title: Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress.
abstract: Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPKα1 (S485), acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.
authors.affiliation: CNR Institute of Neuroscience, Viale G. Colombo 3, 35121 Padova, Italy; Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy. Electronic address: lucia.biasutto@cnr.it.
authors.forename: Lucia
authors.initials: L
authors.lastname: Biasutto
authors.affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd, Manassas, VA 20110, USA.
authors.forename: Antonella
authors.initials: A
authors.lastname: Chiechi
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, 10900 University Blvd, Manassas, VA 20110, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Couch
authors.affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd, Manassas, VA 20110, USA.
authors.forename: Lance A
authors.initials: LA
authors.lastname: Liotta
authors.affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd, Manassas, VA 20110, USA. Electronic address: vespina@gmu.edu.
authors.forename: Virginia
authors.initials: V
authors.lastname: Espina
mesh_terms: D002478:Cells, Cultured; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D018384:Oxidative Stress / Q000502:physiology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods / Q000592:standards; D040901:Proteomics / Q000379:methods / Q000592:standards; D017382:Reactive Oxygen Species / Q000378:metabolism; D012015:Reference Standards; D055213:Retinal Pigment Epithelium / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D015398:Signal Transduction
chemical_list: D010750:Phosphoproteins; D017382:Reactive Oxygen Species
keywords: AFM; AMD; ARPE-19 cells; Age related macular degeneration; Age-related macular degeneration; Atomic force microscopy; ESCRT; Endosomal sorting complex required for transport; Exosomes; FBS; Fetal bovine serum; MVB; Multi vesicular bodies; Oxidative stress; ROS; RPE; RPMA; Reactive oxygen species; Retinal pigment epithelium; Reverse phase protein arrays; TLR; Toll like receptor