Purchase this article with an account.
T. K. Lin, J. Cui, K. Kurji, G. Walker, L. Yu, L. Chen, S. Prasad, J. A. Matsubara; Microarray Analysis Identifies Induction of Inflammatory Gene Expression After Advanced Glycation Endproduct (AGEs) Stimulation of Human RPE in vitro. Invest. Ophthalmol. Vis. Sci. 2009;50(13):701.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
AGEs, products of covalent modification of proteins by glucose, accumulate with increasing age and are linked to several age related disorders including atherosclerosis, Alzheimer’s disease, and cataract formation. AGE adducts are found in drusen and in Bruch’s membrane of patients with age-related macular degeneration (AMD). This study investigates the effects of AGEs on primary culture of retinal pigmented epithelium (RPE) and their putative role in retinal inflammation in AMD.
Cell viability assays were performed in 1% fetal bovine serum (FBS) in Dulbecco’s Modified Eagle’s Medium (DMEM) and AGEs (0.01-50.0 µg/ml). Cell survival was determined at 24h with the MTT assay (3-[4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyltetrazolium bromide). Genome-wide changes in gene expression of RPE stimulated with 10 µg/ml AGEs for 24h were studied with Agilent Oligo microarrays and verified by quantitative RT-PCR. Western blots were performed to correlate gene expression and in vitro changes in protein levels.
MTT assay demonstrated a dose dependent cytotoxicity of AGEs on RPE. AGE stimulation at 0.01, 10.0 or 50.0 µg/ml resulted in 104%, 89%, and 71% RPE cell viability, respectively. RPE cells stimulated with 10 µg/ml AGE resulted in 73 up- and 61 down-regulated genes above the 2.0 fold thresholds. Th1-associated chemokine (C-X-C motif) ligand 11 (CXCL11), a potent ligand for chemokine (C-X-C motif) receptor 3 (CXCR3), was upregulated 12.7 folds. Angiopoietin-like 1 (ANGPTL1) and endothelial cell-specific molecule 1 (ESM1) mRNAs, both associated with highly vascularized tissues, were upregulated 3.3 and 2.2 fold respectively. Matrix metallopeptidase 16 (MMP16), involved in the breakdown of extracellular matrix, was upregulated 2.0 fold. AGE stimulation caused a 3.3 fold reduction in mRNA levels of solute carrier family 30, member 10 (SLC30A10), which encodes a zinc transporter, and 2.5 fold reduction in apolipoprotein D (APOD), which encodes a component of high density lipoprotein.
AGE stimulation causes RPE cells to upregulate chemokines, angiogenic and tissue remodeling associated molecules. These gene products will be discussed in context of chronic retinal inflammation. AGE stimulation also reduced SLC30A10, and may be significant as zinc supplementation has been found to lower the probability of developing neovascular AMD in patient subgroups. A decrease in APOD mRNA deserves further consideration due to its relationship with cholesterol, which is a component of drusen, and as a regulator of oxidative stress.
This PDF is available to Subscribers Only