May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
NADPH oxidase and aldose reductase inhibition attenuates high glucose–induced NF–B activation and apoptosis of human lens epithelial cells.
Author Affiliations & Notes
  • S.K. Srivastava
    Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX
  • A. Bhatnagar
    Department of Medicine, University of Louisville, Louisville, KY
  • B. Friedrich
    Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX
  • K.V. Ramana
    Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX
  • Footnotes
    Commercial Relationships  S.K. Srivastava, None; A. Bhatnagar, None; B. Friedrich, None; K.V. Ramana, None.
  • Footnotes
    Support  NIH Grant EY01677, DK36118
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1698. doi:
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      S.K. Srivastava, A. Bhatnagar, B. Friedrich, K.V. Ramana; NADPH oxidase and aldose reductase inhibition attenuates high glucose–induced NF–B activation and apoptosis of human lens epithelial cells. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1698.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Hyperglycemia–induced oxidative stress followed by lipid peroxidation and generation of lipid–derived aldehydes contributes to stress signals leading to secondary diabetic complications. However, the mechanisms that regulate hyperglycemia –induced stress signals that cause injuries are not clear. The purpose of this study is to investigate the involvement of NADPH oxidase and aldose reductase (AR) in the cytotoxic signals of hyperglycemia leading to apoptosis of human lens epithelial cells (HLEC). Methods: Growth–arrested HLEC were cultured without or with AR and NADPH–oxidase inhibitors followed by stimulation with high glucose (50 mM). The cytotoxicity was assessed by MTT assay, thymidine incorporation, measuring caspase–3 activation, nucleosomal degradation and apoptosis. The levels of HNE and SOD were measured by using specific ELISA kits. Electrophoretic mobility gel shift assays were performed to determine the activation of NF–ΚB and AP1. Western blot analysis was used to measure the activation of MAPK, JNK and PKC using respective antibodies. Results: Inhibition of AR by two structurally unrelated inhibitors – sorbinil and tolrestat prevents the apoptosis and activation of caspase–3 in HLEC, caused by exposure to high glucose. Inhibition of AR attenuates high glucose–induced increase in HNE and SOD levels, activation of PKC, IΚB–α phosphorylation and the stimulation of NF–ΚB and also attenuates the increase in p38 and JNK phosphorylation. Similarly, NADPH oxidase inhibitors, diphenyleneiodonium and apocyanin, prevented the apoptosis of HLEC induced by high glucose and also the generation of HNE, superoxide anions and activation of NF–ΚB. Conclusions: The results indicate that hyperglycemia–induced cytotoxicity in the lens could be due to apoptosis of lens epithelial cells caused by oxidative stress–initiated signals. The stress signals are mediated by AR and NADPH oxidase that regulate transcription factors NF–ΚB and AP1, and activate caspase–3 causing cell death.

Keywords: apoptosis/cell death • diabetes • signal transduction 
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