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Mohammad Dahrouj, Yueying Liu, Craig Crosson, Zsolt Ablonczy; AGE products disrupt RPE barrier function in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1722.
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© ARVO (1962-2015); The Authors (2016-present)
Advanced glycation end-products (AGEs), form during aging, are increased in diabetic patients, and have been correlated with the progression of diabetic retinopathy. However their effect on RPE function is not fully understood. To investigate how AGE products influence RPE function, the effect of glycated-albumin (Glyc-alb) on human RPE monolayers barrier function and subretinal fluid reabsorption in vivo were evaluated.
Transepithelial electrical resistance (TEER) measurements were used to assess the permeability of confluent ARPE-19 and human fetal RPE monolayers. Cells were treated apically with 100 μg/mL albumin or Glyc-alb in the absence or presence of ZM323881 (10 nM), a VEGF-R2 inhibitor. Monolayer TEER was monitored for up to 6 hours post drug-administration. ELISA assays were used to measure VEGF secretion. The rate of reabsorption of subretinal blebs in rabbits was used to evaluate the RPE function in vivo. Albumin or Glyc-alb (1 mg/mL) was injected into the vitreous and subretinal blebs (1-5µL PBS) were created 48 hours later. Rate of bleb reabsorption was measured using high resolution OCT. In selected experiments, ZM323881 (1 µM) was co-injected with Glyc-alb.
Although albumin did not significantly alter TEER, Glyc-alb induced a 20% reduction in TEER. Pretreatment with ZM323881 reversed the effect of Glyc-alb on TEER. Additionally, Glyc-alb caused a significant increase in the rate of VEGF secretion into the apical media from 27.5 to 39.7 pg.mL-1.hr-1. In rabbits, the baseline rate for fluid reabsorption was 8.2 ± 0.6 µL.cm-2.hr-1. The intravitreal injection of albumin did not significantly alter fluid reabsorption; however intravitreal injection of Glyc-alb significantly decreased this rate of reabsorption to 2.2 ± 0.4 µL.cm-2.hr-1. Administration of ZM323881 reversed the effect of Glyc-alb on subretinal fluid reabsorption (5.15 ± 0.6 µL.cm-2.hr-1).
Our data show that Glyc-alb was effective in disrupting RPE function in vitro and in vivo and this response can contribute to the development of retinal edema. The ability of ZM323881 to reverse this response provides evidence that these AGE-induced effects are dependent on VEGF secretion and VEGF-R2 activation. These data provide new information on the cellular mechanisms involved in the development of diabetic macular edema, as well as identifying new therapeutic targets.
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