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Original Investigation | Journal Club

Proteomic Landscape of the Human Choroid–Retinal Pigment Epithelial Complex

Jessica M. Skeie, PhD1; Vinit B. Mahajan, MD, PhD1
[+] Author Affiliations
1Omics Laboratory, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City
JAMA Ophthalmol. 2014;132(11):1271-1281. doi:10.1001/jamaophthalmol.2014.2065.
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Importance  Differences in geographical protein expression in the human choroid–retinal pigment epithelial (RPE) complex may explain molecular predisposition of regions to ophthalmic diseases such as age-related macular degeneration.

Objective  To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions.

Design, Setting, and Participants  Experimental study of choroid-RPE tissue from 3 nondiseased eyes. The choroid-RPE complex underwent biopsy from beneath the foveal, macular, and peripheral retina. Protein fractions were isolated and subjected to multidimensional liquid chromatography and tandem mass spectrometry. A bioinformatic pipeline matched peptide spectra to the human proteome, assigned gene ontology classification, and identified protein signaling pathways unique to each of the choroid-RPE regions.

Main Outcomes and Measures  Mean number of mass spectra, statistically significant differentially expressed proteins, gene ontology classification, and pathway representation.

Results  We identified a mean of 4403 unique proteins in each of the foveal, macular, and peripheral choroid-RPE tissues. Six hundred seventy-one differentially expressed proteins included previously known risk factors for retinal diseases related to oxidative stress, inflammation, and the complement cascade. Gene ontology analysis showed that unique categories in the foveal and macular regions included immune process proteins as well as protein complexes and plasma membrane proteins. The peripheral region contained unique antioxidant activity proteins. Many proteins had the highest expression in the foveal or macular regions, including inflammation-related proteins HLA-A, HLA-B, and HLA-C antigens; intercellular adhesion molecule 1 (ICAM-1); S100; transcription factor ERG; antioxidant superoxide dismutase 1 (SOD1); chloride intracellular channel 6 ion (CLIC6); activators of the complement cascade C1q, C6, and C8; and complement factor H. Proteins with higher expression in the periphery included bestrophin 1 (BEST1), transcription factor RNA binding motif protein 39 (RBM39), inflammatory mediator macrophage migration inhibitory factor, antioxidant SOD3, ion channel voltage-dependent anion-selective channel protein 3 (VDAC3), and complement inhibitor CD55. The complement activation was among the highest represented pathways (P < 7.5e−13).

Conclusions and Relevance  This proteomic data set identifies novel molecular signatures in anatomically sensitive regions of the choroid-RPE complex. The findings give mechanistic insight into choroid-RPE function, reveal important choroid-RPE processes, and prioritize new pathways for therapeutic targeting.

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Figure 1.
Fundus Images of Choroid–Retinal Pigment Epithelial (RPE) Complex Disease Display Region-Specific Diseases

A, Geographical map of the 3 different tissue samples. B, Detailed image of the human choroid-RPE tissue punch biopsy specimens collected for this study. C, Fundus image of neovascular age-related macular degeneration (arrowhead indicates subretinal hemorrhage from choroidal neovascularization; small circle, fovea; large circle, macula area). D, Fundus image of macular geographic atrophy sparing the fovea (large circle indicates the macula area). E, Fundus image of pigment degeneration in the periphery.

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Figure 2.
Differential Expression of Several Proteins in the Human Choroid–Retinal Pigment Epithelial (RPE) Complex

Unbiased clustering of proteins differentially expressed (P < .05) in the 3 different regions of the human choroid-RPE complex. Proteins represented in this cluster analysis were compared using analysis of variance (P < .05). The heatmap is divided into regions. A, Proteins with highest expression in the macula. B, Proteins with highest expression in the periphery. C, Proteins with expression exclusive to the periphery. D, Proteins with expression exclusive to the fovea. E, Proteins with highest expression in the fovea. F, Proteins absent in the macula but present in the fovea and periphery. Numbers indicate the number of proteins in each category. The bar at the bottom depicts a logarithmic color scale for relative expression where orange represents high and black represents low levels.

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Figure 3.
Gene Ontology (GO) Distributions and Pathway Analysis of Differentially Expressed Human Choroid–Retinal Pigment Epithelial (RPE) Proteins by Geographical Location

Analysis reveals unique groups of proteins. A, Differentially expressed proteins were determined by analysis of variance. Proteins with P < .05 were chosen from each geographical region. Proteins were grouped into subcategories of biological processes, molecular functions, and cellular component for each region of choroid-RPE. B, Top ten differentially expressed protein pathways represented in the choroid-RPE. ACM indicates muscarinic acetylcholine receptor M3; EGF, endothelial growth factor; GPCR, G-protein coupled receptor; LRRK2, leucine-rich repeat serine/threonine-protein kinase 2; LTD4, leukotriene D4; and PDGF, platelet-derived growth factor.

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Figure 4.
Proteins With High Expression in the Human Foveal Choroid–Retinal Pigment Epithelial Tissues

Nineteen proteins had statistically significant differences in expression among the 3 different geographical regions, with the highest expression in the fovea. Proteins are divided into categories. A, Matrix proteins. B, Rab proteins. C, Oxidative stress proteins. D, Protein expression regulators. E, Immune modulators. The statistically significant changes are available in eTable 4 in the Supplement. Abundance of protein is displayed as mean number of spectra. ACAA1 indicates acetyl coenzyme A acyltransferase 1; ERG, transcription factor ERG; HM13, minor histocompatibility antigen H13; LAM, laminin; MYH, myosin; NID1, nidogen 1; PRDX1, peroxiredoxin 1; PSMD11, proteasome 26S subunit 11; and TUFM, Tu translations elongation factor. Error bars indicate SEM.

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Figure 5.
Network Diagram of Complement Cascade Proteins With High Representation in the Human Choroid–Retinal Pigment Epithelial (RPE) Complex

Each pie chart shows the relative protein representation in the fovea (pink), macula (blue), and periphery (yellow). The size of the pie chart represents a relative abundance of each complement protein in the data set. Activators (green arrows) and inhibitors (red lines) are also listed in eTable 6 in the Supplement. The 3 different pathways that initiate complement activation are shown. All pathways converge at the formation of C3 and C5 convertases and result in the formation of anaphylatoxins C3a and C5a (blue) and the membrane attack complex in the terminal pathway. The (n) indicates that multiple C9 molecules can be linked to this protein complex. CD55 indicates decay accelerating factor for complement; CD59, complement regulatory protein; CFD, complement factor D; CFH, complement factor H; CFI, complement factor I; CLU, clusterin; MASP, mannan-binding serine peptidase; MBL, mannose-binding lectin; and SERPING1, serpin peptidase inhibitor, clade G (c1 inhibitor), member 1.

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