Induction of angiogenic cytokine expression in cultured RPE by ingestion of oxidized photoreceptor outer segments

Abstract

PURPOSE. Normal aging is associated with accumulation of lipofuscin pigment in the retinal pigment epithelium (RPE). This may occur as a result of phagocytosis and incomplete degradation of oxidized photoreceptor outer segments (POS). This study was undertaken to determine whether phagocytosis of UV-irradiated POS (artificial lipofuscin) would increase expression in the RPE of various chemotactic and angiogenic cytokines. METHODS. ARPE-19 cells were exposed to latex beads (0.76 μm), naïve bovine POS, and UV-irradiated POS (Ox-POS; 2 × 107/mL), and supernatants were collected at 18 and 36 hours. The supernatants were assayed for IL-8, monocyte chemotactic protein-(MCP)-1, and TNF-α by ELISA. Protein synthesis and NFκB activity were inhibited by actinomycin D and SN50, respectively. Phagocytosis and generation of intracellular reactive oxygen species were assessed by flow cytometry. Confocal and electron microscopy studies were also performed to verify phagocytosis and cellular integrity. RESULTS. IL-8 and MCP-1 levels were decreased in the naïve POS group (IL-8: 473.76 ± 66.9 pg/mL, P = 0.0005; MCP-1: 550.1 ± 21.8 pg/mL, P = 0.0001), but were increased in the Ox-POS group (IL-8: 1348.8 ± 164.9 pg/mL; MCP-1: 1772.28 ± 65.19 pg/mL) compared with the control (IL-8: 741.09 ± 39.8 pg/mL; MCP-1: 1413.47 ± 38.4 pg/mL) and latex bead groups (data not shown). TNF-α levels were not affected. At 12 hours (but not at 6 hours), ROS were increased in the Ox-POS group. The cytokine increases observed were dependent on de novo protein synthesis and were NF-κB dependent. CONCLUSIONS. Ingestion by RPE of oxidized bovine POS stimulates expression of the chemotactic and angiogenic factors IL-8 and MCP-1 that have the capability to promote angiogenesis directly, or indirectly through the accumulation of immune cells such as macrophages, which themselves may release angiogenic promoters and degrade Bruch's membrane. This may be of significance in the development of exudative AMD.