Evidence supporting a role for N(ε)-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Müller glia dysfunction and death in diabetic retinopathy

PURPOSE: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy. METHODS: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Müller (Moorfields/Institute of Ophthalmology-Müller 1 [MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Müller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Müller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) were determined by reverse transcriptase PCR (RT–PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide. RESULTS: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Müller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Müller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Müller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-α. Incubation of Müller glia with FDP-lysine-HSA also caused apoptosis at high concentrations. CONCLUSIONS: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Müller glial abnormalities occurring in the early stages of diabetic retinopathy.