Abstract 10295: The iPLA2β-Nox4-H2O2 Feed-Forward Loop Plays an Indispensible Role in Accelerated Monocyte Chemotaxis Induced by Diabetic Condition
Objective: Hyperglycemia is one of the most important metabolic factors in the development of the vascular complications in diabetic patients. A hallmark of diabetic complications is the enhanced recruitment of blood monocytes into inflamed tissues. The calcium-independent phospholipase A2 beta (iPLA2β) was reported to be required for monocyte chemotaxis, however, the underlying mechanism remains unclear. In this study, we identified an intracellular iPLA2β-Nox4-H2O2 feed-forward loop which modulates monocyte chemotaxis under diabetic conditions.
Methods: The human monocyte-like THP-1 cells treated with glucose at 25 mM or a combination of glucose at 25 mM and native LDL at 100 µg/ml (HG+LDL) were used as an in vitro model to determine the effect of metabolic stress on expressions of iPLA2β, Nox4, and H2O2 production. Bromoenol lactone (BEL) and siRNA of iPLA2β were employed as pharmacological and molecular inhibitors to access iPLA2β activity. Nox4 knockdown by siRNA was used to evaluate the effect of Nox4 on H2O2 production.
Results: 1) High glucose (HG) and HG+LDL treatment increased monocyte chemotaxis in response to MCP-1 and this increase was suppressed to normal level by siRNA directed against iPLA2β and BEL, indicating that iPLA2β mediates HG-enhanced monocyte chemotaxis. 2) HG and HG+LDL induced Nox4 expression and H2O2 formation. 3) the iPLA2β inhibitor BEL inhibited HG-upregulated Nox4 and H2O2 while H2O2 in turn promoted iPLA2β expression. 4) H2O2 induction of iPLA2β was blocked by addition of GF-109203X, a specific PKC inhibitor, suggesting that PKC regulates iPLA2β expression. H2O2 formation was restored by adding lysophosphatidic acid (LPA), one of the products of iPLA2β's enzymatic activity. 5) Nox4 knockdown inhibited intracellular H2O2 production while addition of adenovirus containing Nox4 gene restored H2O2 formation.
Conclusion: Hyperglycemia-activated iPLA2β mediates the induction of Nox4 and H2O2 formation, which in turn increases iPLA2β expression. We conclude that iPLA2β, Nox4 and H2O2 form an intracellular positive feedback loop, which contributes to the increased monocyte responsiveness to chemoattractants induced by diabetic conditions.
- © 2011 by American Heart Association, Inc.