A Study Of The Role Of Chaperone-Mediated Autophagy In Atherosclerosis And Underlying Mechanisms

1.BackgroundAtherosclerotic cardiovASCular disease（ASCVD）is the genetic name of disease that based of atherosclerosis,including stroke,coronary heart disease and peripheral artery disease,etc.ASCVD is the primary cause of death and disability in our country,which has caused a heavy social burden and become a major public health event in our country.In spite of constant advances in medicine,the prevention and treatment of atherosclerosis remains to be improved.Therefore,it is of great significance to further study the pathogenesis of atherosclerosis and explore new therapeutic targets for better management of acute cardiovascular and cerebrovascular disease in China.Macrophages are the main cellular population in atherosclerotic plaque and play vital roles in the development of atherosclerosis.Macrophages uptake modified low-density lipoprotein（LDL）and transform into foam cells,which marks the formation of atherosclerotic lesion.The accumulation of macrophage foam cells increases plaque lipid load,inflammatory response,and plaque instability.Therefore,how to reduce lipid accumulation in macrophage foam cells is a potential therapeutic target for the treatment of atherosclerosis and has always attracted the attention of scientists.Recent studies have shown that autophagy-lysosomal system plays an important role in the development of atherosclerosis.Serving as the "waste disposal system" of cells,lysosomes are the main places where the damaged and/or unwanted components are degraded.Autophagy is a lysosomal degradation pathway,which works as a quality control system and is crucial for maintaining the balance of material and energy metabolism of cells.In the past decade,the regulatory role of autophagy in lipid catabolism is an encouraging finding.Autophagy regulates lipid catabolism and promotes cholesterol efflux,hereby reducing intracellular lipid load.These studies gave rise to the concept of "lipophagy,"a process in which lipid droplets are transported in autophagosome to lysosomes,where they undergo catabolism under the role of lysosomal acidic lipase.Promotion of lipophagy is an important pathway to promote lipid metabolism and reverse cholesterol transport（RCT）.Depending on the patterns of cargo delivery to the lysosomal lumen,at least three types of autophagy have been described:macroautophagy,microautophagy,and chaperone-mediated autophagy（CMA）.The three kinds of autophagy coexist in eukaryotic cells and cooperatively maintain cell homeostasis.And among them,macroautophagy and CMA are particularly important for mammalian cells.macroautophagy is commonly known as autophagy,and is a well-studied form of autophagy at present.Over the past 10 years,macroautophagy has been improved to be involved in the pathogenesis of atherosclerosis.Enhancing macroautophagy activity can improve endothelial function,promote lipid metabolism of macrophages and inhibit apoptosis of smooth muscle cells,etc.,which contributes to plaque stability by alleviating plaque inflammation and reducing plaque lipid load.However,little is known about the role of its less-well-studied cousin,CMA,in atherosclerosis.CMA is another autophagic process different from macroautophagy in cargo selection and transportation.macroautophagy is a non-selective form of in bulk degradation of longevity protein,damaged organelles or lipid droplets.CMA,on the other hand,is a highly selective type of autophagy,which degrades only proteins bearing in their amino acid sequence a pentapeptide（KFERQ sequence）.The typical double-membrane structure in macroautophagy is not needed in CMA.Instead,the cargo are recognized by heat shock protein 70（Hsc70）,which subsequently bind to LAMP-2A on the lysosomal membrane and then transport to the lysosome for degradation.The selectivity of CMA makes it suitable and competent for the degradation of key proteins that participate in cellular processes,which underlies the basis physiological functions of CMA,such as regulation of metabolic pathways,immune response and control of cell cycle.CMA dysfunction is linked to many human disease,including aging,neurodegenerative disorders and metabolic disorders.In the present,it remains unknown whether CMA is involved in atherosclerosis or other cardiovASCular diseases.Considering that