The Role And Mechanism Of VSMCs Senescence In CKD-related AS Plaque Instability

Background:Cardiovascular disease is one of the most common complication and leading cause of death in chronic kidney disease(CKD)patients.Atherosclerosis(AS)plaque rupture is an important pathological feture for the occurrence of cardiovascular events,and the thinning of the AS plaque fibrous cap is a key event of plaque instability and plaque rupture.The incidence of acute myocardial infarction in CKD patients is 7.4 times higher than that in the general population,suggesting that AS plaque in CKD patients is more unstable.The activity and quantity of Vascular Smooth Muscle Cells(VSMCs)in AS plaque fibrious cap are the vital factors to maintain the thickness of fibrious cap and the stability of AS plaque.VSMCs senescence is regarded as an important mechanism in AS plaque rupture.It's been shown that CKD is an aging-related disease,and the internal environment of CKD is complex which can lead to cell senescence and aging.The role and mechanism of VSMCs senescence in CKD-related AS plaque instability need to be paid more attention to.The discussion of this problem will help to reveal the reasons for the high incidence of cardiovascular events in CKDCyclicGMP-AMPsynthase(cGAS)is a double-stranded DNA sensor,which plays an important role in innate immunity.Previous studies have shown that cGAS can capture self-leaking or exogenous DNA and lead to the synthesis of CyclicGMP-AMP(cGAMP).cGAMP induces the production of type I interferon(IFN-I)by activating STING-TBK1-IRF3 signal pathway and then resists the attack of pathogens.cGAS-STING plays an important role in cell senescence.After cell injury induced by exogenous factors,cGAS recognizes the leakage of self-DNA to promote IFN-I response causing uncontrollable inflammatory response in the form of autocrine and paracrine which further aggravates the phenotype of cell senescence.But,the role of cGAS-STING signaling pathway activation in VSMCs senescence and AS plaque instability is not clear,especially in the CKD-induced aging environment,whether it plays a key role needs further study.In this study,Apoe-/-mice and human VSMCs were used as the research object respectively in vivo and vitro.Firstly,it was confirmed that the VSMCs in the plaque fibrous cap of AS appeared aging,and the performance of AAS mice was more obvious.Next,the gene expression of VSMCs from AS and AAS mice was detected by gene Microarray.Bioinformatics analysis showed that JAK-STAT1 signal pathway and type I IFN response were significantly activated in CKD group.The activation of cGAS-STING signal pathway induces type I IFN response and plays a key role in VSMCs senescence in vivo and in vitro.Moreover,the changes of protein expression in vascular tissue of AS and AAS mice were detected by TMT proteomics and the results showed that mitochondrial damage was involved in CKD-induced VSMCs senescence,AS plaque instability and the activation of cGAS-STING signal pathway.Further studies found that CKD caused increasingly ROS production in fibrous cap VSMCs,resulting in mitochondrial damage and mtDNA leakage.mtDNA captured by cGAS then activated STING-TBK1-IRF3 signal pathway to induce the increasing production of IFN? which accelerated VSMCs senescence.However,the treatment Klotho(strong antioxidant stress protein)significantly inhibited the AS plaque instability induced by CKD.The effects and mechanisms of CKD-induced AS plaque instability and VSMCs senescence are studied in the following five parts:?The role VSMCs senescence in CKD-related AS plaque instability;?The role of IFN-I response in CKD-induced VSMCs senescence;?The effects of the activation of cGAS-STING signaling pathway in CKD-induced VSMCs senescence IFN-I response and AS plaque instability;?The role of ROS and mitochondrial damage in VSMCs senescence;?Klotho inhibits ROS production to lighten AS plaque instability induced by CKD.Results:1.CKD induces senescence of VSMCs in the fibrous cap of accelerated AS plaques and VSMCs cultured in vitro.Apoe-/-mice were divided into three groups:normal diet group(ND),AS group(sham operation+high-fat diet),AAS group(preparation of 5pm-6 kidney damage CKD model+high-fat diet).In AAS group,AS plaques area was greater,life span was shorter,incidence of myocardial infarction was higher and fibrous cap was thinner accompanied by a decrease in the number of VSMCs than that in AS group.Immunohistochemistry,immunofluorescence double staining and Western blot analysis showed that the expression of SA?G,p53 and p16 significantly increased in AAS group than that in AS group.After incubating hVSMCs with uremic serum,it was further confirmed that the complex internal environment of CKD could significantly increase the expression of SA?G,p53 and p16.These results suggest that VSMCs senescence plays an important role in CKD-induced AS progression and plaque instability2.IFN-I mediates CKD-induced AS plaque instability and VSMCs senescenceWe sorted the VSMCs of mice by flow cytometry and extracted RNA for gene Microarray analysis.It was found that JAK-STAT1-IFN-I signal pathway significantly up-regulated in AAS group and AS group,especially in AAS group.Immunofluorescence double staining and Western blot showed that the expression of IFN?,p-STAT1,MX1 and IRF7 was significantly higher than that in AS group.The above results were also further confirmed in vitro.In vitro,IFN? treatment of hVSMCs could inhibit the proportion of Ki-67+,increase the expression of SA ?G+and p53,and inhibit the expression of ?