Effects And Mechanisms Of RNA Polymerase I Inhibitor-prevented Arterial Injury-induced Neointimal Hyperplasia

BackgroundRestenosis after coronary angioplasty is an important reason for the failure of percutaneous coronary intervention.The fundamental mechanisms of restenosis are vascular elastic retraction,vascular remodeling and neointimal hyperplasia.Since the stents have been used in the clinic,the incidence of restenosis was significantly reduced,as the vascular remodeling and elastic retraction were mostly prevented.In normal condition.vascular smooth muscle cells in vascular walls proliferate at an extremely low rate and most of them are in the G0 phase of cell cycle.Vessel injury would activate smooth muscle cells,make them into cell cycle progression and increase their migration.Underpinning this principle,several proliferation inhibitors have been proved to be effective in suppressing neointima formation when applied locally via drug-eluting stents.Among these agents,rapamycin and its derivative everolimus,which target the mammalian target of rapamycin(mTOR)pathway,are now widely used in the clinic.Although the use of drug-eluting stents significantly reduces the incidence of restenosis,these drugs have not completely eliminated the occurrence of neointima.Several recent studies have shown that patients who accepted drug-eluting stents for percutaneous coronary intervention,appeared "late catch-up" phenomenon:six years mortality,quality of life,unstable angina admission sho-w no significant difference comparing with the patients who accepted bare metal stents.Therefore,development of new therapeutic options by searching for new biological targets and pharmacological agents is still required.The proliferation of vascular smooth muscle cells was observed in neointima formation.Cell proliferation requires the synthesis of an abundance of proteins and ribosome is the site for protein synthesis.In nucleus,RNA polymerase I(Pol I)transcript ribosomal DNA into precursor ribosomal RNA.After multi-step modification,precursor ribosomal RNA form mature 18S,5.8S and 28S ribosomal RNA,after that ribosomal RNA combine with ribosomal protein and shape ribosomal subunits.In several tumor cells,RNA polymerase I is over activated,while inhibition of ribosomal RNA synthesis can inhibit cell proliferation.CX-5461 is a new type of RNA polymerase I specific inhibitor.It was confirmed that CX-5461 could significantly inhibit the proliferation of cells in fast proliferating condition.The compound is currently in early clinical trials of leukemia and solid tumor treatment.Although there is no report on the role of RNA polymerase I in cardiovascular cells,the evidences show that the proliferation of vascular smooth muscle cells may be associated with an increased rate of protein synthesis.Therefore,in the following study,we examined the pharmacological effects of specific RNA I polymerase inhibitor CX-5461 on vascular smooth muscle cells,to explore the possible mechanisms of the inhibition effect of CX-5461 on restenosis and smooth muscle cells proliferation.Objectives1.To explore the effects of CX-5461 on neointimal hyperplasia in carotid artery balloon injury model.2.To investigate the biological mechanisms of CX-5461 on neointimal hyperplasia after vascular injury.Materials and Methods1.Animals and treatment groupsAll animal experiments were approved by the Qilu Hospital Animal Ethics Committee,and performed in accordance with the ARRIVE(Animals in Research:Reporting In Vivo Experiments)guidelines.Male Wistar rats weighing-250-300 g were purchased from Vital River Laboratories(Beijing,China).Rats were randomly divided into 4 groups:sham group,balloon injury group,balloon injury +CX-5461 low dose(0.7 ?M)group,balloon injury +CX-5461 high dose(14 ?M)group.2.Carotid artery balloon injuryRats were anesthetized using pentobarbital sodium(60 mg/kg,i.p.).The entire length of the left common carotid artery and the branches were exposed.The blood flow was stopped by clamping the common carotid artery.An arteriotomy incision was made in the external