Very long chain ceramides production mediated by ELOVL4 stabilizes tight junctions and prevents diabetes-induced retinal vascular permeability

Abstract

Purpose : Increased vascular permeability due to the loss of tight junction integrity is a well-accepted early sign of diabetic retinopathy. Tight junctions involve close apposition of transmembrane proteins between cells. While modifications of tight junction proteins in diabetes have been studied in detail, the role of diabetes-induced lipid abnormalities in retinal vascular tight junction structure in function received little attention.

Methods : The role of very long chain (VLC ≥26) ceramides in tight junctions was addressed using streptozotocin (STZ)-induced diabetes mouse and bovine retinal endothelial cells (BRECs) models of the loss of blood-retinal barrier. ELOVL4 was overexpressed using E1/E3 deleted adenoviral vector for cell culture studies, or AAV2 mut quad vector for mouse studies. Permeability was determined by RITC dextran in confluent monolayers of BRECs or by measuring extravasation of FITC-albumin after 8 weeks of diabetes in mice. Tight junction proteins were assayed by Western blot and immunostaining, ceramide colocalization with tight junction complexes was determined by immunogold electron microscopy and ceramide composition was analyzed by tandem mass spectrometry.

Results : ELOVL4 mediated production of very long chain (VLC ≥26) ceramides in BREC. Ultrastructure and lipidomic analysis revealed that omega-linked acyl-VLC ceramides co-localize with tight junction complexes. In agreement with previous studies, ELOVL4 expression was significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly increased VLC ceramide levels, decreased basal permeability, inhibited VEGF and IL-1β induced permeability and prevented VEGF-induced decrease in occludin expression and border staining of tight junction proteins; ZO-1 and claudin-5. Intravitreal delivery of hELOVL4-AAV2 reduced diabetes-induced increase in vascular permeability.

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

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.