Purpose: To determine whether human X-linked inhibitor of apoptosis (XIAP) enhances the survival of cultured human retinal pigment epithelial cells exposed to H(2)O(2).
Methods: ARPE-19 cells were exposed to H(2)O(2) to induce oxidative cell death. Intracellular reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescein diacetate. MTT assay was performed to quantify mitochondrial stress. Cell apoptosis was determined by TUNEL assay. Human XIAP was delivered with bicistronic expression of green fluorescent protein (GFP), using recombinant adeno-associated virus (AAV-XIAP-GFP). The null vector, containing identical sequences but without XIAP, was used as a control (AAV-NULL-GFP). Transduced cells underwent fluorescence-activated cell sorting. XIAP overexpression was examined by immunostaining and Western blot analysis.
Results: ARPE-19 cells exposed to 0.25 mM H(2)O(2) for 1 hour showed increased TUNEL staining compared with nonstressed cells (17 ± 1.4 vs. 1.8 ± 0.4 cells per 20 × field; P = 0.000006), accompanied by a significant increase in intracellular ROS (207 ± 46% vs. 100 ± 9.5%; P = 0.0002). The AAV-XIAP-GFP transduced cells had 11-fold higher XIAP expression than the AAV-NULL-GFP controls (1300 ± 126% vs. 120 ± 10%; P = 0.0006). XIAP over-expression significantly reduced the number of apoptotic cells after stress compared with the AAV-NULL-GFP controls (3.2 ± 0.6 vs. 18 ± 1.6 cells per 20 × field; P = 0.00003). Mitochondrial stress was reduced by AAV-XIAP-GFP, but did not reach a statistical significance (68 ± 3.5% vs. 74 ± 3.8%; P = 0.24).
Conclusions: Overexpression of human XIAP protects ARPE-19 cells against H(2)O(2)-induced oxidative cell death by acting downstream on the apoptotic pathway. XIAP gene therapy using AAV may provide a means of reducing the effect of oxidative stress to RPE cells in age-related macular degeneration.