The mechanism underlying vascular smooth muscle cell apoptosis induced by atorvastatin may be mainly associated with down-regulation of survivin expression.
OBJECTIVE
Studies have shown that statins may induce vascular smooth muscle cells (VSMCs) apoptosis. But its mechanisms are incompletely understood. In this study, we investigate the effects of atorvastatin and survivin antisense oligonucleotides (ASODN) on VSMCs apoptosis and the relation between survivin and VSMCs apoptosis.


METHODS
Cultured VSMCs were treated with atorvastatin and vascular endothelial grow factor (VEGF). Apoptosis of VSMCs at 6-72 h after treatment with atorvastatin was detected by means of Hoechst33258 staining. Survivin and Fas factors expression were detected by means of immunohistochemistry. Survivin and Fas mRNA expression were detected by means of RT-PCR. In order to determine the relations between survivin and VSMCs apoptosis, we also performed transfection of VSMCs with survivin ASODN using GenePORTER Transfection Reagent and studied the survivin protein expression by means of western blotting.


RESULTS
VSMCs apoptosis after treatment with atorvastatin was increased in a dose- and time-dependent manner. The expression of survivin and survivin mRNA in VSMCs was significantly down-regulated at 24 h and disappeared at 48-72 h after treatment with atorvastatin. Fas and Fas mRNA in VSMCs could only be detected at 72 h and not at 6-48 h after treatment with atorvastatin. We did not observe any effects of VEGF on VSMCs apoptosis, on survivin and survivin mRNA expression, and on Fas and Fas mRNA expression in VSMCs after treatment with atorvastatin. At 48 hours after the start of transfection, survivin protein expression was significantly reduced after transfection with 0.5, 1.0 and 2.0 microg/ml of survivin ASODN and there was no survivin protein expression after transfection with 3.0 and 4.0 microg/ml of survivin ASODN. In contrast, in the GenePORTER only and SODN studies, survivin protein expression was observed with western blotting. Hoechst33258 staining showed that treatment of VSMCs with survivin ASODN resulted in VSMCs apoptosis.


CONCLUSIONS
Atorvastatin induces VSMCs apoptosis in a dose- and time-dependent manner. Transfection of survivin ASODN can directly induce VSMCs apoptosis. The mechanisms of VSMCs apoptosis induced by atorvastatin may be mainly associated with down-regulation of survivin expression in VSMCs. Up-regulation of Fas in VSMCs may play a role in later stages following atorvastatin treatment.
Xu
Yi-Guan
YG
Cardiovascular Department, First Affiliated Hospital of Medical College of Shantou University, Shantou, Guangdong, PR China. menmeno2003@yahoo.com.cn
Zhou
Sheng-Hua
SH
Li
Yu-Guang
YG
Zheng
Chun-Hua
CH
Li
Xu-Ping
XP
Liu
Qi-Ming
QM
Xu
Duan-Min
DM
Chen
Shang
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D000069059:Atorvastatin; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004359:Drug Therapy, Combination; D053222:Fas Ligand Protein / Q000235:genetics / Q000378:metabolism; D006538:Heptanoic Acids / Q000494:pharmacology*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011758:Pyrroles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
C516989:Birc5 protein, rat; D053222:Fas Ligand Protein; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008869:Microtubule-Associated Proteins; D016376:Oligonucleotides, Antisense; D011758:Pyrroles; D012333:RNA, Messenger; D000077022:Survivin; D042461:Vascular Endothelial Growth Factor A; D000069059:Atorvastatin