Abstract

SA.08.06

Functional effects of oxygen free radicals on retinal pigmentepithelium – protective effects of erythropoietin

Zeitz O.¹, Gawad A.¹, Schlichting L.², Klemm M.¹, Richard G.¹, Strauß O.²
1Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf; ²Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf

Objective: Several ocular diseases like glaucoma or age related macular degeneration are associated with the occurence of oxygen free radicals. Own results regarding the impact of oxygen free radicals on retinal pigment epithelial cells will be summarized. As potential protective strategy the natural hormone erythropoietin is tested.
Methods: Hydroxylradicals are generated from the Fenton reaction from H₂O₂ and Fe³⁺. A self designed cell culture system was used to ensure defined exposure times and defined amounts of oxygen free radicals. The radical concentration will be given as multiples of a reference concentration which was examined by NMR spectroscopy previously. Intracellular Calcium (FURA-2-AM), survival rate (life dead assay) and apoptosis rate (TUNEL assay and Annexin V labelling) was measured in cultured ARPE-19 cells. The experiments were done in absence and presence of pharmacological test compounds.
Results: Hydroxyl radical lead to a transient rise in intracellular calcium levels. After several hours apoptosis can be observed. Apoptosis rate reaches its maximum after 6 hours (11.8±1.7% at 3x radical concentration; P<0.05). Scavengers like vitamin C do not have protective properties. Particularly in low concentrations below 1mM they may increase the oxidative damage (cell survival rate without vitamin C 87±1% to 89±2% vs. 79±2% to 82±3% in presence of 0.1 to 1mM vitamin C; n=6 each; P<0.05). Erythropoietin (Epo) reduces apoptosis rate after 6 hours significantly (12.5±0.9% w/o Epo vs. 4.0±0.8% with 5U/ml Epo; P=0.02; n=3 each). Erythropoietin is even effective if it is given to the cells after radical exposure.
Conclusions: Oxygen free radicals influence the calcium metabolism of retinal pigment epithelium cells and may induce apoptosis. Influencing the radical damage by scavengers is a difficult issue since scavenger molecules may be transformed into radicals. This may even increase the oxidative damage. Erythropoietin is antiapoptotic at retinal pigment epithelium cells. It might be a promising approach for future clinical interventions.

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