[The effect of diamide on the expression of macrophage inflammatory protein-1 alpha in endothelial cells].
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.
Yang
Limin
L
Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. liminyang35@hotmail.com
Zhu
Xuewei
X
Zhao
Xia
X
Deng
Zhongduan
Z
D001161:Arteriosclerosis / Q000473:pathology; D002478:Cells, Cultured; D054407:Chemokine CCL4; D003958:Diamide / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology
D054407:Chemokine CCL4; D019402:Macrophage Inflammatory Proteins; D012333:RNA, Messenger; D011838:Radiation-Sensitizing Agents; D003958:Diamide