carotid branch distally,and a 1.25-mm balloon catheter(Medtronic,Minneapolis,MN,USA)was introduced into the common carotid artery.The balloon was slowly inflated to a pre-determined pressure of 3 atm,and the injury was induced by withdrawing the catheter with rotation.The catheter was deflated and the process was then repeated for additional 2 times.For peri-vascular treatment,CX-5461 was first dissolved in DMSO and mixed with 25%Pluronic F-127 gel(from Sigma-Aldrich,Shanghai,China)to final concentrations of 0.7(low dose)or 14 ?M(high dose),and stored at 4° C.Equivalent amount of DMSO was used as vehicle control.After balloon injury,200 ?l of the gel solution was applied over the artery and allowed to solidify.After surgery,a dose of meloxicam(1 mg/kg,s.c.)was given for analgesia.3.Tissue collection and slides makingCarotid arteries were collected 14 days after injury.Rats were anesthetized using pentobarbital sodium.The reagent delivery section of the left common carotid artery was exposed and collected,fixed in 4%paraformaldehyde and embedded in paraffin.Cross-sections of 4-?m thickness were cut,other vessels were preserved in liquid nitrogen.4.Histopathology assayTissue sections were dewaxed and rehydrated,processed Verhoeff-Van Gieson staining(brown elastic plate,the more obvious neointima)and Hematoxylin-Eosin staining.Neutral resin was used for sealing and save the image under the microscope after drying.5.Evan's Blue staining30 minutes before euthanasia,rats were injected intravenously via the tail vein with 0.5 mL of 0.5%Evans blue dye,which specifically stains denuded areas of the arterial wall.Arteries were fixation in 100%methanol and opened longitudinally and save the image under microscope.6.Immunohistochemistry analysisTissue sections were deparaffinized,and heated in a citrate buffer(pH 6.0)in a microwave oven to retrieve antigens.Sections were then treated with 3%(v/v)hydrogen peroxide to quench the endogenous peroxidase activity,and blocked with 5%BSA,Tissues were incubated with various primary antibodies at 4? overnight followed by corresponding biotin-conjugated secondary antibodies at 37? for 120 min.Slides were developed using a Vectastain Elite ABC Kit and diaminobenzidine(DAB)substrate.The nuclear was stained by hematoxylin,and the slides were sealed with neutral resin.Images were taken under microscope.7.Immunofluorescence assayTissue sections were deparaffinized and then treated with 3%(v/v)hydrogen peroxide at 37? for 10 min to quench the endogenous peroxidase activity,and blocked with 5%BSA.Tissues were incubated with various primary antibodies at 4?overnight followed by corresponding Alexa Fluor 488-conjugated secondary antibodies at 37? for 60 min.Nuclear was counterstained with DAPI for 15min.Fluorescent images were taken by confocal microscope.8.Western blot analysisVessels were lysed with RIPA lysis buffer containing DTT and proteinase inhibitor on ice.Protein samples were separated by SDS-PAGE and transferred to PVDF membranes.The membrane was blocked with 5%nonfat milk,incubated with specific primary antibodies overnight,followed by washing in TBST and incubating with secondary antibodies.The membrane was developed with ECL Prime reagents and detected with a LAS-4000 luminescent image analyzer.9.Real-time quantitative PCRVessels were collected 14 days after surgery and kept in liquid nitrogen.Trizol was added to the frozen tissues,and then were ground by grinding machine.Then,chloroform,isopropanol and ethanol were added separately to precipitate RNA.After measuring concentration with Nano-drop Spectrophotometer,RNA was reverse transcript according to the Takara PrimeScript RT reagent Kit.Then conduct Real-time PCR according to the Real-time PCR SYBR Select Master Mix kit,GAPDH or ?