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Vincent Monnier, Michael Robinson, Christopher Strauch, Xingjun Fan; Conditional Expression of hSVCT2 using αA-Crystallin Promoter with Pax6 Consensus Binding Sites for Long-term Studies of Crystallin Ascorbylation and Cataractogenesis in Mouse. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2982. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
: Ascorbic acid (ASA) is present in mM levels in the human lens but absent in mouse lens, and is a powerful glycating agent that has been implicated in age-related cataractogenesis. In previous studies we demonstrated that transgenic expression of hSVCT2 using the αA-Crystallin promoter with chick δ1-crystallin lens enhancer in C57BL/6 mouse lenses recapitulated glycation-related crystallin damage (PNAS 2006 (103):16912). However, understanding of the role of ascorbylation in cataractogenesis was hampered due to presence of congenital cataract. To circumvent this problem we generated a novel mouse by expressing hSVCT2 using an αA-Crystallin promoter with a Pax6 consensus binding site insertion at -282/+34 contained in the CPV14 vector (Zhao et al, IOVS 2004 (45):1930).
hSVCT2 cDNA was introduced into CPV14 vector. Transgenic hemizygous mice in C57BL/6 background were generated (n= 15) and kept alive until 24 months of age. Lenses were analyzed in vivo for opacities at 12 and 24 months using slit lamp examination, and biochemically for levels of ASA, GSH, and formation of advanced glycation enpdroducts (AGEs). Data were compared with C57BL/6 wild type mouse.
Hemizygous CPV14-hSVCT2 transgenic mice have around 1.5-2.0mM of ascorbic acid in the lens, and has no congenital cataract. At 12months old, ~10% of mice lenses started to develop mild opacification (<0.3 µm) compared to WT lenses. At 24 months, over 50% of CPV-hSVCT2 transgenic mice lenses developed large size opacification (>0.3µm) as well as nuclear cataract. Ascorbic acid oxidation and ascorbylation products determined by LC/MS were significantly elevated vs. WT: carboxymethyl-lysine (CML) (~2fold), methylglyoxal-derived hydroimidazolones MG-H1 (2.5 fold) and pentosidine (2.5 fold). Methionine sulfoxide (MetSOX), the oxidation marker was also significantly increased (~ 2fold) vs. wt.
A novel transgenic mouse model of ascorbylation that is free of congenital cataract at birth but progressively displaying lens opacities during aging was successfully generated. The novel model is expected to help us understand the relationship between crystallin damage in vivo, aggregation and cataractogenesis and help develop targeted anticataract drugs.
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