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.

Increased Secretion and Expression of Myostatin in Skeletal Muscle From Extremely Obese Women

“…The remaining 14 are metabolic enzymes, cytoskeletal, or contractile proteins. The presence of intracellular proteins in conditioned culture media has been described in numerous surveys of protein secretion (8,17,23). Several of the proteins secreted by primary human myotubes have been described previously in studies of differentiating mouse myotubes (17) and human adipose (23) and retinal pigment epithelial cells (8).…”

“…Labeling media was supplemented with 13 C 6 -Lys (Invitrogen) for cells derived from extremely obese donors, whereas unlabeled media were supplemented with regular L-Lys for lean cells and 10% dialyzed fetal bovine serum (FBS) and 50 g/ml gentamicin/amphotericin B. Cells were subcultured in collagen I-coated 75-cm 2 flasks to passage 3, which has been shown previously to produce fully labeled cells (8). Differentiation into myotubes was initiated at 90% confluence using labeled and unlabeled differentiation medium containing 2% horse serum with media changes every 2 days for a total of 9 days (16).…”

“…The retention time and protein identification for each peptide were then confirmed by the y ions from each MS/MS and the relative abundance of selected peptide pairs (Figs. 1 and 2) calculated as previously described (8). The search parameters allowed for variable modifications, including amidomethylation of cysteine, oxidation of methionine, and presence of 13 C 6 -Lys.…”

“…The search parameters allowed for variable modifications, including amidomethylation of cysteine, oxidation of methionine, and presence of 13 C 6 -Lys. Secreted proteins had to have a minimum of one 13 C 6 -Lys peptide identified with high confidence and had to be verified by manual inspection for having a consecutive series of y ions (8).…”

“…In this study, we hypothesized that skeletal muscle of extremely obese and insulinresistant women would secrete proteins into circulation that act as prognostic or diagnostic biomarkers of obesityassociated comorbidities. However, a top-down approach toward identifying protein biomarkers in blood is hampered by an abundant background of serum proteins, wherein a secreted protein of interest may be diluted several orders of magnitude (5,8). To overcome these limitations, we adopted a bottom-up approach for characterizing the skeletal muscle secretome using primary human muscle cells and stable isotope labeling by amino acids in culture (SILAC), which allows for the identification of muscle-specific proteins and for the quantification of proteins between samples (5,8).…”