title: [Advanced glycation end products inhibit production of nitric oxide by human endothelial cells through activation of the p38 signal pathway].
abstract: OBJECTIVE
  To study the effect of advanced glycation end products (AGE) modified protein on nitric oxide (NO) production by human endothelial cells and the possible role of signal transduction pathway in this pathological procedure.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) and HUVEC-derived cell line (ECV304) were cultured in vitro with AGE modified human serum albumin (AGE-HSA) or AGE modified bovine serum albumin (AGE-BSA) of the concentrations of 12.5, 25, and 50 micro g/ml for 2, 4, 8, 12, and 24 hours. NO levels in the supernatant at each time point were determined by using Griess reagents. HUVECs and ECV304 cells incubated with HAS or BSA of the same concentrations were used as controls. The phosphorylation activity of cellular p38 mitogen-activated protein kinase (p38-MAPK) was analyzed by Western blot using a phospho-specific antibody after the cells were incubated with AGE-HAS or AGE-BSA of the concentration of 12.5, 25, 50, and 100 micro g/ml for 5, 15, 30, 60, and 120 minutes. HUVECs were incubated with SB 203580, a specific inhibitor of p38, of the concentrations of 0.075, 0.15, and 0.3 micro mol/L for 4 hours, then the culture with SB 203580 was extracted. The endothelial cells were cleansed with RPMI 1640 culture and then were incubated with AGE-HAS or AGE-BSA for 8 hours. The No level in this supernatant was examined after the above-mentioned method.
  
  
  RESULTS
  The NO level in the supernatant of culture of HUVECs incubated with AGE-HAS or AGE-HAS for 2 hours were 92.2% +/- 10.1% and 94.1% +/- 12.5% of the controls respectively. The NO levels reached the lowest values, 72.4% +/- 7.14% and 68.3% +/- 10.9% of the values of the controls respectively after 8-hour incubation with AGE-HAS or AGE-HAS. AGE-HSA and AGE-BSA decreased the NO levels in the supernatant in a dose and time-dependant manner. The unmodified HSA and BSA did not influence the NO production by HUVECs or ECV304 cells. There was no difference in inhibition effect on NO production between AGE-HSA and AGE-BSA (P > 0.05). There was no difference in response to AGE-HSA and AGE-BSA between HUVECs and ECV304 cells (P > 0.05). In the culture of HUVECs or of ECV304 cells incubated with AGE-HSA or AGE-BSA, the p38 phosphorylation activity was obviously enhanced 10 minutes after dose-dependently, reached its peak 30 minutes after, and returned to its basic level in 120 minutes. SB203580 of the concentration of 0.3 micro mol/L completely abolished the inhibitive effect of AGE-albumin on NO production by endothelial cells.
  
  
  CONCLUSIONS
  AGE modified protein inhibits the production of NO by human endothelial cells through activation of the p38 signal pathway. This AGE modification-induced pathobiological cascade may be involved in the pathogenesis of atherosclerosis seen in AGE-associated diseases.
authors.affiliation: Division of Nephrology, Nanfang Hospital, the First Military Medical University Guangzhou 510515, China.
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Guo
authors.forename: Fanfan
authors.initials: F
authors.lastname: Hou
authors.forename: Xun
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Wang
mesh_terms: D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D017127:Glycation End Products, Advanced / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D011725:Pyridines / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D017127:Glycation End Products, Advanced; D007093:Imidazoles; D011725:Pyridines; D009569:Nitric Oxide; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580