-actin as a housekeeping gene.Gene expression was detected and data was analyzed by relative quantitative methods.10.Cell cultureMOVAS was purchased from ATCC company.Cells were cultured with high glucose DMEM containing 10%fetal bovine serum in a humidified culture incubator at 37° C and 5%C02.Subculture was carried out when the density reached about 80%.11.Cell viability assayMTT assay was employed to detecting the viability of cells.MOVAS were seeded in a 96-well plate and given different reagents.Cell viability was assessed with a colorimetric tetrazolium-based assay using CellTiter 96 Aqueous kit according to the manufacturer's direction.12.Statistical analysisData are presented as mean± SD.Data analysis was performed with unpaired t-test or one-way ANOVA followed by post hoc Newman-Keuls test as appropriate.P<0.05 was considered as statistically significant.Results1.EC5 of CX-5461Since there was no previous study on the role of CX-5461 in cardiovascular system,we examined the pharmacological characteristics of CX-5461 on vascular smooth muscle cells.MTT was used to detect the inhibitory effect of CX-5461 on the proliferation of MOVAS cells,and EC50=1.06?M.2.Effects of CX-5461 on neointimal proliferation induced by vascular injuryAccording to EC50 of CX-5461 on MOV AS,we selected two concentrations of CX-5461,namely a sub-EC50(0.7?M,low dose)level and a supra-EC50(14 ?M,high dose)level,for following in vivo studies.We found that 14 days after surgery,even at the low dose,peri-vascular administration of CX-5461 effectively inhibited the neointima formation.CX-5461 at the higher concentration completely abolished the development of neointima(carotid artery intimal area,intima/media area ratio).Histological examination of the arterial cross sections revealed that CX-5461 had no obvious effects on the gross distribution and morphology of medial SMCs.3.Effects of CX-5461 on the cell cycle of vascular wall cellsWe performed immuno-staining of PCNA,cyclin A,and Aurora B(a marker of mitosis)in arterial cross-sections.Aurora B-positive cells were undetectable in the tunica media in either normal or injured arteries.Since nuclear-localization of cyclin A was essential for its cell cycle regulatory activity,we counted cells with cyclin A high-expression which overlapped the nuclei.Balloon injury somehow decreased the prevalence of cyclin A-high cells in the media,probably because of concomitant emigration of the activated smooth muscle,whereas CX-5461 significantly increased the number of cyclin A-high cells in the media.PCNA is regarded as a marker of cell proliferation that is synthesized in G1 phase of cell cycle,and high express in S phase.The immuno-staining results showed that the prevalence of PCNA-high cells in the media were not significantly changed by CX-5461.These data,together with the in vitro results,indicated that CX-5461 induced a cell cycle arrest at the G2/M phase in vivo.4.Effects of CX-5461 on the process of re-endothelializationWe measured re-endothelialization of the denuded area using a method described by Morita and coworkers.We showed that CX-5461 at the low dose did not significantly delay the endothelial recovery process,while at the high dose it inhibited the rate of re-endothelialization by?35%.5.Ribosomal RNA synthesis after balloon injuryUpstream binding factor-1,UBF-1 is a nucleolar transcription factor essential for rDNA transcription.We demonstrated that phosphorylation ofUBF1(upstream binding factor-1)on S484 was significantly increased in the neointimal area and the tunica media of injured arteries,indicating an enhancement in the process of rRNA synthesis.6.CX-5461 increases p53 posttranslational modifications in vivoPhosphorylation and acetylation of p53 lead to full activation of its transcriptional functions.Using antibodies to phospho-p53,acetyl-p53 for immuno-staining,we showed that p53 phosphorylation and acetylation were all significantly augmented in the medial layer of CX-5461-treated arteries.The levels of these markers were also highly expressed in the neointima in untreated arteries.Consistent with this,using qPCR we confirmed that the in vivo expression levels of p21CIP/WAF1 and GADD45 were significantly increased in high dose CX-5461-treated vessels as compared to sham.We also performed western blotting experiments and confirmed that,as compared to sham tissues,phospho-p53,acetyl-p53 and