| BackgroundAtherosclerosis is a chronic inflammatory disease of the vessel wall that involvesmultiple cell types of different origins, as well as the complex interactions andsignaling pathways between them. The possible function of genes in this process has beengiven much attention by scientists and clinicians in recent years. Connexins are membersof a family of proteins encoded by at least20different mammalian genes expressed in awide variety of tissues. Connexins form transmembrane channels called gap junctionswhich connect neighbouring cells and allow passive diffusion of small molecules. In theconnexin family, connexin37is a junction protein that has a key function in initiatinginflammatory response. In our previous study, we found that the C allele of the connexin37gene is associated with susceptibility to coronary heart disease, particularly in the malepopulation. Boerma et al. identified a link between single nucleotide polymorphism (SNP)in the human connexin37gene and thickening of the carotid in the Swedish malepopulation, in which the C allele is over-represented in individuals with atheroscleroticplaques. The C allele of this SNP has also been associated with coronary heart disease inTaiwan, northern China and Switzerland. Subsequently, two studies performed in Japaneseand Caucasian populations revealed that T SNP is a risk factor for acute myocardialinfarction (AMI), particularly in high-risk male individuals. Seifi et al. suggested thatpolymorphism in the Cx37gene potentially has a significant role in the manifestation ofAMI disease in Iranian population. However, Cx37deletion in apolipoprotein E-deficient(ApoE(-/-)) mice increases susceptibility to atherosclerosis.Gene knockout studies have shown that Cx37forms gap junction channels between endothelial cells. Two polymorphic Cx37variants (Cx37-S319and Cx37-P319) have beenidentified with a possible link to atherosclerosis. Although these results are encouraging,the relative contribution and synergistic effects of Cx37on formation and atheroscleroticplaques remain unknown. We hypothesised that Cx37affects atherosclerotic plaques.Small interfering RNAs (siRNAs) have been proven to be efficacious in silencingtarget genes by means of RNA interference (RNAi). This mechanism is conserved innature from yeast to humans. Lentivirus can efficiently deliver siRNA because of theirability to transduce both dividing and nondividing cells stably. A previous study has shownthat pig and nonhuman primate animal atheroscleroses are most similar to the humanmodel. In this study, we constructed one lentiviral vector aimed at knocking down Cx37toelucidate its role in effecting atherosclerotic plaques in pigs. This vector was observed byintravascular ultrasound (IVUS).ObjectivesObject of part1: To construct Cx37siRNA lentiviral vector: According to siRNA Cx37gene design corresponding DNA fragments,cloning it to BLOCK．It Lemiviral Pol IImiRNAi vector. Then validate vitro and in vivo gene silencing effect of Cx37i, choose theappropriate Cx37siRNA with the best gene silencing effect. HEK293cells were culturedby homologous recombination to generate a carry Cx-37siRNA virus suspension.Object of part2: To establish an atherosclerotic pig model by high-fat diet.Object of part3: To compare the volume and nature of the porcine aortic atheroscleroticplaque by intravascular ultrasound two months after localized injection of Cx37siRNAvirus suspension.Materials and MethodsCell cultureHEK293cell lines were cultured using previously reported methods. HEK293cellswere derived from the cell bank of the Chinese Academy of Science under37°C,5%CO2environment and complete medium (United States of America Gibco-BRL Company)culture of HEK293cells. When cell fusion rate reached90%, the cells future generation decreased by1:4and froze in the logarithmic growth phase. Recovery took place when thecells grew poorly.Preparation of lentiviral vectors and target screening for RNAiFirst, three different sequences (sites A, B and C) of Cx37genes in pig were selectedas target for RNA interference. The targeted sequence of mm-Cx37-si-1was as follows:5GGUUAACGGUGCUCUUCAU3location:209, targeted sequence of mm-Cx37-si-2:5CCAAGGACCUACAUGUAGA3location:488, targeted sequence of mm-Cx37-si-3:5CAGACCCUUACCCUGAACA3location:841. P3xflag-Cx37. The P3xflag wasselected as control.Transfection reagent lipofectamine2000was purchased from American InvitrogenCompany. With the wild-type lipofectamine2000plasmid Cx373interference fragment,the negative control and P3xflag-Cx37were cotransfected with RNAi plasmid and mixedat a molar ratio of5:1.Second, cells were collected after72h and washed three times with cool PBS liquid.Disposable PBS residual liquid was then suctioned. Up to0.2ml of RIPA lysis buffercontaining protease inhibitor mixture was added and placed on ice for30min. Exactly1.5ml cells were collected by cell scraper centrifuge tube on ice. Ultrasonic lysis of the cellwas performed for30s at4°C. The supernatant was centrifuged at12000rpm for30min,and then transferred to another microcentrifugal clean tube, after which it was preserved at–20°C.Third, protein