Investigation On Role And Mechanism Of NONO In Vascular Remodeling Disease

1 BackgroundAt present,research has shown that cardiovascular diseases are still the most common causes of death in developed countries.Atherosclerosis is the main pathophysiological cause of cardiovascular diseases.Surgical procedures remain necessary treatments for patients with life-threatening coronary atherosclerosis,such as angioplasty,stenting,atherectomy and bypass surgery.However,restenosis after mechanical injury remains one of the most prominent pathological problems limiting the prognosis of patients.Restenosis is characterized by the formation of neointima due to the thickening of vascular media.Studies have shown that vascular smooth muscle cells(VSMCs),as the main cell type of vascular wall,play an important role in neointima formation.VSMCs in normal blood vessels have a low-secreted and contractile phenotype,while mechanical damage to blood vessels will result in the loss of contractile function of VSMCs,which will be transformed into a synthetic phenotype with high proliferation,high migration and high secretion to accelerate vascular remodeling.Therefore,it is of great significance to explore the underlying factors and molecular mechanisms of VSMCs proliferation,migration and phenotypic switching in the prevention and treatment of vascular restenosis caused by neointima formation.NONO(also known as human p54nrb)is a non-POU domain-containing octomer-binding protein,one member of Drosophila behavior human splicing(DBHS)family which also contains splicing factor proline/glutamine rich(SPFQ),paraspeckle component 1(PSPC1)and long non-coding RNA NEAT 1.The structure of NONO protein contains RNA recognition motifs(RRMs),a NonA/paraspecle domain(NOPS)and coiled helix domains.These motifs indicate that NONO can act as a transcription or splicing factor which is able to participate in protein-protein interaction and protein-nucleic acid interaction and play important biological roles in DNA damage,RNA synthesis and transcriptional regulation.In recent years,NONO has been implicated in breast cancer,melanoma,neuroblastoma,aortic dissection,and atherosclerosis.Previous studies in our laboratory found that overexpression of NONO protein significantly inhibited the formation of plaque fiber cap,inhibited collagen deposition,affected the stability of plaque,and promoted the progression of atherosclerotic plaques in ApoE knockout mice.On the other side,knockdown of NONO stabilized atherosclerotic plaques by promoting collagen deposition and inhibiting the expression levels of inflammatory factors.NONO is a multifunctional nuclear protein which has been shown to be involved in a variety of pathological processes,including the secretion of inflammatory factors in autoimmune diseases.However,the role and mechanism of NONO protein in neointima formation have not been clearly defined.2 Objectives(1)To clarify the role of NONO in the maintenance of normal artery structure(2)To observe the distribution of NONO in neointima and its influence on neointima formation induced by carotid artery ligation(3)To explore the potential mechanisms of NONO in neointima formation3 Materials and Methods3.1 NONO knockout mice obtained by CRISPR/Cas9 techniqueNONOgt/0 mice(also called NONO knockout mice,or NONO KO mice)were obtained from C57BL/6J mice by CRISPR/Cas9 technique created by Professor Jiang Hong in our laboratory.NONO gene locates on X chromosome.NONOgt/+and wild-type(WT)male mice were hybridized to obtain NONOgt/0 mice and their wide-type(NONO+/0,WT)male littermates.3.2 Mouse model of carotid artery ligation(CAL)NONO KO mice and their WT male littermates more than 10 generations were used in this study.9-10-week-old NONOgt/0 mice and the WT male littermates were sacrificed after left carotid artery ligation for 21 days.3.3 Animal tissue collectionMice were anesthetized with pentobarbital sodium intraperitoneally,weighed and fixed.Blood was taken from the apex of the heart,and serum was used to detect the levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL-C)and