Proteomic Analysis of Human Retinal Pigment Epithelium Growing on an "Aged" Substrate

Abstract

Purpose:: Age-related pathological changes in the outer retina have been linked to the formation of advanced glycation endproducts (AGEs) and crosslinking at Bruch’s membrane, although it is largely unknown how such aged substrate alters RPE function. Using a model system to mimic aged Bruch’s membrane this study has investigated alterations to the aged RPE proteome compared to non-aged substrate to asses age-related RPE dysfunction.

Methods:: The human retinal epithelial cell line ARPE-19 was cultured (0-28 days) on a synthetic basement membrane (BM) extract (Matrigel^TM). RPE were exposed to native, unmodified BM or AGE-modified BM (AGE-BM) to mimic age-related adduct formation comparable to that detected on Bruch’s membrane from donor eyes. The RPE proteome derived from control and aged cells were extracted in either CHAPS/Tris buffer or ammonium bicarbonate buffer resolved using IEF either pH 3-10 and 4-7 range. 2DGE profiles were analysed using densitometric annotation and proteins of interest identified by ESI-MS/MS. Subsequently identified candidate proteins were assessed using Western Blot analysis and immunohistochemistry.

Results:: Semi-quantitative 2DGE analysis revealed significant alterations in protein expression levels between glycated and native cell profiles. 2DGE resolved on average 279 spots from cells cultured on BM as compared to 221 spots for cells culture on AGE-BM. Thirty seven proteins which exhibited significant altered expression between treatments were identified by ESI-MS/MS. Proteins identified included calmodulin, ubiquitin carboxyl terminal hydrolase (UCH L1) and pro-activator polypeptide activity which were significantly (each >2.8 fold) upregulated in the AGE-BM treated cells. Significant decreases were also evident on AGE-BM culture at levels >4.9 fold for phosphoglycerate kinase, c-myc promoter binding protein and TNFß precursor. Post-translational modifications were also noted.

Conclusions:: Differential expression was observed for a wide range of RPE proteins, some of which represent regulators of oxidative stresses/angiogenesis and resultant alteration to cell signalling mechanisms. The abundance of protein disulfide isomerase, calmodulin and UCH L1 supports the concept that proteasomal and lysosomal dysfunction are important in RPE dysfunction. This study shows that AGE-modifications on the substrate could be linked to age-related abnormalities in the RPE.

Keywords: proteomics • retinal pigment epithelium • aging