phospho-S/T*Q were all increased in CX-5461(high dose)-treated vessels,which contained virtually no neointima.The higher levels of these markers in balloon-injured vessels were likely to be due to the increased expressions in the neointima,as indicated by immunohistochemistry staining.7.CX-5461 increase ATM/ATR activation in vivoThe phosphorylation of p53 and transition of G2/M were regulated by ATM/ATR(the ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related)pathway.Using antibodies to phospho-S/T*Q,we showed that activation of the ATM/ATR pathway was significantly augmented in the medial layer of CX-5461-treated arteries.Conclusion1.CX-5461 inhibits arterial injury-induced neointimal hyperplasia.2.CX-5461 induces cell G2/M blockade in tunica media of balloon-injury vessel.3.CX-5461 may inhibit cell proliferation by activating the ATM/ATR pathway.BackgroundOur in vivo study showed that RNA I polymerase inhibitor CX-5461 could prevent neointima hyperplasia in balloon injury vessels,probably by activating ATM/ATR pathway and increasing p53 post-translational modification.However,the corresponding cellular mechanisms of the action are not totally clear.CX-5461 is a novel RNA polymerase I specific inhibitor,which can inhibit the over activation of RNA polymerase I in some tumor cells and prevent cell rapid proliferation.In this study,we investigated the pharmacological effects and mechanisms of CX-5461 on vascular smooth muscle cells rapid proliferation.In recent years,more researches regarded cell cycle and its regulators as target in treatment of restenosis.The cell cycle consists of two phases:interphase(the period between the two mitotic stages)and the division stage(the process of cell mitosis).The interphase is divided into four periods:G0,G1.S and G2.During the G1 phase,cells prepare for DNA replication,and there is a checkpoint R in late G1 phase.After that,cells enter the S phase and replicate DNA.in the G2 phase of mitosis a large number of proteins are synthesized.And there is also a checkpoint for the quality of synthetic DNA in the G2 phase.The regulation of cell cycle is essential for the proliferation of vascular smooth muscle cells,so we are here to explore whether CX-5461 has an effect on cell cycle.Nucleolus is the site of ribosome biosynthesis,and the destruction of normal function of nucleolus will produce a unique cellular stress response,called nucleolar stress,resulting in the stabilization of p53 protein.CX-5461 is a specific inhibitor of RNA polymerase I required for ribosomal DNA transcription.In this part of the study,we will investigate whether Pol I inhibition induces nucleolar stress responses in vascular smooth muscle cells.ATM(ataxia telangiectasia mutated gene ataxia telangiectasia-mutated)/ATR(ataxia telangiectasia and Rad-3 associated protein)kinase system is the classical pathway in response of DNA damage.ATM,ATR belong to the PI-3K family,the function is not only the same duplication.UV,hypoxia and other conditions can activate the ATM/ATR pathway and activate downstream target proteins(such as p53 protein,etc.),block the cell cycle,and then repair the damage DNA.Here we investigate the role of CX-5461 in the ATM/ATR pathway in vascular smooth muscle cells.Studies have proved that the clinical application of rapamycin eluting stent will delay the process of re endothelialization,which may lead to postoperative thrombosis,which is an important factor affecting the outcome of interventional surgery.In this study,we also investigated the effect of CX-5461 on vascular endothelial cells.Objectives1.To explore the effects of CX-5461 on rapid proliferation of vascular smooth muscle cells.2.To investigate the mechanisms of CX-5461 on rapid proliferation of vascular smooth muscle cells.3.To explore the effects of CX-5461 on endothelial cells.Materials and Methods1.Cell culture1.1.MOVAS cultureThis part is same as Part ?.1.2.HUVEC cultureHUVECs(Human Umbilical Vein Endolial Cell)were purchased from the ATCC company,and cultured in complete ECM medium supplemented with 5%FBS in a 37° C,5%CO2 humidified