title: Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: role of miR-146b-5p.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Raquel
authors.initials: R
authors.lastname: Echavarria
authors.affiliation: Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mayaki
authors.affiliation: Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Neel
authors.affiliation: Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Sharon
authors.initials: S
authors.lastname: Harel
authors.affiliation: Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Veronica
authors.initials: V
authors.lastname: Sanchez
authors.affiliation: Department of Critical Care, McGill University Health Centre, Montréal, Québec, Canada Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada sabah.hussain@muhc.mcgill.ca.
authors.forename: Sabah N A
authors.initials: SN
authors.lastname: Hussain
mesh_terms: D042683:Angiopoietin-1 / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D018920:Coculture Techniques; D005786:Gene Expression Regulation; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control*; D018836:Inflammation Mediators / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000378:metabolism; D007962:Leukocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014162:Transfection; D020298:U937 Cells
chemical_list: C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D000893:Anti-Inflammatory Agents; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; C495345:TLR4 protein, human; D048029:TNF Receptor-Associated Factor 6; D051197:Toll-Like Receptor 4; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases
keywords: Angiopoietins; Endothelial cells; Inflammation; MicroRNAs; Toll-like receptors