Overexpression of ELOVL4 stabilizes tight junctions and prevents VEGF-induced vascular permeability in diabetic retina and retinal cells.

Abstract

Purpose : We have previously demonstrated that the expression level of elongation of very long chain fatty acids –like 4(ELOVL4) is significantly reduced in the diabetic retina leading to decreased levels of very long chain (VLC ≥26:2) fatty acids. VLC fatty acids produced by ELOVL4 incorporate into VLC ceramides that are known to play an important role in skin permeability barrier maintenance. We hypothesized that, similar to skin, VLC ceramides could play a beneficial role in maintenance of the blood-retinal barrier and reduction of ELOVL4 in diabetes could lead to increased retinal vascular permeability through the decrease in VLC ceramides.

Methods : Human ELOVL4 was overexpressed in bovine retinal endothelial cells (BRECs) by an E1- and E3-deleted adenoviral vector under the control of CMV promoter. Permeability was determined in confluent monolayers by RITC dextran. Tight junction proteins were assayed by Western blot and immunostaining. Ceramide colocalization with tight junction complex was determined by immunogold electron microscopy. Tight junction isolates were analyzed by tandem mass spectrometry. Streptozotocin (STZ) mice 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 CMV-chicken β-actin (smCBA) promoter or empty AAV2 mut quad as a negative control. Retinal vascular permeability was assessed by measuring extravasation of FITC-albumin after 8 weeks of diabetes.

Results : Overexpression of hELOVL4 in BRECs significantly decreased basal permeability by 46% (n=3, P<0.01), as well as VEGF and IL-1β-induced permeability by 47.7% and 36.2%; respectively (n= 3-6, p<0.001, P<0.01; respectively). Importantly, hELOVL4 overexpression prevented VEGF-induced decrease in occludin expression and border staining of tight junction proteins; ZO-1 and claudin-5. Ceramides were found to colocalize with tight junction complexes. Lipidome analysis of tight junction isolates revealed the presence of VLC ceramides. Intravitreal delivery of hELOVL4-AAV2 in STZ mice reduced diabetes-induced increase in vascular permeability by 75% after 8 weeks of diabetes (n= 8-12 per group, p<0.01).

Conclusions : Normalization of retinal ELOVL4 expression could prevent blood-retina barrier dysregulation in DR through increase in VLC ceramides and stabilization of tight junctions.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.