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Nermin Kady, Xuwen Liu, Todd Lydic, Sergey Seregin, Andrea Amalfitano, Vince Chiodo, Sanford L Boye, William W Hauswirth, David A Antonetti, Julia V Busik; Overexpression of hELOVL4 prevents diabetes-induced blood-retinal barrier breakdown through an increase in very long chain ceramides. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4292. doi: https://doi.org/.
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Diabetes induced loss of the blood-retinal barrier occurs early in the progression of diabetic retinopathy. Several mechanisms affecting vascular permeability in the diabetic retina have been identified including VEGF-induced alterations in junctional complex organization and content; however, the role of dyslipidemia in increased retinal permeability has not been studied. Maintenance of water permeability barrier in skin requires very long chain (VLC, ≥ C26) ceramides and ELOVL4 is the elongase responsible for production of VLC fatty acids that are incorporated into VLC ceramides. We have previously demonstrated a decrease in ELOVL4 in diabetic retina. The goal of this study is to determine whether restoration of retinal ELOVL4 levels is sufficient to prevent diabetes induced increase in retinal permeability.
Streptozotocin (STZ) diabetic rats received intravitreal injection of hELOVL4 packaged in adeno-associated virus type 2 containing four capsid Y-F mutations (AAV2 mut quad) under the control of a small chicken β-actin (smCBA) or vehicle. Retinal vascular permeability was assessed by measuring extravasation of FITC-albumin after 8 weeks of diabetes. Human ELOVL4 was overexpressed in human retinal pigment epithelial (RPE) cells or bovine retinal endothelial cells (BREC) by an E1- and E3-deleted adenoviral vector under the control of CMV promoter. Permeability was determined in confluent monolayers by RITC dextran. Lipid extracts were analyzed by tandem mass spectrometry. Tight junction proteins were assayed by Western blot.
Intravitreal delivery of hELOVL4-AAV2 in STZ diabetic rats resulted in a 39% reduction in diabetes-induced vascular permeability after 8 weeks of diabetes. Overexpression of hELOVL4 in RPE caused a 37% reduction in permeability. Overexpression of hELOVL4 in BREC significantly (46%) decreased basal permeability, inhibited VEGF and IL-1β induced permeability and simultaneously increased expression and border staining of tight junction proteins. Lipidome analysis of hELOVL4-overexpressing cells revealed a 2.5-fold increase in 26:0 ceramide relative to controls, while 16:0 ceramide decreased 1.7-fold.
Normalization of retinal ELOVL4 expression could prevent early breakdown of the blood-retina barrier in diabetic animals by modulating retinal sphingolipid metabolism.
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