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Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

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Abstract

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.

Conclusion

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.

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References

  1. Caplice N, Mueske C, Kleppe L, Broze G, Simari R (1997) Expression and regulation of tissue factor pathway inhibitor in arteries and vascular smooth muscle cells. Circulation 96(Suppl I):I–663 (Abstract)

    Google Scholar 

  2. Mehran R, Dangas G, Abizaid AS et al (1999) Angiographic patterns of in-stent restenosis: classification and implications for long-term outcome. Circulation 100:1872–1878

    PubMed  CAS  Google Scholar 

  3. Williams DO, Holubkov R, Yeh W et al (2000) Percutaneous coronary intervention in the current era compared with 1985–1986: the National Heart, Lung, and Blood Institute Registries. Circulation 102:2945–2951

    PubMed  CAS  Google Scholar 

  4. Lefkovits J, Topol EJ (1997) Pharmacological approaches for the prevention of restenosis after percutaneous coronary intervention. Prog Cardiovasc Dis 40:141–158

    Article  PubMed  CAS  Google Scholar 

  5. Koop C, Holzenbein T, Steiner S et al (2004) Inhibition of restenosis by tissue factor pathway inhibitor: in vivo and in vitro evidence for suppressed monocyte chemoattraction and reduced gelatinolytic activity. Blood 103:1653–1661

    Article  Google Scholar 

  6. Singh R, Pan S, Mueske C et al (2001) Role for tissue factor pathway in murine model of vascular remodeling. Circ Res 89:71–76

    Article  PubMed  CAS  Google Scholar 

  7. Zoldhelyi P, Chen ZQ, Shelat H, McNatt JM, Willerson JT (2001) Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries. Pro Natl Acad Sci 98:4078–4083

    Article  CAS  Google Scholar 

  8. Yin XH, Yutani C, Ikeda Y et al (2002) Tissue factor pathway inhibitor gene delivery using HVJ-AVE liposomes markedly reduces restenosis in atherosclerotic arteries. Cardiovasc Res 56:454–463

    Article  PubMed  CAS  Google Scholar 

  9. Hamuro T, Kamikubo Y, Nakahara Y et al (1998) Human recombinant tissue factor pathway inhibitor induces apoptosis in cultured human endothelial cells. FEBS Lett 421:197–202

    Article  PubMed  CAS  Google Scholar 

  10. Tasiou A, Konduri SD, Yanamandra N et al (2001) A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas. Int J Oncol 19: 591–597

    PubMed  CAS  Google Scholar 

  11. Lin YF, Zhang N, Guo HS et al (2007) Recombinant tissue factor pathway inhibitor induces apoptosis in cultured rat mesangial cells via its Kunitz-3 domain and C-terminal through inhibiting PI3-kinase/Akt pathway. Apoptosis 12:2163–2173

    Article  PubMed  CAS  Google Scholar 

  12. Ho YS, Wang YJ, Lin JK (1996) Induction of p53 and p21/WAF1/CIP1 expression by nitric oxide and their association with apoptosis in human cancer cells. Mol Carcinog 16:20–31

    Article  PubMed  CAS  Google Scholar 

  13. Ho YS, Lee HM, Mou TC, Wang YJ, Lin JK (1997) Suppression of nitric oxide-induced apoptosis by N-acetyl-l-cysteine through modulation of glutathione, bcl-2 and bax protein levels. Mol Carcinog 19:101–113

    Article  PubMed  CAS  Google Scholar 

  14. Mignotte B, Vayssiere JL (1998) Mitochondria and apoptosis. Eur J Biochem 252:1–15

    Article  PubMed  CAS  Google Scholar 

  15. Zou H, Li Y, Liu X, Wang X (1999) An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274:11549–11556

    Article  PubMed  CAS  Google Scholar 

  16. Alnemri ES, Livingston DJ, Nicholson DW et al (1996) Human ICE/CED-3 protease nomenclature (letter). Cell 87:171

    Article  PubMed  CAS  Google Scholar 

  17. Walsh K, Smith RC, Kim HS (2000) Vascular cell apoptosis in remodeling, restenosis, and plaque rupture. Circ Res 87:184–188