atherosclerosis is a pathological process driven by lipid metabolism disorder and chronic inflammation,and the regulatory role of CMA in lipid metabolism and immune response,we speculated that CMA may be closely related to the pathogenesis of atherosclerosis.In our present study,we will investigate the presence of CMA and changes in the level of CMA in atherosclerotic plaques,the cells that contribute to this process,the role of CMA in atherosclerosis,whether CMA affects the development of atherosclerosis.Our study will reveal new pathogenesis of atherosclerosis and offer insight into the role of the autophagy-lysosome system in atherosclerosis,which contributes to exploring new target for the treatment of atherosclerosis.2.Objective（1）To investigate the alterations of CMA in atherosclerosis progression;（2）To investigate whether CMA affects the development of atherosclerosis.（3）To investigate the role of CMA in macrophage lipid metabolism,hereby analyzing the mechanism by which CMA affects atherosclerosis.3.Methods3.1 Animal models of progressive atherosclerosisA model of progressive atherosclerosis was generated in ApoE-/-mice by high-fat diet（HFD）feeding for different durations（8,12,18,24 and 38 weeks）to investigate the dynamic changes in LAMP-2A expression.3.2 Human coronary artery samplingHuman coronary atherosclerotic plaques were obtained from autopsy specimens of 5 male body donors with coronary heart disease after sudden coronary death,and the bodies were provided by the Red Cross Society of Shandong Province,China.The coronary arteries were embedded with optimal cutting temperature compound and cut into 7-μm-thick cross sections for histopathological staining.3.3 Macrophage-specific LAMP-2A（L2A）-deficient miceMacrophage-specific LAMP-2A（L2A）-deficient mice on a C57BL/6 background were generated using loxP insertion to selectively delete the exon region in the LAMP-2 gene that encodes the LAMP-2A variant（L2Afl/fl mice）and crossed with Cre-recombinase transgenic mice under the control of the Lysosomal M promoter.L2Afl/fl LysM-cre mice（L2A-m（?）KO mice）were further crossed with ApoE-/-mice（C57BL/6 background）to generate L2Afl/fl/LysM-Cre/ApoE-/-mice（L2A-m（?）KO/ApoE-/-mice）.Eight-week-old L2A-m（?）KO/ApoE-/-mice and their L2Afl/fl/LysM-Cre（-）/ApoE-/-littermates were fed a HFD（21%[wt/wt]fat,1.0%cholesterol,0.3%sodium cholate and 5%saccharose）for 16 weeks.3.4 Blood lipid,blood glucose and body weightSerum levels of total cholesterol,triglyceride,LDL-C,HDL-C and blood glucose in each group were measured.3.5 Histopathological staining（1）HE staining and oil red O stainingThe atherosclerotic lesions in the aorta were quantified by en face analysis and aortic root cross-sectional measurement.The aortas were stained with 0.5%oil red O.For aortic root cross-sectioning,slides were stained with oil red O and hematoxylin-eosin（H&E）.（2）Immunohistochemical（IHC）stainingImmunohistochemical（IHC）staining was used to analyze MOMA-2,LAMP-2A and α-SMA.The positive reactions of tissue sections were developed using an AEC Peroxidase Substrate Kit.Sections reacting with non-immune IgG as well as secondary antibodies were used as negative controls.（3）Immunofluorescent stainingImmunofluorescent staining was used to analyzed CD68 or MOMA-2,LAMP-2A and LAMP1.Sections reacting with non-immune IgG as well as secondary antibodies were used as negative controls.3.6 Production of polyclonal antibodies against LAMP-2B and LAMP-2CRabbit polyclonal antibodies against LAMP-2B and LAMP-2C were produced by AtaGenix Laboratories（Wuhan,China）.Synthetic peptides consisting of the cytosolic tail of mouse LAMP-2B（aa 399 to 413,FISYMIGRRKSRTGY）or that of LAMP-2C（aa 401 to the C-terminus,YLIGRRKTYAGYQTL）were used to produce antibodies that recognize only LAMP-2B or LAMP-2C,respectively.3.7 Macrophage culture and treatmentPrimary peritoneal macrophages were obtained from L2A-m（?）KO mice and their control littermates.Briefly,the mice were injected i.p.with 1 mL of 3%sterile starch,and 3 days later,peritoneal macrophages were isolated with cold PBS and cultured in DMEM supplemented with 10%FCS（Gibco）and 1%antibiotics（Gibco）.3.8 Western blottingSamples were separated,transferred and incubated with primary antibodies against the following:LAMP-2A,LAMP-2B,LAMP-2C,LAMP2,LAMP1,Atg5,Atg7,LC3B,Beclinl,SQSTM1/p62,SR-A、CD36、LAL、perilipin2 and β-actin.3.9 Macrophage Lipid