high-density lipoprotein(HDL-C).The hearts,livers,spleen,kidneys and bilateral carotid arteries of the mice were taken after 0.9%saline through perfusion and stored in 4%paraformaldehyde or liquid nitrogen.The carotid artery tissues were fixed with formaldehyde for 24 hours,washed with flowing water,dehydrated with gradient alcohol and embedded in paraffin.The paraffin slicer sectioned 5 μm continuously.3.4 Tissue protein extractionInvent MinuteTM protein extraction kit was used for tissue protein extraction.Human coronary tissues or mouse tissues and the appropriate volume of protein extraction lysate(lmg tissues corresponding to 10 μl lysate)were put into the centrifuge tube with high-speed centrifugation after fully grinding.A BCA kit was used to test the concentration of tissue protein.Protein was then added with appropriate volume of protein loading buffer and boiled at 99℃ for denaturation.3.5.Staining of tissue sectionsCarotid artery sections were stained with hematoxylin-eosin(H&E),Verhoeff’s alcohol hematoxylin,immunohistochemistry and immunofluorescence to measure the area of neointima,contents of smooth muscle cells,macrophages and endothelial cells in neointima,NONO expression levels,PCNA and Ki67 expression levels,and SPHK1 and S1PR1 expression levels.3.6 Extraction and culture of primary smooth muscle cells of mice5-6 weeks old NONOgt/0 mice and WT male littermates were used to extract primary smooth muscle cells of mice,WT and NONO KO vascular smooth muscle cells(VSMCs).After washing the separated aortas of mice with cold PBS,VSMCs were extracted by tissue adherent method,cultured in DMEM containing 20%fetal bovine serum(FBS).VSMCs were cultured according to the conventional cell culture methods.3.7 Culture of human aortic smooth muscle cells(HASMCs)HASMCs was purchased from ScienCell corporation and cultured in smooth muscle culture medium(SMCM)according to the conventional cell culture methods.3.8 VSMCs cell migrationThe effect of 20ng/ml PDGF-BB on the migration of WT and NONO KO VSMCs was detected by both cell scratch assay and Transwell assay.3.9 VSMCs cell proliferationCell cycle kit and CCK8 were used to determine the effects of 20ng/ml PDGF-BB on cell cycle and cell proliferation of WT and NONO KO VSMCs.3.10 Fluorescence-quantitative reverse transcription-polymerase chain reaction(RT-PCR)Tissue RNA was extracted using RNA extraction kit(Qiagen).Cell RNA was extracted using RNA extraction kit(Fastagen).All steps were based on the manuscript instruction.Nanodrop was used to detect RNA concentration.cDNA was obtained using Takara reverse transcription kit,and RT-PCR was performed with SYBR and specific primers to detect mRNA expression levels of NONO,PCNA,p21,SPHK1,S1PR1,a-SMA,CNN1 and SM22a.3.11 Cellular immunofluorescenceCellular immunofluorescence was used to detect the effect of 20ng/ml PDGF-BB on the expression and nuclear translocation of Erk in WT and NONO KO VSMCs.3.12 Western blot(WB)WB technique was used to detect the protein expression levels of NONO,PCNA,p21,a-SMA,SM22a,p-Mekl,t-Mek1,p-Erkl/2 and t-Erk 1/2 in WT and NONO KO VSMCs3.13 Co-immunoprecipitation(Co-IP)Cell lysis buffer for IP with RIPA and PMSF was used and Co-ip technique was performed to investigate the mutual binding of NONO protein and Erk protein after 20ng/ml PDGF-BB stimulation of WT VSMCs3.14 Human coronary artery tissuesCoronary artery tissues were from the key laboratory of cardiovascular remodeling and function in Qilu Hospital of Shandong University.Coronary arteries were obtained from the transplant heart of the organ donor and treated in the operating room with minimal ischemia time(<15 minutes).The segment of the artery is classified as healthy or diseased by an experienced cardiac surgeon according to the degree of coronary atherosclerosis in the organ donor.The research protocols were approved by the Research Ethics Committee of Qilu Hospital of Shandong University(KYLL-2018(KS)-227).3.15 Statistical analysisAll data were expressed as mean ± standard error of the mean(SEM).SPSS 19.0 