Considerable evidence exists for oxidative damage to extracellular materials during multiple human pathologies. Unlike cells, the extracellular compartment of most biological tissues is less well protected against oxidation than intracellular sites in terms of the presence of both antioxidants (low molecular mass and enzymatic) and repair enzymes. The extracellular compartment may therefore be subject to greater oxidative stress, marked alterations in redox balance and an accumulation of damage due to slow turnover and/or poor repair. The nature and consequences of damage to ECM (extracellular matrix) are poorly understood, despite the growing realization that changes in matrix structure not only have structural consequences, but also play a key role in the regulation of cellular adhesion, proliferation, migration and cell signalling. The ECM also plays a key role in cytokine and growth factor binding, and matrix modifications would therefore be expected to alter these parameters. In the present study, we review mechanisms of oxidative damage to ECM, resulting changes in matrix structure and how this affects cellular behaviour. The role of such damage in the development and progression of inflammatory diseases is also discussed with particular reference to cardiovascular disease.

Introduction

ECM (extracellular matrix) is a complex material consisting of collagens, other proteins, glycoproteins and proteoglycans, which are surrounded by interstitial fluid. Through its close association with surrounding cells, the ECM plays a key role in the regulation of cellular behaviour, as it modulates adhesion, migration and proliferation, through the binding of various growth factors and cytokines and by affecting intracellular signalling. These effects are mediated, in part, by interactions between ECM and transmembrane proteins such as integrins and dystroglycans. ECM generation and its normal turnover have been reviewed in [1,2].

The ECM is constantly changing in response to cues to and from cells and is both a scaffold and a roadmap for developing tissues and organs/tissues undergoing regeneration and repair. Collagens typically provide architecture and strength to the ECM, while proteoglycans and hyaluronan maintain hydration and mechanical properties as well as providing concentration gradients of signalling molecules critical for tissue development, form and function. As a result, ECM modification may modulate ECM properties and function and play a role in human disease. ECM modification has thus been linked to multiple inflammatory conditions, including atherosclerosis, kidney disease, rheumatoid arthritis and osteoarthritis [2,3]. These alterations may arise from both oxidation and enzymatically.

Different tissues have different ECM compositions [1]. Cartilage contains an abundance of type II collagen and the proteoglycan, aggrecan, while the basement membranes of the glomerulus and tubules of the kidney are rich in laminin, type IV collagen and the proteoglycan perlecan. The ECM of vascular tissues is complex, with elastic arteries containing an internal elastic lamina, which is rich in elastin. The medial and adventitial areas of blood vessels are rich in types I and III collagens, while the basal laminas and the basement membrane underlying endothelial cells are rich in type IV collagen, laminin and perlecan.

Collagens possess an unusual and characteristic (Gly-Xaa-Yaa)n sequence, with Xaa and Yaa frequently proline and hydroxyproline respectively; this allows assembly into homo- and hetero-trimeric helices and fibrillar structures. Non-collagenous domains are often found at the N- and C-termini, and also as interruptions between collagenous domains. Elastin contains a high abundance of non-polar amino acids (typically 30% glycine and 11% proline), with this resulting in poorly or insoluble, rubber-like microfibril networks. Classical collagens have a triple-helical structure that provides tensile strength, whereas others, such as type VI collagen, have more complex tertiary and quaternary structures, consistent with its role in the maintenance of basement membrane integrity [1]. Collagens have also been implicated in modulating cell adhesion signals.

The ECM contains multiple complex glycoproteins with simple linear or complex branched, sugar structures. Both fibronectin and laminin contain integrin recognition sequences, with fibronectin composed of two near-identical chains linked by a disulfide bond, whereas laminin is a complex homo- or hetero-trimer consisting of α, β and γ peptide chains. Laminin type 1, with α1, β1, γ1 chains, is well characterized; other laminins with different chain compositions have also been identified in basement membranes, where they interact with other ECM molecules and integrins [1].

Hyaluronan, a GAG (glycosaminoglycan; sugar polymer) consisting of alternating N-acetylglucosamine and hexuronic acid units, plays a key role in maintaining the hydration status of tissues. It is synthesized by plasma membrane enzymes and has a polydisperse molecular mass, with its activity dependent on molecular mass, as well as receptor binding and activation [1].

