FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2893876947> ?p ?o ?g. }

• W2893876947 endingPage "2959" @default.
• W2893876947 startingPage "2948" @default.
• W2893876947 abstract "We hypothesized that autophagy and associated lysosome function serve as a critical modulator during Nod-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome activation on proatherogenic stimuli. We first demonstrated that 7-ketocholesterol stimulated Nlrp3 inflammasome formation and activation as shown by increased colocalization of inflammasome components [Nlrp3 versus apoptosis associated speck-like protein (Asc) or caspase-1] and enhanced cleavage of caspase-1 into active caspase-1 to generate IL-1β in coronary artery smooth muscle cells. Deletion of the CD38 gene (CD38−/−) that regulates lysosome function and autophagic flux also led to Nlrp3 inflammasome formation and activation. In the presence of rapamycin, the effects of either 7-ketocholesterol treatment or CD38 gene deletion were abolished. The autophagy inhibitor spautin-1 and the lysosome function blocker bafilomycin A1 also enhanced Nlrp3 inflammasome formation and activation. In animal experiments, we found that increased colocalization of Nlrp3 versus Asc or caspase-1 enhanced IL-1β accumulation and caspase-1 activity in the coronary arterial wall of CD38−/− mice on the Western diet compared with CD38+/+ mice. This increased colocalization was blocked by treatment with rapamycin but enhanced by chloroquine, a water-soluble blocker of autophagic flux. Morphologic examinations confirmed that the media of coronary arteries was significantly thicker in CD38−/− mice on the Western diet than CD38+/+ mice. In conclusion, the deficiency of autophagic flux promotes Nlrp3 inflammasome formation and activation in coronary artery smooth muscle cells on proatherogenic stimulation, leading to medial thickening of the coronary arterial wall. We hypothesized that autophagy and associated lysosome function serve as a critical modulator during Nod-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome activation on proatherogenic stimuli. We first demonstrated that 7-ketocholesterol stimulated Nlrp3 inflammasome formation and activation as shown by increased colocalization of inflammasome components [Nlrp3 versus apoptosis associated speck-like protein (Asc) or caspase-1] and enhanced cleavage of caspase-1 into active caspase-1 to generate IL-1β in coronary artery smooth muscle cells. Deletion of the CD38 gene (CD38−/−) that regulates lysosome function and autophagic flux also led to Nlrp3 inflammasome formation and activation. In the presence of rapamycin, the effects of either 7-ketocholesterol treatment or CD38 gene deletion were abolished. The autophagy inhibitor spautin-1 and the lysosome function blocker bafilomycin A1 also enhanced Nlrp3 inflammasome formation and activation. In animal experiments, we found that increased colocalization of Nlrp3 versus Asc or caspase-1 enhanced IL-1β accumulation and caspase-1 activity in the coronary arterial wall of CD38−/− mice on the Western diet compared with CD38+/+ mice. This increased colocalization was blocked by treatment with rapamycin but enhanced by chloroquine, a water-soluble blocker of autophagic flux. Morphologic examinations confirmed that the media of coronary arteries was significantly thicker in CD38−/− mice on the Western diet than CD38+/+ mice. In conclusion, the deficiency of autophagic flux promotes Nlrp3 inflammasome formation and activation in coronary artery smooth muscle cells on proatherogenic stimulation, leading to medial thickening of the coronary arterial wall. Nod-like receptor family pyrin domain containing 3 (Nlrp3) belongs to the Nod-like receptor (NLR) family, which can form Nlrp3 complexes with the adaptor protein apoptosis associated speck-like protein (Asc) and pro–caspase-1.1Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.Nature. 2004; 430: 213-218Crossref PubMed Scopus (1405) Google Scholar, 2Zhou R. Tardivel A. Thorens B. Choi I. Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation.Nat Immunol. 