| Background:Atherosclerosis is the primary cause of myocardial infarction,it is the leading cause of deaths in the world.The continuous accumulation of foam cells is a key factor in the initiation and development of plaques.It can promote the formation of new blood vessels in the plaques,leading to the formation of a large number of lipid pools and necrotic cores,which ultimately exacerbates the development of unstable plaques.Therefore,to elucidate the molecular mechanism of foam cell formation during the progression of atherosclerotic plaques and reduce the incidence of acute coronary syndrome is a key issue that cardiovascular doctors need to solve urgently.Atherosclerosis is a chronic inflammatory disease associated with lipids dysfunction.Mounting evidence has shown that lipid metabolism of macrophages in atherosclerotic plaques is impaired.The lipid droplets（LDs）is the major site of cholesterol and triglycerides storage in macrophage foam cells.The term"lipophagy"refers to the removal of lipid droplets from macrophage and smooth muscle cells.Defective lipophagy is involved in the pathological processes of diseases related to metabolic disorders,such as fatty liver,obesity and atherosclerosis.Vascular endothelial cells can inhibit the adhesion of inflammatory cells and the formation of thrombus.Therefore,endothelial dysfunction plays a vital role in the development of atherosclerosis.Endothelial cells feature several adhesion molecules at the cell surface such as vascular cell adhesion molecule-1（VCAM-1）,intercellular adhesion molecule-1（ICAM-1）,and platelet selectin（P-selectin）.These molecules are upregulated by atherogenic stimuli and thereby contribute to the adhesion and infiltration of macrophages.Therefore,vascular endothelium plays a key role in recruiting macrophages to accelerate the progression of atherosclerosis.The roles of macrophage autophagy in the progression and stability of atherosclerotic plaques,macrophage lipid metabolism disorder and foam cell formation will be investigated in vivo and in vitro.【Objective】1.To investigate the role of Sirt6 in the area and stability of atherosclerotic plaques.2.To investigate the role of Sirt6 in macrophage autophagy flux,macrophage adhesion and infiltration and apoptosis of macrophages in the progression of atherosclerosis.3.To clarify the effects of macrophage specific Sirt6 knockout on the accumulation of lipid droplets,the pathway of lipid droplet degradation,the formation of foam cells,and the level ofβoxidation in macrophages.4.Clarify the role of lipophagy in foam cell formation and confirm that Sirt6/Wnt1-mediated lipophagy can improve macrophage lipid metabolism disorder.【Methods】1.Six to Eight-week-old of WT,Apo E-/-,Apo E-/-:Sirt6-/-和Apo E-/-:Sirt6 Tg mice were fed 4-month high-fat-diet（15%fat,1.25%cholesterol and 0.2%bile acid salt）which helps to build the atherosclerosis model.2.HE and Oil red O staining were performed to detect the percentage of plaque area and the positive area of necrotic core.3.The positive area of CD68 andα-SMA in plaque were detected by immunohistochemical staining and the content of collagen in plaque was detected by masson staining.4.Immunofluorescence staining:all sections were blocked with goat serum for 1h and then incubated with CD68 antibodies at 4℃.The sections were then stained with rabbit anti-mouse secondary antibody（1:1,000;Abcam）for 1 hour at room temperature.The tissue slices were washed and mounted with medium containing DAPI.All slices were observed by the Olympus FV1000 laser confocal microscope.5.Cell culture,24 hours incubation of ox-LDL（50μg/m L）or 30 hours incubation of Ac-LDL（50μg/m L）were conducted to facilitate the formation of foam cells.6.The autophagic flux level was investigated by the conduction of GFP-m RFP-LC3adenovirus.7.The autophagosomes and lipid droplets were detected by transmission electron microscopy.8.The level of macrophage apoptotic indux was detected by Tunel staining with fluorescence microscopy.9.The expression of LC3,p62,beclin1,Sirt6,ADFP,PLIN2,Wnt1,β-catenin,and SNF2H were detected by Western blot.10.The interaction of Sirt6 and SNF2H were detected by Immunofluorescence staining and CO-IP experiment.11.The green fluorescent Bodipy493/503 were performed to detect the lipid droplets by confocal microscope.12.Mitochondrial oxygen consumption rate（mt OCR）was performed to detect theβ-oxidation in foam cells.13.Colocalization of lipid droplets and lysosomes was detected by Immunofluorescence staining:macrophages were treated with acetylated low-density lipoprotein for 30 hours.Before observation with a fluorescent microscope,Bodipy（10μg/m L）and Lyso-tracker（50 n M）were Co-stained for 30 min.【Results】1.Sirt6 deficiency augmented atherosclerosis plaque size.Sirt6 overexpression increased the plaque stability score.2.Sirt6 overexpression enhanced autophagy flux in macrophages,reducing contact with endothelial cells,thus leading to reduced infiltration of macrophages.Sirt6 inhibition increased macrophage apoptosis.3.Expression of Sirt6 was observed in macrophage-derived foam cells.Sirt6overexpression decreased the number and size of lipid droplets in macrophages.4.Autophagy contributes to lipid metabolism of macrophages in atherosclerosis.5.Sirt6 overexpression stimulated lipid droplet catabolism in macrophage foam cells via autophagic-lysosome degradation pathway.Sirt6 increases foam cell lipolysis and oxidative metabolism through autophagy.6.Sirt6 interacts with the chromatin remodeler SNF2H to inhibit Wnt1 signaling,thus promoting lipophagy and regulating lipid metabolism disorder in macrophages.【Conclusion】1.The continuous progression and instability of atherosclerotic plaques are related to the defective autophagy and decreased Sirt6 expression.2.Dysregulated lipid metabolism in macrophages is related to decreased Sirt6 expression in the progression of atherosclerosis.3.Sirt6 overexpression in macrophages can increase autophagy levels,reduce the contact with endothelial cells and macrophage infiltration.Furthermore,decreases macrophage apoptosis and stimulates the stability score.4.Sirt6 reduces the number and size of lipid droplets and decreases the expression of lipid droplet-associated protein in macrophages by regulating the Wnt1/β-catenin pathway.It will help develop a new treatment strategy to reduce the incidence of atherosclerosis. |
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