title: Profiling of atherosclerotic lesions by gene and tissue microarrays reveals PCSK6 as a novel protease in unstable carotid atherosclerosis.
abstract: OBJECTIVE
  Carotid plaque instability is a major cause of ischemic stroke, but detailed knowledge about underlying molecular pathways is still lacking. Here, we evaluated large-scale transcriptomic and protein expression profiling in a biobank of carotid endarterectomies followed by characterization of identified candidates, as a platform for discovery of novel proteins differentially regulated in unstable carotid lesions.
  
  
  RESULTS
  Genes highly upregulated in symptomatic versus asymptomatic plaques were selected from Affymetrix microarray analyses (n=127 plaques), and tissue microarrays constructed from 34 lesions were assayed for 21 corresponding proteins by immunohistochemistry. Quantification of stainings demonstrated differential expression of CD36, CD137, and DOCK7 (P<0.05) in unstable versus stable lesions and the most significant upregulation of a proprotein convertase, PCSK6 (P<0.0001). Increased expression of PCSK6 in symptomatic lesions was verified by quantitative real-time polymerase chain reaction (n=233), and the protein was localized to smooth muscle α-actin positive cells and extracellular matrix of the fibrous cap by immunohistochemistry. PCSK6 expression positively correlated to genes associated with inflammation, matrix degradation, and mitogens in microarrays. Stimulation of human carotid smooth muscle cells in vitro with cytokines caused rapid induction of PCSK6 mRNA.
  
  
  CONCLUSIONS
  Using a combination of transcriptomic and tissue microarray profiling, we demonstrate a novel approach to identify proteins differentially expressed in unstable carotid atherosclerosis. The proprotein convertase PCSK6 was detected at increased levels in the fibrous cap of symptomatic carotid plaques, possibly associated with key processes in plaque rupture such as inflammation and extracellular matrix remodeling. Further studies are needed to clarify the role of PCSK6 in atherosclerosis.
authors.affiliation: From the Department of Molecular Medicine and Surgery (L.P., E.H., A.R., M.L., C.O., U.H.), and Department of Medicine (G.P.-B., J.O.), Karolinska Institute, Stockholm, Sweden; Department of Molecular Genetics, Novo Nordisk, Copenhagen, Denmark (L.F.); Department of Surgery, Södersjukhuset, Stockholm, Sweden (P.G.); Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden (F.P.); and Department of Proteomics, Royal Institute of Technology, Stockholm, Sweden (J.O.).
authors.forename: Ljubica
authors.initials: L
authors.lastname: Perisic
authors.forename: Erika
authors.initials: E
authors.lastname: Hedin
authors.forename: Anton
authors.initials: A
authors.lastname: Razuvaev
authors.forename: Mariette
authors.initials: M
authors.lastname: Lengquist
authors.forename: Cecilia
authors.initials: C
authors.lastname: Osterholm
authors.forename: Lasse
authors.initials: L
authors.lastname: Folkersen
authors.forename: Peter
authors.initials: P
authors.lastname: Gillgren
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Paulsson-Berne
authors.forename: Fredrik
authors.initials: F
authors.lastname: Ponten
authors.forename: Jacob
authors.initials: J
authors.lastname: Odeberg
authors.forename: Ulf
authors.initials: U
authors.lastname: Hedin
mesh_terms: D058070:Asymptomatic Diseases; D016893:Carotid Stenosis / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005355:Fibrosis; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D018836:Inflammation Mediators / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000276:immunology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology / Q000276:immunology; D020411:Oligonucleotide Array Sequence Analysis; D058226:Plaque, Atherosclerotic; D043484:Proprotein Convertases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012422:Rupture, Spontaneous; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D046888:Tissue Array Analysis; D015854:Up-Regulation
chemical_list: D016207:Cytokines; D018836:Inflammation Mediators; D012333:RNA, Messenger; C080654:PCSK6 protein, human; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: atherosclerosis; carotid stenosis; muscle, smooth; symptomatic carotid stenosis