| Objective:Vascular aging is a comprehensive effect of vascular cell（endothelial cells,smooth muscle cells,fibroblasts）senescence,which plays an important role in vascular stenosis,atherosclerosis（AS）and other physiological and pathological processes.The senescence of VSMCs that is the major components of the blood vessel wall,which is the main driving force for the occurrence and development of vascular aging-related diseases.Previous studies have confirmed that VSMC senescence exhibit proliferation,migration,inflammation and collagen deposition during neointima formation caused by vascular injury.Although it has been confirmed that vascular cell senescence is involved in the development of vascular proliferative diseases,there are still few studies on the mechanism of aging exaggerates neointima formation after vascular injury.Our previous studies have shown that circ-Sirt1 generated from exons 2-7 of the SIRT1 gene suppressed vascular inflammation and neointimal formation.Several studies have confirmed that excessive proliferation of VSMCs may lead to the occurrence of replicative senescence,suggesting that replicative senescence is highly relevant in the context of VSMC proliferation.However,the effect of circ-Sirt1 inhibiting vascular inflammation on cellular senescence during neointimal hyperplasia remains to be elucidated.In this study,C57BL/6J mice were used to establish a carotid artery stenosis model and VSMCs were used as the research object.The role of circ-Sirt1 on VSMCs senescence and neointimal hyperplasia was explored at the global,cellular and molecular levels.Methods:The carotid artery stenosis model was constructed using C57BL/6J mice.The relationship between circ-Sirt1 and aging-related neointimal hyperplasia was evaluated by SA-β-gal staining,Western blot and q RT-PCR experiments.The proteins that bind to circ-Sirt1 were explored by RNA-pull down and mass spectrometry.The effect of circ-Sirt1 on p53activation and nuclear translocation was explored from the perspective of RNA-protein interaction.Results:1.circ-Sirt1 inhibits VSMC senescence1.1 The expression of circ-Sirt1 is decreased in the arteries of aged mice and the neointimal hyperplasiaTo verify the relationship between circ-Sirt1 and vascular aging,the carotid arteries of young and old mice were used to the experiments.The expression of circ-Sirt1 was detected by q RT-PCR and RNA fluorescence in situ hybridization.The results showed that the expression of circ-Sirt1 in carotid artery tissue of aged mice was decreased compared with young mice,while the expressions of senescence markers p53 and p21 were increased.To investigate the role of circ-Sirt1 in vascular intimal hyperplasia and VSMC senescence,we established a carotid artery stenosis model mice.SA-β-gal staining and immunofluorescence staining showed that SA-β-gal positive staining was significantly increased in the neointima compared with the vascular media,and mainly ACTA2-positive cells.The expression of circ-Sirt1was detected by q RT-PCR and RNA fluorescence in situ hybridization,and the results showed that the expression of circ-Sirt1 decreased during neointima formation.The data suggested that VSMC senescence may be related to the decreased expression of circ-Sirt1.To further confirm the relationship between circ-Sirt1 and senescence,VSMCs were treated with AngⅡ to construct cellular senescence model in vitro.SA-β-gal staining and Western Blot detection showed that SA-β-gal-positive cells were significantly increased,accompanied with increased p53,p21,andγ-H2AX expression and decreased level of PCNA after chronic treatment with AngⅡ.The expression of circ-Sirt1 was detected by q RT-PCR,and it was found that the expression of circ-Sirt1 was decreased in senescent VSMCs.These results suggest that rapidly proliferating VSMCs undergo senescence during the formation of neointima,and the expression of circ-Sirt1 was decreased.1.2 circ-Sirt1 inhibits VSMC senescenceIn order to further explore the relationship between circ-Sirt1 and VSMC senescence,circ-Sirt1 was overexpressed by adenovirus and knocked down by the specific si RNA.SA-β-gal staining and Western blot showed that knockdown of circ-Sirt1 promoted AngⅡ-induced VSMC senescence,while overexpression of circ-Sirt1 inhibited AngⅡ-induced VSMC senescence.Furthermore,similar changes were observed in PDGF-BB-induced VSMC senescence.Our previous studies have confirmed that circ-Sirt1 can upregulate the expression of SIRT1 protein at the post-transcriptional level,which can deacetylate the senescence