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E. Kubo, T. Miyazawa, N. Fatma, D.P. Singh, Y. Akagi; Preferential Expression of Peroxiredoxins 5 and 6 in Rat Diabetic Retinopathy: Assigning the Role in the Attenuation of Hyperglycemia–Induced Pericyte Apoptosis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1708.
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Hyperglycemia–induced oxidative stress is implicated to be involved in pericyte apoptosis, one of the changes observed in the development of diabetic retinopathy. The six mammalian Peroxiredoxins (Prdxs) comprise a novel family of anti–oxidative proteins that reduce hydrogen peroxide and alkyl hydroperoxide, and thus negatively regulate oxidative stress induced apoptosis. In the present study, we investigated the high glucose–associated changes in expression levels of Prdxs, and ability of highly expressed Prdx5 and 6, in prevention of hyperglycemia–induced pericyte apoptosis.
Tissues isolated from streptozotocin (STZ)–induced diabetic rats, and swine pericyte cultured with high–glucose (50mM D–glucose) medium were used to monitor high glucose–associated changes in the Prdxs mRNA and protein expression levels. Ability of Prdx6 or Prdx5 in preventing high–glucose induced apoptosis of pericyte was evaluated using cell survival assay (MTS assay). While DAPI staining and TUNEL assay was done to define apoptotic cell death. 8–OHdG immunochemistry was performed to monitor oxidative stress–induced DNA damage. Using standard molecular subcloning techniques, Prdx 6 and Prdx5 were linked to TAT transduction domain for their efficient delivery to cells/tissues. Western analysis was used to affirm intracellular presence of TAT–linked protein.
Real–time RT–PCR revealed the higher expression of Prdx 5 and 6 mRNA in retina in comparison to other Prdxs (Prdxs1–4). The mRNA and protein expression of Prx5 was increased in retina of STZ induced diabetic rat (12weeks). Immunohistochemical analysis revealed the presence of Prdx5 and 6 in cytoplasm of the ganglion cells, inner nuclear layer and photoreceptor cells. Western analysis disclosed the efficient and concentration dependent transduction of TAT–Prdx5 or TAT–Prdx6 in cells. Survival assay revealed the inhibition of hyperglycemia– induced apoptosis of pericytes was by Prdx5 or Prdx6.
Preferential expression of Prdx5 and Prdx6 in diabetic retina and prevention of high–glucose induced pericyte apoptosis by them suggest that supply of Prdx5 or Prdx6 or their combination might be a promising therapeutic strategy for treatment of diabetic retinopathy
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