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D.O. Zamora, M. Riviere, Y. Pan, S.R. Planck, J.T. Rosenbaum, L.L. David, J.R. Smith; Proteomic Profiling of Human Retinal and Choroidal Endothelial Cells Reveals Heterogeneity Related to Tissue of Origin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5160.
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© ARVO (1962-2015); The Authors (2016-present)
A variety of inflammatory or infectious diseases target specific tissues within the eye. This phenomenon may relate in part to the differential abundance of proteins expressed by endothelial cells (EC) of different vascular beds. We used a proteomics approach to compare protein preparations isolated from retinal and choroidal EC, hypothesizing that the proteome of retinal vascular endothelium would differ from that of the choroidal endothelium.
Paired retinal and choroidal tissues were removed from eyes of three human cadaver donors. The EC were isolated as previously described (IOVS, 2001; 42; 2861–2866) and grown in MCDB–131 medium supplemented with 10% FBS and endothelial growth factors. Confluent cells were cultured for 4 hours in medium with 5% FBS, washed with PBS, scraped from plates and lysed by sonication in buffer containing 2% SDS. Protein was precipitated with acetone and dissolved in pH 8.8 buffer containing 8 M urea and 2 M thiourea. Individual samples of retinal and choroidal EC were labeled with CyDye3 or CyDye5, respectively, and a separate pooled sample was labeled with CyDye2. Differentially labeled proteins from each donor were pooled, mixed with a CyDye2–labeled internal standard and separated by 2–dimensional difference gel electrophoresis. Gels were imaged using a laser scanner (GE Healthcare). Differentially abundant proteins were identified by image analysis of the CyDye–labeled gels. Corresponding spots were excised from accompanying Coomassie–stained gels. Protein identification was performed by trypsin digestion and liquid chromatography/tandem mass spectrometry.
The protein profiles of retinal and choroidal EC were remarkably similar, but exhibited reproducible differences. In comparison to protein isolates from choroidal EC, isolates from retinal EC contained a higher abundance of the intermediate filament protein, vimentin (53 kDa), and the actin–binding protein, transgelin–2 (22 kDa).
These data suggest there are differences in the protein composition of vascular endothelium of the retina and choroid. Differentially expressed proteins are potential therapeutic targets for inflammatory or infectious diseases that specifically involve retina or choroid.
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