Accumulation AssayLDs were stained using Bodipy 493/503 or Oil-red-O for 30 min prior to visualization.3.10 Statistical analysesQuantitative values were presented as mean ± standard error of the mean（SEM）.Categorical data were expressed as percentage（%）.A normal distribution of data was ensured for parametric analysis.The t-test was used to analyze a difference between two groups,and comparison of three or more groups was made by one-way analysis of variance（ANOVA）test with Tukey post hoc analysis.The Wilcoxon rank-sum test was used to assess non-normally distributed continuous variables.Bonferroni correction was used to correct p values for multiple comparisons.The chi-square test or Fisher’s exact test was used to compare categorical data.All analyses were performed with SPSS 16.0（SPSS Inc.,Chicago,IL,USA）.p<0.05 was considered statistically significant.4 Results4.1 CMA was most related to macrophages among the cell types in atherosclerotic plaquesThe CMA marker LAMP-2A was analyzed in lysates of mouse primary peritoneal macrophages,smooth muscle cells（SMCs）and endothelial cells（ECs）.The results showed that the protein level of LAMP-2A was high in macrophages and very low in ECs and SMCs.IHC staining and immunofluorescence analysis of consecutive sections from the aortic roots of ApoE-/-mice for LAMP-2A,α-SMA（specific for SMCs）and MOMA-2（specific for monocyte macrophages）also showed that LAMP-2A mainly colocalized with macrophages.This result was further verified in coronary atherosclerotic plaques obtained from human autopsy specimens.These data suggested that macrophages were the predominant cell type in atherosclerotic plaques that expresses a CMA marker.4.2 CMA was impaired in the progression of atherosclerosisThe CMA marker LAMP-2A was analyzed by IHC staining of consecutive sections of aortic roots taken at each stage of atherogenesis for MOMA-2 and LAMP-2A.The results showed that LAMP-2A levels gradually decreased in mice fed a HFD for 18 weeks and longer or was even undetectable in mice fed a HFD for 32 weeks,which was further verified by immunoblotting of whole-aorta lysates for LAMP-2A.Immunofluorescence analysis also confirmed the marked decline in LAMP-2A in advanced lesions in both mice and humans.4.3 Generation and analysis of macrophage-specific conditional LAMP-2A-knockout miceThe results showed that the LAMP-2A protein was undetectable in both primary peritoneal macrophages from L2A-m（?）KO mice and in aortic root plaques from L2A-m（?）KO/ApoE-/-mice,while the levels of other LAMPs（LAMP-1,LAMP-2B and LAMP-2C）were comparable in the L2A-m（?）KO mice and WT mice.In addition,basal macroautophagy activity and starvation-induced macroautophagy was slightly higher in LAMP-2A-deficient macrophages than in WT macrophages.4.4 Deficient CMA of macrophages promoted development of atherosclerosis in vivoThere was no difference in body weight or blood glucose,serum triglyceride or cholesterol levels between L2A-m（?）KO/ApoE-/-mice and Ctr mice（ApoE-/-mice）.However,L2A-m（?）KO/ApoE-/-mice exhibited markedly more whole-aorta atherosclerosis than Ctr mice.HE and oil-red-O staining of the aortic root also showed a significant increase（52%）in the lesion area in L2A-m（?）KO/ApoE-/-mice compared to Ctr mice.Taken together,these data demonstrated that LAMP-2A deficiency in macrophages accelerated atherosclerotic plaque formation.4.5 LAMP-2A deficiency promotes lipid accumulation in macrophagePeritoneal macrophages isolated from L2A-m（?）KO mice transformed into more foam cells and internalized more lipids than that from control mouse visible with either BODIPY493/503.4.6 PLIN2 degradation is not involved in the regulation of CMA on lipid metabolismMacrophages were treated with MG 132（a proteasome inhibitor）,leupeptin（trypsin-like and cysteine protease inhibitor）,and CQ（a lysosomal protease inhibitor）.Addition of proteasome-specific inhibitor MG 132 significantly increased PLIN2 level after treatment for 24 h.However,the other protease inhibitors had no effect on PLIN2 protein levels.We further analyzed PLIN2 level in macrophages from both control and L2A-m（?）KO mice and found no differences between the two groups.4.7 CMA modulates macrophage lipid metabolism through lipid regulatory enzymesBoth of the SR-A and CD36 were reduced in macrophages of L2A-m（?）KO mice compared with that of WT mice.The protein level of ATP binding cassette sub family A member 1（ABCA1）was also comparable between the two groups.Long-chain-fatty-acid-CoA ligase 1（ACSL1）was increased,and Lysosomal acid lipase（LAL）was reduced in LAMP-2A-deficient macrophage.5 Conclusions（1）Macrophages were the predominant cell type in atherosclerotic plaques that expresse a CMA marker.