incubator.Subculture was carried out when the density reached about 80%.Passages from 3 to 6 of HUVEC were chosen for experiments.1.3.Primary VSMCs cultureA male 150-250g rat was anesthetized using pentobarbital sodium(60 mg/kg,i.p.).The thoracic aorta was collected under aseptic conditions and put into D-Hanks.Clean the vessel and scrap off the intimal.The blood vessel was cut and centrifuged,inoculated with high glucose DMEM medium containing 20%FBS,cultured in a cell incubate in 37? and 5%CO2 for 3-5 days and replaced the medium.2.TUNEL assayCells were seed in 8-well plate and treated with CX-5461 in different concentration.TUNEL was performed using an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit(from Merck Millipore)following the manufacturer's protocol.Make starosporine(100nM)stimulation as positive control.After neutral resin sealing and drying,randomly select 5-10 view each well and calculate the number of positive cells and total number.3.Caspase 3/7 activity assayCells seeded in 96-well plates were incubated with the Caspase-Glo 3/7 substrate reagent at 37? for 60 min.The samples were transferred to a white-walled plate and the luminescence signals were detected using a Varioskan Flash plate reader.4.Cell viability assayThis part is same as Part ?.5.Flow cytometry assayCells were seeded in 6-well plates and detached with trypsin and fixed overnight in cold ethanol.Propidium iodide staining was performed using a kit from Abcam according to the manufacturer's instructions.Cell cycle was analyzed using flow cytometer.6.Western blot analysisCells were lysed in RIPA lysis buffer with DTT and proteinase inhibitor on ice.Protein samples were separated by SDS-PAGE and transferred to PVDF membranes.The membrane was blocked with 5%nonfat milk,incubated with specific primary antibodies overnight,followed by washing in TBST and incubating with secondary antibodies.The membrane was developed with ECL Prime reagents and detected with a LAS-4000 luminescent image analyzer.7.Real-time quantitative PCRCells were collected by trypsin digestion and precipitation.Then,RNA was precipitated by adding chloroform,isopropanol and ethanol and measured the concentration.Reverse transcript RNA according to the Takara PrimeScript RT reagent Kit.Then conduct Real-time PCR according to the Real-time PCR SYBR Select Master Mix kit,GAPDH or beta-actin as a housekeeping gene.Gene expression was detected and data were analyzed by relative quantitative methods.8.Immunofluorescence assayCells were seeded in 8-well plates,treated with different reagents and fixed in 4%paraformaldehyde.After blocked in 5%BSA for 30min and then incubated with various primary antibodies at 4? overnight followed by corresponding Alexa Fluor 488-or 594-conjugated secondary antibodies at 3 7? for 60 min.Nuclear was counterstained with DAPI for 15min.Fluorescent images were taken with the confocal microscope.9.GADD 45 shRNA transfectionThe MOVAS were seeded in 96-well plates;the next day replaced the medium with DMEM containing Polybrene of 5?g/ml dilution of the virus.Replace the medium with fresh DMEM in 24 hours.After 48 hours,the fluorescence was observed under fluorescence microscope,and the efficiency of virus infection was detected.10.Trans-well cell migration assayUse Boy den chambers for trans-well cell migration assay.HUVEC were trypsinized,resuspended in medium without FBS,and counted the numbers to balance the numbers of cells per well.Make sure that there were about 105 cells in each upper well.1%FBS was added to the lower chamber as a chemotactic factor.The mediums in up and lower both contain CX-5461 or DMSO.After 6h incubated,HUVEC under the up chamber were fixed and stained with crystal violet.Photographed under a light microscope 5-19 random fields.11.Monolayer wound healing assayCells were seeded in 6 well plates:scratch quickly on the cells according to a ruler with pipette suction perpendicular to the bottom of the plates when the density reached 100%.The cells were treated with CX-5461 or 5%DMSO respectively,and photographs were taken at 1,3,6,9,12 