Tissue factor pathway inhibitor gene transfer prevents vascular smooth muscle cell proliferation by interfering with the MCP-3/CCR2 pathway

Increased vascular smooth muscle cell (VSMC) proliferation substantially contributes to the pathogenesis of atherosclerosis and intimal hyperplasia after vascular injury. The importance of inflammation in VSMC proliferation is now being recognized. Preventing the inflammatory response is one therapeutic strategy that can be used to inhibit atherosclerosis in the clinic. The present study, using RNA interference and gene transfer techniques, was conducted to investigate the effect of monocyte chemotactic protein-3 (MCP-3) on VSMC proliferation that is a result of TNF-alpha stimulation, and whether overexpression of the tissue factor pathway inhibitor (TFPI) gene could prevent VSMC proliferation by blocking the MCP-3/CC chemokine receptor 2 (CCR2) pathway. Mouse VSMCs were infected in vitro with recombinant adenoviruses containing either mouse MCP-3-shRNA (Ad-MCP-3-shRNA), the TFPI gene (Ad-TFPI), or the negative control, which was shRNA encoding the sequence for EGFP (Ad-EGFP) or DMEM only. The cells were then stimulated with TNF-alpha for different time periods on the third day after gene transfer. The data show that VSMC proliferation in the Ad-MCP-3-shRNA and Ad-TFPI groups was markedly decreased using BrdU ELISA and MU assays; MCP-3-shRNA and TFPI inhibited the expression of MCP-3 and CCR2 after long-term stimulation and inhibited the phosphorylation of ERK1/2 and AKT after short-term stimulation, as shown by ELISA and western blot analysis. This study provides convincing evidence that clarifies the effect of the proinflarnmatory factor MCP-3 in promoting VSMC proliferation. Our data also show, for the first time, that TFPI has an anti-proliferative role in TNF-alpha stimulated-VSMCs at least partly by interfering with the MCP-3/CCR2 pathway and then via suppression of the ERK1/2 and PI3K/AKT signaling pathways. We conclude that TFPI gene transfer may be a safe and effective therapeutic tool for treating atherosclerosis and intimal hyperplasia.