and Graphpad Prism 5 software were used.Comparisons between two groups were assessed by Student’s t-test,and comparisons of three or more groups were evaluated by a one-way analysis of variance(ANOVA)followed by the multiple comparison test when necessary.p<0.05 were considered statistically significant.4 Results4.1 NONO expression level was increased in human coronary stenoses and ligated mouse carotid arteriesBoth mRNA and protein expression levels were higher in stenotic than normal coronary arteries and in the ligated relative to the control carotid arteries.4.2 Increased NONO was primarily expressed by SMCs during neointima formationWe clarified the cellular localization of NONO and SM22a(a marker of SMCs)protein expression in both human stenotic coronary arteries and mice neointima by immunostaining,and found that most SMCs expressed NONO in two kinds of plaques.4.3 Phenotypes of NONO KO mice and their WT littermatesNONO KO mice were obtained from C57BL/6 mice by CRISPR/Cas9 technique.NONOgt/0 male mice had significantly lower body weight,birth rate,and survival rates compared with those in their WT littermates.Besides,NONO 9t/0 mice also had craniofacial morphology changes compared to their WT littermates.The immunoblots demonstrated deficient NONO expression in heart,liver,spleen,lung,kidney,brain and aorta of NONO KO mice.No difference was detected in fasting glucose and the lipid levels,including total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL-C)and high-density lipoprotein(HDL-C)in WT and NONO KO mice.4.4 NONO was not required for maintaining artery structure in miceWe found that loss of NONO decreased body weight of adulthood mice.but had no effect on the structure of uninjured arteries.We harvested carotid arteries at postnatal day 28,35 and 42 for WT and NONOgt/0 mice.No arterial structure difference was found in the medium thickness and internal diameter of vessels by H&E staining.Besides,loss of NONO protein increased expression of PSPC1 and unchanged expression of SFPQ.4.5 NONO deficiency inhibited neointima formation in ligated arteryA significant reduction in neointima formation in NONOgt/0 mice was found compared with their littermates 3 weeks after carotid artery ligation(P<0.05).4.6 Loss of NONO inhibited VSMC proliferation in vivo and in vitroWe found a reduced number of markers of cell proliferation,Ki67+ and PCNA+cells,in the neointima of NONOgt/0 mice compared with that of WT littermates in the neointima of ligated carotid artery by immunostaining(P<0.05).Meanwhile,NONO knockout VSMCs showed less cells in S phase and inhibited cell proliferation compared with controls under stimulation of PDGF-BB(P<0.05).qPCR and Western blot analysis revealed that p21 and PCNA transcript and protein levels were upregulated and downregulated,respectively,after PDGF-BB stimulation(P<0.05).4.7 Loss of NONO inhibited migration and phenotyping switching of VSMCs in vivoand in vitroWe found a reduced number of markers of promoting VSMCs migration or invasion and neointima formation,Sphk1+and S1pr1+ cells,in the neointima of NONOgt/0 mice versus that of their WT littermates through histochemical staining(P<0.05).Meanwhile,NONO knockout VSMCs showed inhibited migration ability compared with controls under stimulation of PDGF-BB(P<0.05).qPCR results showed VSMCs lacking NONO exhibited lower mRNA levels of Sphk1 and S lprl under stimulation of PDGF-BB(P<0.05).qPCR and Western blot analysis revealed that VSMCs lacking NONO showed higher mRNA levels of pro-phenotypic modulation genes,includinga-SMA),CNN1,and TAGLN(P<0.05)4.8 NONO interacted with Erkl/2 to mediate the role of SMCs in neointimal formationImmunofluorescence showed obvious colocalization of endogenous NONO and Erk1/2 in nucleus,and NONO deficient VSMCs showed suppression of nuclear translocation with PDGF-BB stimulation.The co-immunoprecipitation assay revealed that NONO interacted with Erk1/2 in VSMCs from WT mice.NONO KO VSMCs showed significant lower p-Erk1/2 compared with WT mouse VSMCs.5 Conclusions(1)NONO protein exists mainly in smooth muscle cells in neointima,and loss of NONO significantly inhibits the pathological process of vascular remodeling.