Effects Of A20 Gene On Intimal Hyperplasia Of Balloon Injured Rat Carotid Artery And Related Molecular Mechanisms

Object:Percutaneous coronary intervention (PCI) is now one of the most effective methods to treat coronary artery disease, but restenosis (RS) following PCI is a primary problem to limit its long-term benefits. Many investigates confirmed that restenosis was the arterial wall's healing response to mechanical injury and comprised two main processes- neointimal hyperplasia (smooth muscle proliferation/migration, extracellular matrix deposition) and vessel remodeling. Many growth factors, cytokine and adhesion molecule participate this pathophysiological procedure, and just inhibiting certain factor can not suppress lesion formation completely. Therefore, it becomes an effective approach of inhibiting restenosis to suppress the critical gene which regulates cell proliferation, migration and differentiation and so on the final common path.Catheter-based gene therapy has emerged as one of the most promising approaches to prevent coronary artery restenosis, because it appears capable of delivering therapeutic gene specifically to the location of the disease, at a precise site in the arterial wall. The choice of efficacious therapeutic gene and appropriate vector are the crux of the matter for a successful gene therapy.A kind of new protein molecule, zinc finger protein A20 (ZFPA20) has been found in recent-years'researches of cellular endogenous protective mechanism. It is confirmed that zinc finger protein A20 is not only important negative feedback factor to regulate signaling pathway of NF-κB but also essential regulating proteinum to suppress inflammatory responses in vivo. It is product of activated NF-κB sgnaling system induced by LPS or TNF. A20 has a protective effect on damaged cells through terminating the activation of NF-κB induced by TLR4, which is activated by LPS. Previous researches have shown that the activation of NF-κB initiate lesion formation and inflammatory response after vascular injury. A20 has both ubiquitinating and deubiquitinating activities. As an important ubiquitinating modification enzyme, A20 is involed in dealing with NF-κB prosoma, IκBαdegradation and IKK (IκB-kinase) activation. A20 leads to a negative-feedback response that terminates activation of NF-κB though above main mechanisms.One side because of inflammation mechanism of restenosis and regulation the inflammation mechanism of A20, on the other hand, bacause the activation of NF-κB initiates restenosis after vascular injury and A20 negative-feedback inhibits activation of NF-κB, A20 was served as target of happening and development process of restenos. A20 expression in vivo was regulated by gene therapy. The present study was designed to evaluate the transfection efficiency of A20 gene in injured arterial wall and to investigate the effect of in vivo local transfection of zinc finger protein A20 gene on intimal hyperplasia, proliferation and the phenotype transformation of vascular smooth muscle cells and reendothelialization of balloon-injured rat carotid arteries and its possible molecular mechanism.Methods:1. Identification and extraction of recombinant plasmid DNA in large quantity: Smearing the obtained E.coli carrying pCAGGS-GFP/A20 plasmid on ampicillin resistance plate, overnight cultured. puting single colony from plate into 3ml ampicillin resistance LB medium and overnight cultured on 37℃shaking table. After collecting bacteria, extracting recombinant plasmid DNA in small quantity and identificating by using EcoRⅤ/SmaⅠdouble restriction enzyme digestion. The production 1% agarose gel electrophoresis was performed. Endo-free plasmid DNA was extracted in large quantity from E.coli carrying plasmid. Plasmid DNA extraction was diluted in suitable proportion and detected the concentration.2. Establishment of rat carotid injury model and grouping. Balloon catheter denudation of the endothelium of rat common carotid artery was routinely used as a model of restenosis. One hundred and four male Sprague-Dawley rats weighing 350-400g were randomly divided into 4 groups (n=26): the sham group (no injury), the model group (simple injury), the control group (vacant vector and transfection regent group) and the therapeutic group (A20 gene and transfection regent group). Rats were anesthetized with 10% chloraldurat (3ml/kg intraperitoneally, i.p.). After a midcervial incision, the left common carotid artery and its bifurcation were exposed. Two vascular clips were placed at the distal end and in the middle of the injured arterial segment. A cannula was introduced into the common carotid artery via the external carotid artery. The left common carotid artery was balloon-injured three times with a 2F balloon catheter (2F Fogarty, Edwards Lifesciences Corporation), then pulling out Balloon catheter. pCAGGS-GFP/A20 20μl or pCAGGS-GFP 20μl with 40μl Lipofectamine 2000 (Invitrogen Corporation) was gradually instilled into the lumen for 30 min. Just balloon-injured operation in the model group. The sham group was only tied with a ligature at the external carotid artery without the balloon- injured operation. Rat were sacrificed according to various requirement on the different day after operation respectively. The transfection efficiency of plasmid in injured arterial wall was evaluated by using fluorescence microscope 24 hours after transfection. Quantification of intimal hyperplasia was determined by histopathologic examination 14 days after operation. Proliferation index of VSMCs in vivo was assessed by thymidine analogue bromodeoxyuridine (BrdU) labeling technique 10 days after operation. The expression