（2）Progressive atherosclerosis was characterized by dysfunctional CMA.（3）Deficient CMA of macrophages promoted development of atherosclerosis.（4）Deficient CMA promoted lipid accumulation in macrophage,which may underlie the basis of accelerated atherosclerosis progression in CMA-deficient mice.1.Background It has been widely accepted that atherosclerosis is a chronic inflammatory disease.Anti-inflammatory strategies of atherosclerosis is gradually on the stage of history.In recent years,a series of large-scale clinical trial was performed to assess various anti-inflammatory agents for the treatment of atherosclerosis,including human monoclonal neutralizing antibody binding to C-reactive protein（CRP）,tumor necrosis factor（TNF-a）,interleukin 6（IL-6）and interleukin 1 beta（IL-iβ）.Most of these clinical research results are unsatisfactory until 2017,when the results of the CANTOS trial were published on N Engl J Med.The results showed that treatment with the monoclonal IL-iβ-neutralizing antibody canakinumab reduced the risk of recurrent cardiovASCuto events in patients with prior heart attack.The CANTOS trial established IL-iβ as a centol anti-inflammatory therapeutic target for atherosclerosis.However,IL-iβ plays a central role in the body’s immune response,blocking IL-iβ systemically bears the risk of disturbing immune homeostasis and of detrimentally affecting protective immune responses,such as the fatal infections and sepsis present on canakinumab treatment.Therefore,it is necessary and important to explore some other more ideal targets to improve the effects of anti-inflmnmatoiy therapies for the treatment of atherosclerosis.Recent studies emphasize the role of NLRP3 inflammasome/IL-1 p-dependent inflammatory response in the occurrence and development of atherosclerosis.Inflammasomes are central upstream regulatory platform in IL-1 cytokine production,and particularly the NLRP3 inflammasome.NLRP3 inflammasome serves as an important "headquarters11 of the body’s immune response and inflammatoiy response,and is involved in the pathogenesis of many major human diseases.It has been shown that NLRP3 inflammasome activation and secretion of IL-iβ occur in early atherosclerosis.Although the CANTOS trial cannot definitely answer to what extent the NLRP3 inflammasome contributes to atherogenesis,it might still represent a valuable therapeutic target for treatment of atherosclerosis.Further understanding of mechanism of NLRP3 inflammasome activation,especially its negative regulatory mechanism,is an important means to alleviate vASCular inflammation and treat atherosclerosis.Activation of the NLRP3 inflammasome usually requires two independent signals: an initial priming signal activating the TLR/NF-kB pathway to induce the expression of NLRP3 and its precursor IL-ipand IL-18;the second signal promoting NLRP3 inflammasome assembly and acting as a platform for precursor caspase-1 activation.Activated caspase-1 cleaves the precursor forms of IL-iβ and IL-18 to form activated forms of IL-ip and IL-18,which are then secreted extracellular to exert biological effects.Besides the transcriptional regulation of NLRP3 inflammasomes,it is also regulated by posttranscriptional modifications.One of the most important is ubiquitination modification.Ubiquitin is an important pathway to regulate the activation of NLRP3 inflammasome.The core NLRP3 protein of the NLRP3 inflammasome is ubiquitinated in the basal state,and deubiquitination of the NLRP3 protein occurs once the NLRP3 inflammasome is activated.Because proteins degraded through the proteasome or macroautophagy lysosomal pathways first need to be labeled by ubiquitination,deubiquitinated NLRP3 inflammasomes is difficult to be degraded by these two pathways,which is an important mechanism leading to the continued activation of NLRP3 inflammasomes.Effective degradation and clearance of ubiquitinated NLRP3 inflammasomes is an important pathway that negatively regulate NLRP3 inflammasomes activation.Promotional the degradation of NLRP3 inflammasome is a milder and more