Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

TY - JOUR

T1 - Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

AU - Kennett, Eleanor C

AU - Rees, Martin D

AU - Malle, Ernst

AU - Hammer, Astrid

AU - Whitelock, John M

AU - Davies, Michael Jonathan

N1 - Copyright 2010 Elsevier Inc. All rights reserved.

PY - 2010/7/15

Y1 - 2010/7/15

N2 - 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.

AB - 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.

KW - Cell Line

KW - Cell Proliferation

KW - Coronary Artery Disease

KW - Coronary Vessels

KW - Epithelial Cells

KW - Extracellular Matrix

KW - Heparan Sulfate Proteoglycans

KW - Heparitin Sulfate

KW - Humans

KW - Immunohistochemistry

KW - Oxidative Stress

KW - Peroxynitrous Acid

KW - Protein Binding

KW - Protein Multimerization

KW - Tunica Intima

U2 - 10.1016/j.freeradbiomed.2010.04.018

DO - 10.1016/j.freeradbiomed.2010.04.018

M3 - Journal article

C2 - 20416372

SN - 0891-5849

VL - 49

SP - 282

EP - 293

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

IS - 2

ER -