Effect of nuclear factor-kappa B on vascular smooth muscle cell proliferation and neointima formation after angioplasty / 中国组织工程研究

ABSTRACT

BACKGROUND: Inflammatory and proliferating effect after mechanical injury of vascular wall is the major cause of restenosis. Nuclear factor-kappa B (NF-κB) in the NF-κB/Rel family is expressed in a variety of cell types and activates a series of target genes, which are related to the pathophysioiogy of vascular wall.OBJECTIVE: To investigate the effect of antisense and decoy NF-κB oligonucleotides on vascular smooth muscle cell (VSMS) proliferation in vitro and neointimal proliferation and monocyte chemotactic protein-1 (MCP-1) in the balloon-injured carotid artery of rats.DESTGN: Randomized controlled animal trial.SETTTNG: Department of Cardiology, Ruijin Hospital, Medical College, Shanghai Jiaotong University.MATERTALS: Totally 126 male Sprague-Dawley (SD) rats, aged 3 months, weighing 350 to 380 g, were involved in this study. Synthesis of primer and oligonucleotide: they were synthesized and designed by Shanghai Bioengineering Co. Ltd according to literatures and international internet cDNA library.METHODS: This study was carried out in the Laboratory of Cell Biology, Medical College, Shanghai Jiao Tong University and Cardiovascular Laboratory, Ruijin Hospital Affiliated to Shanghai Jiaotong University from May 2001 to March 2003.Rat aortic smooth muscle cells were isolated from May 2001 to March 2003. Rat thoracic aorta vascular smooth muscle cells were cultured by primary-explant method. And the third to fifth generations of VSMCs were involved in the experiment. Proliferating cell nuclear antigen (PCNA) NF-κB p65 protein synthesis in proliferating smooth muscle cells were detected. SD rat carotid artery underwent balloon injury. The involved 126 rats were randomly divided into 7 groups with 18in each group: normal group: normal group (the procedure was the same as other group except for balloon injury), sense group, antisense group, decoy group, scramble group, antisense plus decoy group, model group. Each group includes 6time points (6 hours, and 1,3,5,7,14 days, n =3). Then, the effect of antisense and decoy NF-κB oligonucleotides on intimai proliferation and MCP-1 and NF-κB p65 and extracellular signal regulated kinase(ERK2) expression in the balloon-injured carotid artery of rats were detected.MAIN OUTCOME MEASURES: ①Effect of oligonucleotide of NF-κB p65 on VSMCs proliferation; ② NF-κB p65 gene expression and protein synthesis; ③ Patho-morphological change after carotid balloon-injury. ④ Vascular MCP-1 mRNA Expression in balloon-injured rat carotid artery; ⑤ MCP-1 immunoreactivity in the injured arterial wall detected by immunohistochemistry; ⑥ NF-κBp65 and ERK2 protein synthesis after balloon-injury detected by Western blot in injured rat carotid arteries.RESULTS: ①PCNA protein synthesis increased in proliferating smooth muscle cells. ②NF-κB p65 gene expression was found in the cytoplasm and nucleus of proliferating smooth musclecells by in situ hybridization and NF-κB p 65 protein level increased in proliferating smooth muscle cells by flow cytometry. NF-κB p65 gene expression in antisense group decreased 53.66% compared with in sense group; it decreased 57.35% in decoy group compared with in scramble group. There were all statistical differences(P＜0.05).③ PCNA expression were inhibited in proliferating smooth musclecells by antisense and decoy oligonucleotides. Compared with positive control group, PCNA protein expression in antisense group and decoy group decreased 45.12% and 45.05%,respectively. ④ In model group, sense group and scramble group, vessel intimal area, medial area and intimal area/medial area increased at the 5th day after balloon-injury and reached the maximum at the 7th day after injury. The intimal area/medial area was significantly decreased in the antisense group and decoy group. The effect of antisense plus decoy oligonucleotides was more obvious than that of antisense group and decoy group alone but there were not significant differences among three groups. ⑤ Reverse transcription-polymerase chain reaction showed that MCP-1 mRNA expression was significantly increased 6 hours after balloon-injury, but not evident after 1 day. It was increased at the 3th, 5th and 7th days continuously, but decreased at the 14th day. MCP-1 mRNA expression was decreased at each time point in antisense group, decoy group, antisense plus decoy group (P＜0.05). ⑥Western blot analysis showed that NF-κB p65 was weakly expressed at 6 hours after vascular balloon-injury, increased significantly at 1 day, reached the peak at 7 days and weakened at 14 days, while ERK2 protein was weakly expressed, a little increased at 1 day, reached the peak at 7 days and weakened at 14 days. Treatment of antisense group, decoy group and antisense plus decoy group inhibited protein synthesis more significantly than those of model group, sense group and scramble group (P＜0.05).CONCLUSTON: NF-κB expression increases in proliferating smooth muscle cells. NF-κB modulates genes expression and protein synthesis of MCP-1 and ERK2. Cellular proliferation in vessel wall dynamically changes after balloon angioplasty injury. Antisense and decoy oligonucleotide of NF-κB by local lipofectamine transfer inhibit the expression of regulated target gene.