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C.S. Alge, S. Suppmann, S.G. Priglinger, A.S. Neubauer, U. Welge-Lussen, M. Ueffing, A. Kampik; A Proteomic Approach to Proliferative Vitreoretinopathy . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2950.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). To probe the proteomic shift in the process of RPE dedifferentiation the protein expression pattern of native, differentiated RPE cells was compared to that of cultured, thereby dedifferentiated RPE cells. Methods: Differentiated, native human RPE cells and monolayers of dedifferentiated, cultured primary human RPE cells of passage 3 were processed for 2-D electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9 – 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver stained gel spots were excised, digested in situ and analyzed by MALDI-TOF mass spectroscopy. The resulting peptide mass fingerprints were searched against the NCBInr, MSDB and EnsemblC database to identify the respective proteins. Results: A total of 179 protein spots was analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells including RPE 65, CRALBP, and CRBP were absent in dedifferentiated, cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including Cathepsin D and Clathrin were still present. Dedifferentiated RPE cells displayed a strong shift towards increased expression of proteins associated with cell shape, cell adhesion and stress fiber formation (eg. cytokeratin 19, gelsolin, tropomyosins) and also acquired increased expression of factors involved in translation and tumorigenic signal transduction (eg. annexin I , translation initiation factor (eIF)-5A). Conclusion: The proteomic changes illustrated in this study clearly reflect the dynamic changes in the protein expression pattern associated with dedifferentiation of the RPE. Some of the identified proteins have previously been described to be associated with RPE dedifferentiation or PVR, while other proteins may be newly linked with RPE dedifferentiation and proliferation and have not yet been described in the RPE. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.
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