title: In-Depth Proteomics Identifies a Role for Autophagy in Controlling Reactive Oxygen Species Mediated Endothelial Permeability.
abstract: Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells.
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Francesca
authors.initials: F
authors.lastname: Patella
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Neilson
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Athineos
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Erami
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Kurt I
authors.initials: KI
authors.lastname: Anderson
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Karen
authors.initials: K
authors.lastname: Blyth
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Ryan
authors.affiliation: Cancer Research UK, Beatson Institute , Glasgow G611BD, U.K.
authors.forename: Sara
authors.initials: S
authors.lastname: Zanivan
mesh_terms: D001343:Autophagy / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D010539:Permeability; D040901:Proteomics / Q000379:methods; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species
keywords: ROS; SILAC; autophagy; contact inhibition; endothelial cell; permeability; proteomics