JAMA Ophthalmology Journal Club Slides: Proteomic Landscape of the Choroid-RPE Complex

1. JAMA Ophthalmology Journal Club Slides:Proteomic Landscape of theChoroid-RPE Complex Skeie JM, Mahajan VB. Proteomic landscape of the human choroid–retinal pigment epithelial complex. JAMA Ophthalmol. Published online July 24, 2014. doi:10.1001/jamaophthalmol.2014.2065.

2. Introduction • Differences in geographical protein expression in the human choroid–retinal pigment epithelial (RPE) complex may explain molecular predisposition of specific regions to ophthalmic diseases such as age-related macular degeneration. • Mass spectrometry provides a robust platform for identifying thousands of proteins and defining differences in the subfoveal, macular, and peripheral choroid-RPE proteomes. • Objective: To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions.

3. Introduction Fundus Images of Choroid-RPE Complex Disease Display Region-Specific Diseases

4. Methods • Study Design: The human choroid-RPE was biopsied from beneath the foveal, macular, and peripheral retina. Proteins underwent chromatography and tandem mass spectrometry. • Participants: Three nondiseased human eyes. • Data Analysis: A bioinformatic pipeline matched peptide spectra to the human proteome and identified protein signaling pathways unique to each of the choroid-RPE regions. • Limitations: Limited specimen number and proteomic data set will require prospective validation.

5. Methods Choroid-RPE Proteome Analysis Pipeline A, The human choroid-RPE was dissected into fovea, macula, and periphery samples. B, Fractions of proteins were isolated and digested.C, The peptide fragments were analyzed using multidimensional liquid chromatography and mass spectrometry. D, X!Hunter, X!!Tandem, and the open mass spectrometry search algorithm were used for peptide fragment identification.E, Proteins were further analyzed using bioinformatics.

6. Results • We identified more than 4000 unique proteins in each region of the choroid-RPE complex. • The most abundant proteins included albumin, serpin peptidase inhibitor, vimentin, transferrin, actin, cathepsin D, and complement component C3. • Pathway and functionally related proteins were differentially expressed in the fovea, macula, and peripheral choroid-RPE complex.

7. Results Heatmap of Proteins Expressed in Specific Regions Each row in the heatmap represents a protein. The columns represent the individual tissue samples. Orange indicates high expression; black, low expression. Many proteins are highly specific to a particular region. For example, several antioxidant proteins had higher expression in the periphery (SOD3 and GPX3) and others had higher expression in the fovea (SOD1, GPX1, GPX4, PRDX1, PRDX2, PRDX3, and VTN).

8. Results Top 10 Differentially Expressed Protein Pathways Represented in the Choroid-RPE Bioinformatic software identified protein pathway relationships. Protein signaling pathways were found in the fovea, macula, or peripheral choroid-RPE complex. ACM indicates muscarinic acetylcholine receptor M3; EFG, 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.

9. Results Proteins With High Expression in the Human Foveal Choroid-RPE Tissues

10. Results Network Diagram of the Complement Cascade Proteins With High Representation in the Human Choroid-RPE Complex One of the most highly represented pathways was the complement cascade, providing an important context for interpreting the prior genetic studies, specifically how genome-wide association study risk alleles relate to potential therapeutics. C8G, C6, CFH, and CLU were significantly higher in the fovea than in the macula and the periphery. CD55 was highest in the periphery and almost completely absent in the fovea.

11. Comment • There are significant molecular differences between regions of the human choroid-RPE complex that may account for the regional susceptibility of age-related macular degeneration, central serous retinopathy, retinitis pigmentosa, and inflammatory choroiditis. • Transcription proteins profoundly influence tissue functions by controlling gene expression. V-ets erythroblastosis virus E26 oncogene homolog (ERG) was highest in the foveal choroid-RPE region. • Ion channels maintain fluid homeostasis, and voltage-dependent anion channels 1 and 3 (VDAC1 and VDAC3) displayed highest expression in the periphery, while chloride intracellular channel 6 (CLIC-6) was highest in the foveal choroid-RPE region. • Inflammatory proteins, such as macrophage migration inhibitory factor (MIF) and proteasome subunit beta type 8 (PSMB8), were higher in the periphery.

12. Comment • Molecular control of infection and inflammation regulation is a primary activity within the choroid-RPE complex. Among the most abundant proteins were transferrin and complement component C3 of the innate immune system. • Complement factor H (CFH) inhibits formation of the membrane attack complex, so the Y402H variant potentially causes a loss of function. There was relatively more CFH in the fovea than in the periphery, which may represent a protective mechanism that can be disrupted by loss of function mutations in patients with age-related macular degeneration and high-risk alleles. • CD55, an inhibitor of membrane attack complex formation,showed higher expression in the peripheral choroid-RPE region. Lower CD55 expression in the posterior pole may make the fovea and macula more susceptible to complement-activated damage.

13. Contact Information • If you have questions, please contact the corresponding author: • Vinit B. Mahajan, MD, PhD,Omics Laboratory, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, 200 Hawkins Dr, Iowa City,IA 52242 (vinit-mahajan@uiowa.edu). Funding/Support • This study was supported by the Bright Focus Foundation and by grants 1F32EY022280-01A1 (Dr Skeie) and K08EY020530 (Dr Mahajan) from the National Institutes of Health. Conflict of Interest Disclosures • None reported.