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Characterisation of the advanced glycation endproduct receptor complex in the retinal pigment epithelium

S McFarlane, J V Glenn, A M Lichanska, D A C Simpson and A W Stitt

Queen’s University of Belfast, Institute of Clinical Science, Royal Victoria Hospital, Belfast, UK

Correspondence to:
Correspondence to:
Professor A Stitt
Ophthalmology & Vision Science, The Queen’s University of Belfast, Institute of Clinical Science, Royal Victoria Hospital. Belfast BT12 6BA, UK; a.stitt{at}qub.ac.uk

Aims: Advanced glycation endproducts (AGEs) accumulate with ageing and may have a significant impact on age related dysfunction of the retinal pigment epithelium (RPE). Many of the cellular effects of AGEs in other cell types are mediated through AGE binding proteins. The aim of this study was to characterise the AGE receptor complex in RPE cells in vitro and to focus on the role of the R3 component (galectin-3) as the primary effector of the complex.

Methods: Primary cultures of bovine RPE cells and the human D407 RPE cell line were exposed to AGE modified albumin. Receptor expression was determined using mRNA analysis by quantitative real time RT-PCR and protein characterisation by western blotting. Immunocytochemical analysis examined the cellular localisation of the various components of the AGE receptor complex. The role of the galectin-3 receptor component was examined by transfection and overexpression using the D407 cell line and analysis of soluble AGE-R3 by ELISA.

Results: All three components of the AGE receptor complex were expressed by bovine and human RPE cells. AGE exposure upregulated two components of the receptor complex and also induced significant RPE expression of VEGF mRNA (p<0.05). RPE D407 cells stably transfected to overexpress galectin-3 showed less VEGF induction. In non-transfected RPE which were exposed to AGEs, there was less soluble galectin-3 protein released into the medium (p<0.05), a response that was not evident in transfected cells.

Conclusion: A conserved AGE receptor complex is evident in primary cultures of bovine RPE cells and also in a human cell line. These cells show a pathological response to AGE exposure, an effect which appears to be modulated by the galectin-3 component of the receptor complex.

Abbreviations: AGE, advanced glycation endproduct; AMD, age related macular degeneration; CSLM, confocal scanning laser miscroscope; PBS, phosphate buffered saline; RPE, retinal pigment epithelium

Keywords: retinal pigment epithelium; advanced glycation endproducts; galectin-3

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