Abstract
Purpose: :
Advanced glycation endproducts (AGE), a result of covalent modifications of proteins by glycosylation, accumulate with aging and are linked to several age-related retinal disorders. AGE deposits are found in drusen and in Bruch's membrane of aged and diseased eyes, but what cellular pathways are activated by their presence in outer retina is not fully known. This study investigates the effects of AGE stimulation on global gene transcription, cellular pathways and protein secretion in primary culture of human retinal pigment epithelial cells (RPE). Revealing the effects of AGE on RPE function will help elucidate the pathological changes that occur in outer retina with accumulation of AGE and may allow new insight into the effects of drusen on age-related retinal changes and the pathogenesis of AMD.
Methods: :
Primary cultures of human RPE cells were stimulated with 10 μg/ml AGE for 4h, and genome-wide changes in gene expression were studied with Agilent Oligo microarrays. Primary genes of interest were detected in RNA samples using qRT-PCR with primers designed from Primer Express 2.0 software. Western blot analysis of protein extract was performed with select primary autoantibodies. RPE gene differential expression was analyzed using gene set enrichment analysis (GSEA). Protein levels of secreted cytokines and growth factors were studied using Bio-Plex Luminex arrays.
Results: :
A total of 41 up- and 18 down-regulated RPE genes were differentially expressed. These genes fell into three main categories as assessed by GSEA: inflammation (interferon-induced, immune response and chemokine expression), proteasome degradation and caspase pathways. The highest upregulated gene was CXCL11, a chemokine. Its production in AGE stimulated RPE cells was confirmed using western blot analysis. Cytokine assay of supernatant using Bio-Plex Luminex assay showed the highest increase in pro-inflammatory cytokine GM-CSF secretion along with several anti-inflammatory cytokines including IL-10, IL-1ra and IL-9, while many pro-inflammatory cytokines remained unchanged or underexpressed after AGE stimulation.
Conclusions: :
Our studies provide new insights into the cellular pathways triggered by the accumulation of AGE in the outer retina. RPE cells respond to AGE stimulation by activation of two major cell signaling pathways: the NF-kB pathway and the JAK-STAT signaling pathway. Our results suggest novel mechanisms for AGE modulation of RPE cellular pathways and identify novel targets for drug development that can minimize the progression of age-related retinal diseases.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • inflammation