title: PEP-1-MsrA ameliorates inflammation and reduces atherosclerosis in apolipoprotein E deficient mice.
abstract: BACKGROUND
  Methionine sulfoxide reductase A (MsrA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. However, therapeutic use of exogenous MsrA in oxidative stress-induced diseases is limited, because it cannot enter the cells. The aim of this study is to investigate whether MsrA with PEP-1, a cell penetrating peptide, fused to its N-terminus can protect against oxidative stress in macrophages and can attenuate atherosclerosis in apolipoprotein E deficient (apoE(-/-)) mice.
  
  
  METHODS
  MsrA and the fusion protein PEP-1-MsrA were expressed and purified using a pET28a expression system. Transduction of the fusion protein into macrophages was confirmed by Western blot and immunofluorescence staining. Intracellular reactive oxygen species (ROS) and apoptosis levels were measured by flow cytometry. In in vivo study, MsrA or PEP-1-MsrA proteins were intraperitoneally injected into apoE(-/-) mice fed a Western diet for 12 weeks. Plasma lipids levels, inflammatory gene expression, and paraoxonase-1 (PON1) and superoxide dismutase (SOD) activities were assessed. Atherosclerotic lesions were analyzed by Oil Red O staining and immunohistochemistry.
  
  
  RESULTS
  PEP-1-MsrA could penetrate the cells and significantly reduced intracellular ROS levels and apoptosis in H2O2-treated macrophages. It also decreased TNFα and IL-1β mRNA levels and increased the IL-10 mRNA level in lipopolysaccharide-treated macrophages. In in vivo study, PEP-1-MsrA injection significantly increased plasma PON1 and SOD activities and decreased plasma monocyte chemoattractant protein 1 (MCP-1) level compared to the injection of vehicle control or MsrA. In PEP-1-MsrA injected mice, hepatic PON1 levels were increased, while the expression of TNFα and IL-6 mRNA in the liver was suppressed. Although plasma total cholesterol and triglyceride levels did not change, the aortic atherosclerosis in PEP-1-MsrA treated mice was significantly reduced. This was accompanied by a reduction of total and apoptotic macrophages in the lesions.
  
  
  CONCLUSIONS
  Our study provides evidence that PEP-1-MsrA may be a potential therapeutic agent for atherosclerosis-related cardiovascular diseases.
authors.affiliation: Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. isabel_wuyao@whu.edu.cn.
authors.forename: Yao
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. 2008302210023@whu.edu.cn.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Xie
authors.affiliation: Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. xuyanyong@whu.edu.cn.
authors.forename: Yanyong
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. 2013203010022@whu.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: Cardiology Division of Wuhan University Zhongnan Hospital, Wuhan, China. tongchuanfeng@hotmail.com.
authors.forename: Chuanfeng
authors.initials: C
authors.lastname: Tong
authors.affiliation: Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC, USA. Daping.Fan@uscmed.sc.edu.
authors.forename: Daping
authors.initials: D
authors.lastname: Fan
authors.affiliation: Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. fen.du@whu.edu.cn.
authors.forename: Fen
authors.initials: F
authors.lastname: Du
authors.affiliation: Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. yu.hong@whu.edu.cn.
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics*; D017209:Apoptosis; D043303:Aryldialkylphosphatase / Q000378:metabolism; D050197:Atherosclerosis / Q000235:genetics* / Q000517:prevention & control; D002460:Cell Line; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation / Q000188:drug therapy; D008099:Liver / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008297:Male; D051150:Methionine Sulfoxide Reductases / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010455:Peptides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D001057:Apolipoproteins E; C503924:Pep-1 peptide; D010455:Peptides; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D003543:Cysteamine; D013482:Superoxide Dismutase; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; D043303:Aryldialkylphosphatase