title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains.
abstract: The heparan sulfate (HS) proteoglycan perlecan is a major component of basement membranes, plays a key role in extracellular matrix (ECM) structure, interacts with growth factors and adhesion molecules, and regulates the adhesion, differentiation and proliferation of vascular cells. Atherosclerosis is characterized by chronic inflammation and the presence of oxidized materials within lesions, with the majority of protein damage present on ECM, rather than cell, proteins. Weakening of ECM structure plays a key role in lesion rupture, the major cause of heart attacks and strokes. In this study peroxynitrite, a putative lesion oxidant, is shown to damage perlecan structurally and functionally. Exposure of human perlecan to peroxynitrite decreases recognition by antibodies raised against both the core protein and heparan sulfate chains; dose-dependent formation of 3-nitrotyrosine was also detected. These effects were modulated by bicarbonate and reaction pH. Oxidant exposure resulted in aggregate formation, consistent with oxidative protein crosslinking. Peroxynitrite treatment modified functional properties of perlecan that are dependent on both the protein core (decreased binding of human coronary artery endothelial cells), and the HS chains (diminished fibroblast growth factor-2 (FGF-2) receptor-mediated proliferation of Baf-32 cells). The latter is consistent with a decrease in FGF-2 binding to the HS chains of modified perlecan. Immunofluorescence of advanced human atherosclerotic lesions provided evidence for the presence of perlecan and extensive formation of 3-nitrotyrosine epitopes within the intimal region; these materials showing marked co-localization. These data indicate that peroxynitrite induces major structural and functional changes to perlecan and that damage to this material occurs within human atherosclerotic lesions.
authors.affiliation: The Heart Research Institute, 7 Eliza Street, Newtown, NSW 2042, Australia.
authors.forename: Eleanor C
authors.initials: EC
authors.lastname: Kennett
authors.forename: Martin D
authors.initials: MD
authors.lastname: Rees
authors.forename: Ernst
authors.initials: E
authors.lastname: Malle
authors.forename: Astrid
authors.initials: A
authors.lastname: Hammer
authors.forename: John M
authors.initials: JM
authors.lastname: Whitelock
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davies
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation; D003324:Coronary Artery Disease / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D003331:Coronary Vessels / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000276:immunology / Q000378:metabolism*; D006497:Heparan Sulfate / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017539:Tunica Intima / Q000378:metabolism* / Q000473:pathology
chemical_list: D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D030421:Peroxynitrous Acid; C071927:perlecan