title: Stress responses of human retinal pigment epithelial cells to glyoxal.
abstract: OBJECTIVE
  Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells.
  
  
  METHODS
  Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of Nε-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal. The percentage of apoptotic cells was further quantified by flow cytometry after staining of fixed cells with propidium iodide to determine cells with a subdiploid (fragmented) DNA content. Apoptosis related activation of caspase 3 was determined by Western blotting. Glyoxal induced changes in VEGF-A165amRNA expression and protein production were determined by real-time PCR and by flow cytometry after immunocytochemical staining.
  
  
  RESULTS
  Increasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-A165amRNA expression and VEGF-A protein production were significantly increased after incubation with glyoxal in ARPE-19 cells.
  
  
  CONCLUSIONS
  The glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.
authors: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
authors: Cora
authors: C
authors: Roehlecke
authors: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany. monika.valtink@tu-dresden.de.
authors: Monika
authors: http://orcid.org/0000-0003-3205-1876
authors: M
authors: Valtink
authors: Division of Vascular Endothelium and Microcirculation, Medical Clinic III, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
authors: Annika
authors: A
authors: Frenzel
authors: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
authors: Doris
authors: D
authors: Goetze
authors: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
authors: Lilla
authors: L
authors: Knels
authors: Division of Vascular Endothelium and Microcirculation, Medical Clinic III, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.|CRTD/DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence, Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.
authors: Henning
authors: H
authors: Morawietz
authors: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.|CRTD/DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence, Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.
authors: Richard H W
authors: RH
authors: Funk
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006037:Glyoxal / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012164:Retinal Diseases / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*; D055815:Young Adult
chemical_list: D017127:Glycation End Products, Advanced; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D006037:Glyoxal; D053148:Caspase 3
keywords: Advanced glycation end products (AGEs); Glyoxal; Nε-carboxymethyllysine (CML); Oxidative stress; Retinal pigment epithelium; VEGF