Regulation of monocyte chemoattractant protein-1 expression in macrophages
Regulation of monocyte chemoattractant protein-l expression in macrophages
Johnny Yip Chin Wing
for the Degree of Master of Philosophy at the University of Hong Kong
in August 2003
Homocysteine (Hcy) is a sulfur-containing ammo acid that exists as an intermediate metabolite of methionine. Epidemiological studies have revealed a close correlation between hyperhomocysteinemia and cardiovascular diseases. One of the characteristics of atherosclerosis is recruitment of leukocytes to the arterial wall. Monocyte infiltration is initiated by chemotactic factors and cell adhesion molecules produced by the overlying endothelial cells. These molecules contribute to monocyte adhesion to the endothelium, and their subsequent transmigration into the intima of arterial walls. In the subendothelial spaces, infiltrated monocytes differentiate into macrophages and take up oxidized lipoproteins to become lipid-laden foam cells. Monocyte chemoattractant protein-l (MCP-I) is a potent pro-inflammatory chemokine that stimulates the migration of monocytes into the intima of vessel walls.
It has been demonstrated that Hcy at high levels stimulates the production of MCP-I in vascular cells. The objective of the present study was to determine the effect ofHcy on MCP-I expression in THP-I derived macrophages, and to elucidate the underlying mechanisms involved. Results from reverse transcription-polymerase chain reaction (RT-PCR) suggested that Hcy at pathophysiological concentrations stimulated MCP-I mRNA expression in cultured human macrophages in a dose-dependent manner. This was accompanied by an increase of MCP-I protein production revealed by enzymatic immunoassays. Electrophoretic mobility shift assay (EMSA) indicated that elevated levels ofHcy could enhance the activity ofNF-KB, which is an inducible transcription factor responsible for MCP-I gene expression. Phosphorylation and subsequent degradation oflKB-a, a regulatory protein ofNF-KB, in response to Hcy treatment was monitored by Western immunoblotting. Results from nitro blue tetrazolium (NBT) reduction assay detected a significant increase in superoxide levels in cultured macrophages after Hcy treatment. In addition, cell fractionation analysis indicated that Hcy could stimulate NAD(P)H oxidase activity in macrophages, and the Hcy-induced production of MCP-l mRNA and protein could be blocked by specific inhibitor, diphenylene iodonium (DPI), to this superoxide-generating enzyme. In conclusion, our results have clearly demonstrated that Hcy stimulates the production of MCP-l possibly by activating NAD(P)H oxidase in macrophages. The production of oxygen free radicals in turn enhanced the NF-KB activity and eventually leads to MCP-l gene expression. These findings may help determine the role that hyperhomocysteinemia contributes to the development and progression of atherosclerosis.
Word Count = 364
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:homocysteine monocytes macrophages chemokines
Date of Publication:01/01/2003