The Inhibition Effect And Mechanism Of Human Arresten Gene Transfection On The Intimal Hyperplasia Of Autogenous Vein Graft In Rats

BACKGROUND:Autogenous vein is most commonly used for reconstruction vascular operation as the one of the best curative effect grafted materials. However, the long-term effectiveness of operation is limited by patency stenosis or restenosis rates after autogenous vein graft. Restenosis is a complicated course of multiple factor,multiple link and multiple stage. Intimal proliferation and long-term arteriosclerosis are the pathematology foundation of restenosis after transplantation. The essential component of hyperplasic intima are vascular smooth muscle cells and extracellular matrix, while excessive proliferation and migration to endomembrane are the important pathological base of neointima proliferation, and the crucial link of vascular restenosis resulted by diversified factors after reconstructive vascular operation.; meanwhile balance breakdown between the proliferation and apoptosis of vascular smooth muscle cells provide a chance for intima proliferation. It will be the effective prevention and cure approaches of preventing restenosis after reconstructive vascular operation that to inhibit the excessive proliferation and migration of VSMC. arresten, the NC1 domain of the alpha1 chain of type IV collagen, is a recently reported by Colorado angiogenesis inhibitor derived from vascular basement membrane. Its molecular weight is about 26kD. arresten has recently been shown effective in the inhibition of proliferation and migration of endothelial cells,tubiform formation and induction of endothelial apoptosis. It also can suppress neovascularization and tumor growth and metastases in vivo. We found that arresten may inhibit the proliferation of VSMC in vitro in prophase research, so we suppose that angiogenesis inhibitor arresten may produce a marked effect in the intimal hyperplasia and development of restenosis after reconstructive vascular operation. Hence, to research the effect of arresten on intimal hyperplasia after autogenous vein graft is of important significance and application perspective to elucidate the molecular mechanisms of intimal hyperplasia and vasotransplantation restennosis deeply, provide effective prevention and cure strategy.OBJECTIVE:To construct human arresten gene eukaryotic expression vector, express recombinant plasmid in COS-7 cells and excrete protein in our experiment. Rat vascular smooth muscle cells were cultured from thoracic aorta of male Sprague-Dawley rats using the tissue explants method and subcultured by trypsinization;To express human arresten gene in eukaryotic cell, and investigate its effect on the biological behavior off vascular smooth muscle cells in vitro. To construct the model of autogenous vein graft in rat, transfect recombinant plasmid pSecTag2-AT into vein grafts locally; to explore the effect of arresten on the intimal proliferation of venous autografts.METHODS: (1) We design a pair of specific primer P1 and P2 according to the cDNA sequence of human arresten in recombinant plasmid pGEMArr contrasted in prophase and multiclone encoding sequence in eukaryotic expression vector pSecTag2-B. arresten gene was amplified by PCR using recombinant plasmid pGEMArr as the template. The amplified target gene were purified and reclaimed by QIAquick PCR Purification Kits. The purified amplified conduction after coupled reaction was inserted into the pSecTag2-B vector by T4 DNA Ligase. The recombinant plasmid was identified using restriction analysis by incision enzyme BamHI and EcoRI, and it was named as pSecTag2-AT. The target gene was sequenced using the 377 DNA automatic sequencing meter. The target gene sequence and reading frame of codon was affirmed correctly or not through the analysis of the DNA sequence of the obtained gene using DNAssist and DNAMAN analysis software. The recombinant expression plasmid was transfected into COS-7 cells. RT-PCR was used to detect the expression of arresten mRNA in cells, while Western blot assay was applied to detect expressed arresten protein in concentrated supernatants. (2) Rat vascular smooth muscle cells (VSMC) were cultured and identified successfully in vitro. Its proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. Migration of VSMC was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chemotaxis chamber) with pores of 8μm in diameter. Annexin V/FITC assay was carried out to detect the effect of the arresten protein on apoptosis of VSMC in vitro. (3) The model of autogenous vein graft was prepared on Sprague-Dawley rat strains, and recombinant plasmid pSecTag2-AT was transfected into vein grafts locally. RT-PCR was used to detect the expression of arresten mRNA in blood vessel; the intimal and medial areas and thickness were measured by computerized planimetry under a light microscope to compare the degree of intimal hyperplasia (IH) by the calculated ratio between intima (I) and media (M) after staining with hematoxylin-eosin (HE) and Verhoeff (elastic fibers). Immunohistochemical labeling and morphologic analysis of vein graft sections were used to identify proliferating cell nuclear antigen (PCNA )positive cells and smooth muscle alpha-actin(α-SMA) positive cells; Western blot were used to detect the protein of transforming growth factor--β1 (TGF-β1).RESULTS: (1) arresten gene eukaryotic expression vector was identified correctly using restriction analysis, and the sequence was identified and confirmed by DNAssist and DNAMAN analytical software. Three clonged nucleotide sequence were in the same manner; analysis of sequence comparison showed that sequenced result was as the same as the sequence of human arresten cDNA clonged in prostage. Target gene inserted into expression vector was correct. There was coincidence between arresten gene fragment inserted into vector and reading frame of codon of expression vector. It suggested that arresten gene was clonged into expression vector completely and exactly to assure the correct expression of target protein. Eukaryotic expression plasmid pSecTag2-AT was constructed successfully. RT-PCR revealed that arresten-transferred cells contained a 449bp specific fragment of arresten gene. Successful protein expression in supernatants was confirmed by Western blot. (2) CCK-8 assay showed that the proliferation of VSMC were inhibited significantly by arresten protein as compared with control group (F=40.154, P<0.01). It was certain time and concentration dependence. Transwell's chamber showed that the number of control group, pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53,14.00±3.33, and the differences were statistically significant (F =38.915,P<0.01);apoptosis assay showed that arresten protein promoted the apoptosis of VSMC(F=29.32,P<0.01), and the rate of apoptosis in different time had no statistical significance (P>0.05).(3)RT-PCR revealed that the genome of arresten-transferred tissue contained a 449bp specific fragment of arresten gene; the intimal and medial areas of pSecTag2-AT transfected group was less than that of control group, and the difference were statistically significant (P<0.01), while I/M had no statistics difference. A less intimal thickness of pSecTag2-AT transfected group was seen compared with the control group and pSecTag2 transfected group.α-SMA staining suggested that VSMC were in the hyperplasic intima; the number of PCNA-positive-stained cells and expression index was lower as compared with that of the control group, and the differences were also statistically significant (P<0.01). Western blot revealed that protein level of TGF-β1 decreased obviously compared with the control group and pSecTag2 transfected group.CONCLUSIONS: We constructed eukaryotic expression vector of human arresten gene successfully, expressed recombinant plasmid in COS-7 cells and excreted protein. arresten protein expressed in eukaryotic cells can inhibit proliferation and migration of VSMC effectively and promote the apoptosis of VSMC in vitro. Local transfection of human arresten gene can inhibit the intimal hyperplasia of venous autografts and improve patency level of blood vessel. These showed the good clinical application perspective for prevention and cure of restenosis after transplantation of blood vessel.