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

J. V. Glenn, H. Mahaffy, S. Dasari, M. Oliver, M. Chen, M. E. Boulton, H. Xu, W. J. Curry, Alan W. Stitt

Research output: Contribution to journalArticle

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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 (AGEBM), 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 AGEmodification 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.

Original languageEnglish (US)
Pages (from-to)349-359
Number of pages11
JournalGraefe's Archive for Clinical and Experimental Ophthalmology
Volume250
Issue number3
DOIs
StatePublished - Mar 2012
Externally publishedYes

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Retinal Pigment Epithelium
Proteomics
Bruch Membrane
Proteins
Proteome
Basement Membrane
Retina
Immunohistochemistry
Advanced Glycosylation End Products
Public Opinion
Electrospray Ionization Mass Spectrometry
Densitometry
Electrophoresis, Gel, Two-Dimensional
Macular Degeneration
Hydrolases
Ubiquitin
Heat-Shock Proteins
Lipids

Keywords

  • Advanced glycation endproducts
  • Bruch's membrane
  • RPE
  • Ubiquitin carboxyterminal hydrolase-1

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate. / Glenn, J. V.; Mahaffy, H.; Dasari, S.; Oliver, M.; Chen, M.; Boulton, M. E.; Xu, H.; Curry, W. J.; Stitt, Alan W.

In: Graefe's Archive for Clinical and Experimental Ophthalmology, Vol. 250, No. 3, 03.2012, p. 349-359.

Research output: Contribution to journalArticle

Glenn, J. V. ; Mahaffy, H. ; Dasari, S. ; Oliver, M. ; Chen, M. ; Boulton, M. E. ; Xu, H. ; Curry, W. J. ; Stitt, Alan W. / Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate. In: Graefe's Archive for Clinical and Experimental Ophthalmology. 2012 ; Vol. 250, No. 3. pp. 349-359.
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