Thrombin Regulates Expression of Monocyte Chemoattractant Protein-1 in Vascular Smooth Muscle Cells

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Abstract
Thrombin, a serine protease generated at sites of vascular injury, plays a role in the pathogenesis of atherosclerosis and restenosis after angioplasty. Adherence of monocytes to the endothelium and migration into the subendothelial space is an important early event in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) may be an important mediator of monocyte recruitment to the tissue in this and other diseases. We have characterized the expression of MCP-1 in vascular smooth muscle cells (VSMCs) isolated from human renal artery and studied its regulation by thrombin. Serum-deprived cells release monocyte chemotactic activity that is neutralized (80%) by an MCP-1 antibody. The antibody recognized a 13- and 15-kD protein in smooth muscle cell–conditioned medium. Thrombin stimulates MCP-1 gene expression in a concentration- and time-dependent manner. An increase over basal levels was observed with concentrations of thrombin as low as 0.05 U/mL. The maximal effect occurred at 5 U/mL. The stimulatory effect was detected within 1 hour, reached a maximum at 3 hours, and was still present at 8 to 24 hours after the addition of thrombin. A concentration- and time-dependent effect of thrombin on MCP-1 gene expression was also found in rat VSMCs. The thrombin protease inhibitor hirudin blocked thrombin-induced MCP-1 expression. Thrombin stimulated the release of MCP-1 protein in conditioned medium of human VSMCs as measured by radioimmunoassay and chemotactic assay. Thrombin also increased monocyte chemotactic activity in short-term organ cultures of rat aortic rings and in first passage cells. The effect of thrombin on MCP-1 was mimicked by a thrombin receptor–activating peptide (NH2-Ser-Phe-Leu-Leu-Arg-Asn-Pro-COOH). These data describe an important biological activity of thrombin in VSMCs and provide a novel mechanism whereby locally released thrombin may contribute to the pathogenesis of atherosclerosis or restenosis after angioplasty.