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Free Radic Biol Med. 2012 Dec 15;53(12):2344-56. doi: 10.1016/j.freeradbiomed.2012.10.002. Epub 2012 Oct 9.

Targeted subendothelial matrix oxidation by myeloperoxidase triggers myosin II-dependent de-adhesion and alters signaling in endothelial cells.

Author information

1
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.
2
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.
3
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.
4
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.
5
Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, Austria. Electronic address: ernst.malle@medunigraz.at.
6
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.

Abstract

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.

KEYWORDS:

(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride; (•)NO(2); 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4–d]pyrimidine; 4-aminobenzoic acid hydrazide; BAH; ECs; Endothelial dysfunction; Extracellular matrix; FAK; Free radicals; HBSS; HOCl; HOSCN; Hank's balanced salt solution; MLC-2; MPO; Met; Myeloperoxidase; NO; NO(2)(−); PBS; PP2; Redox signaling; Y-27632; bovine aortic endothelial cells; focal adhesion kinase; hypochlorous acid; hypothiocyanous acid; methionine; myeloperoxidase; myosin light chain II; nitric oxide; nitrite; nitrogen dioxide radical; phosphate-buffered saline, SCN(−), thiocyanate

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