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S. Johnen, C. Maltusch, M. Stöcker, L. Wickert, A. K. Salz, P. Walter, G. Thumann; Effect of PEDF Fusion Proteins on the Expression of Zinc Transporter Proteins. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2164.
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Zinc plays essential roles in almost all aspects of metabolism, from catalysis to structural determinants to regulatory functions. A number of investigators have shown that zinc may be important to maintain proper vision and in fact it appears that zinc may be one factor in delaying and/or preventing age-related macular degeneration (AMD). On the other hand, it has also been shown that zinc is a component of drusen in aging and AMD eyes. Since zinc is a highly charged, hydrophilic ion, it is unable to cross biological membranes by simple diffusion and requires specialized zinc transporter proteins for cellular uptake and release. Here we show that recombinant PEDF alone and EGFP tagged PEDF secreted by transfected cells affect the expression of the zinc transporter proteins ZnT2 and ZIP2 in cultured ARPE-19 cells.
Recombinant PEDF and PEDF-EGFP fusion proteins were purified by Ni-NTA metal affinity chromatography and subsequently identified by immunoblotting. A confluent monolayer of ARPE-19 cells was treated with defined concentrations of the purified fusion proteins ranging from 1 to 3 µg/ml. After 48 hours the cells were harvested for RNA isolation and the gene expression of the two zinc transporter proteins was determined by RT-PCR.
Treatment of ARPE-19 cells with purified recombinant PEDF or PEDF-EGFP fusion proteins resulted in increased mRNA levels of the zinc transporter proteins ZnT2 and ZIP2, similar to the effects of commercially available PEDF.
Non-virally transfected, autologous pigment epithelial cells that express PEDF might be a useful substitute for RPE transplantation to the subretinal space in AMD patients. The genetically modified cells express a fully functional human PEDF, as evidenced by the effect on the expression of zinc transporter proteins and should thus be able to provide a subretinal environment conducive to re-establish a proper and functional choroid-RPE-neural retina complex that is necessary for visual function.
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