(2)Loss of NONO inhibits the neointimal formation via inhibiting the proliferation,migration,phenotypic switching and phosphorylation level of Erkl/2 in VSMCs.1 BackgroundAbdominal aortic aneurysm(AAA)is an enduring vascular degenerative condition with serious complications,such as sudden death.At least 200,000 people die from AAA with aortic rupture,accounting for 90%of all AAA deaths annually worldwide.Surgical restoration is the restricted choice for patients with a huge AAA Unfortunately,effective medications for patients with small AAAs or the replacements of surgical therapy are still lacking.AAA is pathologically described by the dilatation of abdominal aorta resulted from destructive extracellular matrix(ECM)and inflammatory response.AngiotensinⅡ(Ang Ⅱ)is the key effective hormone in the rennin-angiotensin system,which prompts matrix degradation,vascular remodeling as well as chronic inflammation in the abdominal aorta.Inactivation of transforming growth factor β(TGF-β)cascade accelerates the development and rupture of AAA via the inhibition of ECM synthesis and promotion of matrix degradation.Prolyl-4-hydroxylase al(P4Hal)is the rate limiting factor of P4H enzyme,which is regulated by TGF-β1,and is the key enzyme for collagen synthesis.In addition,inflammatory response also has an imperative role in the advancement of AAA.Previous studies have reported that in the tissue of AAA,inflammatory cells were markedly accumulated which released inflammatory factors and activated matrix metalloproteinase(MMPs)to degrade the ECM and accelerate the development of AAA.Our group firstly found that tumor necrosis factor a(TNF-α)directly repressed P4Hal manifestation through the apoptosis of signal-regulated kinase 1-c-Jun N-terminal kinase-non-POU-domain containing octamer-binding protein(ASK1-JNK-NONO)cascade to inhibit collagen synthesis in atherosclerosis.Furthermore,we also found that NONO might interconnect with NF-κB p65 to regulate inflammation in atherosclerosis.The nuclear protein NONO/p54nrb is a multifunctional and highly conserved protein engaging in almost every step of gene regulation,including DNA repair,RNA transport and transcriptional regulation.A previous study has found that NONO is an integral component in the cAMP signaling cascade that is necessary for the stimulation of cAMP responsive genes,such as TNF-α and IL-6.NONO interacts with NF-κB and this led to the enhanced inflammation and atherosclerotic plaque vulnerability.Moreover,NONO decreases P4Hal expression to suppress ECM synthesis in human aortic smooth muscle cells(HASMCs)and atherosclerotic plaques.These researches suggest that NONO may participate in the progression of AAA,however,there is no study to confirm it.In the current study,we hypothesized that knockdown of NONO might inhibit AAA formation by increasing collagen synthesis and deposition and inhibiting the inflammatory response.2 Objectives(1)To clarify the distribution and effects of NONO in AAA(2)To observe the influence of NONO knockdown on the pathological process of AAA(3)To explore the potential mechanism of NONO in AAA3 Materials and Methods3.1 Isolation as well as culture of vascular smooth muscle cells(VSMCs)The primary mouse VSMCs were abstracted from the media of mouse aortas of ApoE-/-mice in Dulbecco’s modified eagle medium(DMEM,GIBCO,NY)complemented with 20%fetal bovine serum(FBS,GIBCO,NY),100 U/mL of penicillin plus 100 μg/mL of streptomycin at 37℃ in 5%CO2.Briefly,6-week-old ApoE-/-male mice were used to obtain the aortas and the vascular adventitia was removed under a microscope.After washing the aortas with PBS along with 100 U/mL of penicillin as well as 100 μg/mL of streptomycin three times,the aortas were cut into about 1mm length explants by a sterile Venus.The explants were individually plated into a 6 cm culture dish and cultured in DMEM containing 20%FBS for 7 days before the next change of medium.The acquired cells reserved SMC