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1 From the Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor; and the 2 Center for Ophthalmic Research, Department of Ophthalmology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.
PURPOSE. Lens epithelium-derived growth factor (LEDGF) has been shown to be a growth and survival factor and to be present in a wide variety of cell types. The purpose of this study was to determine whether LEDGF enhances survival of human retinal pigment epithelial (RPE) cells when challenged by oxidative stress or by ultraviolet (UVB) irradiation in a culture system.
METHODS. Primary RPE cells were cultured in standard Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum. Protein blot analysis with antibodies to LEDGF was used to detect LEDGF in RPE cells. Initially, RPE cells were cultured in the standard medium for 1 day to allow attachment to the culture plates and then cultured in serum-free DMEM, with and without LEDGF. The trypan blue exclusion method was used to test RPE cell viability. Single-cell electrophoresis was used to evaluate single strand breaks of genomic DNA after exposure to H2O2 or irradiation by UVB.
RESULTS. LEDGF was present in RPE cells, predominantly in the nucleus. RPE cells grew for 1 week and survived for 3 weeks in the presence of LEDGF. In the absence of LEDGF, they increased in number for the first week and gradually died in the following 2 weeks. LEDGF protected RPE cells against H2O2 exposure and UVB irradiation. DNA damage induced by H2O2 exposure or UVB irradiation was lower in the presence than in the absence of LEDGF. The expression of heat shock protein (Hsp)27 was elevated by LEDGF.
CONCLUSIONS. LEDGF enhanced survival of RPE cells in culture when challenged by oxidative stress and UVB irradiation. LEDGF protected DNA from single-strand breakage and upregulated the expression of Hsp27. These results suggest that LEDGF may be a potential agent for protecting RPE cells under various stress conditions.
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