concentrations were determined by BCA method using Pierce s BCAprotein assay reagent kit. Solutions A and B (50:1) were mixed and placed at normaltemperature for30min. Then,200μLper hole for96holes were added, as well as differentconcentrations of10μL standard sample BSA (1:10dilution). A blank hole with10μLdouble-distilled water adjusted to zero was placed at37°C after30min in the enzymeinstrument to determine the570nm absorbance value (OD value). The standard was drawnin the standard curve. The concentration of total protein sample was calculated. Western blotSDS-PAGE concentration was10%. For each sample, the volume was30μg. Ananti-tag antibody protein flag was used, and4°C overnight incubation was undertaken tomonitor the expression quantity of GAPDH in the samples, The flag antibodies were asfollows: rabbit,1:2000; GAPDH: rabbit,1:10000antibody. All cells were divided into fivegroups. The experimental groups comprised overexpression of empty plasmid group, Cx37plasmid, and Cx37overexpression of recombinant plasmid+different interferencefragments. The transfection reagent was lipofectamine2000. The plasmids comprisedP3xflag and P3xflag-Cx37-0.4μg. The interference fragment was50nM and thetransfection time was72h. According to Western blot results, the best interferencefragment was selected. The preparation of lentiviral vectors expressing Cx37involved theuse of BLOCK-It Lentiviral Pol II miR RNAi Expression System with green fluorescenceprotein (GFP)(Catalog No. K4948-00; Invitrogen). A scrambled siRNA sequence (namedmock-siRNA) with no known homology to mammal genes served as control.According to the analysis results of siRNACx37gene silencing, Cx372,3fragments in vitro, the mouse mononuclear macrophage cell line RAW263.7as targetcells, mock-LV, Cx372i、3i lentiviral vector transfection of mouse macrophage cell lineRAW264.7. After five days virus transfection cells were collected.Fluorescencequantitative RT—PCR and Western blot were used to detect the expression ofthe transfected cells Cx37mRNA level, protein level, furtherscreening of effectiveinhibition of Cx37expression by small interfering RNA vector.Modelling of pigsSixty male pigs were obtained from Wuzhishan, China. The pigs were provided withnormal diet and water. After1week, the pigs received high-fat diet (5%lard,1%sugar,3%cholesterol and0.2%propylthiouracil) and fed three times daily. All pigs were consistentlyprovided with high-fat diet for10months. This study was carried out in strict accordancewith the recommendations found in the Guide for the Care and Use of Laboratory Animalsof the National Institute of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of NanjingMedical University Wuxi People s Hospital.Abdominal aortic angiography and IVUS analysisAt the end of month8, abdominal aortic angiography and IVUS were performed onall pigs after anaesthesia. All IVUS images were acquired using a20-MHz Volcano EagleEye IVUS catheter (Volcano Therapeutics, Inc., Rancho Cordova, CA, USA). Once theabdominal aorta lesion had been identified, the IVUS catheter was inserted distal to thelesion and automatically pulled back to assess the severity and length of the lesion (rate of0.5mm/sec). The location of the IVUS catheter was determined using continuousfluoroscopy throughout the time of pullback, as well as by recording anatomical landmarksobserved during IVUS imaging. To create adequate images, an average of two pullbacksper artery was performed. Subsequently, the best play loop was selected based on imagingresolution and quality. Continuous EKG monitoring was performed during the procedure togate IVUS frames for analysis. IVUS-virtual histology (VH) data were recorded on theimaging system hard drive, and then extracted and archived for analysis. Analysis wasbased on border contour calculation from greyscale. Tissue maps provided by the software(dark green for fibrous tissue, light green for fibrofatty tissue, red for necrotic core andwhite for dense calcium) were used to analyse each independent frame. Once the totallength of each lesion had been determined, a20mm vascular segment containing thevascular lesion was selected for analysis. This segment was then divided into equal2.0mmsubsections, generating a total of10series of cross-sections per vascular segment. Then,the abdominal cavity was opened. An incision was made on the left paracolic sulci and sideperitoneum, the descending colon and its mesentery to the right to free, and free of theabdominal aorta. Wire positioning should be upstream to block blood flow. In making anincision in the aortic plaque, we need to find the location of the needle and inject it byapproximately30°in the Cx37siRNA virus suspension and mock-siRNA virus suspensionor saline, then into the sutured vascular and abdominal areas. All pigs were maintained ona high-fat diet and divided into three groups. Cx37siRNA group received an infusion of 10lentiviral of Cx37viral suspension. Nontreatment group pigs were randomly dividedinto mock and saline subgroups. The mock subgroup received an infusion of10pL ofmock viral suspension. The control subgroup did not receive virus