AGEs decrease cystatin C secretion and alter its polarity in the retinal pigment epithelium - contribution to age-related retinal proteolytic imbalance

The potent cysteine proteinase inhibitor cystatin C is one of the most abundantly expressed proteins of the retinal pigment epithelium (RPE) (top 2%). The RPE function is essential for supporting the neuroretina. High basolateral secretion levels of cystatin C suggest an important role in regulating proteolysis by RPE in relation to maintaining the structure and function of its specialized surrounding tissues, such as the Bruch’s membrane (BM)/choroid. A variant of cystatin C (variant B) was associated with an increased risk of developing exudative age-related macular degeneration (AMD) and presents reduced secretion compared to the wild type. In order to further investigate the functional role of cystatin C in the retinal context, we analysed the expression and secretion of wild type cystatin C in response to a common age-related stress, the accumulation of advanced glycation end-products (AGEs). Primary human fetal RPE cells and human RPE cell lines ARPE19 and D407 were cultured for 14 days on in vitro Bruch’s membrane mimics (placental ECM/matrigel), that were either untreated, or treated with glycolaldehyde to induce AGEs formation. Expression/secretion of cystatin C and potential targets, cathepsins B, L, and S in/from RPE were analysed 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 decreased by up to 35% (independent T test, p=≤0.041) compared to cells on untreated BrM mimics. Secretion levels were also similarly reduced from approximately 56ng/ml/24h in normal conditions, to 36ng/ml/24h in the presence of AGEs (p=