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[The effect of diamide on the expression of macrophage inflammatory protein-1 alpha in endothelial cells]
Limin Yang1, Xuewei Zhu, Xia Zhao
1Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. liminyang35@hotmail.com
Objective:
To study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells.
Methods:
After exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers.
Results:
Incubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group.
Conclusions:
Diamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.
Insights
Diamide, a lipid peroxidation inducer, stimulates endothelial cells to produce macrophage inflammatory protein-1 alpha (MIP-1 alpha). This protein exhibits chemotactic activity, attracting monocytes and potentially contributing to atherogenesis.
Area of Science:
- Endothelial Cell Biology
- Immunology
- Molecular Biology
Context:
- Endothelial cells (ECs) play a crucial role in vascular inflammation.
- Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a key chemokine involved in immune cell recruitment.
- Lipid peroxidation, induced by agents like diamide, can affect cellular function and inflammatory responses.
Purpose:
- To investigate the effect of diamide on MIP-1 alpha expression in human umbilical vein endothelial cells.
- To determine the chemotactic activity of MIP-1 alpha produced by diamide-treated ECs.
- To explore the potential role of diamide-induced MIP-1 alpha in atherogenesis.
Summary:
- Diamide exposure significantly increased MIP-1 alpha mRNA and protein expression in cultured ECs.
- Conditioned medium from diamide-treated ECs demonstrated enhanced monocyte chemotactic activity.
- This chemotactic activity was significantly reduced by anti-MIP-1 alpha antibodies, confirming MIP-1 alpha's role.
Impact:
- Diamide acts as a stimulus for ECs to produce bioactive MIP-1 alpha.
- The findings suggest a mechanism by which diamide may contribute to atherogenesis by recruiting monocytes.
- This research highlights the importance of lipid peroxidation in modulating endothelial cell chemokine production and inflammatory cell recruitment.