infusion. After2months,abdominal aortic angiography and IVUS were performed in all pigs. The expression ofsemi-quantitative RT-PCR Cx37mRNA was detected. The expression of Cx37protein wasdetermined by Western blot. In the current study, all animals used were handled and treatedin accordance with the guidelines of the National Institute of Health.Serum lipid level measurementDetection of serum lipid was conducted by orbital venous sinus blood. The plasmalevels of total cholesterol (TC), plasma triglyceride (TG), low-density lipoproteincholesterol, and high-density lipoprotein cholesterol (HDL-C) were measured by oxidasemethod (Center Laboratory of Wuxi People s Hospital, Wuxi, China).Statistical analysisData were expressed as mean±SD. Continuous variables between two groups werecompared using independent t-tests and Chi-squared tests. Multiple groups were comparedby ANOVA. All tests were conducted using SPSS17.0software for Windows (Lei AnTechnology Company, Beijing City, China). Proportions were compared using Fisher sexact test with expected frequency of <5. Chi-squared testing was applied in all other cases.P<0.05indicated statistical significance.ResultsBody weight， serum lipid profile and liver and kidney functionsNo significant difference in body weight among the Cx37siRNA group andnontreatment group (mock subgroup, saline subgroup) was found, which indicated thesafety of virus transfection in these animals. Likewise, serum cholesterol and triglyceridelevels in the treatment group did not differ significantly from those in the nontreatmentgroup (mock subgroup, control subgroup). This finding suggests that the therapeutic effectsof gene transfer were independent of serum lipid levels. Gene silencing in vitroWe screened the most effective targeting sites for Cx37genes using Western blotanalysis. HEK293cell line was transfected with lentiviral-based vectors expressing threedifferent Cx37siRNAs. In addition, gene silencing analysis showed that the Cx37-site2，3lentivirus was the most effective vector in blocking Cx37expression. According tothe results, we choose the siRNA Cx372,3fragment in vitro. In vitro results showed thatsiRNA372,3gene fragment of CX37mRNA level of silence effects were52%,83%, inhibitory effect of protein levels respectively is50%,67%. Consequently,Cx37siRNA3lentiviruses were selected for further in vivo studies.Efficient transfection of lentivirusPrevious local virus delivery to precollar-induced aortic atherosclerosis of pigsresulted in efficient transfection to aortic atherosclerosis. GFP expression provides anefficient and convenient means to check the transfection efficiency of lentivirus. Therefore,we detected GFP fluorescence in aortic atherosclerosis1week after transfection. SiRNAtransfection and greater fluorescence were observed2weeks after transfection. When thestudy was terminated, GFP was still visible. It was already weak2months aftertransfection. These results demonstrated the efficient in vivo transfection of lentivirus bysiRNA in aortic atherosclerosis. Virus local transfection did not affect normal functioningof the animals and did not result in weight change (34.89+4.16kg in transfected mice vs.34.54+3.87kg in mock-siRNA and31.96±4.84kg in saline).Gene silencing in vivoTo evaluate the efficacy of lentivirus-mediated gene silencing in vivo, we tested aorticatherosclerosis of pigs for Cx37mRNA and protein expression levels by real-time PCRand Western blot analysis. Cx37mRNA levels in the Cx37siRNA group were lower by34%, those in the mock-siRNA group by61%, and those in the saline group by94%(P <0.05). The mock group showed no significant change in Cx37compared with the controlgroup. Western blot analysis showed that Cx37protein was lower in the Cx37RNAi groupthan in the other groups (0.21±0.07vs.0.65±0.06vs.0.54±0.07). Therefore, the local application of siRNA lentivirus can be efficiently silenced (Figures4to6).Effects of Cx37siRNA on atherosclerosis plaqueThe plaque necrosis on10-month percentages (after injection Cx37siRNA) decreasedin the Cx37siRNA groups compared with8-month (before injection Cx37siRNA)percentages (5.26±2.11vs.7.83±1.03%, P<0.05). In the mock-siRNA and saline groups, nodifferences in eight-month percentages of plaque necrosis were observed (P=0.074,0.061,respectively). In the Cx37siRNA group, plaque volumes decreased relative to theeight-month percentages (21.03±6.24vs.31.23±10.23, P<0.01). By contrast, plaquevolumes increased relative to their eight-month percentages (38.54±13.56vs.32.12±11.21mm3,37.36±14.21vs.30.21±12.02mm3, P=0.031, P=0.027) in the mock siRNA andsaline groups.Conclusion1. In this study, we successfully constructed lentiviral vectors targeting porcine Cx37gene by RNAinterference technology.2. In this study, we successfully establish an atherosclerotic pig model by high-fat diet.3. In Vivo results show that, Cx37gene silencing can effectively inhibit the expression ofCx37gene in the porcine aortic atherosclerotic plaque. We successfully evaluated theimpact of siRNA silence Cx37on stabilization and reduction of plaque by intravascularultrasound. |
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