OBJECTIVE

To investigate the effects of advanced glycation end products (AGE) on secretion of monocyte chemoattractant protein-1 (MCP-1) by human endothelial cells and its signal transduction pathway.

METHODS

Human umbilical vein endothelial cells (HUVECs) and HUVEC-derived cell line (ECV304) were cultured in vitro with indicated concentration of AGE modified human serum albumin (AGE-HSA) or AGE modified bovine serum albumin (AGE-BSA). The production of MCP-1 was evaluated by Western blotting and enzyme-linked immunoadsorbent assay (ELISA). The MCP-1 mRNA expression was assayed by reverse-transcription polymerase chain reaction (RT-PCR). Intracellular oxidative stress was detected by flow cytometry. The phosphorylation activity of cellular p38 mitogen-activated protein kinase (p38-MAPK) was analyzed by Western blotting using a phospho-specific antibody.

RESULTS

AGE-HSA and AGE-BSA, but not their unmodified form, upregulated the expression of MCP-1 mRNA and protein dose- and time-dependently. The MCP-1 concentration in the supernatant of HUVECs incubated with 50 micro g/ml AGE-HSA for 12 hours increased from 48.3 pg/ micro g +/- 0.6 pg/ micro g protein to 148.1 pg/ micro g +/- 12.6 pg/ micro g protein (P < 0.01). AGE modified proteins were associated with enhanced oxidative stress and p38-MAPK phosphorylation activity. Incubation of HUVECs with 50 micro g/ml AGE-HSA for 30 minutes resulted in increase of p38-MAPK phosphorylation activity by 91% +/- 14% (P < 0.01). Antioxidant or SB 203580, a specific inhibitor of p38, could block the over-expression of MCP-1.

CONCLUSIONS

AGE modified proteins stimulate endothelial cells to produce MCP-1 through activation of the p38 signal pathway. This effect may contribute to the pathogenesis of atherosclerosis seen in AGE-associated diseases.