regulator p53.Western blot experiments confirmed that circ-Sirt1 can up-regulate SIRT1 protein expression and promote the deacetylation of p53 in AngⅡ-induced senescent VSMCs.To confirm whether the inhibitory effect of circ-Sirt1 on VSMC senescence completely depends on SIRT1,we used the SIRT1-specific inhibitor Ex527 to inactivate SIRT1 in VSMCs overexpressing circ-Sirt1,and then examined the effect of Ex527 on the anti-aging effect of circ-Sirt1 by SA-β-gal staining and Western blot.We found that Ex527 could not completely inhibit the anti-aging effect of circ-Sirt1.The data suggested that circ-Sirt1 inhibits p53-mediated cellular senescence,but it is not completely dependent on SIRT1.1.3 circ-Sirt1 inhibits VSMC proliferation and migrationPrevious studies have confirmed that AngⅡ and PDGF-BB can not only induce cell senescence,but also promote the proliferation and migration of VSMCs.To determine the effect of circ-Sirt1 on VSMC migration,we treated VSMCs with AngⅡ or PDGF-BB on the basis of overexpression of circ-Sirt1.Cell wounding assay showed that circ-Sirt1 could inhibit the proliferation and migration of VSMCs induced by AngⅡ or PDGF-BB.2.The molecular mechanism of circ-Sirt1 inhibiting VSMC senescence2.1 circ-Sirt1 interacts with p53More recently,accumulated researches have demonstrated that circ RNAs bind and sequester specific proteins to appropriate subcellular positions,and participate in modulating certain protein-protein and protein-RNA interactions.To further study the molecular mechanism of circ-Sirt1 inhibiting aging,we attempted to identify the proteins that interact with circ-Sirt1 in AngⅡ induced senescent VSMCs.Through RNA pull-down combined with mass spectrometry analysis,we searched for the proteins that interact with circ-Sirt1in senescent VSMCs,and then we performed GO enrichment analysis on potential interacting proteins,finally we found the transcriptional regulator p53,which inhibits cell cycle progression.The interaction between circ-Sirt1and p53 was confirmed by RNA pull-down combined with Western blot and RIP experiments.These results suggest that circ-Sirt1 may inhibit VSMC senescence by interacting with p53.2.2 Identification of mutual binding between circ-Sirt1 and p53In order to find out the possible regions where circ-Sirt1 interacts with p53,Mfold was used to predict the secondary structure of circ-Sirt1,and RNA composer software was used to build the tertiary structure model of circ-Sirt1on the basis of the secondary structure.Molecular docking of circ-Sirt1 and p53 was carried out by NPDock software based on the molecular space structure.The results showed that the interaction region between circ-Sirt1 and p53 was located at nt 729-792.To validate the prediction,we constructed the circ-Sirt1 vector mutating p53 binding sites within binding region,synthesized blocking oligo that were complimentary to nt 729-792,and performed a pull-down experiment.We showed that the biotinylated probe pulled down significantly higher levels of p53 when the cells were overexpressed circ-Sirt1.But the interaction of p53with circ-Sirt1 was weakened in VSMCs transfected with circ-Sirt1 mutant or blocking oligo.We further verified the interaction by RIP assay.2.3 circ-Sirt1 inhibits p53 nuclear translocation induced by AngⅡAs above mentioned,overexpression of circ-Sirt1 significantly decreased the p53 protein level in AngⅡ-treated VSMCs.We further determined that the expression of p53 m RNA was reduced when circ-Sirt1 was overexpressed.Studies in tumor-derived cells have demonstrated that p53 protein directly binds to its own promoter to induce the transcription of itself and downstream target genes responsible for cell cycle arrest and DNA repair.We predicted p53 responsive element and confirmed the DNA binding for p53transcriptional autoactivation in senescent VSMCs using Ch IP.These results lead us to speculate whether the interaction between circ-Sirt1 and p53 results in a cytoplasmic sequestration of p53,which in turn affects the expression of p53 and downstream target genes.To validate this hypothesis,we detected circ-Sirt1 expression in the cytoplasmic and nuclear fractions of VSMCs using q RT-PCR and fluorescence in situ hybridization.We showed that circ-Sirt1was primarily expressed in the cytoplasm of VSMCs.Immunofluorescence staining showed that AngⅡ could induce the nuclear translocation of p53.Overexpression of circ-Sirt1 significantly inhibited p53 nuclear translocation.These results suggest that circ-Sirt1 may interact with p53 in the cytoplasm to