    PubMed  CAS  Google Scholar 

  18. Kamenz J, Seibold W, Wohlfrom M et al (2000) Incidence of intimal proliferation and apoptosis following balloon angioplasty in an atherosclerotic rabbit model. Cardiovasc Res 45:766–776

    Article  PubMed  CAS  Google Scholar 

  19. Lim S, Jin CJ, Kim M et al (2006) PPARgamma gene transfer sustains apoptosis, inhibits vascular smooth muscle cell proliferation, and reduces neointima formation after balloon injury in rats. Arterioscler Thromb Vasc Biol 26:808–813

    Article  PubMed  CAS  Google Scholar 

  20. Malik N, Francis SE, Holt CM et al (1998) Apoptosis and cell proliferation after porcine coronary angioplasty. Circulation 98:1657–1665

    PubMed  CAS  Google Scholar 

  21. Atsuchi N, Nishida T, Marutsuka K et al (2001) Combination of a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI gene transfer additively reduces neointima formation in balloon-injured rabbit carotid arteries. Circulation 103:570–575

    PubMed  CAS  Google Scholar 

  22. Li P, Nijhawan D, Budihardjo I et al (1997) Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489

    Article  PubMed  CAS  Google Scholar 

  23. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90:405–413

    Article  PubMed  CAS  Google Scholar 

  24. Hu Y, Benedict MA, Ding L, Nunez Q (1999) Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. EMBO J 18:3586–3595

    Article  PubMed  CAS  Google Scholar 

  25. Slee EA, Harte MT, Kluck RM et al (1999) Ordering the cytochrome c-initiated caspase cascade; hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144:281–292

    Article  PubMed  CAS  Google Scholar 

  26. Hakem R, Hakem A, Duncan GS et al (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94:339–352

    Article  PubMed  CAS  Google Scholar 

  27. Kuida K, Haydar TF, Kuan CY et al (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94:325–337

    Article  PubMed  CAS  Google Scholar 

  28. George J, Gondi CS, Dinh DH, Gujrati M, Rao JS (2007) Restoration of tissue factor pathway inhibitor-2 in a human glioblastoma cell line triggers caspase-mediated pathway and apoptosis. Clin Cancer Res 13:3507–3517

    Article  PubMed  CAS  Google Scholar 

  29. Crook NE, Clem RJ, Miller LK (1993) An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J Virol 67:2168–2174

    PubMed  CAS  Google Scholar 

  30. Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) X-linked IAP is a direct inhibitor of cell death proteases. Nature 388:300–304

    Article  PubMed  CAS  Google Scholar 

  31. Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) The c-IAP-1 and c-IaP-2 proteins are direct inhibitors of specific caspases. EMBO J 16:6914–6925

    Article  PubMed  CAS  Google Scholar 

  32. Deveraux QL, Roy N, Stennicke HR et al (1998) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17:2215–2223

    Article  PubMed  CAS  Google Scholar 

  33. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES (1998) Autoactivation of procaspase-9 by apaf-1-mediated oligomerization. Mol Cell 1:949–957

    Article  PubMed  CAS  Google Scholar 

  34. Sato Y, Kataoka H, Asada Y et al (1999) Overexpression of tissue factor pathway inhibitor in aortic smooth muscle cells inhibits migration induced by tissue factor/factor VIIa complex. Thromb Res 94:401–406

    Article  PubMed  CAS  Google Scholar 

  35. Kamikubo Y, Nakahara Y, Takemoto S et al (1997) Human recombinant tissue factor pathway inhibitor prevents the proliferation of cultured human neonatal aortic smooth muscle cells. FEBS Lett 407:116–120

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, No. 194, XueFu Road, NanGang District, Harbin, 150086, Heilongjiang Province, China

    Yu Fu, Zhaoying Zhang, Gaigai Zhang, Yue Liu, Ying Cao, Jinfeng Yu, Jing Hu & Xinhua Yin

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  1. Yu Fu
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  2. Zhaoying Zhang
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  3. Gaigai Zhang
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  8. Xinhua Yin
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Correspondence to Xinhua Yin.

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Fu, Y., Zhang, Z., Zhang, G. et al. Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells. Apoptosis 13, 634–640 (2008). https://doi.org/10.1007/s10495-008-0199-4

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