Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Abstract

Purpose The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Methods Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Results Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. Conclusions This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.

Advanced glycation end products‐related modulation of cathepsin L and NF‐κB signalling effectors in retinal pigment epithelium lead to augmented response to TNFα

“…Glycolaldehyde was used to induce AGE formation on the basement membrane matrix as glycolaldehyde‐derived AGE adducts involving reactive α‐oxaloaldehydes have been observed in human BrM . The use of glycolaldehyde to induce AGE formation on matrix in vitro, along with the degree of AGE adduct formation and crosslinking have been described previously . A progressive rise in AGE has been observed in the BrM with age .…”

“…The cellular model used in this study exploited the presence of AGE in the ECM of RPE cells to mimic one aspect of the ageing process of these cells. Glycolaldehyde was used to induce AGE formation on the basement membrane matrix as glycolaldehyde‐derived AGE adducts involving reactive α‐oxaloaldehydes have been observed in human BrM . The use of glycolaldehyde to induce AGE formation on matrix in vitro, along with the degree of AGE adduct formation and crosslinking have been described previously .…”

Aging is not a disease: Distinguishing age-related macular degeneration from aging

“…Recently, it was suggested that AGEs may be generated from bisretinoids within RPE (Yoon et al, 2012). Accumulation of AGEs within Bruch's membrane has been shown to alter the RPE proteome, potentially reducing the monolayer's degradative capacity (Glenn et al, 2012). …”

Mechanisms for Countering Oxidative Stress and Damage in Retinal Pigment Epithelium

“…Several E3 ligases have been detected including parkin, which is expressed in photoreceptors as well as across the retina (Esteve-Rudd et al, 2010), and KLHL7, which is expressed in ganglion cells and the inner nuclear layer (Friedman et al, 2009). PGP 9.5, a Ub carboxy-terminal hydrolase, is only present in retinal ganglion and horizontal cells (Bonfanti et al, 1992), but the Ub hydrolase, UCH-L3, is enriched in photoreceptor inner segments (Sano et al, 2006) and Ub carboxy-terminal hydrolase-1 (UCH-L1) was detected in the RPE layer of human retinal sections (Glenn et al, 2011) as well as in horizontal, bipolar, amacrine, and ganglion cells (Esteve-Rudd et al, 2010). Recently, it has been demonstrated that Ub-specific protease 2 (USP2) is rhythmically expressed in the retina and controls the circadian clock at the evening light to dark transition (Scoma et al, 2011).…”