features(purity>90%).Cells at passages 3-9 were used for experiments.3.2 Preparation of lentiviral vectors and target screening of siRNAThe siRNA negative control(si-NC)was a scramble siRNA sequence with unidentified homology to mammal genes(GeneChem,Shanghai).The siRNA sequence of the mouse NONO gene was documented in Supplementary material[10].The siRNA sequence was also described and confirmed previously as follows,CCCACCAACAACTGAACGTTTCTCGAGAAACGTTCAGTTGTTGGTGGG.The replication-incompetent lentivirus(LV)was created by siRNA expression vector with pHelper 1.0 as well as pHelper 2.0 into 293T cells.At 48 h after transfection,viral supernatant was harvested and filtered via a 0.45 cellulose acetate filter.The titer of the viral vectors was detected by fluorescence-activated cell sorting analysis of GFP-positive 293T cells(GeneChem,Shanghai).sh-NONO-LV was transduced into VSMCs(5×105 cells in 12-well plates)at a multiplicity of infection(MOI)of 100.At day 3 after transduction,cells were collected for western blot in order to detect the efficiency and screen the most effective sh-RNA of NONO.Besides,sh-NONO were transduced into VSMCs for different treatments.3.3 AnimalsAll animal experimental protocols were permitted by the Ethics Committee and the Scientific Investigation Board of Shandong University Qilu Hospital according to the Animal Management Rules of the Chinese Ministry of Health.8-week-old apolipoprotein E-knockout(ApoE-/-)male mice were bought from Beijing Weitonglihua Animal Experimental Center.All animals were kept on a 12-hr/12-hr light-dark cycle at 22℃ room temperature with the high-fat food(0.25%cholesterol plus 15%cocoa butter)and water freely available for 8 weeksDuring the first part of the in vivo experimentation,20 ApoE-/-male mice receiving high-fat diet were arbitrarily distributed into the control group(n=10)as well as AAA group(n=10).After 4 weeks of high-fat diet in all ApoE-/-male mice,mice in the control group continued to be fed with high-fat fed for 4 weeks to obtain the control mice,and mice in the AAA group were treated with continuous subcutaneous infusion of Ang Ⅱ(1.44 mg/kg per day,Lintai Biological Technology Company,Xi’an,China)by an osmotic pump(Alzet model 2004,Alza Corp.,USA)for 4 weeks[3]in order to examine the expression of NONO on Ang II-influenced AAA.During the second part,20 ApoE-/-male mice receiving high-fat diet were arbitrarily distributed into four groups.The first group of samples were collected after 4 weeks of high-fat diet without lentivirus injection by tail vein and subcutaneous infusion of Ang II.The second group of samples were collected after 4 weeks of high-fat diet with sh-NC lentivirus injection by tail vein at the beginning of feeding(2×107 TU/mice).The third group of samples were collected after 4 weeks of high-fat diet with sh-NC lentivirus injection by tail vein at the beginning of feeding(2×107 TU/mice)and 2 weeks of subcutaneous injection of Ang II(1.44mg/kg/d)(n=5).The fourth group of samples were collected after 4 weeks of high-fat diet with sh-NC lentivirus injection by tail vein at the beginning of feeding(2×107 TU/mice)and 4 weeks of subcutaneous injection of Ang II(1.44mg/kg/d)(n=5).All samples were used to inspect the transfection competence of lentivirus by detecting GFP fluorescence.In the third part,100 ApoE-/-male mice receiving high-fat diet were arbitrarily separated into 2 groups:one was the control group without Ang II infusion(n=25),and another was the Ang Ⅱ group.The control group received no lentivirus injection and no infusion of Ang Ⅱ.Mice treated with Ang II were further arbitrarily distributed into 3 groups(n=25/group):No treatment group(NT group)received infusion of AngⅡ after 4 weeks of 0.9%saline injection;Sh-NC group received infusion of Ang Ⅱfollowing 4 weeks of sh-NC(2×107TU per mice)injection,and sh-NONO group received infusion of Ang II following 4 weeks of sh-NONO(2×107TU per mice)injection.3.4 Body weight and serum lipid profileThe body weight of animals was measured at the beginning and at the end of the experiment by an