| Background:According to the World Health Organization,with the occurrence of High blood pressure,Diabetes,Obesity and Hypercholesteremia,there is a rise in the tendency of atherosclerosis which will give rise to Myocardial infarction,Stroke and Sudden death.Thus,to achieve the ultimate aim of decreasing the mortality of atherosclerosis,it is absolutely necessary to make a point of the role by which cell signaling conducts that involved in the pathogenesis of atherosclerosis.In general,atherosclerosis has a detrimental effect on human,health.Circulation research and other reports suggest that atherosclerosis is a chronic inflammatory disease which is the leading cause of death in developing countries.Emerging evidence favors the notion that macrophage autophagy plays a prominent role in the pathogenesis of vulnerable plaque,suggesting the therapeutic potential of targeting autophagy in atherosclerosis.Here ApoE-/-mice were crossed with Mst1 knockout or Mst1 Tg mice to generate ApoE-/-: Mst1-/-and ApoE-/-: Mst1 Tg mice.All animals were fed high-fat-diet for 4 months to induce arterial atherosclerosis.Murine macrophage RAW264.7 cells were subjected to ox-LDL(50μg/m L)in an effort to examine the cellular mechanisms.A significant increase in the levels of Mst1 and p-Mst1 was observed in the aorta of ApoE-/-mice.Mst1 knockout significantly reduced atherosclerotic area,decreased lipid core area and macrophage accumulation as compared with ApoE-/-mice.Along the same line,Mst1 overexpression increased plaque area,lipid core and macrophage accumulation as compared with ApoE-/-mice.Mst1 deficiency significantly increased levels of Beclin1 and LC3 II,while decreased that of p62 in aortic atherosclerosis.Moreover,in vitro data indicated that Mst1 knockdown prompted more typical autophagosomes upon ox-LDL challenge.Mst1 knockdown also enhanced autophagic flux as evidenced by GFP-mRFP-LC3 staining,increased LC3-II expression and decreased p62 expression in the presence of bafilomycin A1.Mst1 knockdown decreased,while Mst1 overexpression increased macrophage apoptosis upon ox-LDL exposure.In conclusion,Mst1 deficiency diminishes atherosclerosis and stabilizes atherosclerotic plaques in ApoE-/-mice.Mst1 may participate in atherosclerosis progression through inhibition of macrophage autophagy and promotion of macrophage apoptosis.【Objective】 1.To investigate the role of Mst1 in the process of atherosclerosis by which will provide anew therapy target for atherosclerosis.2.To investigate the clear mechanism in the pathogenesis of stabilizing theatherosclerosis plaque 3.To strike a balance between macrophage autophagy and macrophage apoptosis inwhich the process of atherosclerosis will be inhibited.【Methods】 1.Animal handling,ApoE-/-mice purchased from Jackson Laboratories(Bar Harbor,Maine,USA)were crossed with Mst1 knockout or Mst1 Tg mice to generate ApoE-/-: Mst1-/-and ApoE-/-: Mst1 Tg mice.Eight-week-old wild type(WT),ApoE-/-,Mst1 Tg,Mst1-/-,ApoE-/-: Mst1 Tg and ApoE-/-: Mst1-/-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,Mice were executed and the aorta was isolated.HE staining was performed according to the manufacturer’s instruction.The aortic sections were fixed with 4% PFA,washed with 60% isopropanol and stained with Oil red O(0.5% in 60% isopropanol)for 15 min,followed by counterstaining with haematoxylin.The reference luminal diameter,minimal luminal diameter,percent area stenosis and Oil red O positive staining area percentage were measured using the National Institutes of Health Image J software.3.Immunofluorescence staining,all the sections were blocked with 1% goat serum albumin for 1 hour and then incubated with mouse monoclonal macrophage CD68 antibody(Ab955,1:200;Abcam)at 4℃ overnight.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.4.Determination of plasma IL-6 and MCP-1 Activity,serum samples were collected from mice that were treated with high-fat-diet for 4 months.The concentrations of IL-6 and MCP-1 were measured by enzyme-linked immunosorbent assay(ELISA)according to the manufacturer’s instruction.Values were expressed as pg/m L.5.Cell culture,twenty-four hours incubation of ox-LDL(50μg/mL)was conducted to facilitate the formation of foam cells.6.The autophagic flux level were investigated by the conduction of GFP-mRFP-LC3 adenovirus.7.The autophagosomes were detected by transmission electron microscopy.8.The level of macrophage apoptotic indux were detected by Tunel staining with fluorescence microscopy.9.The expression of LC3,p62 and beclin1 and other autophagy related proteins were detected by Western blot.