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E. An, J. Lin, K. Csaky, Y. Hathout; Effect of TNF-alpha on ARPE-19 Secreted Protein. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2198.
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Tumor necrosis factor-α (TNF-α) is one of the major inflammatory cytokines which is known to be involved in the pathogenesis of various vitreoretinal diseases including age related macular degeneration. To study the effect of cytokines on retinal pigment epithelium cells, we investigated the effect of TNF-α on ARPE19 to test whether RPE protein secretion is altered under inflammatory mediated injury which may help understanding AMD pathology (e.g. drusen formation and choroidal neovascularization).
All proteins in ARPE19 cells were metabolically labeled by growing the cells in medium containing 13C6-Arg and 13C6, 15N2-Lys. This strategy will enable us to distinguish RPE secreted proteins from serum protein contaminants. LC-MS/MS analysis and ZoomQuant quantification were used to assess differential protein secretion in TNF-α treated RPE cell versus non-treated cells. Differentially secreted proteins were evaluated by the Ingenuity computational pathway analysis software to identify possible global functions of the secreted protein pattern.
A total of 58 proteins were identified and quantified in the spent media of TNF-α treated RPE versus non-treated RPE cells. A significant Ingenuity pathway network was obtained for 18 among 21 upregulated proteins in TNF-α treated cell. The most relevant function extracted from this network was related to angiogenesis. For example Plasminogen activator inhibitor 1 precursor which promotes angiogenesis was elevated in TNF-α treated RPE cells by almost a factor of 2 when compared to non treated cells. Other upregulated secreted proteins that have angiogenic activity include pigment epithelium-derived factor, Thrombospondin-1, Laminin alpha-5 chain, Laminin gamma-1 chain and complement C3. While other proteins such as Tolloid-like protein 2, Semaphorin-7A, Vimentin, Glucose-6-phosphate isomerase, Desmoglein-2, Collagen alpha-1(VI) chain, and inter-alpha-trypsin inhibitor heavy chain were down regulated following treatment with TNF-alpha.
These preliminary data clearly show that RPE cells secret several proteins that are involved in the regulation of angiogenesis. Alteration in the secretion of these proteins may bring insight into understanding choroidal neovascularization seen in some AMD patients.
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