effective approach than direct intervention against IL-iβ or upstream targets that regulate NLRP3 inflammasome production.Compared with proteasome or macroautophagy,CMA is a more efficient and specific protein degradation system.There is no evidence suggesting that substrate recognition by CMA is modified by ubiquitination,but rather depends on the Hsc70 sequence exposed in the protein.Studies have shown that not only abnormally synthesized and post-translationally damaged are CMA substrates,properly folded and fully fixnctional proteins can also become CMA substrates in specific cellular scenarios and that their timely selective degradation by CMA contributes to the regulation of the cellular pathways.In recent years,it has been reported that CMA is involved in many cell life processes by regulating the degradation of key proteins participated in cellular activities.However,it has not been reported whether NLRP3 inflammasomes can be degraded by the CMA pathway,and whether this process is related to the accelerated plaque progression in CMA deficient mice.In this light,we proposed the following scientific question: Can CMA regulate the activation of NLRP3 inflammasome by mediating its degradation,thereby influencing the inflammatory response and atherosclerosis? This study will contribute to improve the regulation mechanisms of NLRP3 inflammasome and provide new targets for anti-inflammatory treatment of atherosclerosis.2.Objective（1）To investigate the effect of CMA on the activation of NLRP3 inflammasome,and to clarify the molecular mechanism of CMA’s influence on atherosclerosis;（2）To investigate the effect of CMA on the degradation of NLRP3 inflammasome,and to clarify the mechanism of CMA regulating the activation of NLRP3 inflammasome.3.Methods3.1 Macrophage-specific LAMP-2A（L2A）-deficient mice Macrophage-specific LAMP-2A（L2A）-deficient mice on a C57BL/6background were generated using loxP insertion to selectively delete the exon region in the LAMP-2 gene that encodes the LAMP-2A variant(L2A^fl/fl mice)and crossed witii Cre-recombinase transgenic mice under the control of the Lysosomal M promoter.LysM-cre mice were fUrther crossed with ApoE^-/- mice to generate L2A^fl/fl/LysM-Cre/ApoE"/mice.3.2ELISA The concentrations of mouse JLA,IL-18 and TNF-a in the serum and cellular supernatants were measured using ELISA kits.3.3Enrichment of active Caspase-1 and IL-ip in the cellular supernatant Cleaved Caspase-1（p10 + p12）and IL-ip（p17）in the cellular supernatant were enriched and assessed by Western blotting.3.4Immunofluorescent staining Immunofluorescent staining was used to analyzed the level of NLRP3 and IL-iβ in atherosclerotic plaque,the formation of NLRP3/ASC specks and the colocalization between the NLRP3 protein and the CMA components.3.5Western blotting Samples were separated,transferred and incubated with primary antibodies against the following: LAMP-2A、LAMP1、NLRP3、ASC、pro-Caspase 1、Cleaved Caspasel（p10+p12）、pro-IL-iβ、CleavedIL-ip（p17）、Flag、AldoA、Hsc70、Cath D,JNK,p-JNK3 p38 MAPK,p-p38 MAPK,p44/42 MAPK（Erk1/2）,p-p44/42MAPK（Erk1/2）,NF-kB p65,p-NF-icB p65（Ser536）, iKBa , p-lKBa（Ser32）, histone H3 and P-actin.3.6Lysosome isolation Lysosomes were isolated from primary peritoneal macrophages using a Lysosome Isolation Kit.3.7Plasmid construction and transfection Flag-tagged LAMP-2A（Flag-LAMP-2A）,Flag-Hsc70,Myc-tagged NLRP3（Myc-NLRP3）or mutant Myc-NLRP3（Myc-NLRP3 Mut）, Myc-Caspase-1 and Myc-ASC plasmids were transfected into HEK293 T cells with Lipofectamine 2000 according to the manufacturer’s instructions.3.8Coimmunoprecipitation assay Coimmunoprecipitation（Co-IP）was performed using Immunoprecipitation Kits.Macrophages treated with or without LPS were used to detect the endogenous interaction between NLRP3 inflamasomes and CMA components Hsc70 or LAMP-2A.3.9RT-PCR Total RNA of macrophage was extracted and mRNA expression levels of IL-iβ,IL-18,TNF-a and NLRP3 were detected,respectively.3.10 Statistical analyses Quantitative values were presented as mean 士 standard error of the mean（SEM）.Categorical data were expressed as percentage（%）.A normal distribution of data was ensured for parametric analysis.The t-test was used to analyze a difference between two groups,and comparison of three or more groups was made by one-way analysis of variance（ANOVA）test with Tukey post hoc analysis.The Wilcoxon rank-sum test was