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

[摘要] Purpose: Recent evidence suggests thatneuroglial dysfunction and degeneration contributes to the etiology andprogression of diabetic retinopathy. Advanced lipoxidation end products(ALEs) have been implicated in the pathology of various diseases,including diabetes and several neurodegenerative disorders. The purposeof the present study was to investigate the possible link between theaccumulation of ALEs and neuroretinal changes in diabetic retinopathy. Methods: Retinal sections obtained fromdiabetic rats and age-matched controls were processed forimmunohistochemistry using antibodies against several well definedALEs. In vitro experiments were also performed using a human Müller(Moorfields/Institute of Ophthalmology-Müller 1 [MIO-M1]) glia cellline. Western blot analysis was used to measure the accumulation of theacrolein-derived ALE adduct Nε-(3-formyl-3,4-dehydropiperidino)lysine(FDP-lysine) in Müller cells preincubated with FDP-lysine-modifiedhuman serum albumin (FDP-lysine-HSA). Responses of Müller cells toFDP-lysine accumulation were investigated by analyzing changes in theprotein expression of heme oxygenase-1 (HO-1), glial fibrillary acidicprotein (GFAP), and the inwardly rectifying potassium channel Kir4.1.In addition, mRNA expression levels of vascular endothelial growthfactor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα)were determined by reverse transcriptase PCR (RT–PCR). Apoptotic celldeath was evaluated by fluorescence-activated cell sorting (FACS)analysis after staining with fluorescein isothiocyanate (FITC)-labeledannexin V and propidium iodide. Results: No significant differences inthe levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and4-hydroxyhexenal-derived ALEs were evident between control and diabeticretinas after 4 months of diabetes. By contrast, FDP-lysineimmunoreactivity was markedly increased in the Müller glia of diabeticrats. Time-course studies revealed that FDP-lysine initiallyaccumulated within Müller glial end feet after only a few months ofdiabetes and thereafter spread distally throughout their inner radialprocesses. Exposure of human Müller glia to FDP-lysine-HSA led to aconcentration-dependent accumulation of FDP-lysine-modified proteinsacross a broad molecular mass range. FDP-lysine accumulation wasassociated with the induction of HO-1, no change in GFAP, a decrease inprotein levels of the potassium channel subunit Kir4.1, andupregulation of transcripts for VEGF, IL-6, and TNF-α. Incubation ofMüller glia with FDP-lysine-HSA also caused apoptosis at highconcentrations. Conclusions: Collectively, these datastrongly suggest that FDP-lysine accumulation could be a major factorcontributing to the Müller glial abnormalities occurring in the earlystages of diabetic retinopathy.

[学科分类] 生物化学/生物物理