title: 4-HNE increases intracellular ADMA levels in cultured HUVECs: evidence for miR-21-dependent mechanisms.
abstract: OBJECTIVE
  To investigate whether 4-hydroxynonenal (4-HNE) regulates asymmetric dimethylarginine (ADMA) metabolism through pathway independent of direct adduct formation with ADMA metabolizing enzyme and the involvement of microRNA (miRNA) miR-21 in human umbilical venous endothelial cells (HUVECs).
  
  
  METHODS
  Cultured HUVECs were treated with 4-HNE (at concentrations of 1, 5, and 10 µM, respectively) or 1‰ DMSO (vehicle control) for 24 h. MiR-21 inhibitor (final concentration of 100 nM) was transfected at 1 h before 4-HNE treatment. HUVECs were also transfected with miR-21 (at concentrations of 50 nM and 100 nM) and cultured for 12, 24, and 48 h, respectively. DDAH mRNA and miR-21 expression in the HUVECs were determined by semi-quantitative real time PCR. DDAH1 and DDAH2 protein expression were analyzed by Western blot. ADMA in the cell medium and cell lysates were analyzed by ELISA. ADMA metabolizing activity of the cell lysates was also determined.
  
  
  RESULTS
  MiR-21 decreased DDAH1 and DDAH2 expression and ADMA metabolic activity significantly, while increased intracellular ADMA accumulation significantly in HUVECs. 10 µM 4-HNE treatment for 24 h increased the expression of miR-21 and intracellular ADMA concentration, decreased the expression of DDAH1/2 mRNA and protein, decreased ADMA metabolizing activity of the cell lysates significantly. MiR-21 inhibitor reversed the inhibitory effects of 4-HNE on DDAH1 expression completely, and partially reversed the changes in ADMA metabolizing activity and intracellular ADMA accumulation challenged by 10 µM 4-HNE.
  
  
  CONCLUSIONS
  4-HNE down-regulates DDAH1 expression and increases intracellular ADMA accumulation in HUVECs through a miR-21-dependent mechanism.
authors.affiliation: Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, Hunan, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Ji-Peng
authors.initials: JP
authors.lastname: Zhou
authors.forename: Da-Bin
authors.initials: DB
authors.lastname: Kuang
authors.forename: Jie
authors.initials: J
authors.lastname: Tang
authors.forename: Yuan-Jian
authors.initials: YJ
authors.lastname: Li
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Chen
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D000581:Amidohydrolases / Q000235:genetics; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000447:Aldehydes; D017931:DNA Primers; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; C018524:N,N-dimethylarginine; D001120:Arginine; D000581:Amidohydrolases; C059999:dimethylargininase; C027576:4-hydroxy-2-nonenal