GW24-e1023 Advanced Glycation End Product Modified Low Density Lipoprotein Activates Mast Cells via Toll-Like 4 Receptor Pathway. (1st October 2013) - Xinna, Zhao
Abstract : Objectives: To investigate the effects of AGE-LDL, in murine bone marrow-derived mast cells (mBMMCs), on mast cell degranulation and their possible signal pathways. Methods: Carotid plaques were excised during Carotid Endarterectomy of diabetic and nondiabetic patients. Toluidin blue and AGE-specific antibody and CD117 antibody were used to explore the localization and distribution of AGEs and mast cells in arterial segments. Bone marrow-derived cells were harvested from male, 6-8 weeks old C57/BL6 mice, then cultured in medium supplemented with recombinant murine interleukin-3 (rmIL-3) and recombinant murine stem cell factor (rmSCF). Mast cell purity was examined in 4-6 weeks by toluidin blue staining and fluorescence activated cell sorting (FACS). Degranulation of MCs, stimulated by different doses of AGE-LDL, was quantified by assaying the secretion of histamine and-hexoaminidase. Expression of a receptor for AGE-LDL, TLR4, was assessed by real-time PCR, flow cytometry and westernblot. Bone marrow-derived mast cells from TLR4 -/- mice, activated by AGE-LDL, were used to measure the secretion of histamine and-hexoaminidase. Underlying signal pathways involved were assessed by detecting expression of NF-κB, p38 MAPK, Erk1/2 and JNK phosphorylation, on AGE-LDL-stimulated bone marrow-derived MCs from both wild-type (WT) and TLR4 -/- mice, using western blot and use of NF-κB, p38MAPK and Erk1/2 inhibitors. Results: (1) MCs are present in human coronary plaques, especially in the rupture-prone shoulder region and in the fibrous capsusceptible to erosion, partially colocalized with AGEs. (2) High purity (>99%) of BMMC population were reached after being cultured in supplement medium with rmIL-3 and rmSCF for 4-6 weeks. (3) AGE-LDL dose-dependently induced mast cell histamine and-hexoaminidase release with maximal effects being obtained within 30min. (4) TLR4 mRNA and protein expression was upregulated. AGE-LDL stimulated exocytosis was remarkably reduced in BMMCs from TLR4 knockout (KO) mice, compared with those from WT mice. (5) AGE-LDL activated a phosphorylation of p38, ERK1/2 kinases and NF-B, but not JNK, in BMMCs from WT mice. These results were absent in TLR4 -/- mast cells. p38MAPK, ERK1/2 and NF-B inhibitors can reduce AGE-LDL-induced mast cell exocytosis of histamine and beta-hexoaminidase. Conclusions: These results indicate that AGE-LDL activates mast cell degranulation via a TLR4-mediated signal pathway. This mechanism may partly explain the increased risk of atherosclerosis and acute cardiovascular events observed in diabetics.