Department of Life Science and GT5 research program, Ewha Womans University, Seoul, Korea.
Cardiovasc Res. 2013 Aug 1;99(3):483-93. doi: 10.1093/cvr/cvt107. Epub 2013 Jun 6.
NADPH oxidase (Nox) isozymes that generate intracellular reactive oxygen species (ROS) and Toll-like receptor 2 (TLR2), an inflammatory mediator, are both involved in the development of atherosclerotic lesions. To identify the molecular connection between TLR2 and Nox isozymes in vascular remodelling, we analysed generation of ROS and pro-inflammatory cytokines in aortic smooth muscle cells from Nox1-deficient mice in response to the synthetic triacylated lipoprotein Pam3CSK, a TLR2 agonist.
We showed that TLR2 signalling stimulates progression of the pro-inflammatory phenotype in mouse aortic smooth muscle cells (MASMCs) through activation of Nox1. We demonstrated the interaction of TLR2 with Nox1 using yeast two-hybrid and co-immunoprecipitation assays. MASMCs from Nox1-deficient mice failed to generate of ROS in response to Pam3CSK4, indicating that Nox1 is essential for TLR2-dependent production of ROS. We also found that Pam3CSK4 stimulated migration of MASMCs from wild-type mice in a Transwell system, but MASMCs from Nox1-deficient mice failed to show this response. Wild-type MASMCs produced matrix metalloprotease 2 in response to Pam3CSK4, whereas Nox1-deficient MASMCs failed to generate this protease. Moreover, stimulation of MASMCs with Pam3CSK4 resulted in increased expression of the pro-inflammatory cytokine macrophage inflammatory protein 2 in a Nox1-dependent manner, leading to enhanced monocyte-endothelial cell adhesion and trans-endothelial migration of U937 cells.
These data suggest that Nox1 plays an important role in TLR2-mediated intracellular H2O2 generation, activation of matrix metalloprotease 2, and secretion of pro-inflammatory cytokines, which in turn stimulate MASMC migration and vascular remodelling.
产生细胞内活性氧物种 (ROS) 的 NADPH 氧化酶 (Nox) 同工酶和炎症介质 Toll 样受体 2 (TLR2) 均参与动脉粥样硬化病变的发展。为了确定 TLR2 与 Nox 同工酶在血管重塑中的分子联系，我们分析了缺乏 Nox1 的小鼠主动脉平滑肌细胞 (MASMC) 对合成的三酰化脂蛋白 Pam3CSK（TLR2 激动剂）产生 ROS 和促炎细胞因子的情况。
我们表明，TLR2 信号通过激活 Nox1 刺激小鼠主动脉平滑肌细胞 (MASMC) 前炎症表型的进展。我们使用酵母双杂交和共免疫沉淀测定证明了 TLR2 与 Nox1 的相互作用。缺乏 Nox1 的 MASMC 对 Pam3CSK4 没有产生 ROS，表明 Nox1 是 TLR2 依赖性 ROS 产生所必需的。我们还发现 Pam3CSK4 刺激野生型小鼠的 MASMC 在 Transwell 系统中迁移，但缺乏 Nox1 的 MASMC 没有表现出这种反应。野生型 MASMC 对 Pam3CSK4 产生基质金属蛋白酶 2，而缺乏 Nox1 的 MASMC 则无法产生这种蛋白酶。此外，Pam3CSK4 刺激 MASMC 导致依赖 Nox1 的方式增加促炎细胞因子巨噬细胞炎症蛋白 2 的表达，从而增强单核细胞-内皮细胞粘附和 U937 细胞的跨内皮迁移。
这些数据表明，Nox1 在 TLR2 介导的细胞内 H2O2 产生、基质金属蛋白酶 2 的激活和促炎细胞因子的分泌中发挥重要作用，进而刺激 MASMC 迁移和血管重塑。
