ABSTRACTA LIPIDOMICS STUDY OF DIABETIC RETINOPATHY REVEALS A NOVEL ROLE FOR FATTY ACID ELONGASE ELOVL4 IN THE MAINTENANCE OF RETINAL VASCULAR INTEGRITYByTodd A. Lydic Diabetic retinopathy (DR) is a blinding microvascular complication of diabetes mellitus for which no cure or preventative intervention exists. Chronic retinal inflammation is thought to play a major role in the development of diabetic retinal lesions, including endothelial injury and subsequent dysfunction of the blood-retinal barrier. Dyslipidemia is a major complication of diabetes which has been positively associated with the development of DR, yet no potential link between retinal lipid profiles and diabetes induced retinal vascular degeneration has been explored. We developed a mass spectrometry based lipidomics platform suitable for sensitive analysis and structural elucidation of diverse classes of complex retinal lipid species, and applied this lipidomics approach to the study of diabetic animal models to identify novel pathways involved in the development of diabetic retinal lesions. Lipidomics analysis of retina lipids in a streptozotocin (STZ) rat model of type 1 diabetes revealed multiple aberrations of retinal lipid metabolism at both early (six weeks) and late (36 weeks) time points of experimental diabetes. Dysregulated lipid metabolism in diabetic retina included a significant decrease in retinal glycerophospholipid polyunsaturated fatty acid (PUFA) content, including decreased docosahexaenoic acid (DHA, 22:6n3) and 32:6n3 very long chain PUFA (VLCPUFA). Investigation of the potential mechanisms of altered retinal lipid profiles in diabetes led to the novel finding that diabetes decreases the expression of several retinal fatty acid elongases, including dramatic downregulation of the most abundant retinal elongase, elongation of very long chain fatty acids 4 (ELOVL4). The alteration of rat retina elongases and glycerophospholipid content was coupled with increased retinal gene expression of inflammatory markers including pro-inflammatory cytokines and intracellular adhesion molecule-1 (ICAM-1).Decreased ELOVL4 expression or function has been associated with multiple retinal disorders, including blood-retinal barrier breakdown, in animal and human studies. This led us to hypothesize that ELOVL4 modulates diabetes-induced retinal vascular degeneration. The exact role of ELOVL4 in fatty acid synthesis has remained nebulous; however, ELOVL4 has been suggested to produce VLCPUFA. Using an adenovirus and capsid-modified Adeno-Associated Virus serotype 2 (AAV2) to overexpress ELOVL4 in human retinal pigment epithelial (RPE) and human retinal endothelial cell (HREC) culture models of the blood-retinal barrier, we found that ELOVL4 did not affect glycerophospholipid VLCPUFA content. Instead, ELOVL4 increased levels of C26 fatty acids incorporated into ceramide while decreasing shorter chain ceramides in these cells. Altered sphingolipid content by ELOVL4 decreased RPE cell activation in response to stimulation by the pro-inflammatory cytokine IL-1â. Intravitreal delivery of ELOVL4-AAV2 to retinas of STZ diabetic rats increased retina very long chain ceramides, reduced retinal vascular activation, blunted diabetes-induced retinal vascular permeability, and increased endothelial expression of blood-retinal barrier components despite having no mitigating effect on retinal inflammation. Taken together these data indicate that ELOVL4 prevents early stage vascular degeneration in diabetic retina through modulation of sphingolipid metabolism. Retinal delivery of ELOVL4 by AAV2 vectors may represent an effective intervention to prevent early vascular lesions of DR.
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