title: Asymmetric dimethylarginine impairs fibrinolytic activity in human umbilical vein endothelial cells via p38 MAPK and NF-κB pathways.
abstract: BACKGROUND
  Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of nitric oxide (NO) synthase. An increased synthesis and/or a reduced catabolism of ADMA might contribute to the onset and progression of thrombosis. The present study was designed to evaluate the effect of ADMA on fibrinolytic factors in endothelial cells, and to investigate the cellular mechanisms.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of ADMA for various periods; Then HUVECs were preincubated with NO precursor (L-arginine), MAPK inhibitors, or NF-κB inhibitor (PDTC) before ADMA treatment to repeat the experiment. Protein levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and NF-κB activity were measured by ELISA; mRNA levels of tPA and PAI-1 were assayed by qRT-PCR; The activation of MAPK was characterized by western blot analysis.
  
  
  RESULTS
  (1) ADMA decreased tPA antigen levels in time- and concentration-dependent manners, with the maximum effect of 30 μmol/L ADMA for 48h (control 109.01 ± 4.15 ng/ml vs ADMA 86.76 ± 5.95 ng/ml, p<0.01); (2) 30 μmol/L ADMA elevated antigen levels of PAI-1 in a time-dependent manner, with the maximum effect of 30 μmol/L ADMA for 48 h (control 2721.12 ± 278.02 ng/ml vs. ADMA 3435.78 ± 22.33ng/ml, p<0.05); (3) ADMA reduced tPA mRNA levels and increased PAI-1 mRNA levels; (4) L-arginine, SB203580 (p38 MAPK inhibitor) and PDTC attenuated the effects of ADMA on tPA and PAI-1 significantly. (5) ADMA induced a rapid phosphorylation of p38 MAPK, and stimulated NF-κB activity greatly.
  
  
  CONCLUSIONS
  ADMA may accelerate thrombosis development by impairing fibrinolytic activity in vascular via inhibiting nitric oxide production and then activating its downstream p38 MAPK and NF-κB pathways.
authors.affiliation: The First Affiliated Hospital of Shantou University Medical College, Shantou, 515041, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhang
authors.forename: Naping
authors.initials: N
authors.lastname: Chen
authors.forename: Wenbing
authors.initials: W
authors.lastname: Qiu
authors.forename: Xinwu
authors.initials: X
authors.lastname: Xu
authors.forename: Dongming
authors.initials: D
authors.lastname: Wang
authors.forename: Philip S
authors.initials: PS
authors.lastname: Tsao
authors.forename: Hong
authors.initials: H
authors.lastname: Jin
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D016328:NF-kappa B; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C051321:SERPINE1 protein, human; C018524:N,N-dimethylarginine; D001120:Arginine; D048051:p38 Mitogen-Activated Protein Kinases; D010959:Tissue Plasminogen Activator