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R.A. Kowluru, A. Kowluru, M. Kanwar; Small Molecular Weight G–Protein Ras and Retinal Capillary Cell Death in Diabetes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):947.
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We have recently shown that the expressions of small molecular weight G–protein, H–Ras, and its effector protein, Raf–1, are increased in the retina in diabetes, and specific inhibitors of Ras function inhibit glucose–induced apoptosis of retinal capillary cells. In this study, we have utilized molecular approaches to further confirm the contributory roles for H–Ras in the development of diabetic retinopathy.
Bovine retinal endothelial cells were transfected using the Effectene transfection reagent kit (Qiagen) with the plasmids of either wild type (WT), constitutively–active (V12) or dominant–negative (N17) H–Ras. Glucose–induced NO release, NF–kB activation, degree of apoptotic cell death were determined in retinal endothelial cells overexpressing the WT, V12 or N17 mutants of Ras.
Overexpression of V12 Ras potentiated glucose–induced NO levels, NF–kB activation and the degree apoptosis in retinal endothelial cells. In contrast, glucose–mediated increases in NO levels, NF–kB activation and apoptotic cell death were nullified in the cells overexpressing the inactive N17 mutant of H–Ras.
Our findings demonstrate that H–Ras is important in the activation specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, and suggest that this G–protein plays an important role in the pathogenesis of retinopathy in diabetes.
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