2010; 11: 136-140Crossref PubMed Scopus (1854) Google Scholar The formation of this large multiprotein complex, called Nlrp3 inflammasome, leads to the activation of caspase-1, which is required for maturation and production of mature IL-1β and IL-18 by cleavage of their precursor.3Dinarello C.A. Donath M.Y. Mandrup-Poulsen T. Role of IL-1beta in type 2 diabetes.Curr Opin Endocrinol Diabetes Obes. 2010; 17: 314-321PubMed Google Scholar, 4Martinon F. Mayor A. Tschopp J. The inflammasomes: guardians of the body.Annu Rev Immunol. 2009; 27: 229-265Crossref PubMed Scopus (1863) Google Scholar, 5Neven B. Callebaut I. Prieur A.M. Feldmann J. Bodemer C. Lepore L. Derfalvi B. Benjaponpitak S. Vesely R. Sauvain M.J. Oertle S. Allen R. Morgan G. Borkhardt A. Hill C. Gardner-Medwin J. Fischer A. de Saint Basile G. Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cell-mediated autoinflammatory disorders CINCA/NOMID, MWS, and FCU.Blood. 2004; 103: 2809-2815Crossref PubMed Scopus (252) Google Scholar It has been reported that various human inflammatory disorders may result from the dysregulated production of IL-1β derived from Nlrp3 inflammasomes, including atherosclerosis,6Duewell P. Kono H. Rayner K.J. Sirois C.M. Vladimer G. Bauernfeind F.G. Abela G.S. Franchi L. Nunez G. Schnurr M. Espevik T. Lien E. Fitzgerald K.A. Rock K.L. Moore K.J. Wright S.D. Hornung V. Latz E. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.Nature. 2010; 464: 1357-1361Crossref PubMed Scopus (2570) Google Scholar inherited cryopyrin-associated periodic syndromes,7Mateeva V. Kadurina M. Cryopyrin-associated periodic syndrome caused by a novel mutation in the NLRP3 gene.Exp Dermatol. 2014; 23: 2Google Scholar gouts,8Martinon F. Petrilli V. Mayor A. Tardivel A. Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome.Nature. 2006; 440: 237-241Crossref PubMed Scopus (3760) Google Scholar glomerular sclerosis,9Abais J.M. Xia M. Zhang Y. Boini K.M. Li P.L. Redox regulation of NLRP3 inflammasomes: ROS as trigger or effector?.Antioxid Redox Signal. 2015; 22: 1111-1129Crossref PubMed Scopus (511) Google Scholar and type 2 diabetes.3Dinarello C.A. Donath M.Y. Mandrup-Poulsen T. Role of IL-1beta in type 2 diabetes.Curr Opin Endocrinol Diabetes Obes. 2010; 17: 314-321PubMed Google Scholar The formation and activation of Nlrp3 inflammasomes by the adipokine visfatin may be critical for instigating endothelial inflammatory response, leading to endothelial dysfunction and arterial inflammation and thereby initiating atherosclerosis during obesity.10Xia M. Boini K.M. Abais J.M. Xu M. Zhang Y. Li P.L. Endothelial NLRP3 inflammasome activation and enhanced neointima formation in mice by adipokine visfatin.Am J Pathol. 2014; 184: 1617-1628Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar However, so far it remains unknown how the inflammasome activation is regulated within vascular cells and what mechanism controls inflammasome products to be transported and metabolized given that they are not synthesized through classical protein synthesis pathways through the endoplasmic reticulum. Autophagy is a cellular process through which cytoplasmic components, such as damaged organelles and protein aggregates, are delivered to lysosomes for degradation and recycled back.11Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (3918) Google Scholar, 12Feng Y. He D. Yao Z. Klionsky D.J. The machinery of macroautophagy.Cell Res. 2014; 24: 24-41Crossref PubMed Scopus (1318) Google Scholar Basal-level autophagy in various tissues maintains constitutive turnover of cytosolic components. However, starvation autophagy is stimulated to recycle nutrients and generate energy for cell survival under stress (oxidative insult) conditions.11Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (3918) Google Scholar As an evolutionarily conserved process, autophagy plays an essential role in various cellular processes, such as the clearance of pathogens and cell death, thereby being importantly implicated in human physiology and diseases.13Lavandero S. Troncoso R. Rothermel B.A. Martinet W. Sadoshima J. Hill J.A. Cardiovascular autophagy: concepts, controversies, and perspectives.Autophagy. 2013; 9: 1455-1466Crossref PubMed Scopus (134) Google Scholar Because different pharmacologic compounds can modulate autophagic machinery, this process is also a potential therapeutic target for a variety of diseases.14Rubinsztein D.C. Codogno P. Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases.Nat Rev Drug Discov. 2012; 11: 709-730Crossref PubMed Scopus (1130) Google Scholar Given the critical role of autophagy in turnover of organelles or other intracellular molecules via elimination of damaged and aged cellular components, it may also be involved in the control of Nlrp3 inflammasome formation and activation because various organelle stressors activate Nlrp3 inflammasomes, such as damaged mitochondria, phagosome-lysosome, and endoplasmic reticuli if they are not entirely degraded via the autophagic process. In addition, several stimulatory metabolites activate Nlrp3 inflammasomes in vascular tissues, such as cholesterol crystals and monosodium urate crystals, and their action mechanism is associated with lysosome injury, which may change the autophagic process.15Elliott E.I. Sutterwala F.S. Initiation and perpetuation of NLRP3 inflammasome activation and assembly.Immunol Rev. 2015; 265: 35-52Crossref PubMed Scopus (525) Google Scholar, 16Latz E. Xiao T.S. Stutz A. Activation and regulation of the inflammasomes.Nat Rev Immunol. 2013; 13: 397-411Crossref PubMed Scopus (2015) Google Scholar Therefore, autophagy may suppress Nlrp3 inflammasome activation via reducing the activation of Nlrp3 inflammasome induced by organelle stress,17Jabir M.S. Hopkins L. Ritchie N.D. Ullah I. Bayes H.K. Li D. Tourlomousis P. Lupton A. Puleston D. Simon A.K. Bryant C. Evans T.J. Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy.Autophagy. 2015; 11: 166-182Crossref PubMed Scopus (108) Google Scholar, 18Lamkanfi M. Kanneganti T.D. Van Damme P. Vanden Berghe T. Vanoverberghe I. Vandekerckhove J. Vandenabeele P. Gevaert K. Nunez G. Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes.Mol Cell Proteomics. 2008; 7: 2350-2363Crossref PubMed Scopus (237) Google Scholar, 19Zhou R. Yazdi A.S. Menu P. Tschopp J. A role for mitochondria in NLRP3 inflammasome activation.Nature. 2011; 469: 221-225Crossref PubMed Scopus (3447) Google Scholar whereas the loss of autophagy may result in inflammasome activation, leading to different inflammatory disorders, such as atherosclerosis, pneumonia, diabetes, sepsis, and colitis.20Guo W. Sun Y. Liu W. Wu X. Guo L. Cai P. Shen Y. Shu Y. Gu Y. Xu Q. Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer.Autophagy. 2014; 10: 972-985Crossref PubMed Scopus (195) Google Scholar There is also evidence that inducers of autophagy may attenuate the production of mature IL-1β and IL-18 via the Nlrp3 inflammasome pathway.21Martins J.D. Liberal J. Silva A. Ferreira I. Neves B.M. Cruz M.T. Autophagy and inflammasome interplay.DNA Cell Biol. 2015; 34: 274-281Crossref PubMed Scopus (45) Google Scholar, 22Harris J. Hartman M. Roche C. Zeng S.G. O'Shea A. Sharp F.A. Lambe E.M. Creagh E.M. Golenbock D.T. Tschopp J. Kornfeld H. Fitzgerald K.A. Lavelle E.C. Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.J Biol Chem. 2011; 286: 9587-9597Crossref PubMed Scopus (607) Google Scholar Nlrp3 inflammasome activation has been reported to mediate early inflammatory responses after vascular injury and that increased production of IL-1β and IL-18 are associated with human plaque vulnerability and atherogenesis.23Paramel Varghese G. Folkersen L. Strawbridge R.J. Halvorsen B. Yndestad A. Ranheim T. Krohg-Sorensen K. Skjelland M. Espevik T. Aukrust P. Lengquist M. Hedin U. Jansson J.H. Fransen K. Hansson G.K. Eriksson P. Sirsjo A. NLRP3 inflammasome expression and activation in human atherosclerosis.J Am Heart Assoc. 2016; 5: e003031Crossref PubMed Scopus (186) Google Scholar, 24Shi X. Xie W.L. Kong W.W. Chen D. Qu P. Expression of the NLRP3 inflammasome in carotid atherosclerosis.J Stroke Cerebrovasc Dis. 2015; 24: 2455-2466Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Studies from our group reported that the CD38 signaling pathway is critical for autophagy maturation and deficient CD38 signaling and consequent dysregulation of autophagy may lead to arterial wall thickening as shown in CD38−/− mice.25Xiong J. Xia M. Xu M. Zhang Y. Abais J.M. Li G. Riebling C.R. Ritter J.K. Boini K.M. Li P.L. Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes.J Cell Mol Med. 2013; 17: 1598-1607Crossref PubMed Scopus (28) Google Scholar, 26Bao J.X. Zhang Q.F. Wang M. Xia M. Boini K.M. Gulbins E. Zhang Y. Li P.L. Implication of CD38 gene in autophagic degradation of collagen I in mouse coronary arterial myocytes.Front Biosci (Landmark Ed). 2017; 22: 558-569Crossref PubMed Scopus (9) Google Scholar Zhang et al27Zhang Y. Xu M. Xia M. Li X. Boini K.M. Wang M. Gulbins E. Ratz P.H. Li P.L. Defective autophagosome trafficking contributes to impaired autophagic flux in coronary arterial myocytes lacking CD38 gene.Cardiovasc Res. 2014; 102: 68-78Crossref PubMed Scopus (43) Google Scholar reported that CD38 gene deletion impaired autophagic flux and caused defective autophagosome trafficking in coronary artery smooth muscle cells (CASMCs), which is attributed to atherogenesis. However, it remains unknown how autophagic flux derangement leads to atherosclerotic pathology in coronary arteries and whether it is associated with Nlrp3 inflammasome activation. This study was designed to answer these essential questions. CD38 wild-type (CD38+/+) and CD38 knockout (CD38−/−) C57BL/6J male mice (8 to 12 weeks old) were used in this study. All mice were maintained in the environmentally controlled room (25°C and approximately 40% to 50% humidity) with a 12-hour light/dark cycle. Both CD38+/+ and CD38−/− mice were randomly separated into different experimental groups and fed with normal diet (ND) or Western diet (WD) for 8 weeks. All procedures were performed in accordance with the NIH's Guide for the Care and Use of Laboratory Animals28Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar and were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University. CASMC isolation from mice has been described previously.29Adhikari N. Shekar K.C. Staggs R. Win Z. Steucke K. Lin Y.W. Wei L.N. Alford P. Hall J.L. Guidelines for the isolation and characterization of murine vascular smooth muscle cells. A report from the International Society of Cardiovascular Translational Research.J Cardiovasc Transl Res. 2015; 8: 158-163Crossref PubMed Scopus (21) Google Scholar Briefly, 2% isoflurane was used to anesthetize the mice. Then, the carotid arteries were removed and put into phosphate-buffered saline (PBS) on ice. The adventitia was removed from the artery using angled forceps under the microscope. The tissue was washed three times with PBS and was cut into pieces that were approximately 1 to 2 mm using microdissecting scissors in the cell culture hood. The dissected tissues were washed two to three times with cell culture medium and then added into a cell culture dish without medium for 2 hours. Then, fresh medium was added into the tissue culture dish, and tissues were incubated in a humidified 37°C, 5% carbon dioxide incubator. Dulbecco's modified Eagle's medium supplemented with 10% FBS and 2% antibiotics was used to culture tissues. CASMCs were isolated when they spread out from the dissected tissue. After 5 to 10 days, CASMCs were cloned by picking those cells from cell-yielding islands in the dish. The identification and purity of CASMCs in culture were confirmed as described previously.30Xu M. Zhang Y. Xia M. Li X.X. Ritter J.K. Zhang F. Li P.L. NAD(P)H oxidase-dependent intracellular and extracellular O2*- production in coronary arterial myocytes from CD38 knockout mice.Free Radic Biol Med. 2012; 52: 357-365Crossref PubMed Scopus (31) Google Scholar Western blot analysis was performed as described previously.31Yuan X. Wang L. Bhat O.M. Lohner H. Li P.L. Differential effects of short chain fatty acids on endothelial Nlrp3 inflammasome activation and neointima formation: antioxidant action of butyrate.Redox Biol. 2018; 16: 21-31Crossref PubMed Scopus (65) Google Scholar Briefly, cell culture dish was placed on ice and the cells were washed with ice-cold PBS. Total protein was extracted using ice-cold lysis buffer with protein inhibitor. The cell suspension was maintained on ice for 30 minutes. After centrifugation at 12,300 × g for 15 minutes at 4°C, the supernatant was transferred to a new tube kept on the ice. Protein concentration was measured and resuspended to 2 μg/μL in loading buffer. The equal amount of protein was loaded into the wells of SDS-PAGE gel along with the molecular weight