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Faye Drawnel, Derrick Feenstra, Kristin Sokoll, Gergö Sipos, Julia Hesselmann, Ashwath Jayagopal; Pro-inflammatory phenotypes of retinal microvascular endothelial cells and monocytes upon RAGE Activation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2531.
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Hyperglycemia-induced oxidative stress is a key pathologic process in diabetic retinopathy. However, diabetes also induces a chronic low grade inflammatory state through engagement of the cell surface receptor for advanced glycation endproducts (RAGE), by glycated proteins, or by lipids. These stresses combine in the retina, activating microglia, altering retinal vascular function, and inducing an inflammatory response, leading to sight-threatening complications. Here, we examined the effect of hyperglycemia or RAGE activation on human retinal microvascular endothelial cells (HRMEC) and human CD14+ monocytes.
HRMEC were cultured on transwell filters for 7 days. HRMEC phenotype was visualized by immunofluorescence staining and confocal microscopy for markers PECAM and VWF. Primary CD14+ monocytes were maintained in culture and CD14 surface expression monitored using flow cytometry. Hyperglycemia was mimicked by exposure of cell cultures to high glucose (25.5 mM), or mannitol as an osmotic control (20.5 mM) for 4 days. RAGE was activated by treatment with AGE-modified BSA or BSA alone for 4 days. Cytokines were profiled using luminex and qPCR. Cellular phenotype was characterized using immunofluorescence and flow cytometry.
Treatment of HRMEC with high glucose for 4 days did not produce release of inflammatory cytokines. However, treatment with AGE-BSA induced production of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFNγ and TNFα (p < 0.05) into the apical and basolateral compartments. At the RNA level, expression of ICAM and VCAM were also increased by AGE-BSA treatment only (p < 0.05). AGE-BSA treatment of HRMEC produced limited breakdown of the cellular barrier as evidenced by interrupted ZO-1 staining. However, endothelial cell phenotype markers (PECAM and VWF) were preserved. In response to RAGE activation, CD14+ monocytes also released inflammatory cytokines and adopted an M1-like phenotype.
RAGE activation constitutes a strong pro-inflammatory stimulus in both HRMEC and CD14+ monocytes, whilst short term glucose over-load does not. Pro-inflammatory effects of hyperglycaemia are likely mediated by glucose-dependent AGE formation, which occurs over weeks-months in vivo. Future efforts will focus on improving our understanding of how inflammation and glucose-induced cellular stress interact to produce phenotypes related to Diabetic Retinopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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