title: Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.
abstract: Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to BM was characterized for isolated murine BM, and purified murine laminin-111. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Endothelial cell adhesion to isolated laminin-111 exposed to 10 μM or higher levels of ONOOH was significantly decreased (~25%) compared to untreated controls. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture.
authors.affiliation: The Heart Research Institute, Newtown, NSW, Australia; Faculty of Medicine, The University of Sydney, NSW, Australia.
authors.forename: Georg
authors.initials: G
authors.lastname: Degendorfer
authors.affiliation: Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Denmark.
authors.forename: Christine Y
authors.initials: CY
authors.lastname: Chuang
authors.affiliation: Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Graz, Austria.
authors.forename: Astrid
authors.initials: A
authors.lastname: Hammer
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, Graz, Austria.
authors.forename: Ernst
authors.initials: E
authors.lastname: Malle
authors.affiliation: The Heart Research Institute, Newtown, NSW, Australia; Faculty of Medicine, The University of Sydney, NSW, Australia; Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Denmark. Electronic address: davies@sund.ku.dk.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davies
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology*; D001485:Basement Membrane / Q000473:pathology*; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D007797:Laminin; D016877:Oxidants; D030421:Peroxynitrous Acid
keywords: 3-nitrotyrosine; Endothelial cells; Extracellular matrix; Laminin; Peroxynitrous acid; Protein oxidation