Overexpression ANT1 Gene In Vascular Smooth Muscle Cells Induced Apoptosis With Bax/Bcl-2 Expression Relations

Background and Objective:Restenosis after percutaneous coronary intervention has been the main reason for the failure of atherosclerotic therapy. Neointimal formation and vascular reshape are the main reasons to the occurrence of restenosis. Imbalances of smooth muscle cell apoptosis and proliferation plays a key role in the formation of neointimal. Numerous factors participate in smooth muscle cell apoptosis, and there are many regulating molecular ways. They include mitochondrial mediated signaling pathways and death receptor mediated signaling pathways, etc. Mitochondria is the center of cellular energy metabolism, which plays an important role in cell apoptosis. Stimulus leads to cell apoptosis by increasing the mitochondrial membrane permeability and the reduction of mitochondrial membrane potential. Then apoptosis related proteins were released, such as CytC, apoptosis-inducing factors (AIF) etc. The union of CytC and protease activated factor (Apaf-1) activates cysteine proteolytic enzymes caspase 9 and caspase 3 ways, and eventually induced cell apoptosis.Adenine nucleotide transposition enzymes are one of the transport proteins located in the mitochondrial inner membrane. This gene maintains evolution conservative and plays a key role in regulating cell apoptosis.Many studies showed that ANT1 induced cell apoptosis is related to mitochondrial permeability transition pore (MPTP). ANT1, a necessary ingredient of MPTP interacts with Cyclophilin D, which is the major component of MPTP. The interaction reduced the mitochondrial membrane potential, triggers cell apoptosis of mitochondrial way. ANT1 can induce myocardial cells and tumor cell apoptosis in vitro; meanwhile it can reduce tumor volume tumors in vivo. Therefore, we first constructed adenine nucleotide translocase 1(ANT1)with GFP tag adenovirus vector and transfected the vascular smooth muscle cells(VSMCs) in rat. After adenovirus-mediated ANT1 (Ad-ANT1)or adenovirus-empty(Ad-GFP) transfection, the apoptosis and proliferation of VSMCs was tested, meanwhile the Bax/Bcl-2 expression were detected. Hence, the function of ANT1 in smooth muscle cell apoptosis was evaluated. Hopefully ANT1 maybe a new therapeutic target for the imbalance between vascular smooth muscle cell proliferation and apoptosis in the pathogenesis of restenosis after PCI.Methods:1,Construction of the ANT1 genes recombinant adenovirus vector with the GFP tag. Recombinant shuttle plasmid pShuttle-GFP-CMV-ANT1 and pShuttle-GFP-CMV were ligased with pAdxsi vector after enzymes digestion.The recombinant adenovirus vector was transfected into HEK293 cells by using LiporeetaminelTM 2000 and packaged for the recombinant adenovirus particles. Replication-deficient recombinant adenovirus which carries the ANT1 gene was identified by RT-PCR and amplified.2,VSMCs derived from the thoracic aortic explants of Sprague Dawley rats were primarily cultured, and confirmed by microscope andα-SM-actin immunocytochemistry. After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP) transfection into VSMCs, the transfected efficiency was observed in different time by fluorescence microscope. The ANT1 mRNA and protein expression were detected by RT-PCR and Western Blotting respectively.3,After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP)transfection,the apoptosis of VSMCs stained by Hoechst 33258 was observed by laser confocal microscopy.The proportion of VSMCs apoptosis marked by Annexin-V was detected by flow cytometric analysis or TUNEL assay. The viability of VSMCs was measured by CCK8 assay and cell count. The changes in mitochondrial membrane potential (△Ψm) were detected by fluorescent probe TMRE. 4,After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP) transfection into VSMCs,the Bax/Bcl-2 mRNA and protein expression were detected by RT-PCR and Western Blotting respectively.Results:1,Cultured rat thoracic aortic cells were identified as VSMCs by morphology andα-SM-actin immunocytochemistry and were used in the following experiments.2,The recombinant adenovirus vector was constructed and amplification successfully, The titer of 2×l011 pfu/ml was obtained and the optimal multiplicity of infection(MOI) was 80-100.After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP) transfection into VSMCs , the infection efficiency was 80% by fluorescence microscope. The expression of ANT1 mRNA and protein in VSMCs after transfection with Ad-ANT1 by RT-PCR and Western Blotting are increased significantly. They can be used in the following experiments.3,Apoptosis test Morphologic detection: After Ad-ANT1 transfection,VSMCs stained by Hoechst33258 by laser confocal microscopy showed that cell apoptosis specific features. Chromatin was dense which located mainly in the nuclear membrane. Cell nucleuses become pyknotic and apoptotic peak. There is no significant morphologic change in the Ad-GFP group. The levels of mitochondrial membrane potential (?Ψm) was decreased in Ad-ANT1 group compared with Ad-GFP group ,which was detected by fluorescent probe TMRE. Flow cytometric analysis and TUNEL assay showed a significant increase in the percentage of VSMCs apoptosis after adenovirus-mediated ANT1 (Ad-ANT1) transfection compared with cells of adenovirus-empty(Ad-GFP)group.4,Proliferation activity detection: CCK8 assay and cell count both showed the proliferation activity of Ad-ANT1 group was decreased significantly compared with Ad-GFP group.5,The Bax mRNA and protein expression were detected by RT-PCR and Western Blotting respectively. ANT1 is upregulated in VSMCs after Ad-ANT1 transfection compared Ad-GFP.No differences were observed in the mRNA and protein levels of Bcl-2 in the two groups. Conclusions:1,The recombinant adenovirus ANT1 vector was constructed successfully and can be expressed efficiently in vascular smooth muscle cells in vitro.2,Overexpression of ANT1 in VSMCs induced VSMCs apoptosis and inhibited VSMCs proliferation.3,ANT1 overexpression can induce cell apoptosis in VSMCs. The underlying mechanism may partly be attributed to up-regulate Bax expression and the rise of Bax/Bcl-2 ratio.