electronic balance(Shimadzu Corp.,Kyoto,Japan).Blood samples were acquired through cardiac puncture prior to euthanasia.The serum levels were measured by an enzymatic assay,containing total cholesterol,triglycerides,low-density lipoprotein cholesterol as well as high-density lipoprotein cholesterol.3.5 Measurement of blood pressure(BP)Systolic blood pressure(SBP)was obtained using a tail-cuff method with a photoelectric device(Natsume,Japan)before Ang Ⅱ infusion and before anesthesia in all mice following the beginning of the experimentation.BP was stated as a mean of 3 successive recordings.3.6 AAA quantificationAortas were extracted and fixed in 4%paraformaldehyde overnight.The adventitia was detached under the microscope.The highest diameter of the abdominal aorta was obtained using Image-Pro Plus 6.0 software for aneurysm quantification.AAA was defined as the 150%diameter increase criterion.Besides,any dissection leading to intramural hematoma(even if the actual dilatation is only 110%)should be counted in the AAA mouse model[15].The severity of AAA was classified as described previously:None:no aneurysm,Type Ⅰ(lumen dilatation without intraluminal thrombus),Type Ⅱ(lumen dilatation with thrombus),Type Ⅲ(conspicuous bulbous form of Type Ⅱ with thrombus)and Type IV(multiple aneurysms with thrombus,with some aneurysms overlapping)[16].Two autonomous researchers were in charge for the measurements.3.7 Tissue preparation and histopathological analysisAortic fragments from the aortic arch to the renal arteries were detached,fixed in 4%paraformaldehyde overnight and then paraffin-embedded.10 tissue sections,5μm thick,were derived from the AAA portion with the largest diameter in each mouse,which were used for histological as well as morphological investigationAortic fragments of all groups were used for various staining procedures.Hematoxylin and eosin(H&E)staining was implemented to assess morphology of AAA.Verhoeff staining was done to visualize the elastic fiber integrity of the abdominal aorta through a Verhoeff-Van Gieson staining kit(Abcam).Masson staining as well as Sirius Red staining were implemented to detect the aortic collagen content.Sections of aneurysm were stained for immunohistochemical analysis of macrophages(CD68,abeam,ab125212,1:100),smooth muscle cells(a-SM actin,a-SMA,abeam,ab7817,1:200),collagen I(abeam,ab34710,1:200),collagen III(abeam,ab7778,1:200),P4Hα1(abeam,ab59497,1:200),MMP2(abeam,ab37150,1:200),MMP9(abclonal,A2095,1:200),IL-1β(abeam,ab9722,1:200),MCP-1(abeam,ab9669,1:200),IL-6(abeam,ab7737,1:200)as well as TNF-α(abeam,ab1793,1:400).Following the incubation with the suitable horseradish peroxidase-conjugated secondary antibodies,fragments were incubated with 3’,3’-diaminobenzidine,counterstained,then stained with hematoxylin,desiccated with gradient alcohol and protected with coverslips.Sections reacting with non-immune IgG as well as secondary antibodies were considered as negative controls by Image J software(NIH,USA)was used to calculate the ratio of positive staining region to total plaque area.3.8 Cell treatmentVSMCs were cultured in DMEM complemented with 20%FBS with antibiotics.First,VSMCs were enthused by Ang II(MCE,1×10-7 mol/1)for the Ang II stimulation study.Cells were harvested for measurement of NONO expression levels.Second,VSMCs cultured in 6-well plates were transfected by lentivirus(MOI=100,per group)comprising sh-NC or sh-NONO overnight.The Ang Il group was administered physiological saline of the similar volume.Three days following the transduction,cells were treated with 1×10-7 mol/1 Ang II for 24 hr,and then collected for additional investigation.All experimentations were done thrice plus the mean values were obtained.3.9 Western blot techniqueTotal protein was extracted from the aortas of ApoE-/-mice or VSMCs.The aortic segments from the aortic arch to the renal arteries were collected and used for western blotting.The protein concentrations were quantified by a Protein Assay Kit(Thermo fisher,USA).Equivalent quantities of protein were separated by 