Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes |
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Authors: | Renu A Kowluru Anjan Kowluru Mamta Kanwar |
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Institution: | (1) Department of Ophthalmology, Kresge Eye Institute, 4717 St. Antoine, Detroit, MI, USA;(2) Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA;(3) Department of Ophthalmology, Kresge Eye Institute, 4717 St. Antoine, Detroit, MI 48201, USA |
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Abstract: | We have demonstrated 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 the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal
capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial
cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with
the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase
in apoptosis, nitric oxide (NO) levels and activation of NF-κB and caspase-3 were determined in these genetically manipulated
cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined
by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by
40%, NO levels by about 50%, and activated NF-κB and caspase-3 by about 30–40% compared to the untransfected cells incubated
in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic
cell death, NO levels and NF-κB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate
that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary
cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of
diabetic retinopathy. |
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Keywords: | apoptosis diabetic retinopathy endothelial cells small molecular weight G-protein H-Ras |
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