used to assess non-normally distributed continuous variables.Bonferroni correction was used to correct p values for multiple comparisons.The chi-square test or Fisher5 s exact test was used to compare categorical data.All analyses were performed with SPSS 16.0（SPSS Inc.,Chicago,BL,USA）.p< 0.05 was considered statistically significant.4 Results4.1Deficient CMA promoted release of IL-1 p and IL-18 in macrophages and in vivo The results showed that serum EL-iβ and IL-18 levels,but not TNF-a levels,were increased in L2A-m（?）KO/ApoE（-/-） mice compared to similarly treated ApoE^-/- mice.This difference was more pronounced in L2A-m（?）KO mice injected with lipopolysaccharide（LPS）.In vitrol results showed that IL-iβ and E.-18 secretion was significantly increased in LAMP-2A-deficient macrophages in response to NLRP3 inflammasome stimulation.In contrast,TNF-a secretion was not influenced by LAMP-2A deletion.4.2LAMP-2A deficiency had on influence on the MAPK and iKBa/NF-KB pathways The results showed that the mRNA levels of IL-iβ,IL-18 and TNF-a in LAMP-2A-deficient and control macrophages responded similarly to LPS（or in the presence of ATP）.LAMP-2A depletion had no effect on the LPS-induced phosphorylation of JNK,ERK or p38,nor did it affect NF-kB p65 nuclear translocation within 40 min.4.3Deficient CMA promoted NLRP3 inflammasome activation Immunoblot analysis showed that the level of NLRP3 was considerably increased in LAMP-2A-deficient macrophages compared with Ctr macrophages treated with LPS with or without ATP.There were also no differences in the intracellular levels of pro-caspase-l5 ASC or pro-IL-ip.The secretion of cleaved caspase-1（p10）and mature IL-ip（p17）in the cellular supernatant was significantly elevated in LAMP-2A-deficient macrophages following LPS and ATP treatment.Immunofluorescence showed an increase in the number of cells with NLRP3/ASC specks as well as the number of specks per cell in LAMP-2A-deficient macrophages compared with Ctr macrophages.LAMP-2 A deficiency led to an obvious increase in NLRP3 and IL-ip expression in whole aortic lysates and in aortic root sections.4.4NLRP3 interacted with Hsc70 and LAMP-2A The results showed that NLRP3 colocalized with both Hsc70 and LAMP-2A in macrophages treated with LPS and ATP.This colocalization was also observed in human coronary atherosclerotic plaques.Co-IP further confirmed the direct interaction between endogenous NLRP3 and Hsc70 or LAMP-2A.Neither Caspase-1 nor ASC immunoprecipitated with LAMP-2A.This result was also found in HEK293 T cells transfected with plasmids for the expression of Flag-LAMP-2A and Myc-NLRP3,Myc-Caspase-1 or Myc-ASC.These data demonstrate that NLRP3,but not Caspase-1 or ASC? could interact with CMA components.4.5NLRP3 was a substrate of CMA CMA substrates are characterized by the presence of KFERQ-like motifs in their amino acid sequences.Four KFERQ-like motifs were found in the amino acid sequence of human NLRP3 but none were found in Caspase-1 or ASC.Mouse NLRP3 contains five KFERQ-like motifs,Caspase-1 contains two and ASC contains none.Therefore,among these proteins,NLRP3 is most likely a CMA substrate.All of the four CMA motifs in human NLRP3(³⁵⁵ LEKLQ ³⁵⁹,⁶⁰³ QIRLE ⁶⁰, ⁷⁹⁸ QKLVE ⁸⁰² and ⁹⁹¹EVLKQ ⁹⁹⁵)were mutated by replacing Q and the residue next to it with AA.Co-IP showed that NLRP3 mutant lost the ability to interact with LAMP-2A.4.6Deficient CMA inhibited NLRP3 inflammasome degradation All of the three components of the NLRP3 inflammasome were enriched in lysosomes from staved mice.Notably,lysosomes from staved mice contained more Hsc70 and LAMP-2A.Immunoblot analysis of lysosomal fractions showed that only the NLRP3 protein in lysosomes had been degraded in a CMA-dependent manner.In addition,immunoblot analysis of extracts from WT or L2A-KO mouse peritoneal macrophages treated with CHX（to inhibit the transcription of protein）for various durations further confirmed that LAMP-2A deficiency inhibited NLRP3 protein degradation but not the degradation of Caspase-1 or ASC.5 Conclusions（1）Activation of NLRP3 inflammasome was regulated by CMA.CMA defects promoted the activation of NLRP3 inflammasome.（2）NLRP3 protein was one of the substrates of CMA.Defects in CMA inhibited the degradation of NLRP3 protein.（3）Macrophage CMA dysfunction accelerated plaque progression by promotion of NLRP3 inflammasome-mediated inflammation.