggren
P O
PO
Diczfalusy
U
U
Kallin
B
B
Björkhem
I
I
Orrenius
S
S
Nilsson
J
J
D001011:Aorta; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006651:Histocytochemistry; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D008854:Microscopy, Electron; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D009543:Nifedipine / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D002843:Chromatin; D005456:Fluorescent Dyes; D006888:Hydroxycholesterols; D014409:Tumor Necrosis Factor-alpha; C007997:25-hydroxycholesterol; D007371:Interferon-gamma; D014700:Verapamil; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D009543:Nifedipine; D002118:Calcium; D016257:Fura-2