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Hyperglycemia-Induced Reactive Oxygen Species Toxicity to Endothelial Cells is Dependent on Paracrine Mediators

Julia V. Busik^#, Susanne Mohr, and Maria B. Grant

¹Department of Physiology, Michigan State University, East Lansing, MI
²Department of Medicine, Case Western Reserve University, Cleveland, OH, and
³Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL

Objective: This study determined the effects of high glucose exposure and cytokine treatment on generation of reactive oxygen species (ROS) and activation of inflammatory and apoptotic pathways in human retinal endothelial cells (HREC).

Research Design and Methods: Glucose consumption of HREC, human retinal pigment epithelial cells (HRPE), and human Müller cells (HMC) under elevated glucose conditions was measured and compared to cytokine treatment. Production of reactive oxygen species (ROS) in HREC was examined using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), spin trap-EPR, and MitoTracker Red staining following high glucose and cytokine treatment. The activation of different signaling cascades including the MAPK pathways, tyrosine phosphorylation pathways, and apoptosis by high glucose and cytokines in HRECs was determined.

Results: HREC, in contrast to HRPE and HMC, did not increase glucose consumption in response to increasing glucose concentrations. Exposure of HREC to 25mM glucose did not stimulate endogenous ROS production, activation of nuclear factor kappa B (NFB), ERK, p38 and JNK, tyrosine phosphorylation, IL-1β or TNF production, and only slightly affected apoptotic cell death pathways compared to normal glucose (5mM). In marked contrast, exposure of HRECs to pro-inflammatory cytokines IL-1β or TNF increased glucose consumption, mitochondrial superoxide production, ERK and JNK phosphorylation, tyrosine phosphorylation, NFB activation, and caspase activation.

Conclusion: Our in vitro results indicate that HREC respond to cytokines rather than high glucose suggesting that in vivo diabetes-related endothelial injury in the retina may be due to glucose-induced cytokine release by other retinal cells and not a direct effect of high glucose.

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