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K. Engelmann, C. Roehlecke, M. Valtink, R.H. W. Funk; Induction of Apoptosis by Glyoxal in Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2896.
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Glyoxal is a highly reactive glycating agent involved in the formation of advanced glycation end products (AGEs) which are known to induce apoptosis. AGE–mediated apoptosis may be an important mechanism in the initiation of age–related macular degeneration (AMD). We investigated the in vitro effect of glyoxal on cultures of retinal pigment epithelium (RPE).
The human retinal pigment epithelial cell line ARPE–19 was incubated with varying concentrations of glyoxal. Subdiploid DNA content (sub–G1 assay), binding of Annexin V, enhanced expression of active caspase–3, reactive oxygen intermediates (ROS) and reduced intracellular pH using flow cytometry and ELISA was monitored to investigate apoptotic changes in the RPE cells.
Cytofluorometrical, immunocytochemical and Western blot analysis revealed a dose–dependent accumulation of the AGE product εN–(carboxymethyl)lysine (CML) in cells after incubation with glyoxal in RPE cells. In vitro treatment of RPE cells resulted in glyoxal induced apoptosis. Protein expressions of the anti–apoptotic molecule osteopontin, stress–related proteins such as SOD–Cu/Zn and SOD–Mn, heme oxygenase (HO–1), Hsp–27, Cathepsin D, VEGF, and Ezrin were increased after incubation with glyoxal (flow cytometry, Western blot).
Interestingly, glyoxal exposure induced alteration of all types of cellular proteins which was not confined to the apoptotic cells as revealed by double labelling of active caspase–3 and/ or caspase–cleavage fragment of cytokeratin–18 as markers of apoptosis. Therefore, this study shows that glyoxal induced AGE–load can be an important factor of RPE damage in AMD.
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