Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: role of miR-146b-5p

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.