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GW24-e1023 Advanced Glycation End Product Modified Low Density Lipoprotein Activates Mast Cells via Toll-Like 4 Receptor Pathway
  1. Zhao Xinna,
  2. Duan Yanan,
  3. Huang Xiao,
  4. He Zhiqing,
  5. Wu Zonggui,
  6. Liang Chun
  1. Department of Cardiology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China

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.

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