-SMA.On the contrary,the proportion of SA?G+ cells in VSMCs of IFNAR1-/-/Apoe-/-mice decreased significantly,and the senescence of VSMCs induced by CKD serum incubation was significantly reduced by down-regulating the expression of IFN-I receptor(IFNAR1)and STAT1 in hVSMCs by siRNA.These results suggest that the activation of IFN-I and its downstream signal pathway STAT1 play an important role in CKD-induced VSMCs senescence.3.cGAS-STING signal pathway mediates IFN-I response that promotes CKD-induced VSMCs senescence and plaque instability.Immunofluorescence staining,flow cytometry and Western blot detection showed that the expression of p-TBK1 and p-IRF3 in VSMCs from AS group,while the change in AAS group was more obvious than that in AS group.The expression of p-TBK1 and p-IRF3 also significantly increased in vitro.We used siRNA transfection to knock down the expression of cGAS,STING and IRF3 in VSMCs.Flow cytometry showed that the increased expression of IFNP,the increase of the proportion of SA3G+cells and the down-regulation of a-SMA expression induced by uremic serum were significantly reduced.We also found the above changes in AS plaques of cGAS-/-/Apoe-/-,STING-/-/Apoe-/-,IRF3-/-Apoe-/-and CKD/Tagln-Sting mice.In double knockout mice,the AS plaque area decreased and the thickness of fibroud cap increased significantly.These results suggest that cGAS-STING signal pathway plays an important role in IFN-I response,subsequent VSMCs senescence and plaque instability.CKD can aggravate the activation of cGAS-STING signal pathway,while inhibition of cGAS-STING signal pathway can significantly improve VSMCs senescence and plaque instability induced by CKD4.Mitochondrial damage in VSMCs leads to activation of cGAS-STING signal pathway activation and IFN-I responseIt was found in mmunofluorescence staining that the co-localization of DNA and cGAS in the fibrous cap VSMCs of AS and AAS mice,especially in AAS mice.At the same time,the co-localization of DNA and cGAS was also confirmed in the hVSMCs incubated with the uremic serum.Then we used ChIP to detect the co-precipitated DNA fragments with cGAS The results showed that after uremic serum incubation,there was obvious enrichment of mtDNA in VSMCs.To deplete mtDNA,EtdBr was used in VSMCs.EtdBr could significantly inhibit the increase of serum-induced expression of IFN3,the increase of the proportion of SA?G+cells and the down-regulation of a-SMA expression in patients with CKD.In vivo,transmission electron microscopy showed that the structure of VSMCs mitochondria in the plaque fibrous cap of AS and AAS groups was damaged and the MPTP opening of mitochondrial permeability transition pore was increased,but the above mentioned mitochondrial damage was more obvious in AAS group.In vitro,obvious mitochondrial damages and MPTP opening were observed in VSMCs incubated with uremic serum,and further studies showed that ATP production and mitochondrial oxidative phosphorylation function decreased significantly in VSMCs.However,pretreatment of hVSMCs with cyclosporine(CsA),an inhibitor of MPTP,could effectively inhibit the increase of serum-induced expression of IFN?,the increase of the proportion of SA?G+cells and the down-regulation of ?-SMA expression.These results suggest that the endogenous mtDNA release caused by mitochondrial damage may be an important inducement of cGAS-STING signal pathway activation and IFN-I response in VSMCs5.The increase of VSMCs ROS production caused by CKD induces mitochondrial damage.The increase of ROS production was detected by DHE staining in the plaques of AS and AAS mice,and the co-localization of 8-OH-dG staining and cGAS was also observed in the fibrous cap VSMCs,especially in the AAS group.DCFH probe fluorescence intensity of VSMCs was significantly enhanced after 6 or 12 hours of uremic serum incubation suggesting that cellular ROS significantly increased,and ROS production was earlier than MPTP changing.Pretreatment with N-acetylcysteine(NAC),a ROS scavenger,could significantly reduce CKD-induced mitochondrial damage,the increase of IFN? expression and VSMCs senescence.Therefore,the increase of VSMCs ROS production caused by environmental disorders in CKD plays a key role in mitochondrial damage and subsequent IFN-I response6.Exogenous supplementation of Klotho protein significantly inhibited CKD-induced VSMCs ROS production,senescence and plaque instabilityImmunofluorescence and Western blot showed that the expression of Klotho in VSMCs from AAS mice was significantly decreased.In vitro,uremic incubation also decreased the expression of Klotho in hVSMCs.Klotho+/+/Apoe-/-mice and Klotho+/-Apoe-/-mice were used to create CKD models.The results showed that Klotho+/-/Apoe-/-mice had larger AS plaques,thinner fibrous cap thickness and increased ROS production.It is suggested that CKD could lead to the down-regulation of Klotho expression in VSMCs,while Klotho deficiency could aggravate CKD-induced VSMCs aging and plaque instability.In contrast,treatment of Klotho protein significantly inhibit the progression of atherosclerotic plaque,excessive production of ROS,thinning of fibrous cap,the decrease of VSMCs in fibrous cap and VSMCs senescence induced by CKD.These results suggest that Klotho protein can reduce CKD-induced accelerated AS progression,VSMCs senescence and plaque instability by inhibiting ROS production.To sum up,this study found that CKD accelerate the progress of AS,leading to VSMCs aging and plaque instability.Studies have shown that IFN-I response caused by the activation of cGAS-STING signal pathway plays an important role in VSMCs senescence,while the increase of ROS production induced by CKD and the subsequent mitochondrial damage are the key factors for activating cGAS-STING signal pathway and IFN-I response.Targeted intervention of cGAS-STING signaling pathway and IFN-I response,or exogenous supplement of Klotho protein can significantly improve VSMCs aging and plaque instability induced by CKD,which provides a new idea and target for the prevention and treatment of CKD-related AS plaque instability and cardiovascular events.