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.