Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells.
OBJECTIVE
To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on vascular smooth muscle cells (VSMCs).


METHODS
Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression of cytochrome c, procaspase-3, cleaved caspase-3, cleaved caspase-9 and inhibitor of apoptosis protein-1(IAP-1) were examined by western blot and RT-PCR.


RESULTS
TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th day after gene transfer, the apoptotic rates in the TFPI group were 11.95%, 71.96% and 37.83%, respectively, whereas those in the LacZ group were 1.34%, 1.83% and 6.37%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. Cytochrome c, cleaved caspase-3 and cleaved caspase-9, which are all involved in mitochondrial pathway, were detected in the cytoplasm on the 3rd, 5th and 7th day after TFPI gene transfer. Procaspase-3 expression was significantly decreased over time in the TFPI group (each P < 0.05), which were not seen in the Ad-LacZ and DMEM groups. The expression of IAP-1 mRNA in the TFPI group was also decreased compared with the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd and 7th day after gene transfer.


CONCLUSIONS
The results demonstrated that overexpression of TFPI gene might induce VSMC apoptosis in vitro through the mitochondrial pathway; meanwhile, IAP-1 expression is decreased. These findings indicated that TFPI might inhibit restenosis by inducing apoptosis in VSMCs.
Fu
Yu
Y
Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, No. 194, XueFu Road, NanGang District, Harbin, 150086, Heilongjiang Province, China.
Zhang
Zhaoying
Z
Zhang
Gaigai
G
Liu
Yue
Y
Cao
Ying
Y
Yu
Jinfeng
J
Hu
Jing
J
Yin
Xinhua
X
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D051381:Rats
D051034:Inhibitor of Apoptosis Proteins; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; C494836:Birc3 protein, rat; D053148:Caspase 3; D053453:Caspase 9