Proteoglycans are classified on the basis of the polysaccharide (GAGs) chains attached to their protein core. The GAG chains of heparan sulfate proteoglycans, a major class found in many tissues, are composed of alternating substituted hexosamine and uronic acid subunits, modified by sulfate groups at various positions. This modifies the mechanical properties of the material, its water retention capacity and the binding/localization of growth factors [e.g. FGF2 (fibroblast growth factor 2)] [4]. Damage to the heparan sulfate side chains of perlecan can therefore release bound FGF2 [4]. Mice that cannot synthesize heparan sulfates do not progress past the earliest stages of embryo development [5]. Proteoglycan levels are therefore critical to the maintenance of signalling gradients. These results indicate that ECM modification modulates cell behaviour, tissue structure and function and suggests oxidative damage may regulate ECM properties. Indirect modification by oxidants may also occur as a result of modulation, by oxidation, of enzymes involved in matrix turnover. Oxidation can activate pro-enzymes to active forms [e.g. MMPs (matrix metalloproteinases)] [6], inactivate enzyme inhibitors (e.g. tissue inhibitors of MMPs or TIMPs [7]) or alter structures such that the ECM becomes more prone to proteolysis. Oxidative and enzymatic damage may therefore be synergistic and interdependent.

Sources of extracellular oxidants

As most oxidants have restricted diffusion radii due to their rapid reaction with biological molecules, most oxidants generated within cells (e.g. via electron leakage from the electron transport chains of mitochondria and the endoplasmic reticulum to O2, yielding O2•−) cannot diffuse out. The oxidants formed extracellularly may therefore be different to those generated intracellularly. Some of these sources are reviewed below.

Activation of neutrophils, and other leucocytes (e.g. monocytes, eosinophils and some macrophages) results in the assembly of NADPH oxidase complexes (NOxs) that reduce O2 to superoxide radicals (O2•−), which are released extracellularly [8]. Related complexes are present in many cells. O2•− is poorly reactive and unlikely to cause major damage, but dismutation [spontaneous or catalysed by SODs (superoxide dismutases)] yields H2O2, which can generate further oxidants.

Concurrent with NOx activation, neutrophils, monocytes and some macrophages release the haem enzyme MPO (myeloperoxidase) (reviewed in [911]). Eosinophils release the related enzyme EPO (eosinophil peroxidase) (reviewed in [9]). These enzymes bind to ECM materials and may therefore localize damage at such sites. The resting Fe3+ form of these enzymes reacts with H2O2 to generate Compound I (reaction 1), which subsequently oxidizes halide or pseudohalide ions (primarily Cl and SCN with MPO; SCN and Br for EPO) to the hypohalous acids (HOCl, HOBr and HOSCN; reaction 2, X = Cl, Br, SCN). Other materials are oxidized via one-electron reactions (‘peroxidase cycle’, reactions 3 and 4). The enzymology and role of these peroxidases in disease have been reviewed in [911].

 
formula
(1)
 
formula
(2)
 
formula
(3)
 
formula
(4)

Chloramines (RNHCl) and bromamines (RNHBr) formed from reaction of HOCl and HOBr with amines, can diffuse through cell membranes as they have t1/2 of minutes to hours [12]. These species, as well as hypohalous acids, can react with metal ions or O2•− to generate radicals (reactions 5 and 6; X = Cl, Br) [1315].

 
formula
(5)
 
formula
(6)

Nitric oxide (NO) is generated from arginine by NOS (nitric oxide synthase) enzymes [16]. These enzymes are widely distributed, and occur in both constitutive [e.g. in endothelial cells, eNOS (endothelial NOS); and neuronal cells, nNOS (neuronal NOS)], and inducible forms [iNOS (inducible NOS) in macrophages] [16]. eNOS and nNOS generate pico- to nano-molar levels of NO, whereas iNOS can generate micromolar concentrations [16]. NO has a relatively long lifetime and can diffuse across membranes, a property critical for its signalling activity via interaction with smooth muscle cell guanylate cyclase [16]. Rapid reaction of NO with O2•− yields the powerful oxidant peroxynitrite (ONOO/ONOOH, pKa 6.8; reaction 7 [17]). The neutral (ONOOH) form of this oxidant is highly reactive and undergoes two-electron reactions with target species. ONOOH also undergoes homolytic fission to give HO and NO2 in yields ≤30% (reaction 8). ONOO also reacts with CO2 to give an adduct (ONOOCO2; reaction 9) that decomposes to NO2 and CO3•− (in ~35% yield; reaction 10).

 
formula
(7)
 
formula
(8)
 
formula
(9)
 
formula
(10)

Extracellular metal ions are bound tightly to macromolecules, with this resulting in negligible ‘free’ pools under normal circumstances [18]. During disease, metal ion transporter and storage proteins can be overwhelmed or become faulty with resulting increases in redox-active ‘inappropriately-bound’ metal ion levels; low-micromolar concentrations of iron and nanomolar levels of copper have been detected in advanced human atherosclerotic lesions [19]. GAGs bind metal ions with a high affinity owing to their polyanionic character and hence may be targets for metal-ion-induced oxidants. Other peroxides (e.g. on amino acids/peptides/proteins and lipids) may yield alkoxyl/peroxyl radicals via reactions analogous to reaction 11 (e.g. [20]).

 
formula
(11)

High blood glucose and reactive aldehydes present in people with diabetes can modify ECM components via glycation/glycoxidation reactions. These reactions result in the formation of AGEs (advanced glycation end-products) that can accumulate with age or disease [21].

