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Luminita I Paraoan, Paul Kay, Paul Hiscott, Arvydas Maminishkis, Yit C Yang; AGE-related Modulation of Cystatin C Expression, Secretion and Polarity in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1331.
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Cystatin C is a potent cysteine proteinase inhibitor and one of the most abundantly expressed proteins of the retinal pigment epithelium (RPE) (top 2%). High basolateral secretion levels suggest a role in maintaining the structure and function of extracellular features within the Bruch’s membrane (BM)/choroid. A variant of cystatin C (variant B) is associated with an increased risk of developing exudative age-related macular degeneration (AMD) and presents reduced secretion compared to the wild type. The aim of this study was to analyze the effects of age, and the accumulation of advanced glycation end-products (AGEs) on the expression and secretion of wild type cystatin C.
Confluent monolayers of human fetal RPE (hfRPE) cells were cultured on AGE containing BM mimics in vitro. After 2-10 weeks, cystatin C expression and secretion (total and polarized) were analyzed in cell lysates and conditioned media by qPCR, Western Blotting and ELISA. Barrier properties of monolayers were monitored by transepithelial resistance (TER) measurements.
Cystatin C expression was reduced almost two-fold following culture in the presence of AGEs, at both the mRNA and protein level (independent T test, p= <0.05). Secretion levels were also similarly reduced from approximately 56ng/ml/24h in normal conditions, to 36ng/ml/24h in the presence of AGEs (p= <0.05). Long term exposure to AGEs significantly reduced the TER of hfRPE monolayers, and abolished the basal polarity of cystatin C secretion (paired T test, p=<0.05).
Cystatin C expression/secretion is reduced in conditions characterized by extracellular AGEs accumulation, a common occurrence associated with natural aging and AMD pathology. Reduced concentration of such a highly expressed and ubiquitous proteinase inhibitor at its sites of action could impact efficient protein turnover, contributing to extracellular structural changes commonly observed in the BM/choroid during the progression of AMD.
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