title: Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors.
abstract: Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.
authors.affiliation: Center for Genetic Medicine, Children's National Medical Center, and Program in Biochemistry and Molecular Genetics, Institute of Biomedical Science, The George Washington University, Washington, DC, USA.
authors.forename: Eunkyung
authors.initials: E
authors.lastname: An
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Lu
authors.forename: Jessica
authors.initials: J
authors.lastname: Flippin
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Devaney
authors.forename: Brian
authors.initials: B
authors.lastname: Halligan
authors.forename: Eric P
authors.initials: EP
authors.lastname: Hoffman
authors.forename: Eric
authors.initials: E
authors.lastname: Hoffman
authors.forename: Nataly
authors.initials: N
authors.lastname: Strunnikova
authors.forename: Karl
authors.initials: K
authors.lastname: Csaky
authors.forename: Yetrib
authors.initials: Y
authors.lastname: Hathout
mesh_terms: D000368:Aged; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006801:Humans; D008266:Macula Lutea / Q000648:ultrastructure; D008268:Macular Degeneration / Q000378:metabolism* / Q000473:pathology; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D015593:Retinal Drusen / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D014019:Tissue Donors
chemical_list: D011506:Proteins; D020543:Proteome