marker. The gel was run at 100 V for 2 hours at room temperature and then transferred to a polyvinylidene difluoride membrane at 100 V for 1 hour at 4°C. The membrane was blocked with 5% nonfat milk in Tris-buffered saline and Tween 20 buffer at room temperature for 30 minutes. The membrane was incubated with primary antibodies against pro–caspase-1 or cleaved caspase-1 (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer overnight at 4°C, followed by incubation with the secondary antibody labeled with horseradish peroxidase for 1 hour at room temperature. The membrane was developed with the Odyssey FC Imaging System after being washed three times with Tris-buffered saline and Tween 20 at room temperature. β-Actin (1:8000 dilution, Santa Cruz) was reported to serve as a loading control. The intensity of the bands was quantified using ImageJ version 6.0 (NIH, Bethesda, MD; https://imagej.nih.gov/ij). Cells cultured in eight-well plates or coronary arteries on frozen slides were rinsed in PBS for 5 minutes and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 minutes at room temperature. The samples were washed three times in PBS for 5 minutes and were incubated for 10 minutes with PBS that contained 0.1% Triton X-100. Then the samples were washed three times for 5 minutes in PBS and incubated with 3% bovine serum albumin (BSA) in PBS for 30 minutes to block unspecific binding of antibodies. The samples were incubated with the diluted primary antibody in 3% BSA overnight at 4°C in a humidified chamber followed by incubation with either Alexa-488– or Alexa-555–labeled secondary antibody in 3% BSA for 1 hour at room temperature in the dark room. The sections were washed three times with PBS for 10 minutes each in the darkroom to decant the secondary antibody solution. Finally, the sections were mounted with a drop of mounting medium with DAPI and sealed with nail polish for taking images using the confocal laser scanning microscope (710 LSM; Zeiss, Dublin, CA). Image ProPlus software version 6.0 (Media Cybernetics, Bethesda, MD) was used to measure the colocalization of Nlrp3 with Asc or caspase-1. Pearson correlation coefficients (PCCs) were expressed to summarize the data of colocalization efficiency as described previously.32Abraham N.G. Sodhi K. Silvis A.M. Vanella L. Favero G. Rezzani R. Lee C. Zeldin D.C. Schwartzman M.L. CYP2J2 targeting to endothelial cells attenuates adiposity and vascular dysfunction in mice fed a high-fat diet by reprogramming adipocyte phenotype.Hypertension. 2014; 64: 1352-1361Crossref PubMed Scopus (55) Google Scholar, 33Xia M. Zhang C. Boini K.M. Thacker A.M. Li P.L. Membrane raft-lysosome redox signalling platforms in coronary endothelial dysfunction induced by adipokine visfatin.Cardiovasc Res. 2011; 89: 401-409Crossref PubMed Scopus (51) Google Scholar The mouse hearts were perfused with PBS and fixed in 10% formalin at room temperature. The samples were embedded in paraffin, and 6-μm slides were cut from the embedded blocks. The slides were heated on the hot plate at 65°C for 10 minutes and were put in xylene for 10 minutes to remove the paraffin. Rehydration was performed in graded ethanol (100%, 95%, 75%) and water. Antigen retrieval was performed in sodium citrate buffer (pH 6.0) at 98°C for 15 minutes. Three percent hydrogen peroxide in methanol was used to quench the endogenous peroxidase activity. The samples were blocked with 2.5% horse serum for 1 hour at room temperature. Incubation with the indicated primary antibodies anti–IL-1β (1:100) diluted in PBS was performed overnight at 4°C. After rinsing three times with PBS, the samples were incubated with biotinylated secondary antibodies and a streptavidin-peroxidase complex for 30 minutes at room temperature. Then the samples were sequentially developed with 3,3′-diaminobenzidine solution for 5 minutes. Finally, the sections were counterstained in hematoxylin for 5 minutes, dehydrated in graded ethanol (75%, 95%, 100%), and mounted with Permount. Negative controls were prepared without the primary antibodies. The area percentage of the positive staining was calculated using Image Pro-Plus software version 6.0.34Zhang C. Yi F. Xia M. Boini K.M. Zhu Q. Laperle L.A. Abais J.M. Brimson C.A. Li P.L. NMDA receptor-mediated activation of NADPH oxidase and glomerulosclerosis in hyperhomocysteinemic rats.Antioxid Redox Signal. 