10%polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride(PVDF)membranes.Following the blocking for 1 hr at room temperature(RT)with 5%skim milk,overnight incubations of membranes was done at 4℃ with appropriate primary antibodies.Mouse anti-NONO(Santa Cruz,sc-166702x,1:1000),rabbit anti-collagen Ⅰ(Cell Signaling Technology,84336S,1:1000),rabbit anti-collagen Ⅲ(abcam,ab7778,1:1000),goat anti-P4Hα1(abcam,ab59497,1:1000),rabbit anti-MMP2(abcam,ab37150,1:1000),rabbit anti-MMP9(abclonal,A2095,1:1000),rabbit anti-IL-1β(abcam,ab9722,1:1000),rabbit anti-monocyte chemotactic protein 1(MCP-1)(abcam,ab9669,1:1000),rabbit anti-TNF-a(abcam,ab1793,1:1000).Besides,rabbit anti-Phospho-NF-κB p65(p-p65)(abcam,ab76302,1:1000),and rabbit anti-NF-KB p65(Cell Signaling Technology,8424,1:1000)were used.Following washing,membranes were incubated with the analogous horseradish peroxidase-conjugated secondary antibody(Jackson,USA)for 1hr at RT,then washed and detected by chemiluminescent HRP substrate(Millipore,USA).GAPDH immunoblot analysis was used to verify equal sample loading3.10 Co-immunoprecipitationCell extracts used for co-immunoprecipitation were prepared from VSMCs and co-immunoprecipitation analysis were carried out as described previously[5].Briefly,VSMCs were cultured in a 150 mm2 culture bottle.Cells with 80%～90%confluency were cultured in serum-free medium for 24 hr for synchronization.Next,cells were stimulated with or without 1×10-7 mol/1 Ang II for 1 hr and lysed in 1 ml cold RIPA buffer containing 10 μl PMSF(100 mM/1).Specific antibodies(anti-NONO and anti-NF-κB p65)or pre-immune IgGs were incubated with cell lysates overnight before getting immersed by Protein A/G-plus Agarose beads(Santa Cruz,USA)Precipitating immune complex was unconfined by boiling with 1X SDS electrophoresis sample buffer.Bound proteins were identified by western blot technique.3.11 ImmunofluorescenceVSMCs were cultured,transfected with si-NONO and stimulated with Ang Ⅱ as explained earlier.Cells were fixed using 4%paraformaldehyde for 20 min and permeabilized in PBS with 0.1%Triton X-100.Following the blocking with goat serum for 30 min at RT,samples were incubated with rabbit anti-NF-KB p65(Cell Signaling Technology,8424,1:100)and mouse anti-NONO(Santa Cruz,sc-166702x,1:100)antibodies overnight at 4℃.Alexa 488-conjugated goat anti-mouse IgG as well as Alexa 594-conjugated goat anti-rabbit IgG(Invitrogen,USA)were used as the secondary antibodies.IgG as well as secondary antibodies were considered as negative controls.A droplet of Prolong Gold anti-fade reagent with DAPI(Vector Laboratories,USA)was added to fix the coverslip.Images were captured via laser scanning confocal microscopy(LSM 710,Zeiss,Germany).3.12 Statistical analysisSPSS v19.0(SPSS Inc.,IL)was used for data analysis.Data are presented as mean ± SD.For the comparison between two groups,an independent-samples t-test was used.One-way ANOVA analysis was used for multiple comparisons when the homogeneity of variance assumptions is fulfilled otherwise the equivalent non-parametric test was used.Chi-square test was used for multiple comparisons.The status of mice was observed and Kaplan-Meier curves were plotted during the experiment.P<0.05 signified statistical significance.4 Results4.1 NONO expression in AAA as well as Ang Ⅱ-induced VSMCsThe expression of NONO was suggestively augmented in AAA tissues of ApoE-/-mice compare to the control group(P<0.05).NONO protein also elevated in Ang Ⅱ-stimulated VSMCs compared with the control VSMCs(P<0.05).Double immunofluorescence of NONO in SMC,endothelial cells or macrophages in AAA tissues revealed that all the above cells expressed NONO protein with no significant difference.4.2 Efficiency of lentiviral transfection in vivo and in vitroThe lentivirus with GFP reporter gene was transfected into AAA tissues by tail intravenous injection in ApoE-/-mice,and then GFP fluorescence was examined in AAA tissues at 4,6 and 8 weeks.GFP fluorescence was significantly detected in AAA at 4,6 and 8 weeks after transfection.AAA tissues were transfected with sh-NONO-LV and