and 24 hours to observe the cell migration.Mark the cells in the same position in advance.12.Statistical analysisData are presented as mean ± SD.Data analysis was performed with unpaired t-test or one-way ANOVA followed by post hoc Newman-Keuls test as appropriate.P<0.05 was considered as statistically significant.Results1.Effects of CX-5461 on proliferation in MOVASWe chose different concentrations of CX-5461 according to its EC50,and detected the effects of CX-5461 on MOVAS proliferation.The experimental results showed that:0.07?M.0.7?M,2?M,14?M CX-5461 could inhibit the proliferation of MOVAS,and the inhibitory effect was dose dependent.2.Effects of CX-5461 on apoptosis in MOVASCaspase 3/7 activity assay and TUNEL assay were used to detect apoptosis of MOVAS treated with CX-5461.The results showed that:among the control group,CX-5461 0.07?M 0.7?M,2?M,14?M groups,there was no significant difference in MOVAS apoptosis.Therefore,we hypothesized that the inhibitory effect of CX-5461 on the proliferation of MOVAS was not dependent on apoptosis.3.Effects of CX-5461 on Aurora B in smooth muscle cellsBy immunofluorescence assay,we found that CX-5461 treatment decreased the level of Aurora B in vascular smooth muscle cells.These results suggested that CX-5461 treatment reduced the proportion of vascular smooth muscle cells in the mitotic phase.4.Effects of CX-5461 on cell cycle in smooth muscle cellsThe cell cycle was detected by flow cytometry,and the results showed that the percentage of MOVAS cells treated with CX-5461 in G2/M phase was significantly increased than that of control group,suggesting that CX-5461 treatment induced cell cycle arrest in G2/M phase.In order to simulate the in vivo situation,we chose the primary rat aortic smooth muscle cells(Vascular smooth muscle cell,VSMC)to detect the cell cycle again.In the experiments,we set PDGF-BB(20ng/ml)group to make primary VSMCs proliferate rapidly.The results showed that:in PDGF-BB untreated VSMCs,compared with the control group,the effect of CX-5461 on cell cycle was not obvious;and in the PDGF-BB group,compared primary VSMCs in CX-5461 group with control group,the percentage of cells in G2/M phase increased significantly.Immunofluorescence staining showed that vascular smooth muscle cells treated with CX-5461,the ratio of high expression of PCNA cells had no significant difference compared to the control group:in CX-5461 treatment group,the ratio of cyclin A located in the nuclei was significantly higher than that of the control group.The above results indicated that CX-5461 treatment resulted in G2/M cycle arrest in vascular smooth muscle cells.5.CX-5461 does not induce p53 stabilization but increases p53 phosphorylationTo clarify whether the cell cycle arrest induced by Pol 1 inhibition was related to induction of nucleolar stress,we monitored sub-nuclear localization of nucleophosmin(NPM/B23)in primary VSMCs.While CX-5461 at 700 nM did not significantly alter NPM/B23 localization in the nucleoli.CX-5461 at 14 ?M induced NPM/B23 relocation to the nucleoplasm,indicating the presence of nucleolar stress.However,we could not detect p53 protein stabilization in CX-5461-treated cells with either western blotting or immunofluorescence,suggesting that the effects of CX-5461 could not be fully explained by induction of the canonical nucleolar stress pathway.Interestingly,we found that CX-5461 treatment induced prominent p53 phosphorylation in primary VSMCs.Since phosphorylation increases p53 activity,we determined whether p53 had a crucial role in CX-5461-induced effects on cell proliferation by pretreating primary VSMCs with the selective p53 inhibitor pifithrin-a;we demonstrated that the G2/M blocking effect of CX-5461 was diminished by pifithrin-a.Moreover,we demonstrated that CX-5461 significantly increased the expression levels of p53 target genes p21CIP/WAF1 and GADD45.Whereas p21CIP/WAF1 had a critical role primarily in regulating G1/S transition,GADD45 had been shown to have an important role in mediating G2/M blockade,we treated MOVAS cells with adenoviruses expressing