Targeted subendothelial matrix oxidation by myeloperoxidase triggers myosin II-dependent de-adhesion and alters signaling in endothelial cells.
During inflammation, myeloperoxidase (MPO) released by circulating leukocytes accumulates within the subendothelial matrix by binding to and transcytosing the vascular endothelium. Oxidative reactions catalyzed by subendothelial-localized MPO are implicated as a cause of endothelial dysfunction in vascular disease. While the subendothelial matrix is a key target for MPO-derived oxidants during disease, the implications of this damage for endothelial morphology and signaling are largely unknown. We found that endothelial-transcytosed MPO produced hypochlorous acid (HOCl) that reacted locally with the subendothelial matrix and induced covalent cross-linking of the adhesive matrix protein fibronectin. Real-time biosensor and live cell imaging studies revealed that HOCl-mediated matrix oxidation triggered rapid membrane retraction from the substratum and adjacent cells (de-adhesion). De-adhesion was linked with the alteration of Tyr-118 phosphorylation of paxillin, a key adhesion-dependent signaling process, as well as Rho kinase-dependent myosin light chain-2 phosphorylation. De-adhesion dynamics were dependent on the contractile state of cells, with myosin II inhibition with blebbistatin attenuating the rate of membrane retraction. Rho kinase inhibition with Y-27632 also conferred protection, but not during the initial phase of membrane retraction, which was driven by pre-existing actomyosin tensile stress. Notably, diversion of MPO from HOCl production by thiocyanate or nitrite attenuated de-adhesion and associated signaling responses, despite the latter substrate supporting MPO-catalyzed fibronectin nitration. These data show that subendothelial-localized MPO employs a novel "outside-in" mode of redox signaling, involving HOCl-mediated matrix oxidation. These MPO-catalyzed oxidative events are likely to play a previously unrecognized role in altering endothelial integrity and signaling during inflammatory vascular disorders.
Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Electronic address: m.rees@unsw.edu.au.
Martin D
MD
Rees
Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Electronic address: lei.dang@unsw.edu.au.
Lei
L
Dang
Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Electronic address: thuan@unsw.edu.au.
Thuan
T
Thai
Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Electronic address: dylan.owen@unsw.edu.au.
Dylan M
DM
Owen
Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, Austria. Electronic address: ernst.malle@medunigraz.at.
Ernst
E
Malle
Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. Electronic address: shane.thomas@unsw.edu.au.
Shane R
SR
Thomas
D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology*; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000378:metabolism; D024462:Myosin Type II / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction; D059008:Time-Lapse Imaging; D014657:Vasculitis / Q000201:enzymology
D005353:Fibronectins; D006997:Hypochlorous Acid; D000205:Actomyosin; D006861:Hydrogen Peroxide; D009195:Peroxidase; D024462:Myosin Type II