Effects And Molecular Mechanisms Of CBP Silencing On Neointimal Hyperplasia In Balloon Injured Rat Carotid Artery

Objectives:1. To construct a recombinant lentiviral vector encoding shRNA targeting rat CREB binding protein (CBP) gene, and observe the effects of CBP gene silencing in rat vascular smooth muscle cells (VSMCs).2. To investigate the inhibitory effects of lentivirus-mediated CBP gene silencing on AngiotensinⅡ(AngⅡ) induced VSMCs proliferation, and elucidate its molecular mechanisms.3. To examine the effects of lentivirus-mediated CBP gene silencing on neointimal hyperplasia and endothelialization in balloon injured rat carotid artery, and to identify the underlying mechanisms.Methods:1. Four shRNA sequences targeting CBP gene were designed and synthesized, and the best one was cloned into the pFU-GW-iRNA vector to construct the lentiviral expression vector contained CBP shRNA (LV-CBP shRNA). LV-CBP shRNA confirmed by PCR and DNA sequencing, were subsequently cotransfected with packaging plasmids into 293T cells by Lipofectamine 2000. The titer of virus was deteced according to the expression of enhanced green fluorescent protein (EGFP). CBP mRNA expression in VSMCs with or without lentivirus transfection was measured by real-time PCR.2. We carry out primary culture of SD rats’aorta pectoralis VSMCs by attaching growth. The 3-8 generation cells were applied in the study. Stably lentivirus transfected VSMCs of CBP siRNA, NC siRNA and untreated VSMCs were stimulated by AngⅡ(100 nmol/1). Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assay. Protein and mRNA expression of CBP and relevant cytokines were examined by Western blot, enzyme-linked immunosorbant assay (ELISA) and real-time PCR, respectively. We also used luciferase reporter gene and electrophoretic mobility shift assay (EMSA) to detect Nuclear factor kappaB (NF-κB) transcriptional activity and DNA binding. Meanwhile, NF-κB p65 subunit nuclear translocation was confirmed by immunofluorescence and immunoblotting.3. Male SD rats were randomly divided into four groups:Sham group, PBS group, CBP-siRNA/Lentivirus group and NC-siRNA/Lentivirus group to establish the model of balloon-injury in left common carotid artery, and the rats were sacrificed at 28 days after injury and in vivo gene transfer. Confocal microscopy was used to observe the efficiency of lentiviral transfection. The expression of CBP and acetylated NF-κB p65 were determined by real-time PCR and Western blot. PCNA and CD31 protein expression was detected by immunohistochemistry. Morphometric analysis was used to measure neointimal hyperplasia. Meanwhile, we used Evans blue dye staining to evaluate endothelialization.Results:1. PCR analysis and DNA sequencing demonstrated that the LV-CBP shRNA was constructed successfully. The titer of concentrated lentivirus was 2×109 TU/ml. Compared with non-transfectd VSMCs and LV-NC shRNA, the level of CBP mRNA in LV-CBP shRNA decreased 88.5%,92.7%, respectively.2. It was shown that lentiviral-mediated CBP-shRNAs at different multiplicities of infection (MOI=100,150) both significantly suppressed AngⅡ-induced CBP expression in VSMCs (P<0.05). Knockdown of CBP markedly inhibited AngⅡ-stimulated VSMCs proliferation and the related cytokines (TNF-αand IL-6) production. However, this inhibitory effect of CBP silencing was not enhanced in VSMCs at MOI of 150 compared with MOI of 100 (P>0.05). Then we went further to study the mechanism. We observed that CBP siRNA showed the potent inhibition on AngⅡ-induced NF-κB p65 transcriptional activity in VSMCs (P<0.05). Similarly, no significant difference was found between CBP-siRNA lentivirus treatment groups. Furthermore, CBP gene silencing had no effect on AngⅡ-stimulated NF-κB nuclear translocation or binding to its DNA consensus sequence (P>0.05).3. Recombinant lentivirus exhibited high transfection level in carotid arteries. At 28 days after operations, lentivirus siRNA targeting CBP markedly decreased CBP mRNA and protein expression compared with PBS and NC-siRNA/Lentivirus groups (P<0.05). CBP gene silencing significantly reduced the neointimal area and intima/ media ratio (P boh<0.05). Furthermore, compared with PBS and NC-siRNA/Lentivirus groups, PCNA and acetylation of NF-κB p65 expression were both obviously downregulated in CBP-siRNA/Lentivirus group (P both<0.05). Endothelial cell marker CD31 immunostaining and Evans blue dye staining analysis both showed that CBP knockdown significantly accelerated endothelialization (P both<0.05).Conclusions:1. The recombinant lentiviral vector encoding shRNA targeting CBP has been successfully constructed. LV-CBP shRNA has obvious effect on CBP gene silencing in VSMCs.2. Lentivirus-mediated CBP gene silencing could suppress AngⅡ-induced proliferation in VSMCs. The antiproliferative mechanism of CBP knockdown appears to be partly due to downregulation of NF-κB transcriptional activity, and not through reduction in NF-κB nuclear translocation and DNA binding.3. Lentivirus-mediated CBP gene silencing could efficiently suppress neointimal formation and promote endothelialization in balloon injured rat carotid artery, and the mechanism is involved with downregulation of NF-κB p65 acetylation.