April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Caspase-14 Expression Impairs Retinal Pigment Epithelial Cell Barrier Function
Author Affiliations & Notes
  • Selina Beasley
    Oral Biology/Anatomy, Georgia Regents University, Augusta, GA
    Vision Discovery Institute and Department of Ophthalmology, Georgia Regents University, Augusta, GA
  • Mohamed Elsherbiny
    Oral Biology/Anatomy, Georgia Regents University, Augusta, GA
    Vision Discovery Institute and Department of Ophthalmology, Georgia Regents University, Augusta, GA
  • Sylvia Megyerdi
    Oral Biology/Anatomy, Georgia Regents University, Augusta, GA
    Vision Discovery Institute and Department of Ophthalmology, Georgia Regents University, Augusta, GA
  • Sally El-shafey
    Oral Biology/Anatomy, Georgia Regents University, Augusta, GA
    Vision Discovery Institute and Department of Ophthalmology, Georgia Regents University, Augusta, GA
  • Nader Sheibani
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI
  • Mohamed Al-Shabrawey
    Oral Biology/Anatomy, Georgia Regents University, Augusta, GA
    Vision Discovery Institute and Department of Ophthalmology, Georgia Regents University, Augusta, GA
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2244. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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)).

Results: 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).

Conclusions: 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.

Keywords: 499 diabetic retinopathy • 426 apoptosis/cell death • 701 retinal pigment epithelium  
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