【Results】 1.Mst1 deficiency diminishes atherosclerosis in ApoE-/-miceApoE-/-,ApoE-/-: Mst1 Tg and ApoE-/-: Mst1-/-mice treated with high-fat-diet for 4 months developed marked atherosclerotic plaques in aorta.Measurement of plaque burden by HE staining revealed a 72.2% reduction in the atherosclerotic area in the ApoE-/-: Mst1-/-group as compared with the ApoE-/-group(3.29% ± 1.16 vs 12.56% ± 2.71).Consistently,the percentage of plaque area in the ApoE-/-: Mst1 Tg mice was increased by 62% as compared with those in ApoE-/-mice(20.64% ± 7.02 vs 12.56% ± 1.16).The minimal luminal diameter was increased,while percent area stenosis was decreased in the ApoE-/-: Mst1-/-group as compared with the ApoE-/-group.ApoE-/-: Mst1 Tg mice exhibited decreased minimal luminal diameter and increased percent area stenosis as compared with the ApoE-/-group.Interestingly,Mst1 overexpression or knockout had no significant effects on lipid profiles.2.Mst1 deficiency stabilize atherosclerotic plaque in ApoE-/-miceThe lipid core of atherosclerotic plaques was measured by Oil Red staining.Interestingly,Mst1 knockout caused significant decrease in Oil Red staining positive area as compared with the ApoE-/-mice(6.804% ± 0.707 vs 15.33% ± 1.387).Consistently,ApoE-/-: Mst1 Tg mice exhibited significantly increased Oil Red staining positive area in comparison with the ApoE-/-mice(24.85% ± 2.583 vs 15.33% ± 1.387).Macrophages engulf ox-LDL to form foam cells in atherosclerotic plaques.Aorta sections analyzed by immunofluorescence labeled with CD68 demonstrated that macrophage in the plaque of ApoE-/-: Mst1-/-mice was about 42.95% lower than the ApoE-/-mice(10.61% ± 1.105 vs 6.054% ± 0.757,P<0.05).ApoE-/-: Mst1 Tg mice exhibited increased CD68 positive area as compared with ApoE-/-mice(10.61% ± 1.105 vs 6.054% ± 0.757,P<0.05).Macrophages also secrete inflammatory factors and accelerate the death of smooth muscle cells leading to the deterioration of plague stability.To assess the function of macrophage,we measured IL-6 and MCP-1 expression in ApoE-/-mice treated with high-fat-diet for 4 months.Interestingly,Mst1 knockout decreased IL-6 expression and Mst1 overexpression increased IL-6 expression as compared with the ApoE-/-group.The present study also demonstrated that depletion of Mst1 resulted in reduced serum levels of IL-6(21 ± 4.743 pg/m L versus 42.6 ± 7.925 pg/mL,P<0.05)and MCP-1(312 ± 41.32 pg/m L versus 360 ± 41.23 pg/mL,P<0.05)in ApoE-/-: Mst1-/-mice as compared with that in ApoE-/-mice.Consistently,Mst1 overexpression increased the release of IL-6(66.6 ± 10.01 pg/m L versus 42.6 ± 7.925 pg/mL,P<0.05)and MCP-1(428.4 ± 47.73 pg/mL versus 360 ± 41.23 pg/m L,P<0.05)in ApoE-/-: Mst1 Tg mice as compared with that in ApoE-/-mice.3.Mst1 inhibition enhances autophagy flux in macrophageTransmission electron microscopy(TEM)revealed more typical lipid bilayer-containing autophagosomes in the macrophages transfected with Ad-sh-Mst1 subjected to 50 ug/mL ox-LDL administration.In consistence,significantly reduced number of autophagosomes was observed in Ad-Mst1 transfected macrophage as compared with the ox-LDL treated group.GFP-mRFP-LC3 staining was conducted to simultaneously examine the formation of autophagosomes and autolysosomes.In macrophages expressing GFP-mRFP-LC3,LC3 associated with autophagosomes can be visualized as puncta that are both red and green(appearing yellow in the merged image),whereas autolysosomes are visualized as puncta that are red only.Increased number of both yellow and red puncta was found in the Ad-sh-Mst1 transfected macrophages subjected to oX-LDL administration.Enhanced autophagic flux was also evidenced by increased LC3-II expression and decreased p62 expression in the presence of bafilomycin A1,a lysosomal inhibitor used to evaluate autophagic flux.Consistently,Ad-Mst1 transfected macrophage exhibited less yellow or red puncta,decreased LC3-II expression and increased p62 expression in the presence or absence of bafilomycin A1.4.Mst1 inhibition increases macrophage autophagyTo explore the role of Mst1 in atherosclerosis,expression of Mst1 and p-Mst1 were analyzed in the aorta of ApoE-/-mice.An increase in Mst1 and p-Mst1 expression was found in aorta of ApoE-/-mice as compared with that in WT mice.Ad-sh-Mst1 transfection significantly decreased,while Ad-Mst1 transfection markedly increased Mst1 and p-Mst1 expression in aorta of ApoE-/-mice.Interestingly,Ad-sh-Mst1 transfection up-regulated Beclin1,p-Beclin1 and LC3-II expression while inhibiting p62 in ApoE-/-mice.5.Mst1 inhibition decreases macrophage apoptosis subjects to ox-LDL administrationTUNEL-positive macrophages were less frequently observed in the ox-LDL + Ad-sh-Mst1 group as compared with the ox-LDL group.Quantitative analyses demonstrated that the number of TUNEL-positive macrophage was significantly less in the ox-LDL+Ad-sh-Mst1 group than in the ox-LDL group(0.146 ± 0.018 vs 0.20 ± 0.03,P = 0.009).A significant increased expression of p136-BAD and Bcl-2,decreased expression of Bax and Bax / Bcl-2 ratio were found in the ox-LDL treated macrophages transfected with Ad-sh-Mst1.Ad-Mst1 administration decreased p136-BAD and Bcl-2 expression,increased the expression of Bax and Bax / Bcl-2 ratio in macrophages.【Conclusion】This study provide a new therapy target for atherosclerosis,Mst1 participates in the process of atherosclerosis and lead to the plaque instsbility by inhibiting autophagy.Striking a balance beween macrophage autophagy and apoptosis and modulating them properly can prohibit the pathogenesis of atherosclerosis. |
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