oa Hyperglycemia-induced stress granule formation in mouse microvascular endothelial cells

Background and Objective: Cells exposed to stress conserve energy for the repair of cellular damage by inhibiting translational initiation. The stress stimuli can trigger several stress response pathways leading to global translational attenuation, chiefly by the phosphorylation of eIF2α and disruption of the 43S assembly, which correlates to the compartmentalization of untranslated polyadenylated mRNA in discrete cytoplasmic ribonucleoprotein complexes known as Stress Granules (SGs). In a diabetic milieu, endothelial cells (ECs) that line the lumen of the blood vessels are constantly exposed to high glucose (HG) concentration (stress), which contributes to increased oxidative stress in these cells that in turn is responsible for high rates of cardiovascular complications among diabetic individuals. However, the effect of high glucose as a stress in relation to SG assembly in ECs remains unclear. The central objective of the present study is to evaluate the role of HG-induced oxidative stress in SG assembly in ECs and whether antioxidant treatment could aid in the reversal of these effects. Methods: Mouse microvascular endothelial cells (MMECs) cultured in DMEM were exposed to normal (NG, 11mM) and high (HG, 40mM) glucose for 0-48h. DHE staining was performed to evaluate oxidative stress in ECs upon HG exposure. Fixed cells were probed for SG marker proteins (G3BP and TIAR) followed by immunofluorescence confocal microscopy. Immunoblotting was performed using protein lysate samples and was probed for p-PERK, PERK, p-eIF2α and eIF2a. N-acetyl cysteine was used as the anti-oxidant in order to study the role of oxidative stress in HG induced SG formation. Results: DHE staining indicated that oxidative stress significantly increased in HG exposed MMECs while this effect was reversed upon in NAC treatment. NAC treatment also markedly decreased HG-induced SG assembly. Conclusion: The data suggest that HG-induced oxidative stress plays a major role in SG assembly in ECs. However, more studies are warranted to evaluate the process of assembly and dis-assembly of SGs in ECs upon HG exposure, which may provide a better understanding of the role of SG formation in balancing EC apoptosis and survival in a diabetic milieu. This project was funded by QNRF-NPRP # 08-165-3-054 & 4-910-3-244.