Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes.
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-kappaB 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-kappaB 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-kappaB 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.
Kowluru
Renu A
RA
Department of Ophthalmology, Kresge Eye Institute, 4717 St. Antoine, Detroit, MI 48201, USA. rkowluru@med.wayne.edu
Kowluru
Anjan
A
Kanwar
Mamta
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000473:pathology*; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D016328:NF-kappa B; D009569:Nitric Oxide; D019908:Proto-Oncogene Proteins c-raf; D053148:Caspase 3; D016283:Proto-Oncogene Proteins p21(ras); D005947:Glucose