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Harshini Chakravarthy, Todd Lydic, Peter Jajou, Svetlana Bozack, Matthew Faber, Walter Esselman, Susanne Mohr, Julia Busik; The role of sphingolipid metabolism in diabetes-induced retinal VEGF secretion. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1134. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated that activation of the central enzyme of sphingolipid metabolism, Acid Sphingomyelinase (ASM) plays an important role in the pathogenesis of diabetic retinopathy. Vascular endothelial growth factor (VEGF) has been the focus of much research due to its role in induction of vascular permeability and neovascularization in the diabetic retina. This study was designed to elucidate the role of sphingomyelinase activation in VEGF production by human retinal pigment epithelial cells (RPE) and Müller cells under hyperglycemic conditions.
Human RPE and Müller cells were treated with 2 ng/mL IL-1β or 25 mM glucose for upto 48 hours. Cells treated in normal (5 mM) glucose containing medium served as controls. Following treatment, ASM, IL-1β and VEGF mRNA and protein levels were measured by qPCR and ELISA respectively. Acid sphingomyelinase (ASM) inhibition was achieved by siRNA, Caspase-1 activation was inhibited using 100 µM minocycline. Müller cells isolated from diabetic donors were cultured in normal 5 mM glucose containing medium for upto 48 hours and ASM, IL-1β, ICAM-1 and VEGF mRNA levels were analyzed by quantitative PCR.
In agreement with our previous studies, high glucose induced a 9.3-fold increase in IL-1β mRNA in RPE cells and 12-fold increase in Müller cells. IL-1β increased VEGF production 3.2-fold after 24 hours in Müller cells. Minocycline prevented increase in ceramide levels seen in high glucose conditions 2.9-fold confirming that IL-1β stimulation causes changes in sphingolipid metabolism in Müller cells. Inhibition of ASM completely blocked high glucose-induced increase in VEGF levels in both RPE and Müller cells. As measured by quantitative PCR, ASM was upregulated more than 20-fold in diabetic Müller cells and inhibition of this pathway by siRNA transfection leads to reduction in VEGF levels.
Our results indicate that upregulation of VEGF secretion by diabetes-induced sphingomyelinase activation in RPE and Müller cells could represent an unexploited mechanism in development of diabetic retinopathy. We propose that inhibition of caspase-1/IL-1β and ASM pathways could improve the outcome of standard anti-VEGF therapy in the treatment of diabetic retinopathy.
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