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PE Canning, E Haslett, S McFarlane, C McFarlane, G Burke, A Lichanska, A Hussain, J Marshall, A Ghaffer, AW Stitt; The Effect of AGEs on Permeability of the Inner and Outer Blood Retinal Barrier . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3319.
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Purpose: Breakdown of the inner and outer blood retinal barriers (BRB) is an important pathophysiological event in diabetes and age-related disorders. Advanced glycation endproducts (AGEs) accumulate with age and at an accelerated rate in diabetes and these adducts have been immunolocalised in retinal vascular basement membranes (BMs) and Bruch's membrane. We hypothesised that AGE-modification of BM components cold play an important role in compromise of the inner and outer BRB, either by tight-junction disruption or enhanced caveolae-mediated permeability. Methods: Human ARPE-19 cells and bovine retinal microvasular endothelial cells (RMECs) were seeded onto membrane inserts and exposed to either AGE-modified BM (AGE-BM) or AGE-albumin in the culture medium. Transcellular electrical resistance readings monitored the confluency of cell monolayers. Outer BRB function was determined by measuring the inward (choroid to vitreous) permeability of various fluorescently labelled tracers, whilst inner BRB function measured tracer leakage in the opposite direction. The hydraulic conductivity of AGE-BM and native BM was quantified. Tight junction formation was assessed by confocal microscopy, whilst caveolin-1 (a marker for caveolae) expression, was assessed by western blotting and flow cytometry. Results: AGE-BM hydraulic conductivity was decreased in a step-wise fashion in comparison to native BM controls, an effect partially reversed by prior incubation with the AGE-inhibitor aminoguanidine. ARPE-19 and RMEC monolayers grown on AGE-BM showed a dose dependent decrease in cell permeability to various tracers. In contrast, cell monolayers exposed to AGE-albumin in the culture medium showed a dose dependent increase in cell permeability. Enhanced endocytic uptake of tracer was also observed. These findings coincided with increased expression of caveolin-1. Conclusion: AGE-BM shows altered hydraulic conductivity, although there are also dysfunctional changes to endocytic/transcytotic processes within RPE and RMECs. This study has demonstrated that AGEs may cause perturbations in the inner and outer BRBs.
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