2010; 13: 975-986Crossref PubMed Scopus (38) Google Scholar Caspase-1 activity was performed with the similar protocol described previously.35Chen Y. Pitzer A.L. Li X. Li P.L. Wang L. Zhang Y. Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.J Cell Mol Med. 2015; 19: 2715-2727Crossref PubMed Scopus (75) Google Scholar The FAM-FLICA Caspase-1 Assay Kit (ImmunoChemistry Technologies, LLC, Bloomington, MN) was used to label active caspase-1 enzyme in the frozen sections of coronary artery in the heart. The sections were washed three times in PBS for 5 minutes at room temperature and were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 minutes at room temperature. The samples were washed three times in PBS for 5 minutes and were incubated for 10 minutes with PBS that contained 0.1% Triton X-100. Then the samples were washed three times for 5 minutes in PBS and incubated with 3% BSA in PBS for 30 minutes to block unspecific binding of antibodies. The sections were incubated with anti-rabbit α-smooth muscle actin (α-SMA) (1:500; Abcam, Cambridge, UK) overnight at 4°C. After being washed three times in PBS, the sections were co-stained with Alexa-555–labeled anti-rabbit secondary antibody and fluorescent-labeled inhibitor of caspases (FLICA) reagent (1:10) in the kit for 1.5 hours at room temperature. The sections were washed three times with PBS for 10 minutes each in the darkroom to decant the secondary antibody solution and FLICA reagent. Finally, the samples were mounted with a drop of mounting medium with DAPI and sealed with nail polish for taking images using a confocal laser scanning microscope (710 LSM). Image Pro-Plus software version 6.0 was used to measure the colocalization of von Willebrand factor with FLICA. PCCs were expressed to summarize the data of colocalization efficiency as described previously.32Abraham N.G. Sodhi K. Silvis A.M. Vanella L. Favero G. Rezzani R. Lee C. Zeldin D.C. Schwartzman M.L. CYP2J2 targeting to endothelial cells attenuates adiposity and vascular dysfunction in mice fed a high-fat diet by reprogramming adipocyte phenotype.Hypertension. 2014; 64: 1352-1361Crossref PubMed Scopus (55) Google Scholar Hematoxlin and eosin staining was performed to observe the morphologic changes as described previously.36Zhang R.Z. Qiu H. Wang N. Long F.L. Mao D.W. Effect of Rheum palmatum L. on NF-kappaB signaling pathway of mice with acute liver failure.Asian Pac J Trop Med. 2015; 8: 841-847Crossref PubMed Scopus (24) Google Scholar Briefly, hearts were separated from the base of the aorta and transversely cut into two parts. The half that contained the apex was stored at −80°C and the other half was immersed in 10% neutral buffered formalin for >48 hours. The formalin-fixed heart was embedded with paraffin and then cut into 6-μm serial sections for histopathologic evaluation. For hematoxylin and eosin staining, sections were deparaffinized heated on the hot plate at 65°C for 10 minutes and were put in xylene for 10 minutes. Rehydration was performed in graded ethanol (100%, 95%, 75%) and water. After being washed with running water, the sections were immersed in the hematoxylin and hydrochloride alcohol. As soon as the color turned to antiblue, the sections were stained with eosin. After that, sections were washed with running water and dehydrated with water and different graded ethanol (75%, 95%, 100%). Finally, the slides were mounted with Permount (xylene based) and were photographed under the microscope. Medial thickening was measured using Image Pro-Plus software version 6.0. The medial area was determined by subtraction of the lumen area from the outer medial area. The three thickest vessels were chosen to calculate the means of vessel medial thickness and medial thickness-to-lumen radius ratio for each mouse.37Liu J.L. Bishop S.P. Overbeck H.W. Morphometric evidence for non-pressure-related arterial wall thickening in hypertension.Circ Res. 1988; 62: 1001-1010Crossref PubMed Scopus (33) Google Scholar Data are presented as means ± SEM. Significant differences between and within multiple groups were examined using