the transfection conferred about 50%reduction in the NONO protein expression(P<0.01).Similar results were also observed in VSMCs transfected with or without sh-NONO-LV(P<0.01).4.3 Serum Lipid Levels and Blood PressureSerum lipid levels were detected and no difference was found among the four groups in the third part of the in vivo experimentation.Systolic blood pressure(SBP)was suggestively augmented in the Ang Ⅱ-infused ApoE-/-mice compare to their baseline levels or in control group.However,NONO knockdown had no effects on SBP.4.4 Effect of NONO downregulation on the occurrence of AAA in Ang Ⅱ-infused ApoE-/-MiceIn ApoE-/-mice,in the third part of in vivo experimentations,the incidence of AAA was 80%(P<0.05),84%(P<0.05)and 48%(P<0.05)in the no treatment,sh-NC and sh-NONO groups,respectively,compared with that in the control group which had no AAA.Based on the severity classification method,we observed that Ang Ⅱ induced more type Ⅲ and Ⅳ forms in no treatment and sh-NC groups,whereas Ang Ⅱinduced more type Ⅰ and Ⅱ forms in sh-NONO group.Thus,NONO knockdown suggestively reduced the occurrence of AAA in the Ang Ⅱ-infused ApoE-/-mice.Moreover,the greatest abdominal aortic diameter was suggestively augmented in AngⅡ-infused ApoE-/-mice compare to the control group(P<0.05).NONO knockdown obviously reduced the diameter in the Ang Ⅱ-infused ApoE-/-mice(P<0.05),while there was not any noteworthy alteration in the diameter between the NT and sh-NC groups.Kaplan-Meier curves showed that AngⅡ infusion lead to the decreased percent survival in ApoE-/-mice.4.5 Effect of NONO down-regulation on Ang Ⅱ-induced histological as well as morphological modifications in ApoE-/-mouse aortasH&E as well as Verhoeff staining indicated that Ang Ⅱ infusion induced positive remodeling during the pathological process of AAA in ApoE-/-mice including hypertrophy and breakdown of the adventitia,destruction of the aortic media,and discontinuity of elastin fibers.These pathological changes were largely attenuated in the sh-NONO group compared with those in the NT or sh-NC groups.However,no difference was found in the morphology of the abdominal aorta between the NT and sh-NC group.Furthermore,contents of collagen as well as SMCs in the aortic wall were significantly augmented in the sh-NONO group(P<0.05),whereas the content of macrophages was decreased in the sh-NONO compared with those in the NT or sh-NC group(P<0.01).Additionally,there was not any substantial difference among the NT and sh-NC groups on contents of collagen,SMCs,and macrophages.4.6 Effect of NONO down-regulation on the collagen deposition and degradation in vivo as well as in vitroIn the in vivo experiment,immunohistochemical staining and Sirius red staining showed that NONO knockdown significantly augmented deposition of Sirius Red,contents of Collagen Ⅰ(P<0.01),Collagen Ⅲ(P<0.01)and P4Hα1(P<0.01),and decreased collagen degradation MMP2(P<0.01)and MMP9(P<0.01)in aorta compared with those in the NT or sh-NC groups.No substantial alteration was found among the NT and sh-NC groups on collagen deposition.In the in vitro experiment,knockdown of NONO also obviously augmented the manifestation levels of Collagen Ⅰ(P<0.01),Collagen Ⅲ(P<0.01)as well as P4Hal(P<0.01)proteins,and reduced levels of MMP2(P<0.01)and MMP9(P<0.05)in VSMCs with Ang Ⅱ stimulation.4.7 Effect of NONO on inflammation infiltration in vivo as well as in vitroImmunohistochemical staining analysis showed that NONO knockdown significantly reduced the manifestation levels of IL-1β(P<0.01),MCP-1(P<0.01),IL-6(P<0.01)as well as TNF-α(P<0.01)in vivo,whereas no obvious difference was found between the NT and sh-NC groups.Furthermore,knockdown of NONO also significantly augmented the protein levels of IL-1β(P<0.05),MCP-1(P<0.05)as well as TNF-α(P<0.01)in VSMCs with Ang Ⅱ stimulation.4.8 Effect of NONO on NF-κB translocation as well as phosphorylation in VSMCs with Ang Ⅱ stimulationTranscription factor NF-κB has an imperative part in the pathological course of atherosclerosis through regulating inflammatory cytokines,for inst...