a GADD45 shRNA.GADD45 shRNA alone significantly increased the basal rate of cell proliferation.In the presence of GADD45 shRNA.CX-5461-induced cytostatic effect was blunted.6.Pol I inhibition activates the ATM/ATR pathway in smooth muscle cellsTo elucidate whether activation of the ATM/ATR pathway was involved in the effects of Pol I inhibition,we performed western blotting and immunofluorescence analyses in primary VSMCs using an antibody recognizing the phospho-S/T*Q motif of ATM/ATR substrates.CX-5461 increased the phosphorylation levels of ATM/ATR substrates.Next we pretreated VSMCs with the selective ATM inhibitor KU-55933 and the ATR inhibitor VE-821.We found that VE-821,but not KU-55933.abolished the cell cycle blocking effect of CX-5461 as well as CX-5461-induced p53 phosphorylation.7.Pol I inhibition induces p53 acetylationImmunohistochemical staining of vessels showed that CX-5461 treatment could enhance the acetylation level of p53.We tested the effect of CX-5461 on the level of p53 acetylation.CX-5461 significantly increased the level of p53 acetylation in primary VSMCs.We also confirmed the effect of CX-5461 on p53 acetylation in MOVAS cells.8.Effects of CX-5461 on migration and proliferation in HUVECIn cultured human umbilical vein endothelial cells,CX-5461 at 0.7 and 14 ?M significantly reduced cell proliferation,CX-5461 had no significant effect on the monolayer wound healing response,but significantly inhibited cell migration(by-30%)at 14?M assayed with Boyden chamber.Conclusion1.CX-5461 inhibits the proliferation but does not induce apoptosis in MOVAS.2.The inhibitory effect of CX-5461 on the proliferation of vascular smooth muscle cells can't be completely explained by the classical theory of nucleolar stress.The not effect does not cause the stability of p53,but enhance the level of phosphorylation and acetylation of p53.3.CX-5461 induces G2/M blockade in proliferating smooth muscle cells.4.CX-5461 inhibits cell proliferation by activating ATR,not ATM.5.CX-5461 inhibits HUVEC proliferation,and the inhibitory effect of low dose treatment on HUVEC migration is not obvious.BackgroundOur previous researches suggest that the Pol inhibitor I CX-5461 inhibits the proliferation of vascular smooth muscle cells through inducing cell cycle arrest,but does not cause cell apoptosis as its effect on tumor cells.ATR/p53 pathway takes part in the inhibition process of Pol inhibitor on VSMCs proliferation.The activation of ATR kinase depends on many factors,including DNA fracture or stimulation without DNA fracture,such as hypoxia,oxidative stress,mechanical stimulation.Studies have shown that oxidative stress caused by excessive generation of ROS played an important role in the process of tumor development and progression(to promote cell proliferation and migration,enhance anti-apoptotic activity,but also can cause DNA damage).In cardiovascular system,oxidative stress is one of the stress responses caused by mechanical injury and inflammation of angioplasty in vascular walls.In the process of restenosis,oxidative stress promotes the abnormal proliferation and migration of VSMCs.Oxidative stress is induced by the imbalance of endogenous antioxidants and oxides,which lead to the continuous generation of reactive oxygen species(ROS).NF-E2 related factor 2(Nuclear factor-erythroid 2-related factor 2,Nrf2)is a transcription factor that could protect vascular cells against the oxidative stress and inflammatory responses.Pol I inhibitor activates ATR-p53 pathway to inhibit proliferation of vascular smooth muscle cells,but does not cause apoptosis,perhaps because some of the protective pathways are activated in cells.Experimental evidence has shown that destructing nucleus normal function by silencing WDR-46 protein in Caenorhabditis elegans would activate the detoxification gene expression that mediated by the SKN-1/Nrf-CEP1/p53 pathway.Therefore,we hypothesized that Pol I inhibitor might activate the Nrf2 pathway in vascular smooth muscle cells to protecting cells from apoptosis.Objectives1.To explore the effects of Pol I