Abstract
We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce H2O2 production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.
Highlights
It has been well established that vascular inflammation plays an important role in the generation and progression of atherosclerosis[1,2,3]
To evaluate the function of NADPH oxidase (Nox)[4] in the formation of atherosclerotic plaques, reactive oxygen species (ROS) generation was examined in primary mouse vascular SMCs (VSMCs) (Supplementary Fig. S1) in response to flagellin by measuring the fluorescence of 2’,7’-DCF-DA by confocal microscopy
The atherosclerotic lesions associated with partial ligation of the left carotid artery (LCA) or with injection of recombinant FliC (rFliC) combined with partial ligation of the LCA were significantly suppressed in Nox[4] KO mice. These results indicated that deficiency of Nox[4] significantly inhibits the development of atherosclerosis induced by partial ligation and rFliC injection
Summary
It has been well established that vascular inflammation plays an important role in the generation and progression of atherosclerosis[1,2,3]. TLR isozymes in vascular cells, including endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages, recognize endogenous and exogenous ligands and trigger the expression of downstream proinflammatory molecules, including monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), Several lines of evidence indicate that NADPH oxidase (Nox)-induced reactive oxygen species (ROS) generation serves as a second messenger in various cellular events[8,9]. We previously reported that Nox isozymes become linked with TLRs in ECs, SMCs, and macrophages in response to TLR agonist stimulation. Stimulation of ECs with lipopolysaccharide results in Nox[4] activation through direct interaction with the COOH-terminus of TLR4, leading to the production of proinflammatory cytokines and adhesion molecules, including IL-8, MCP-1, and ICAM-110,11. Oxidized low-density lipoprotein (mmLDL) is known to interact with CD14 and activate
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