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T. Miyazawa, Y. Takamura, S. Aoki, Y. Akagi, D.P. Singh; Over-Expression of Aldose Reductase Gene Induces Apoptosis in Human Lens Epithelial Cells: Involvement of Polyol Pathway Dependent Osmotic and Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3477.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cataract is a major complication in diabetic patients. Several factors and pathways are involved in diabetic complication. One of major contributory factors is aldose reductase (AR). Activation of AR generates osmotic and oxidative stress via polyol pathway. We studied the effect of AR-over-expression to polyol accumulation; oxidative stress (hyperglycemic) and osmotic stress, and its effect in hLECs survival in vitro, and also showed the association between increased polyol pathway and cell death. Methods: cDNA of human AR gene was isolated from human lens cDNA library using PCR with AR specific primers. A construct (GFP-AR) was build using eukaryotic expression vector (pEGFP-C). hLECs were transfected using lipofectamin (Gibco). Western analysis was done to monitor AR, GFP-AR and GFP protein expressions. Liquid chromatography-tandemm mass spectrometry (LC/MS/MS) was used to determine sorbitol and fructose in LECs. MTS and TUNEL assays were used to detect apoptotic cell death. Colorimetric GSH assay was performed in 96 well plates to detect cytosolic GSH levels. Results: Cytoplasmic localization of GFP-AR was detected in 80% cells. A band of 90 kDa was monitored in over-expressing cells using anti-human AR antibody. Intracellular sorbitol and fructose concentrations rapidly elevated (from 0.338 to1.05 and 1.42 to 4.3 nM/mg cell protein, respectively) in these cells cultured for 72hrs in 50 mM glucose. Cell survival gradually decreased in AR over-expressing cells from 72 to 120 hrs incubation with 50 mM glucose (p<0.001) and number of TUNEL positive cells were increased by16.6% over control. GSH level was also decreased significantly in these cells. Conclusions: Present findings reveal that over expression of AR increases polyol pathway dependent osmotic stress and oxidative stress that results into increased apoptosis in LECs. Modulation of polyol pathway in the cultured cells over- expressed with AR, provides an experimental model to evaluate and develop new AR inhibitors to postpone diabetic cataract.
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