title: High-mobility group box 1 protein is implicated in advanced glycation end products-induced vascular endothelial growth factor A production in the rat retinal ganglion cell line RGC-5.
abstract: OBJECTIVE
  High-mobility group box 1 protein (HMGB1) has been reported to be a potent proangiogenic factor induced by inflammatory stress. In this study, we explore the role of HMGB1 in advanced glycation end products (AGEs)-induced vascular endothelial growth factor A (VEGF-A) production in rat retinal ganglion cell line 5 (RGC-5) cells.
  
  
  METHODS
  The VEGF-A protein and mRNA levels in conditioned medium of RGC-5 cells incubated with AGE-modified BSA (AGE-BSA) were examined with real-time PCR and enzyme-linked immunosorbent assay (ELISA), and BSA-treated cells were used as controls. The expression of HMGB1, c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) was assessed with immunofluorescence and western blot analysis. Reactive oxidative species (ROS) were detected with flow cytometry measurements of peroxide-dependent oxidation of 2'-7'-dichlorofluorescein-diacetate (DCFH-DA). N-Acetyl-L-cysteine (NAC), glycyrrhizin (GZ), and SP600125 were used to block ROS, HMGB1, and JNK, respectively.
  
  
  RESULTS
  Compared with the BSA controls, the RGC-5 cells incubated with AGE-BSA showed a dose- and time-dependent increase in VEGF-A mRNA and VEGF-A protein secretion in the supernatant, with the highest levels achieved at 24 h. AGE-BSA stimulated a significant release of HMGB1 in the supernatant and a significant increase of intracellular ROS production at 3 h. NAC blocked HMGB1 production in a dose-dependent manner. Blocking with GZ, NAC, and JNK significantly suppressed AGE-induced VEGF-A production.
  
  
  CONCLUSIONS
  HMGB1 is implicated in the production of VEGF-A in retinal ganglion cell line-5 (RGC-5). Blocking HMGB1, ROS, or the JNK pathway may attenuate VEGF-A production, suggesting HMGB1 and related signaling molecules play a role in diabetic retinopathy.
authors.affiliation: Department of Ophthalmology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Jong-Jer
authors.initials: JJ
authors.lastname: Lee
authors.forename: Chang-Chun
authors.initials: CC
authors.lastname: Hsiao
authors.forename: I-Hui
authors.initials: IH
authors.lastname: Yang
authors.forename: Ming-Huei
authors.initials: MH
authors.lastname: Chou
authors.forename: Chia-Lin
authors.initials: CL
authors.lastname: Wu
authors.forename: Yin-Chu
authors.initials: YC
authors.lastname: Wei
authors.forename: Chih-Hsin
authors.initials: CH
authors.lastname: Chen
authors.forename: Jiin-Haur
authors.initials: JH
authors.lastname: Chuang
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D019695:Glycyrrhizic Acid / Q000494:pharmacology; D024243:HMGB1 Protein / Q000096:biosynthesis* / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000873:Anthracenes; D017077:Culture Media, Conditioned; D017127:Glycation End Products, Advanced; D024243:HMGB1 Protein; C104200:Hbp1 protein, rat; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; C432165:pyrazolanthrone; D012710:Serum Albumin, Bovine; D019695:Glycyrrhizic Acid; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D000111:Acetylcysteine