The Study On The Effect And Mechanism Of NANOG In VSMC Phenotype Switch In Thoracic Aortic Dissection

Background and Objective:Thoracic aortic dissection(TAD)is a medical emergency with very high mortality.When TAD occurred,the aortic intima was ruptured and the blood flowed into the aortic media to form a false lumen.Patients always got an acute death because of the false lumen broken since there was no outflow tracts in the false lumen.50%patients died within 48hours after TAD occurred if there was no timely intervention.Some genetic disease like Marfan syndrome and Ehlers-Danlos syndrome can result in TAD,however,only about10%TAD was resulted from genetic diseases.The pathogenesis of TAD was still unclear and there were no effective prevention and drug treatment for TAD.Based on these,study on the pathogenesis of TAD is helpful for the early prevention and drug treatment of TAD.Aortic media is the main functional structure of the aortic wall,the aortic media abnormity is the pathogenetic base of TAD.The main cell type of aortic media is vascular smooth muscle cells(VSMCs)and VSMCs can transform between contractile and synthetic phenotypes.Compared with contractile VSMCs,synthetic VSMCs showed enhanced proliferation and migration abilities,expressed increased levels of synthetic markers osteopontin(OPN),matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9)and decreased levels of contractile markers?-smooth muscle actin(?-SMA)and smooth muscle 22?(SM22?).Most VSMCs showed contractile phenotype to maintain the normal function of aortic wall under physiological conditions.It was reported VSMCs phenotype switch from contractile to synthetic played important role in the pathophysiologic processes of TAD.However,it is still unclear about the molecular mechanism of VSMCs phenotype switch in TAD.NANOG is a homeodomain protein which has an important function in maintaining the proliferation and pluripotency of embryonic stem cells.NANOG expression also maintains the self-renew and dedifferentiation status in embryonic stem.In contrast,NANOG deficiency induced cell differentiation.VSMCs phenotype switch was regarded as a dedifferentiation process,so the function of NANOG indicated that it might be related with VSMCs phenotype switch in TAD.But role of NANOG in VSMCs phenotype switch is still unknow.OPN is highly expressed in TAD aortic media and synthetic VSMCs.Studies showed OPN contributed VSMCs phenotype switch,high level OPN enhanced proliferation and migration abilities of VSMCs,up-regulated the expression of synthetic marker MMP2 and down-regulated the expression of contractile marker?-SMA.It was also reported OPN expression was regulated by NANOG in embryonic stem cells,which indicated that NANOG might induced VSMCs phenotype switch through regulating OPN in TAD.In the present study,we investigated the effect and mechanism of NANOG in VSMCs phenotype switch in TAD by a series of histology and molecular biology experiments.Research Methods:(A)Specimen Collection and VSMCs Culture1.Specimen collection:TAD aortic tissues were obtained from 20 TAD patients who underwent surgical repair.Control aortic tissues were obtained from 10 donors who had no vascular diseases.2.VSMCs isolation and culture:Primary VSMCs were isolated from TAD and control specimens.Specimens were stripped the intima and adventitia,then cut into about 4mm~2 tissue blocks and inoculated in a 24 well plate.After that,200?l M231medium with smooth muscle growth supplements was added in each well.When cells grew to confluence,tissue blocks were transferred to another plate for re-primary culture and VSMCs were passaged.(B)The Effect and Mechanism of NANOG in VSMC Phenotype Switch1.Adenovirus transfection:According to the MOI,add appropriate volume of adenovirus into the half volume medium to make transfection medium.When VSMCs grew to about 70%confluence,add the transfection medium and incubated at 37?for 2hours,then complement the medium and replaced the medium after overnight incubation.Then check the transfection result 48 hours after adenovirus transfection with the fluorescence microscope.2.siRNA transfection:Lipofectamine 2000 was used for siRNA transfection according to the manufacturer`s instruction.3.Immunohistochemistry:Aortic specimens was first fixed with 10%formalin.After dehydration and paraffin embedding,specimens were made into sections.After