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

Cardiovasc Res. 2015 Jun 1;106(3):465-77. doi: 10.1093/cvr/cvv120. Epub 2015 Mar 30.

Abstract

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 (TNFα, IL1β, IL6, and IL8), and increased NFκB 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.

Keywords: Angiopoietins; Endothelial cells; Inflammation; MicroRNAs; Toll-like receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiopoietin-1 / pharmacology*
  • Anti-Inflammatory Agents / pharmacology*
  • Cell Adhesion / drug effects
  • Coculture Techniques
  • Gene Expression Regulation
  • Human Umbilical Vein Endothelial Cells / drug effects*
  • Human Umbilical Vein Endothelial Cells / immunology
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Inflammation / genetics
  • Inflammation / immunology
  • Inflammation / metabolism
  • Inflammation / prevention & control*
  • Inflammation Mediators / metabolism
  • Interleukin-1 Receptor-Associated Kinases / genetics
  • Interleukin-1 Receptor-Associated Kinases / metabolism
  • Leukocytes / drug effects
  • Leukocytes / immunology
  • Leukocytes / metabolism
  • Lipopolysaccharides / pharmacology
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Signal Transduction / drug effects
  • TNF Receptor-Associated Factor 6 / genetics
  • TNF Receptor-Associated Factor 6 / metabolism
  • Time Factors
  • Toll-Like Receptor 4 / agonists
  • Toll-Like Receptor 4 / antagonists & inhibitors*
  • Toll-Like Receptor 4 / metabolism
  • Transfection
  • U937 Cells

Substances

  • ANGPT1 protein, human
  • Angiopoietin-1
  • Anti-Inflammatory Agents
  • Inflammation Mediators
  • Lipopolysaccharides
  • MIRN146 microRNA, human
  • MicroRNAs
  • TLR4 protein, human
  • TNF Receptor-Associated Factor 6
  • Toll-Like Receptor 4
  • IRAK1 protein, human
  • Interleukin-1 Receptor-Associated Kinases