inhibitor CX-5461 on Nrf2 pathway.2.To explore the role of Nrf2 on apoptosis effect of CX-5461 in VSMCs.Materials and Methods1.Cell cultureHela and MOVAS cells were purchased from ATCC Company.Cells were cultured with high glucose DMEM containing 10%fetal bovine serum in a humidified culture incubator at 37° C and 5%C02.Subculture was carried out when the density reached about 80%by 0.25%trypsin.2.Cell viability assayCells were seeded in a 96-well plate and given different reagents.Cell viability was assessed with a colorimetric tetrazolium-based assay using CellTiter 96 Aqueous kit according to the manufacturer's direction.3.Construction of Nrf2-reponsive reporter plasmidWe designed two Nrf2-r-esponsive reporter constructs,which contain Kpn I and Bql ? enzyme sites.The 2 sequences were synthesized and inserted between the Kpn? and Bgl? sites of pGL4.26.which contains a Photinus pyralis luciferase gene(Luc)and a mammalian selectable marker for hygromycin resistance.4.Extraction of plasmidMelt DH5a cells and mix with plasmid on ice.After 30min icy incubating,put them into 42° C water bath for 90s.quickly transfer them onto the ice.Add SOC medium and incubate at 37 0 C for 90min.Put appropriate bacteria liquid to a solid LB plate,and incubate at 37° C f-or one night.Select a mono-clone to a 15ml centrifuge tube with LB medium,and incubate for 12-16h.Centrifuge the liquid and extract bacterial with QIAGEN miniprep kit.5.Agarose gel electrophoresisSelect appropriate concentration of agarose gel according to DNA molecular weight.Fill the electrophoresis tank with TAE buffer,and place the gel in the tank.The DNA samples were mixed with DNA loading buffer in a 5:1 ratio before adding into the gel pores.The DNA samples were separated by agarose gel electrophoresis(100V).Detect the DNA bands under UV.6.DNA extractionCut the target DNA segment in the gel under UV light and recollect the DNA with QlAquick Gel Extraction kit.Store the DNA samples at-20° C.7.Transient transfectionSeed the cells in a sterile culture plate,and make transfection at about 70%in density.Dilute the DNA into 50 ? 1 Opti-Mem medium(24-well),and put 2 ? 1 in the same volume of Opti-MEM.Mix them together and incubate at room temperature for 30min.The mixed solution was added into the cell medium without antibiotic by drop,and then continued to culture.8.Selection of stable expression cell linesThe Hela cells were seeded in 10cm culture plates,and the constructed plasmids were transiently transfected into cells.Change the medium with DMEM containing 200 ? g/ml Hygromycin in 48h after transfection.When the number of cells became more,select single cell clusters to new plates and continue to culture with medium containing Hygromycin B.Repeat the above steps and subculture as the cells in good condition.9.Luciferase assaySeed the cells in 96-well plates and given different reagents.Disregard the medium and wash the cells with PBS.Lysis buffer was added to the cells(40 ? l per well)and repeat freezing and thawing at-80° C for 3 times to lyse cells.Transfer the lysis buffer with cells to a white-walled plate and add luciferase substrate to the samples(50 ? l per well),detect the luciferase values after mixing well.10.Western blot assayCells were homogenized in cold lysis buffer and protein concentrations were measured with BCA kit.Protein samples were separated by SDS-PAGE and transferred to PVDF membranes.The membrane was blocked with 5%nonfat milk,incubated with Nrf2 primary antibody overnight,followed by washing in TBST and incubating with secondary antibodies.The membrane was developed with ECL Prime reagents and detected with a LAS-4000 luminescent image analyzer.11-Caspase 3/7 assayCells seeded in 96-well plates were incubated with the Caspase-Glo 3/7 substrate reagent at 37? for 60 min.The samples were transferred to a white-walled plate and the luminescence signals were detected using a Varioskan Flash plate reader.12.Immunofluorescence assayCells were seeded in 8-well plates,treated with different reagents and fixed in ice methanol.After incubated in 5%BSA for 30m...