of A20, nuclear factor-kappaB p65 (NF-κBp65) and toll like receptor 4 (TLR4) of carotid arteries in different groups was confirmed by immunohistochemical staining, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis at different time point.3. Thirty six male Sprague-Dawley(SD) rats were randomly divided into three groups (n=12): the sham group(no injury), the control group (vacant vector and transfection regent group) and the therapeutic group (A20 gene and transfection regent group). Rats were sacrificed two weeks after operation to evaluate reendothelialization by Evans blue dye staining. Ultrastructural change of injured artery was observed with transmission electron microscope at day 3 and 7 after balloon injury.4. Cultured rat aorta smooth muscle cells (SMCs) were identified by specificα-actin antibody. VSMCs were pretreated by emodin (5～50μg/l) for 30min and then were stimulated by LPS (20mg/l) for 24h. VSMCs proliferation was assessed by MTT and BrdU corporation. The quantification of A20 mRNA expression was measured by RT-PCR. The quantification of A20, NF-κBp65 protein expression was measured by Western blot analysis.Results:1. After the pCAGGS-GFP/A20 plasmid was digested by EcoRⅤ/SmaⅠdouble restriction enzyme, the production was identified by 1% agarose gel electrophoresis, plasmid size and enzyme digested result was right.2. Much green fluorescence distribution of zinc finger protein A20 gene existed in the remnant intima and media In vivo 24 hours after transfection by using fluorescence microscope, which confirmed the transgene efficiency in injured vascular wall.3. Significant intimal hyperplasia was observed in the model and control group at day 14 after injury. A20 gene transfection markedly reduced the neointimal area (47.8% reduction; P< 0.05) and intimal to media area ratio (42.9% reduction; P< 0.05) in the therapeutic group. Proliferation index of VSMCs (BrdU index) at day 10 after operation decreased more significantly in the therapeutic group (9.6±2.3%) than in the control group (26.7±5.1%,P<0.01). The A20 positive cells ratio of the therapeutic group was much more than that of the control group (P<0.05), whereas, the NF-κBp65 and TLR4 positive cells ratio of the therapeutic group was much less than that of the control group at day 10 after injury (P<0.05). A significantly higher level of A20 mRNA, protein expression was measured in the therapeutic group than in control group at every time point after operation (P<0.05). A significantly lower level of NF-κBp65 and TLR4 mRNA, protein expression was observed in the therapeutic group than in control group at every time point after operation (P<0.05).4. At 2 weeks after balloon injury, compared with control group ,the arteries of animals treated with A20 gene transfection showed a greater area of recovered endothelium, as evidenced by Evans blue staining. Quantification of Re-endothelialization, based on the percentage of re-endothelialization area in the originally injured area re-endothelialized (control 55.6±3.3% vs. therapeutic 79.1±3.7%, P<0.05) revealed a significant acceleration of endothelial recovery in the A20 gene therapeutic group compared with the control group.5. Representative contractile phenotype of VSMC was shown in the sham group and synthetic phenotype was shown at day 7 after balloon injury in the control group. Synthetic phenotype and transitional phenotype was shown at day 3 after balloon injury in the control group. At day 7 VSMC showed relatively more myofilament and less organelles and looked like contractile phenotype in the therapeutic group.6. Compered with the vacant group, the control group showed significantly higher MTT and BrdU corporating OD value. We found that emodin induced a concentration-dependent decrease in the level of MTT and BrdU corporating OD value. Compered with the control group, low-dose groups have not significant difference, high-dose group showed significant difference. The expression of A20 mRNA and protein,NF-κBp65 protein were up-reglulated after VSMCs were stimulated by LPS. The expression of A20 mRNA and protein were more increased in a concentration- dependent manner after VSMCs were pretreated by emodin on different concentration. While the expression of NF-κBp65 protein were down-regulated.Conclusion:1. Local endogenous zinc finger protein A20 expression was increased in balloon-injured rat carotid artery, and possesed evident chronergy. It is confirmed that zinc finger protein A20 is critical for the regulation of inflammatory responses. It is the important regulation protein for inhibiting inflammatory responses in vivo.2. Local transfection of A20 gene resulted in the high expression of zinc finger protein A20 mRNA and protein overexpression in balloon- injured rat carotid artery,3. Local transfection of A20 gene suppressed intimal hyperplasia and VSMC proliferation of balloon-injured rat carotid artery. Its possible molecular mechanism is that A20 is critical for the regulation of inflammatory responses through terminating the activation of NF-κB induced by injury.4. Local transfection of A20 gene accelerated reendothelialization and inhibited the phenotype transformation of VSMC of balloon-injured rat carotid artery. Its possible molecular mechanism is that A20 is critical for the regulation of inflammatory responses through terminating the activation of NF-κB induced by injury.5. EMD can inhibit LPS-mediated rat aorta VSMC proliferation..the possible mechanism is that EMD can suppress activation of NF-kB via increasing the expression of inflammatory reaction regulatory protein A20 in vivo ,which has confirmed further the effect of A20 on the VSMC proliferation.