Role of the sphingomyelin/ceramide pathway in diabetic retinopathy

Diabetic retinopathy (DR) stands as a sight threatening disease without effective therapeutic options. Early DR is recognized to be a persistent low-grade chronic inflammatory disease. Acid (ASMase) and neutral (NSMase) sphingomyelinases (SMases) are important early responders in inflammatory cytokine signaling and key regulatory enzymes of sphingolipid metabolism. Sphingolipids are a major component of plasma membrane microdomains and sphingomyelin hydrolysis by SMases to bioactive lipid ceramide represents a prerequisite for inflammatory cytokines signaling. This study addresses the role of SMases in retinal vascular endothelium inflammation and development of microangiopathic lesions in the retinas affected by retinopathy. We first demonstrated that inhibition/gene silencing of both SMases decrease cytokine-induced cellular adhesion molecules expression in human retinal endothelial cells (HREC); yet a more pronounced anti-inflammatory effect was observed when ASMase inhibition/gene silencing was attained. Similarly, docosahexaenoic acid (DHA), the major ω3 polyunsaturated fatty acid in the retina and well known anti-inflammatory agent, downregulated cytokine-induced inflammation in HREC. Interestingly, DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity in HREC, further underlining the role of SMases as mediators of the inflammatory process. SMases pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HREC. To further demonstrate the role of SMase pathway in inflammation, the caveolae/lipid microdomains sphingolipid composition was then characterized. In support of SMases inhibition and displacement of ASMase from caveolae microdomains by DHA, we found a significant decrease in ceramide/sphingomyelin (Cer/SM) ratio in the caveolae fraction isolated from HREC treated with DHA; moreover, DHA prevented the TNFα-induced increase in the Cer/SM ratio in caveolar membrane microdomains and intracellular inflammatory signaling. To address the role of SMases in retinal inflammation and vessel loss in DR, an in vivo model of streptozotocin (STZ)-induced diabetes in rat was employed. ASMase, but not NSMase was upregulated in the diabetic retinas with an inflammatory status and microangiopathy lesions. DHA-enriched diet restored ASMase in diabetic retina to the control levels and prevented retinal inflammation and vessel loss. More importantly, DHA supplementation specifically prevented vascular ASMase upregulation in the retinas of a rat model with vasodegenerative phase of DR. To directly investigate the role of ASMase in development of retinal acellular capillaries and ocular neovascularization, we next used accelerated models of retinopathy by inducing ischemia/reperfusion (I/R) injury and oxygen-induced retinopathy (OIR) in wild type (ASMase+/+) and ASMase−/− mouse retina. ASMase−/− mouse retina was significantly protected against retinal inflammation, vascular degeneration and ocular neovascularization. In summary, this is the first study to show that ASMase, a key regulatory enzyme of sphingolipid metabolism, is a novel and fundamental mediator and a promising therapeutic target for the prevention of retinal vascular inflammation and diabetic retinopathy.