analysis of variance followed by Duncan's multiple-range test. Sigmaplot software version 12.5 (Systat Software, San Jose, CA) was used to perform the statistical analysis. Differences were considered statistically significant at P < 0.05. Because autophagy and associated lysosome function serve as critical modulators in Nlrp3 inflammasome formation on proatherogenic stimulation, inflammasome formation in 7-ketocholesterol (7-Ket)–treated mouse CASMCs from wild-type (CD38+/+) mice and mice with deletion of CD38 gene (CD38−/−) were first analyzed to determine whether lysosome dysfunction associated with CD38 deficiency is involved in the formation of Nlrp3 inflammasomes. By confocal microscopy, the colocalization (yellow spots within cells) of Nlrp3 with Asc or caspase-1 was found to be increased on 7-Ket stimulation and in cells with CD38 deletion, indicating the aggregation or assembly of these inflammasome molecules (Figure 1, A and C ). Figure 1, B and D, shows the PCCs of Nlrp3 with Asc or caspase-1, representing their colocalization efficiency. Both 7-Ket stimulation and CD38 deletion significantly increased Nlrp3 inflammasome formation. However, an autophagy enhancer, rapamycin (Rap), significantly decreased the colocalization of Nlrp3 versus Asc or caspase-1 induced by 7-Ket stimulations in both CD38+/+ and CD38−/− CASMCs. Therefore, the induction of autophagy by Rap may prevent the formation of the Nlrp3 inflammasome, but the deficiency of autophagy induced the formation of the Nlrp3 inflammasome. Next, Nlrp3 inflammasome activation was analyzed by measurement of the active caspase-1 level (19 kDa) by Western blot analysis, spectrometric assay of caspase-1 activity, and enzyme-linked immunosorbent assay quantitation of IL-1β production in CASMCs. 7-Ket significantly increased cleavage of pro–caspase-1 into bioactive caspase-1 in CD38+/+ CASMCs, which was blocked by Rap treatment (Figure 2A). In CD38−/− CASMCs, even under the basal condition, active caspase-1 levels were increased (Figure 2B), suggesting that deletion of the CD38 gene may activate the Nlrp3 inflammasome, thereby causing cleavage of pro–caspase-1 into bioactive caspase-1. 7-Ket had no further effect on the level of active caspase-1 in CD38−/− CASMCs, whereas Rap caused a significant decrease both under control conditions and on 7-Ket stimulation (Figure 2, A and B). Increased caspase-1 activity and IL-1β production was observed in 7-Ket–stimulated CD38+/+ CASMCs, and increased activation of the Nlrp3 inflammasome was substantially blocked by Rap (Figure 2, C and D). In addition, deletion of the CD38 gene caused a significant increase in caspase-1 activity and IL-1β production, which indicates that the CD38 gene is implicated in Nlrp3 inflammasome activation. In these CD38−/− CASMCs, treatment with 7-Ket did not show a significant increase in caspase-1 activity and IL-1β production, whereas Rap reduced caspase-1 activity and IL-1β production. It seems that improving lysosome function and autophagic flux by Rap treatment antagonized the activating effects of 7-Ket and CD38 gene deletion on Nlrp3 inflammasomes. Whether inhibition of autophagosome generation and direct suppression of lysosome function are associated with Nlrp3 inflammasome activity was tested. Both inhibition of autophagosome formation by spautin-1 (Spa-1) and suppression of vacuolar H(+)-ATPase in the lysosome by bafilomycin A1 (Baf) markedly increased colocalization of Nlrp3 with Asc or caspase-1 in wild-type CASMCs, whether these ce" @default.
• W2893876947 created "2018-10-05" @default.
• W2893876947 creator A5003769038 @default.
• W2893876947 creator A5018188465 @default.
• W2893876947 creator A5019509284 @default.
• W2893876947 creator A5035736739 @default.
• W2893876947 creator A5041993697 @default.
• W2893876947 date "2018-12-01" @default.
• W2893876947 modified "2023-10-14" @default.
• W2893876947 title "Protective Role of Autophagy in Nlrp3 Inflammasome Activation and Medial Thickening of Mouse Coronary Arteries" @default.
• W2893876947 cites W1503602955 @default.
• W2893876947 cites W1523693806 @default.
• W2893876947 cites W1638567831 @default.
• W2893876947 cites W1790743604 @default.
• W2893876947 cites W1803405992 @default.
