title: Increased levels of legumain in plasma and plaques from patients with carotid atherosclerosis.
abstract: The cysteine protease legumain has been shown to be up-regulated in unstable atherosclerotic plaques. This study aims to further elucidate legumain in atherosclerosis, by examining legumain in plasma and carotid plaques from patients with carotid stenosis. Furthermore, legumain secretion from monocyte-derived macrophages treated with atherogenic lipids during macrophage polarization was studied.
  
  
  
  Plasma levels of legumain from patients with carotid stenosis (n = 254), healthy controls (n = 91), and secreted from monocyte-derived macrophages were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting of legumain were performed on isolated plaques and legumain localization was visualized by immunohistochemistry and fluorescence microscopy. Monocyte-derived macrophages polarized to M1 or M2 macrophages were treated with VLDL, oxLDL or cholesterol crystals (CC) and the level of legumain analysed.
  
  
  
  Patients with carotid stenosis had significantly higher levels of plasma legumain compared with healthy controls (median 2.0 versus 1.5 ng/ml, respectively; p = 0.003), although there was no correlation between the level of legumain and the degree of stenosis, and legumain was not an independent factor to identify patients with carotid plaques. Moreover, patients with symptoms the last 2 months had higher expressions of mature legumain, cystatin C and E/M, and the macrophage markers CD80 (M1) and CD163 (M2). Legumain co-localized with both M1 and M2 macrophages within plaques, whereas legumain mRNA expression was significantly higher (p < 0.0001) in plaques compared to non-atherosclerotic arteries (controls). Furthermore, in vitro studies showed significantly increased secretion of legumain from pro-inflammatory M1 compared to pro-resolving M2 macrophages (p = 0.014), and particularly in M1 treated with CC. In plaques, legumain was localized to structures resembling foam cells.
  
  
  
  Legumain is increased in both plasma and plaques of patients with carotid stenosis and might be a new and early biomarker of atherosclerosis.
authors.affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Ngoc Nguyen
authors.initials: NN
authors.lastname: Lunde
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Hospital for Rheumatic Diseases, Lillehammer, Norway.
authors.forename: Sverre
authors.initials: S
authors.lastname: Holm
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; K.G. Jebsen Inflammation Research Centre, University of Oslo, Oslo, Norway.
authors.forename: Tuva B
authors.initials: TB
authors.lastname: Dahl
authors.affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Inass
authors.initials: I
authors.lastname: Elyouncha
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Bjørnar
authors.initials: B
authors.lastname: Sporsheim
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
authors.forename: Ida
authors.initials: I
authors.lastname: Gregersen
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Department of Neurology, Oslo University Hospital Rikshospitalet, Oslo, Norway; Østfold Hospital Trust, Kalnes, Norway.
authors.forename: Azhar
authors.initials: A
authors.lastname: Abbas
authors.affiliation: Department of Neurology, Oslo University Hospital Rikshospitalet, Oslo, Norway.
authors.forename: Mona
authors.initials: M
authors.lastname: Skjelland
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Terje
authors.initials: T
authors.lastname: Espevik
authors.affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway. Electronic address: rigmor.solberg@farmasi.uio.no.
authors.forename: Rigmor
authors.initials: R
authors.lastname: Solberg
authors.affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Harald Thidemann
authors.initials: HT
authors.lastname: Johansen
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; K.G. Jebsen Inflammation Research Centre, University of Oslo, Oslo, Norway.
authors.forename: Bente
authors.initials: B
authors.lastname: Halvorsen
mesh_terms: D000368:Aged; D015415:Biomarkers / Q000097:blood; D002339:Carotid Arteries / Q000000981:diagnostic imaging / Q000201:enzymology*; D016893:Carotid Stenosis / Q000097:blood* / Q000000981:diagnostic imaging / Q000201:enzymology; D016022:Case-Control Studies; D000066670:Cell Plasticity; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003460:Crystallization; D003546:Cysteine Endopeptidases / Q000097:blood*; D005260:Female; D005487:Foam Cells / Q000201:enzymology; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000201:enzymology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D058226:Plaque, Atherosclerotic; D015854:Up-Regulation; D000099240:Asparaginyl Endopeptidase
chemical_list: D015415:Biomarkers; D002784:Cholesterol; D003546:Cysteine Endopeptidases; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D000099240:Asparaginyl Endopeptidase; C099252:oxidized low density lipoprotein
keywords: Asparaginyl endopeptidase (AEP); Atherosclerosis; Carotid; Cholesterol crystals; Cystatins; Inflammation; Legumain; Macrophage polarization; Plaque; Proteases