Diabetes regulates small molecular weight G-protein, H-Ras, in the microvasculature of the retina: implication in the development of retinopathy.
Retinopathy, a largely microvascular complication, affects over 80% of patients with diabetes for 20 years. The purpose of this study is to investigate the effect of diabetes on the activation of H-Ras, a small molecular weight G-protein that regulates cell fate, in the retinal microvessels. Microvessels were prepared from freshly isolated retina from streptozotocin diabetic rats or 30% galactose-fed rats by hypotonic lysis method. Ras activation was quantified by Raf-1 binding assay, and the activation of the signaling proteins, Raf-1 and mitogen activated protein (MAP) kinase, by quantifying their gene transcripts (RTPCR) and/or by protein expression (western blot). Two months of diabetes or experimental galactosemia activated H-Ras (Raf-binding assay) in the retinal microvessels by over 40% and 70% respectively compared to the values obtained from normal rat retinal microvessels. In the same diabetic rats the gene transcripts of H-Ras and its effector protein Raf-1 were elevated by 30% and 135% respectively with their protein expressions elevated by about 25% each, and this was paralleled by similar increases in the protein expressions of H-Ras and Raf-1 in experimentally galactosemic rats. Diabetes increased the gene expression of Ras-Raf-1 downstream signaling protein MAP kinase by over 50%, and that of nuclear transcriptional factor by 25-30%. This activation of H-Ras in retinal microvessels implies that its signaling pathway, in part, could be contributing to the microvascular pathology characteristic of diabetic retinopathy. Comparable activation of H-Ras and its signaling cascade in the retinal microvessels from experimentally galactosemic rats suggests that H-Ras activation is not due to insulin deficiency. Regulation of Ras function could provide important target in the complex approach to inhibit the pathogenesis of diabetic retinopathy.
Kanwar
Mamta
M
Kresge Eye Institute, Wayne State University, Detroit, MI, USA.
Kowluru
Renu A
RA
D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism*; D003930:Diabetic Retinopathy / Q000209:etiology* / Q000235:genetics / Q000378:metabolism*; D011905:Genes, ras; D020935:MAP Kinase Signaling System; D008297:Male; D055806:Microvessels / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012171:Retinal Vessels / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D016328:NF-kappa B; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins