title: Osteoprotegerin causes apoptosis of endothelial progenitor cells by induction of oxidative stress.
abstract: OBJECTIVE
  Elevated serum osteoprotegerin (OPG) levels represent an independent risk factor for atherosclerotic disease, although the underlying mechanism is not clear. The aim of this study was to investigate the association of serum OPG levels and circulating endothelial progenitor cell (EPC) numbers, and to explore the effect of OPG on EPC apoptosis and its underlying mechanisms.
  
  
  METHODS
  Flow cytometry was used to enumerate EPCs in the peripheral blood of 91 patients with systemic lupus erythematosus (SLE). Cultured EPCs, isolated from peripheral blood, were challenged with OPG, and apoptosis was evaluated by TUNEL staining. Expression of apoptosis-related proteins was measured by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. Reactive oxygen species (ROS) were detected by flow cytometry, and the expression of NADPH oxidase (NOX) and MAP kinases (MAPK) was measured by qPCR and Western blotting.
  
  
  RESULTS
  The serum OPG level was independently associated with reduced numbers of EPCs in patients with SLE. In vitro treatment with OPG significantly induced apoptosis of EPCs; this effect was mediated by syndecan 4. OPG-induced apoptosis was abolished by the ROS scavenger N-acetylcysteine and the NOX inhibitor diphenyleniodonium. OPG increased ROS production through activation of NOX-2 and NOX-4 and triggered phosphorylation of ERK-1/2 and p38 MAPK. Quenching of ROS by knockdown of NOX-2 or NOX-4 transcripts inhibited phosphorylation of ERK-1/2 and p38 MAPK. Moreover, inhibitors of ERK-1/2 and p38 MAPK decreased ROS production and subsequent EPC apoptosis, indicating a feed-forward loop between NOX and MAPK to amplify ROS production related to apoptosis.
  
  
  CONCLUSIONS
  Elevated OPG levels increase apoptosis of EPCs by induction of oxidative stress.
authors.affiliation: Catholic Research Institutes of Medical Sciences, Seoul, Republic of Korea.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Yune-Jung
authors.initials: YJ
authors.lastname: Park
authors.forename: Ki-Jo
authors.initials: KJ
authors.lastname: Kim
authors.forename: Jin-Jung
authors.initials: JJ
authors.lastname: Choi
authors.forename: Wan-Uk
authors.initials: WU
authors.lastname: Kim
authors.forename: Chul-Soo
authors.initials: CS
authors.lastname: Cho
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D020868:Gene Silencing; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000473:pathology; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D053244:Osteoprotegerin / Q000097:blood / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014162:Transfection
chemical_list: D053244:Osteoprotegerin; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D019255:NADPH Oxidases; D020929:Mitogen-Activated Protein Kinase Kinases