Effects Of Adenovirus-Mediated Gene Transfer Of TIMP-4 On Biological Behaviour Of Vascular Smooth Muscle Cells

Background:The major mechanism of restenosis (RS) after balloon angioplasty and stent implantation is the neointimal formation and hyperplasia. Vascular smooth muscle cell (VSMC) migration is therefore a key step in the development of intimal lesions. The process of migration requires degradation of basement membrane (BM) and extracellular matrix (ECM) surrounding the cell. The ECM homeostasis is the basis of maintaining the integrity of the vessel wall. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are identified to be the most important proteinases in maintaining the ECM homeostasis. After vascular injury, VSMCs are stimulated by some cytokines and undergo profound changes, including phenotypic modulation from contractile pheno-type to synthetic phenotype. Once up-regulated MMPs degrade ECM, the migration of VSMCs break through the limitation of ECM and across the internal elastic lamina to form the intima. It is believed that the MMPs, as a possible "on-off switch" molecular, play an important role in postinjury RS, so it is essential in maintaining the proteolytic balance between MMPs and TIMPs. It have been identified in different animal models that overexpression of TIMP-1,-2,or-3 can respectively prevent VSMC migration and/or proliferation and inhibit the neointimal development after vascular injury. TIMP-4 is a new member of TIMPs, which shows a specific expression in cardiovascular tissues. It is unclear whether TIMP-4 can influence the biological behaviour of VSMC until now. This study is designed to investigate the effects of recombinant replication-deficient adenovirus(Ad) encoding TIMP-4 on cultured VSMC function, and explore the role of TIMP-4 overexpression in RS after angioplasty and stent implantation.Methods: ①Recombinant Ad were generated by liposome cotransfected of pDC315.TIMP-4 with adenovirus genomic plasmid pBHGlox△E1,3cre in 293 cell. The viral stocks were generated in 293 cells and concentrated by cesium chloride ultracentrifugation. An Ad vector expressing the bacterialβ-galactosidase gene (Ad.LacZ) was amplified and purified and used as controls. ②Primary cultured VSMCs were isolated from the thoracic aortas of Wistar rats, which were identified by morphology and immunocytochemistry. ③The TIMP-4 mRNA and protein were evaluated by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western blot analysis. ④The TIMP-4 activities were assayed by gelatin zymography and reverse zymography. ⑤The Millicell chamber were coated with a barrier of reconstituted basement membrane proteins (Matritgel) to detect the migration of VSMCs. Proliferation of VSMCs were detected by the MTT colorimetric assay, incorporation of [3H]-thymidine and flow cytometry.Results: ①A replication-deficient adenovirus type 5 (Ad5) carrying the cDNA for hTIMP-4 under the transcriptional control of the cytomegalovirus (CMV) immediate early promoter was constructed via site specific recombination. Plaque titration on 293 cells showed titers of 5×1010 pfu/ml and 7.5×1010 pfu/ml for Ad.TIMP-4 and Ad.LacZ respectively. The multiplicity of infection were both 100.②Primary cultured VSMCs identified by morphology and immunocytochemistry. Among the passage 6 to 8 of cultured cell, nearly 100% were VSMCs. ③Expression of the hTIMP-4 mRNA and protein in rat VSMC infected with Ad.TIMP-4 was demonstrated by RT-PCR, immunocytochemistry and western blot analysis. Cells expressing TIMP-4 protein increased significantly with the duration of infection and reached 86.5% at 2 days. Human TIMP-4 mRNA and protein were not detected in uninfected and Ad.LacZ-infected cells. ④Both Gelatin zymography and reverse zymography demonstrated that TIMP-4 secreted by Ad.TIMP-4-infencted VSMCs maintained normal biological activity. ⑤The migration assay revealed a 78.7% reduction in the number of VSMCs migrating across the Matrigel barrier in VSMCs infected with Ad.TIMP-4 at an MOI of 100 compared with Ad.LacZ. The optical density (OD) of MTT and incorporation of [3H]...