dewaxing,antigen retrieval,inactivating the endogenous peroxidases and blocking,sections were incubated with primary antibody and then incubated with horseradish peroxidase-conjugated secondary antibody.DAB developing solution was used for imaging.4.qRT-PCR and Western blot:qRT-PCR was used to detect the mRNA level of the target gene and Western blot was used to detect the protein level of the target protein in the sample.5.Cell proliferation:CCK8 assay was used for the assessment of cell proliferation.6.Scratch wound-healing Assay:Scratch wound-healing assay was used for cell migration assessment in lateral direction.7.Transwell migration assay:Transwell migration assay was used for cell migration assessment in vertical direction.8.Apoptosis Assay:We use ultraviolet irradiation to induce VSMCs apoptosis.VSMCs were stained with Annexin V/PI and then tested with flow cytometry.9.TUNEL assay:VSMCs were fixed with 4%paraformaldehyde and then incubated with the mixture of terminal deoxynucleotidyl transferase enzyme and biotin-11-dUTP after membrane puncture.VSMCs were finally incubated with fluorescent labeling buffer and tested with fluorescence microscope.10.Immunofluorescence:VSMCs were fixed with 75%ethanol.After membrane puncture,blocking,incubate with primary and secondary antibody,nuclear staining,VSMCs were observed under fluorescence microscope.11.Chromatin immunoprecipitation(ChIP):We did ChIP assay following the manufacturer`s instruction of EpiQuik ChIP Kit.(C)Statistical AnalysisStudent t test was used to evaluate the statistical differences.statistical analysis was completed in SPSS.Data were presented as mean±SD.P<0.05 was considered statistically significant.Research Results:(A)NANOG was high expressed in TAD aortic media and VSMCs accompanying VSMCs phenotype switchqRT-PCR,Western blot and immunohistochemistry results showed NANOG was high expressed both in mRNA and protein levels in TAD specimens and VSMCs.The results also showed expression of VSMCs synthetic markers OPN and MMP2 was up-regulated while the expression of VSMCs contractile markers?-SMA and SM22?was down-regulated.Our further experiments on VSMCs verified these findings in TAD aortic tissue.TAD VSMCs immunofluorescence results showed NANOG staining was enhanced in nucleus accompanying with strong OPN staining and weak SM22?staining in cytoplasm.Results in this part showed NANOG might participate in the regulation of VSMCs phenotype switch in TAD.(B)NANOG promoted VSMCs phenotype switch from contractile to syntheticNANOG overexpression in VSMCs up-regulated the expression of synthetic markers OPN and MMP2 while down-regulated the expression of contractile markers?-SMA and SM22?.CCK8 results showed NANOG overexpression enhanced VSMCs proliferation ability.Scratch wound-healing assay and Transwell migration assay results showed NANOG overexpression enhanced VSMCs migration ability both in lateral direction and vertical direction.In apoptosis assay,NANOG overexpression enhanced VSMCs anti-apoptosis capability.TUNEL assay results showed NANOG overexpression decreased VSMCs apoptosis rate.Further experiments showed down-regulate NANOG expression in TAD VSMCs up-regulated the expression of contractile markers?-SMA and SM22?while down-regulated the expression of synthetic markers OPN and MMP2.Results in this part showed NANOG overexpression promoted VSMCs phenotype switch and the function of NANOG might be through OPN.(C)NANOG promoted VSMCs phenotype switch by directly up-regulating OPNChIP results showed NANOG directly bound to the promoter region of OPN.Rescue experiments showed down-regulate OPN in NANOG overexpression VSMCs up-regulate the expression of contractile markers?-SMA and SM22?while down-regulated the expression of synthetic marker MMP2.CCK8 assay,Scratch wound-healing assay,Transwell migration assay,apoptosis assay and TUNEL assay results showed down-regulate OPN in NANOG overexpression VSMCs inhibited NANOG induced enhanced proliferation,migration and anti-apoptosis capabilities.Results in this part showed NANOG promoted VSMCs phenotype switch from contractile to synthetic via directly up-regulating OPN through binding to its promoter region.Research Conclusions:The present study suggests that NANOG is high expressed in TAD aortic media and VSMCs.Increased NANOG promotes VSMCs phenotype switch from contractile to synthetic by directly up-regulating OPN.