affect its nuclear translocation activation.2.4 circ-Sirt1 inhibits NF-κB nuclear translocation induced by AngⅡPrevious studies by our group have confirmed that circ-Sirt1 can directly interact with NF-κB p65 to inhibit its nuclear translocation activation.To determine whether the inhibitory effect of circ-Sirt1 on NF-κB p65 exists during cellular senescence,we detected the nuclear translocation activation of NF-κB p65 by immunofluorescence staining.The results showed that AngⅡ could promote the nuclear translocation of NF-κB p65,and overexpression of circ-Sirt1 could inhibit the nuclear translocation of NF-κB p65.Based on the activation effect of NF-κB on p53 confirmed by previous studies,Ch IP experiments confirmed that overexpression of circ-Sirt1 inhibited the binding of NF-κB to the response element in the p53 promoter.The above results suggest that the inhibitory effect of circ-Sirt1 on VSMC senescence may be related to the inhibition of NF-κB p65 activation.3.Overexpression of circ-Sirt1 inhibits vascular aging-related neointimal hyperplasia in vivo3.1 circ-Sirt1 inhibits vascular aging-related neointimal hyperplasiaTo explore whether the intervention of circ-Sirt1 can be a potential strategy for preventing neointimal hyperplasia by repressing senescence,an Ad-vector or Ad-circ-Sirt1 was infected into mice carotid arteries after ligation in vivo.We showed that circ-Sirt1 expression was significantly increased in carotid arteries infected with Ad-circ-Sirt1.Neointimal hyperplasia induced by injury was obviously repressed by overexpression of circ-Sirt1.Importantly,the number of SA-β-gal positive cells was decreased in Ad-circ-Sirt1-infected arteries compared to the vehicle control 14 days after ligation injury,accompanied by down-regulated expression of senescence markers p53 and p21.These results confirm that circ-Sirt1 can inhibit vascular aging-related neointimal hyperplasia.3.2 circ-Sirt1 is involved in the formation and development of vascular aging-related ASTo explore whether similar regulatory mechanisms exist in the development of AS,We constructed AS model by Apo E-/-mice.The results of Oil Red O staining showed that Apo E-/-mice on Paigen diet for 8 weeks showed obvious Oil Red O staining in the aortic sinus and aortic arch,indicating that the modeling was successful.Immunofluorescence staining and SA-β-gal staining showed that p53 and SA-β-gal positive staining areas were significantly increased in AS lesions of Apo E-/-mice,and co-localized with ACTA2.Compared with WT mice,the expression of circ-Sirt1 was decreased in AS vascular tissue.It is suggested that the regulatory mechanism of circ-Sirt1 inhibiting the activation of p53 in vascular smooth muscle cells to delay senescence may also exist in the occurrence and development of AS.3.3 The expression of circ-Sirt1 is decreased in the patients with vascular remodeling diseasesVascular remodeling diseases,including AS,hypertension,pulmonary hypertension and aneurysm,are a serious threat to human health.VSMC senescence caused by multiple factors plays an important role in vascular remodeling diseases.Therefore,in order to further explore the role of circ-Sirt1 in clinically aging-related vascular diseases,we collected renal arterial specimens from patients with or without AS or hypertension who underwent nephrectomy at the Fourth Hospital of Hebei Medical University,China.The AS renal-artery wall exhibited neointimal hyperplasia.Immunofluorescence revealed that positive staining of p53 in human renal arterial neointima was higher than normal controls,accompanied with increased SA-β-gal activity.The circ-Sirt1 level was significantly decreased in the vascular tissue of patients with AS or hypertension.Together,these findings suggest that impaired circ-sirt1 is involved in neointimal formation and AS development via eliciting senescence.Targeting circ-sirt1 may be considered as a new therapeutic strategy for retarding aging-associated vascular diseases.Conclusions:1.The expression of circ-Sirt1 is decreased in the arteries of aged mice and the neointimal hyperplasia.2.circ-Sirt1 inhibits the senescence and migration of VSMCs.3.circ-sirt1 is mainly expressed in the cytoplasm and inhibits the activation of p53 nuclear translocation by directly interacting with p53 in the cytoplasm.4.Overexpression of circ-Sirt1 inhibits aging-related neointimal hyperplasia and AS in vivo.5.The expression of circ-Sirt1 is decreased in the patients with vascular remodeling diseases. |
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