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Selina Beasley, Mohamed Elsherbiny, Sylvia Megyerdi, Sally El-shafey, Nader Sheibani, Mohamed Al-Shabrawey; Caspase-14 Expression Impairs Retinal Pigment Epithelial Cell Barrier Function. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2244.
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We recently showed that caspase-14 is a novel molecule expressed in the retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR). Here we evaluated the impact of caspase-14 expression on apoptosis, barrier properties, and phagocytic activity of retinal pigment epithelial (RPE) cell.
Human RPE (ARPE-19) cells were transfected with caspase-14 cDNA or control vector, followed by studying the barrier and phagocytic function, and the changes in the activity of other caspases (caspase1, 3, 5, 8, and 9). RPE cell permeability was evaluated by FITC-Dextran Flux assay. The levels and distribution of actin stress fibers (F-actin) were examined by Immunofluorescence staining. The effect of caspase14 expression on the activity of other caspases was determined by using a specific caspase activity assay kit. We also tested the expression of caspase 4 by Western blotting. Phagocytic activity was examined using a phagocytic activity kit. Moreover we tested the impact of high glucose treatment (30 mM) on the levels of caspase14 expression in RPE cells compared to normal glucose (5 mM) or osmotic control (Mannitol (25 mM) + glucose (5mM)).
We found that caspase-14 expression promoted FITC-dextran leakage through the ARPE-19 confluent monolayer. This was associated with a significant increase in the expression and disorganization of F-actin stress fibers and the activity of caspase-3 and caspase-9 (p<0.05). There was also significant increase in the levels of caspase 4 in caspase 14 expressing ARPE-19 cells. There was no significant difference in the phagocytic activity between caspase 14 expressing RPE and control cells. HG treatment significantly increased caspase-14 expression in RPE cells compared to NG treatment or osmotic control (P<0.05).
Our findings suggest that caspase-14 expression and/or activity may compromise RPE barrier function disrupting outer retinal barrier during diabetes. The effect of caspase 14 on RPE barrier function is probably mediated through enhancement of apoptotic pathways.
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