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Background: High-mobility group box 1 (HMGB1) is an endogenous molecule released during cell stress and death termed damage-associated molecular patterns (DAMPs). HMGB1 activates the pattern recognition receptor, toll-like receptor 4 (TLR4), and induces sterile inflammation. However, how HMGB1 and TLR4 affect restenosis, the major complication following balloon and stent intervention clinically, remains unknown. We tested the hypothesis that HMGB1 released following acute arterial injury promotes intimal hyperplasia (IH), a hallmark of restenosis, via TLR4 signaling pathway.Methods and Results: Wire injury of the carotid artery in C57BL/6 wild-type (WT) mice significantly increased intima-to-media ratio in 4 weeks. Global deletion of HMGB1 using an inducible knockout mouse strain prevented IH and vessel remodeling. IH decreased by over 50% in WT mice treated with a HMGB1 neutralizing antibody. Of the mouse strains deficient in putative receptors and co-regulator for HMGB1 (TLR4-/-, TLR2-/-, RAGE-/- and CD14-/-), TLR4-/- mice showed the greatest inhibition of IH after injury. Both TLR4 adaptors MYD88 and TRIF synergistically participated in the inflammatory response to vascular injury. HMGB1 antibody-treated mice and TLR4-/- mice showed a marked decrease in monocytic recruitment following injury. Mice with selective depletion of TLR4 from macrophages (TLR4-/--Mø) exhibited similar level of IH inhibition and macrophage infiltration, compared to the global TLR4-/- mice. In vitro, disulfide HMGB1 concentration-dependently promoted smooth muscle cell (SMC) migration and MCP-1/CCR2 expression, which were abolished by treatment with TLR4 inhibitory peptide. Moreover, conditioned media from HMGB1-treated macrophage induced SMC proliferation, which was blunted by blocking TLR4 on macrophage, but not SMCs. Finally, HMGB1 increased cytokine (TNF-α and IL-6), chemokine (MCP-1) and mitogen (PDGF-A) levels in macrophage in a TLR4-dependent manner.Conclusion: These findings demonstrate, for the first time, that HMGB1 released following acute arterial injury promotes restenosis via SMC migration and MCP-1/CCR2 production as well as macrophage-released TNF-α, IL-6, MCP-1 and PDGF-A in SMC through the TLR4-MyD88/TRIF signal cascade.