• W2893876947 cites W1821547577 @default.
• W2893876947 cites W1900592574 @default.
• W2893876947 cites W1908261460 @default.
• W2893876947 cites W1963705367 @default.
• W2893876947 cites W1971915082 @default.
• W2893876947 cites W1972483153 @default.
• W2893876947 cites W1973968003 @default.
• W2893876947 cites W1974885063 @default.
• W2893876947 cites W1975032025 @default.
• W2893876947 cites W1985124617 @default.
• W2893876947 cites W1985538633 @default.
• W2893876947 cites W1991692801 @default.
• W2893876947 cites W1993513350 @default.
• W2893876947 cites W2002394315 @default.
• W2893876947 cites W2009435405 @default.
• W2893876947 cites W2009800174 @default.
• W2893876947 cites W2018607502 @default.
• W2893876947 cites W2019256890 @default.
• W2893876947 cites W2028545985 @default.
• W2893876947 cites W2036791933 @default.
• W2893876947 cites W2043260789 @default.
• W2893876947 cites W2043291430 @default.
• W2893876947 cites W2044621729 @default.
• W2893876947 cites W2051043379 @default.
• W2893876947 cites W2054593239 @default.
• W2893876947 cites W2062526619 @default.
• W2893876947 cites W2068633454 @default.
• W2893876947 cites W2076062022 @default.
• W2893876947 cites W2077549620 @default.
• W2893876947 cites W2084777655 @default.
• W2893876947 cites W2104388295 @default.
• W2893876947 cites W2105640184 @default.
• W2893876947 cites W2114208319 @default.
• W2893876947 cites W2114769428 @default.
• W2893876947 cites W2117738774 @default.
• W2893876947 cites W2123118757 @default.
• W2893876947 cites W2125922781 @default.
• W2893876947 cites W2129155846 @default.
• W2893876947 cites W2130225602 @default.
• W2893876947 cites W2152174954 @default.
• W2893876947 cites W2155463348 @default.
• W2893876947 cites W2158671450 @default.
• W2893876947 cites W2159495392 @default.
• W2893876947 cites W2160854976 @default.
• W2893876947 cites W2230823892 @default.
• W2893876947 cites W2316915051 @default.
• W2893876947 cites W2395774853 @default.
• W2893876947 cites W2548362415 @default.
• W2893876947 cites W2560056218 @default.
• W2893876947 cites W2743254891 @default.
• W2893876947 cites W2756493627 @default.
• W2893876947 cites W2789375475 @default.
• W2893876947 doi "https://doi.org/10.1016/j.ajpath.2018.08.014" @default.
• W2893876947 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6334256" @default.
• W2893876947 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30273598" @default.
• W2893876947 hasPublicationYear "2018" @default.
• W2893876947 type Work @default.
• W2893876947 sameAs 2893876947 @default.
• W2893876947 citedByCount "25" @default.
• W2893876947 countsByYear W28938769472019 @default.
• W2893876947 countsByYear W28938769472020 @default.
• W2893876947 countsByYear W28938769472021 @default.
• W2893876947 countsByYear W28938769472023 @default.
• W2893876947 crossrefType "journal-article" @default.
• W2893876947 hasAuthorship W2893876947A5003769038 @default.
• W2893876947 hasAuthorship W2893876947A5018188465 @default.
• W2893876947 hasAuthorship W2893876947A5019509284 @default.
• W2893876947 hasAuthorship W2893876947A5035736739 @default.
• W2893876947 hasAuthorship W2893876947A5041993697 @default.
• W2893876947 hasBestOaLocation W28938769471 @default.
• W2893876947 hasConcept C126322002 @default.
• W2893876947 hasConcept C126348684 @default.
• W2893876947 hasConcept C142724271 @default.
• W2893876947 hasConcept C164705383 @default.
• W2893876947 hasConcept C185592680 @default.
• W2893876947 hasConcept C190283241 @default.
• W2893876947 hasConcept C203522944 @default.
• W2893876947 hasConcept C2776820930 @default.
• W2893876947 hasConcept C2776914184 @default.
• W2893876947 hasConcept C2777209026 @default.
• W2893876947 hasConcept C2778742706 @default.
• W2893876947 hasConcept C2779559920 @default.
• W2893876947 hasConcept C55493867 @default.

• next