Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: role of miR-146b-5p
Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: role of miR-146b-5p
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DOI：
10.1093/cvr/cvv120
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发表时间：
2015-06-01
影响因子：
10.8
通讯作者：
Hussain, Sabah N. A.
中科院分区：
文献类型：
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作者：
Echavarria, Raquel;Mayaki, Dominique;Hussain, Sabah N. A.
目的细菌脂多糖（LPS）通过激活TLR 4信号通路诱导内皮细胞的天然免疫炎症反应。在这里，我们调查的影响，血管生成素-1（Ang-1）对LPS诱导的TLR 4信号和miR-146家族的microRNAs的作用，在Ang-1对TRL 4 signaling.Methods和结果白细胞粘附人脐静脉内皮细胞（HUVECs）的影响，采用荧光显微镜检测。使用实时PCR定量HUVEC中粘附分子、促炎细胞因子、miR-146 a和miR-146 b-5 p的表达。使用免疫印迹法测量TLR 4信号传导蛋白水平。HUVEC暴露于LPS 4-6 h诱导强烈的炎症反应，包括增强的白细胞粘附、粘附分子表达（VCAM 1、ICAM 1、E-SELECTIN）的上调、增强的细胞因子产生（TNF α、IL 1 β、IL 6和IL 8）和增加的NF κ B荧光素酶报告基因活性。在LPS暴露前向培养基中加入Ang-1 24 h可显著减弱这些反应。长期Ang-1暴露显著降低IRAK 1和TRAF 6蛋白水平，但对TLR 4、MYD 88、IRAK 4或TAK 1表达无影响。Ang-1可显著上调miR-146 b-5 p水平，但对miR-146 a或miR-146 b-3 p表达无影响。用miR-146 b-5 p模拟物转染HUVEC显著减弱LPS诱导的炎症反应以及IRAK 1和TRAF 6表达。在转染miR-146 b-5 p抑制剂的HUVECs中，Ang-1对LPS诱导的炎症反应及IRAK 1和TRAF 6表达无影响。这种抑制通过miR-146 b-5 p选择性靶向IRAK 1和TRAF 6蛋白而发生。
Aims Bacterial lipopolysaccharides (LPS) induce innate immune inflammatory responses in endothelial cells by activating toll-like receptor 4 (TLR4) signalling. Here, we investigate the effects of angiopoietin-1 (Ang-1) on LPS-induced TLR4 signalling and the role of the miR-146 family of micro RNAs in the effects of Ang-1 on TRL4 signalling.Methods and results Leucocyte adhesion to human umbilical vein endothelial cells (HUVECs) was detected using fluorescence microscopy. Adhesion molecule, pro-inflammatory cytokine, miR-146a, and miR-146b-5p expressions in HUVECs were quantified using real-time PCR. TLR4 signalling protein levels were measured using immunoblotting. Exposure of HUVECs to LPS for 4-6 h induces robust inflammatory responses, including enhanced leucocyte adhesion, up-regulation of adhesion molecule expression (VCAM1, ICAM1, E-SELECTIN), enhanced cytokine production (TNF alpha, IL1 beta, IL6, and IL8), and increased NFkB luciferase reporter activity. Addition of Ang-1 to the culture medium for 24 h prior to LPS exposure significantly attenuates these responses. Prolonged Ang-1 exposure significantly decreases IRAK1 and TRAF6 protein levels but has no effect on TLR4, MYD88, IRAK4, or TAK1 expressions. Ang-1 triggers significant up-regulation of miR-146b-5p levels but has no effect on miR-146a or miR-146b-3p expressions. Transfection of HUVECs with a miR-146b-5p mimic significantly attenuates LPS-induced inflammatory responses and IRAK1 and TRAF6 expressions. In HUVECs transfected with a miR-146b-5p inhibitor, Ang-1 has no effect on LPS-induced inflammatory responses or IRAK1 and TRAF6 expressions.Conclusion Ang-1 disrupts TLR4 signalling, resulting in inhibition of LPS-induced inflammatory responses in endothelial cells. This inhibition occurs through selective targeting of IRAK1 and TRAF6 proteins by miR-146b-5p.