Abstract
Purpose
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-A165a mRNA 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-A165a mRNA 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.
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (funding of the Excellence Initiative by the German Federal and State Governments, Institutional Strategy, measure “support the best”, to HM and RHWF, MO 1695/4-1 and MO 1695/5-1 to HM). The authors thank T. Schwalm for technical assistance and E.D. Schleicher for kindly providing anti-CML antiserum. We also thank Kathy Eisenhofer for critically reading the manuscript.
Contributors
CR, MV, LK, and RHWF designed the study and were involved in data production, data evaluation, and manuscript writing. AF and HM designed and performed experiments on VEGF165 determination, evaluated the data and were involved in manuscript writing. DG performed caspase 3 Western blotting, evaluated the data and was involved in manuscript writing.
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (funding of the Excellence Initiative by the German Federal and State Governments, Institutional Strategy, measure “support the best” to HM and RHWF, MO 1695/4-1 and MO 1695/5-1 to HM).
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Roehlecke, C., Valtink, M., Frenzel, A. et al. Stress responses of human retinal pigment epithelial cells to glyoxal. Graefes Arch Clin Exp Ophthalmol 254, 2361–2372 (2016). https://doi.org/10.1007/s00417-016-3463-2
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DOI: https://doi.org/10.1007/s00417-016-3463-2