Superoxide regulation in diabetes-novel iPLA₂ pathways in human neutrophils
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Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting from defects in insulin secretion, action or both. The complications arising from poorly controlled diabetes mellitus are characterized by increased oxidative stress and inflammation. Neutrophils are key mediators of innate immunity which primarily protect the host from invading pathogens. In diabetes, neutrophils acquire a primed phenotype and are responsible for superoxide mediated host tissue damage. Neutrophil NADPH oxidase mediates superoxide release and is activated by PKC induced phosphorylation of cytosolic subunits like p47phox, p67phox, p40phox and Rac-2 and translocation to their membrane components gp91 and p22phox_ PLA2 derived arachidonic acid is required in the activation of the assembled NADPH oxidase. There is a gap in the current literature regarding the role of PLA2 in NADPH oxidase activation, therefore, we hypothesize that PLA2 is involved in the priming of neutrophils in diabetes. The aim of this study was to identify the specific isoform involved in superoxide generation in neutrophils and investigate the PLA2 activity in neutrophils from diabetic subjects. Experiments with chemical inhibitors revealed that iPLA2, but not cPLA2 was the primary isoform involved in superoxide generation in neutrophils. The specificity of BEL, an iPLA2 inhibitor, was confirmed by ruling out the involvement of PAP-1, inhibition of p47phoxp hosphorylation and apoptosis. To confirm the findings, from chemical inhibition experiments, knockdown experiments for iPLA2 were conducted in HL60-neutrophil like cells. The results from these findings showed that knock down of iPLA2 by siRNA resulted in a 30% reduction in superoxide generation. Superoxide generation was increased in neutrophils from diabetic subjects with poor glycemic control. This process was inhibited by BEL in both healthy and diabetic subjects. It was found that the PLA2 activity from membrane fractions of neutrophils was calcium independent, suggesting the role of iPLA2 and this was increased in diabetic subjects. The iPLA2 expression was increased both at the message and protein levels in neutrophils from diabetic subjects. In order to understand the mechanism involved behind priming of iPLA2 in diabetes, in vitro hyperglycemia experiments were conducted. There was a significant increase in superoxide generation in cells cultured with high glucose, RAGE ligand; S1008 and combination of high glucose and S 1008. This increase in superoxide generation or a priming effect was prevented by BEL, suggesting that iPLA2 mediates the priming event. This dissertation unravels the novel role of iPLA2 in superoxide generation from neutrophils and demonstrates it's priming effect in diabetes.
Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2008 (Dept. of Periodontology and Oral Biology).Includes bibliography: leaves 87-109.
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