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M.J. Radeke, P.T. Johnson, K.E. Peterson, L.V. Johnson, D.H. Anderson; Characterization of the Human Retinal Pigmented Epithelium Secretome by Proteomic and Genomic Analysis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5130.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:. The RPE plays a key role in the maintenance of the retina. It does so by phagocytosis of shed portions of photoreceptor outer segments, regulating the flow of metabolites and catabolites, and directly providing factors that maintain and regulate the retina. Dysfunction of these processes has been implicated in retinal degenerative diseases such as AMD. To better understand the molecular mechanisms involved, we have begun to identify the complement of secreted proteins synthesized by cultured human RPE using proteomic and genomic methods. Methods: Proteins from serum free media conditioned by confluent primary neonatal human RPE cells were fractionated by 2D gel electrophoresis. The identities of the secreted proteins were determined using nanoLC–ESI–MSMS. An additional survey of the secreted transcriptome was carried out using microarray analysis. RNA was purified from cultured neonatal human RPE cells and probes were generated using reverse transcriptase, T7 RNA amplification, and SPIA–RNA amplification. These probes were used to profile the expression of genes coding for secreted proteins by screening a whole human genome microarray. Genes coding for secreted proteins were identified based on their assigned ontologies and a consensus expression pattern was generated from the array results obtained using the different methods. Results:Over 200 of the most abundant protein spots have been analyzed by mass spectrometry. The majority of the proteins identified represent either structural proteins associated with extracellular matrix such as collagens I, II, and III, fibronectin, and perlecan or proteins associated with extracellular matrix homeostasis such as TIMP1 and TIMP2, lysyl oxidase homolog 1, and prolyl–4–hydroxylase. There were also a number of proteins thought to be confined to intracellular compartments. Their presence may result from regulated secretion or cell lysis. Transcriptional analysis was consistent with the proteomic results. In addition, the microarry data revealed a much greater diversity of potential RPE secreted proteins including growth factors, cytokines and proteins involved in local inflammatory processes. Conclusions: The results obtained in these studies represent the initial steps in fully characterizing the RPE secreted proteome. Furthermore, in the future, contrasting the secretome of these relatively undifferentiated RPE cells in culture to differentiated RPE in culture and in situ should yield potential insight into normal RPE function and into dysfunction resulting in disease.
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