Proteomic profiling of human retinal and choroidal endothelialcells reveals molecular heterogeneity related to tissue of origin

[摘要] Purpose: The ocular vascular endothelium plays a key role in thedevelopment of several leading retinal causes of blindness in Westernnations. Choroidal endothelial cells are integral to the subretinalneovascular lesions that characterize the exudative form of lateage-related macular degeneration (AMD), and retinal endothelial cellsparticipate in the initiation of diabetic retinopathy and posterioruveitis. Vascular endothelial cells at different sites exhibitconsiderable molecular diversity. This diversity has implications forunderstanding the pathogenesis of tissue-specific diseases and for thedevelopment of targeted therapies to treat these conditions. Previouswork from our group has identified significant differences in the genetranscript profiles of human retinal and choroidal endothelial cells.Because the proteome ultimately determines the behavior of any givencell, however, it is critical to determine whether molecular differencesexist at the level of protein expression.Methods: Retinal and choroidal endothelial cells were separatelyisolated from five sets of human eyes by enzymatic digestion with typeII collagenase followed by anti-CD31 antibody-conjugated magnetic beadseparation. Cells were washed to remove serum peptides in the culturemedium, and lysed by sonication in buffer containing 2% sodium dodecylsulfate. Protein was then precipitated with acetone. Retinal andchoroidal endothelial samples from each donor were labeled with Cy3 andCy5, respectively, mixed with a Cy2-labeled pooled protein sample tofacilitate spot matching across gels, and separated by two-dimensionaldifference gel electrophoresis (2D-DIGE). Following a globalnormalization, differentially abundant protein spots that were visiblein at least four of five donor gels were detected by the significanceanalysis of microarrays method, with false discovery rate set at 5%.Corresponding spots were excised from additional DIGE-labeled orCoomassie-stained 2D electrophoretic gels. Protein identification wasperformed by liquid chromatography and tandem mass spectrometry.Results: Of 123 protein spots detected by 2D-DIGE that qualified forstatistical analysis, we found 31 spots that demonstrated a significantdifference in abundance between retinal endothelial samples versuschoroidal endothelial samples. For 17 proteins, over 50% of the spectralcounts could be matched to a single protein in the digested spot. Elevenproteins were more abundant in retinal endothelial cells (i.e.,inorganic pyrophosphatase, protein disulfide isomerase A3, calreticulin,peroxiredoxin-4, protein disulfide isomerase, serpin B9, F-actin cappingprotein subunit β, coactosin-like protein, vimentin, cathepsin B,and a high molecular weight form of annexin A3). Six proteins were moreabundant in choroidal endothelial cells (i.e., glutathione peroxidase 1,ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), heat-shockprotein beta-1, superoxide dismutase (Cu-Zn), nucleoside diphosphatekinase A, and a low molecular weight form of annexin 3).Conclusions: Our data indicate that the proteomes of retinal andchoroidal vascular endothelial cells are different. Severaldifferentially expressed proteins are implicated in the regulation ofangiogenesis; these include cathepsin B and UCH-L1, proteins withtranscripts that were also differently expressed according tomicroarray. Our observations further suggest that angiogenesis withinthe retina, a component of severe diabetic retinopathy and posterioruveitis, may be controlled by different mechanisms to those regulatingchoroidal neovascularization, as occur in exudative AMD. Future studiesto establish the role of these angiogenic proteins in disease maysuggest potential new targets for tissue-specific therapies.

[学科分类] 生物化学/生物物理