In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemotaxis, Leukocyte/physiology ; Diamide/*pharmacology ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Lipid Peroxidation ; Macrophage Inflammatory Protein-1/*biosynthesis ; Macrophage Inflammatory Protein-1/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Sulfhydryl Reagents/pharmacology ; Umbilical Veins/cytology

Atherosclerosis is an inflammatory disease. Platelet-endothelium interaction plays an important role in the pathophysiology of atherogenesis. We investigated the role of activated platelets for secretion of interleukin (IL)-1beta, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha and expression of intercellular adhesion molecule (ICAM)-1 on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were incubated with non-stimulated or ADP-activated platelets for 6 hr. Secretion of interleukin (IL)-1beta, MCP-1 and MIP-1alpha and surface expression of ICAM-1 were measured by ELISA and flow cytometry. In the presence of activated platelets, the secretion of IL-1beta, MCP-1, and MIP-1alpha and surface expression of ICAM-1 were significantly increased compared with non-activated platelets. The present study shows that activated platelets may contribute to expression of various inflammatory mediators on endothelial cells.
Blood Platelets/metabolism* ; Cells, Cultured ; Cells, Cultured ; Endothelium, Vascular/secretion* ; Endothelium, Vascular/cytology ; Human ; Intercellular Adhesion Molecule-1/biosynthesis ; Interleukin-1/secretion* ; Macrophage Inflammatory Protein-1/secretion* ; Monocyte Chemoattractant Protein-1/secretion* ; Platelet Activation/physiology*

Atherosclerosis is an inflammatory disease. Platelet-endothelium interaction plays an important role in the pathophysiology of atherogenesis. We investigated the role of activated platelets for secretion of interleukin (IL)-1beta, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha and expression of intercellular adhesion molecule (ICAM)-1 on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were incubated with non-stimulated or ADP-activated platelets for 6 hr. Secretion of interleukin (IL)-1beta, MCP-1 and MIP-1alpha and surface expression of ICAM-1 were measured by ELISA and flow cytometry. In the presence of activated platelets, the secretion of IL-1beta, MCP-1, and MIP-1alpha and surface expression of ICAM-1 were significantly increased compared with non-activated platelets. The present study shows that activated platelets may contribute to expression of various inflammatory mediators on endothelial cells.
Blood Platelets/metabolism* ; Cells, Cultured ; Cells, Cultured ; Endothelium, Vascular/secretion* ; Endothelium, Vascular/cytology ; Human ; Intercellular Adhesion Molecule-1/biosynthesis ; Interleukin-1/secretion* ; Macrophage Inflammatory Protein-1/secretion* ; Monocyte Chemoattractant Protein-1/secretion* ; Platelet Activation/physiology*