Pro-inflammatory LPS drives production and release of the chemokine MCP-1 in human coronary artery smooth muscle cells

The chemokine monocyte chemoattractant protein-1 (MCP-1) plays an important role as chemoattractant for monocytes in atherosclerosis. It is established that MCP-1 is produced by vascular smooth muscle cells, but the underlying mechanisms for its release are not identified. Here, we investigate production and secretion of MCP-1 in primary human coronary artery smooth muscle cells. We demonstrate that the cells express MCP-1 using RT-qPCR, immunocytochemistry and ELISA, and the ELISA analysis shows that they contain high basal levels of MCP-1 compared to human THP-1 monocytes included as positive control representing an immune cell. Immunocytochemistry discloses co-staining for MCP-1 and the ER marker calreticulin, suggesting that they may co-exist. The cellular production of MCP-1 is stimulated by the bacterial endotoxin LPS demonstrated both on mRNA and protein levels. Conditioned medium contains higher amounts of MCP-1 than fresh medium, and pro-inflammatory LPS and TNF-α stimulate release of MCP-1 from the cells. LPS does not enhance the secretion of MCP-1 at an early time point (60 min) neither in the presence nor in the absence of protein synthesis inhibition with cycloheximide, and it has no effect on intracellular [Ca2+] within 0–60 min, suggesting that LPS has no direct effect on the secretory process of MCP-1. We conclude that human coronary artery smooth muscle cells contain high levels of MCP-1, and that pro-inflammatory stimulus triggers secretion of this important chemokine indirectly via activation of MCP-1 production.