武汉云克隆科技股份有限公司

Abstract

BACKGROUND AND PURPOSE:

Calcific aortic stenosis is a chronic inflammatory disease, and aortic valve interstitial cells (AVIC) play an important role in valvular inflammation. Whereas AVIC from stenotic aortic valves exhibit an augmented response to Toll-like receptor 4 (TLR4) stimulation, the underlying mechanism is unclear. This study tested the hypothesis that an excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the inflammatory response to lipopolysaccharide (LPS) in AVIC of stenotic valves.

METHODS AND RESULTS:

Human AVIC were isolated from normal and stenotic leaflets. Nuclear factor kappa-B (NF-κB) activation and production of interleukin-8, monocyte chemoattactrant protein-1, and intercellular adhesion molecule-1 were analyzed after treatment with LPS. The role of Notch1 in the inflammatory response was determined using inhibitor, siRNA, and specific ligand. Cells from diseased valves produced greater levels of chemokines and intercellular adhesion molecule-1 that are associated with enhanced NF-κB activation. Interestingly, diseased cells exhibited augmented Jagged1 release and Notch1 activation after TLR4 stimulation. Inhibition and silencing of Notch1 each resulted in greater suppression of the TLR4-induced inflammatory response in diseased cells. Conversely, activation of Notch1 with a specific ligand, Jagged1, enhanced the LPS-induced inflammatory response in normal AVIC. Further, Notch1 intracellular domain was coimmunoprecipited with the inhibitor of NF-κB kinase after LPS stimulation, and inhibition of Notch1 abrogated the difference in the level of NF-κB activation between diseased and normal cells.

CONCLUSION:

Notch1 enhances the inflammatory response to TLR4 stimulation in human AVIC through modulating NF-κB activation. Excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the TLR4 response in AVIC of stenotic valves.

摘要

背景和目的:

钙化性主动脉瓣狭窄是一种慢性炎性疾病，主动脉瓣间质细胞（AVIC）在瓣膜炎症中起着重要的作用。尽管来自狭窄主动脉瓣的间质细胞对Toll样受体4（TLR4）的刺激有增强的应答反应，但其潜在机制尚不清楚。这项研究验证了一种假设，即TLR4和Notch1通路之间的过度交互作用，是狭窄主动脉瓣间质细胞中对脂多糖（LPS）的炎症应答反应增强的原因。

方法和结果:

从正常和主动脉瓣狭窄病人分离得到人主动脉瓣间质细胞。在使用LPS处理后分析NF-κB的激活和白介素8、单核细胞趋化蛋白1和细胞间粘附分子1的产生。用抑制剂、siRNA和特异性配体鉴定了Notch1在炎症反应中的作用。来自病变瓣膜的细胞产生了更高水平的趋化因子和细胞间粘附分子1，这些因子与增强NF-κB活性有关。有趣的是，在TLR4刺激后，病变细胞增强了Jagged1的释放和Notch1的激活。在病变细胞中，Notch1的抑制和沉默更强的抑制了TLR4诱导的炎症应答反应。相反地，在正常的主动脉瓣间质细胞中，用特定的配体Jagged1激活Notch1，增强了LPS诱导的炎症反应。此外，在LPS刺激后，Notch1 胞内结构域与NF-κB激酶的抑制剂免疫共沉淀，而对Notch1的抑制则消除了病变与正常细胞之间NF-κB激活水平的差异。

结论:

通过调节NF-κB的激活，Notch1增强了人主动脉瓣间质细胞对TLR4刺激的炎症反应。TLR4和Notch1通路之间的过度交互作用是增加TLR4在狭窄主动脉瓣的间质细胞里应答的原因。

使用试剂原文信息：Jagged1 ELISA kit was purchased from Uscn Life Science Inc..