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N. Fatma, P. Sharma, E. Kubo, D.A. Beier, D.P. Singh; Antioxidant Protein 2 (aop2) Knockout Mice: Loss of Resistance to Heat and Oxidative Stress in Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):309.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Oxidative stress is a major risk factor for cataractogenesis. AOP2 is a lens epithelium-derived growth factor (LEDGF) – inducible protein that protects the cells from oxidative stress. However, role of AOP2 is not clear in lens. To this end, we utilized cultured lenses and lens epithelial cells (LECs) from normal and AOP2 knock out mice. We demonstrated the ability of AOP2 to protect eye lenses and/or LECs against oxidative and heat stresses. Methods: Lenses were removed from Aop+/+ and Aop-/-mice, and cell lines were generated as described previously (Singh et al. 1999). RT-PCR and western analysis were performed to validate the expression. Cells and/or lenses were subjected to either oxidative (10 - 200µM H2O2 for variable time periods), heat (42°C-45°C for 3-16 hrs) or serum depletion for a predefined period. MTS assay and Trypan blue exclusion test were used to assess cell viability, and TUNEL and DAPI staining to detect apoptotic cell death. Cells over expressing GFP-AOP2 and mutated GFP-AOP2 (C-47-I or S) were used to define the contribution of cysteine-47 (C-47). Histologic sections of lenses were examined after HE staining. Results: mLECs either from Aop2-/- or Aop2+/+ survived and grew well in DMEM+ 10% FCS. Aop-/- cells died after serum depletion (24hrs). Aop-/- cells were more susceptible to heat and oxidative stress and underwent apoptosis. C-47 of AOP2 was important as mutation abolished the protective effect of AOP2. Over-expressing AOP2 could confer the resistance against stress. Lenses from the Aop2-/- were more susceptible to H2O2 insults than Aop2+/+ and developed opacities in superficial cortex. Histology of Aop2-/- lenses was abnormal; LECs in the bow region had migrated into the posterior subcapsular space and denucleation was impaired. Conclusions: These results emphasize the importance of AOP2 in cellular defence against oxidative and heat stresses. Histological studies of lenses indicate the possible role of AOP2 in lens cell differentiation.
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