Abstract
Homocysteinaemia is an independent risk factor for atherosclerosis. The development of atherosclerosis involves monocyte chemoattractant protein 1 (MCP-1)-mediated monocyte recruitment to the lesion site. The action of MCP-1 is mostly via its interaction with MCP-1 receptor (CCR2), which is the major receptor for MCP-1 on the surface of monocytes. The objective of the present study was to investigate the effect of homocysteine on CCR2 expression in human THP-1 monocytes. Cells were incubated with various concentrations of homocysteine for 6, 12, 24 and 48 h. The expression of CCR2 mRNA was determined by nuclease protection assay and the CCR2 protein was measured by Western immunoblotting analysis. The binding of MCP-1 to CCR2 as a functional receptor on the monocyte surface was determined by flow cytometry. Homocysteine (0.05-0.2 mM) significantly enhanced the expression of CCR2 mRNA (129-209% of the control) and CCR2 protein (up to 183% of control) in these cells after 24 h of incubation. Stimulation of CCR2 expression was associated with a parallel increase in the binding activity of CCR2 (129-191% of control) as well as an enhanced chemotactic response of homocysteine-treated monocytes. Further investigation revealed that the levels of superoxide were significantly elevated in cells incubated with homocysteine for 12-48 h. The addition of superoxide dismutase, a scavenger of superoxide, to the culture medium abolished the stimulatory effect of homocysteine on CCR2 expression as well as the binding activity of the receptor. The stimulatory effect of homocysteine on the expression of CCR2 mRNA and the levels of CCR2 protein was also observed in human peripheral blood monocytes. In conclusion, the present study has clearly demonstrated that homocysteine stimulates CCR2 expression in monocytes, leading to an enhanced binding activity and chemotatic response. Homocysteine-induced superoxide formation might serve as one of the underlying mechanisms for this effect.
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