ECM components can be damaged in UV-exposed tissues such as the skin and eye [1]. Visible light also induces damage in the presence of sensitizers, such as AGE materials, degraded haem and porphyrin intermediates, tryptophan metabolites, some vitamins (e.g. riboflavin) and other materials, including some drugs. Atmospheric pollutants (e.g. sulfur and nitrogen oxides, ozone, diesel particles, smoke and particulates) and some drugs can also induce damage in exposed tissues [18].

Extracellular fluids typically contain lower levels of antioxidants that protect against or decrease oxidative damage than are present within cells. Similarly, the levels of protective enzymes are also typically lower in extracellular fluids. In contrast, plasma contains relatively high levels of metal ion-binding proteins (e.g. transferrin), with these usually only 20–30% loaded. Such chelation usually prevents catalytic radical formation [18]. The effectiveness of these metal ion-binding proteins is, however, compromised in some diseases.

Extracellular repair of ECM damage is also limited, with cellular uptake, catabolism and reuse/excretion the major processes. Modified hyaluronan (via CD44 receptors) and glypican can be taken up by cells for recycling [22]. Plasma does not contain methionine sulfoxide reductase activity, but does contain disulfide reductase/protein disulfide-isomerase activities (possibly on cell surfaces), and so oxidized cysteine residues may be repaired. Other protein modifications are not repaired, and the major fate of these materials is catabolism. The formation of cross-linked materials can result exacerbate the accumulation of modifications [21].

Mechanisms of oxidation of ECM materials

Radical-mediated carbohydrate oxidation occurs primarily via relatively unspecific hydrogen atom abstraction at the large number of C–H bonds on GAGs to yield α-hydroxyalkyl radicals [C(OH)RR′]. These radicals undergo acid- and base-catalysed rearrangement reactions when there are adjacent C–OH and C–OR bonds [23]; the latter can result in glycosidic bond cleavage. These reactions compete with reaction with O2 to give peroxyl radicals (ROO) [23]; the latter rapidly eliminate HO2 to give carbonyl products [24]. This release of HO2 can result in chain reactions that exacerbate damage [24]. The presence of carboxyl, sulfate, amino and substituted amino groups on GAGs can modify oxidation at particular C–H bonds, with evidence for specific attack detected with some polymers [2426].

HOCl and HOBr react preferentially with the (free or substituted) nitrogen functions of amino sugars, with this generating chloramines, chloramides and chlorosulfonamides from free amines, N-acetyl and N-sulfate groups respectively (and corresponding N-bromo species) [27]. These can give rise to polysaccharide cleavage via both non-radical and radical pathways.

Peroxynitrite reacts with carbohydrates via a mechanism consistent with the generation of HO [25,26,28]. In contrast, NO2 (from peroxynitrite decomposition) does not induce marked polymer fragmentation, consistent with its lower reactivity [26,28]. CO3•− generated from ONOOCO2 reacts rapidly with GAGs and model compounds to give sugar radicals and polymer fragments [26,29]. HNO2 from peroxynitrite decomposition also cleaves carbohydrate bonds, but this reaction is slow, and may be less biologically significance.

Protein oxidation is highly complex, with multiple products generated from the 20 common side chains and the peptide backbone (reviewed in [30,31]). For proteoglycans and glycoproteins, the sugar chain can also act as a competing target. Damage to the protein core appears to occur via mechanisms similar to those detected with globular proteins (e.g. [30,31]).

With HOCl and HOBr, side-chain damage predominates, with cysteine, methionine, histidine, lysine, tryptophan, tyrosine and arginine side-targeted preferentially [32,33]. In contrast, peroxynitrite targets predominantly cysteine, methionine, tyrosine, tryptophan and histidine residues [34]. With some of these materials, specific products are formed that can be used as biomarkers of the presence of specific oxidants. Thus tyrosine is converted into 3-chlorotyrosine by HOCl (and 3-bromotyrosine by HOBr), and peroxynitrite converts tyrosine and tryptophan into 3-nitrotyrosine and 6-nitrotryptophan respectively [35,36]. Other tyrosine oxidation products (e.g. dopa and dityrosine) are not specific for particular oxidants [30,31]. The yield of 3-nitrotyrosine is often enhanced by the presence of CO2, with this ascribed to the formation of both CO3•− and NO2 from ONOOCO2. The former is more selective than HO, though still very reactive, with this resulting in higher yields of tyrosine phenoxyl radicals and 3-nitrotyrosine [34].

Oxidation of the ECM proteoglycan perlecan

The following section focuses on damage to perlecan, as an example of ECM modification by oxidants. Damage to other matrix components (e.g. collagen, elastin, fibronectin, laminin and GAGs) is reviewed elsewhere [3].

Perlecan is the major heparan sulfate proteoglycan expressed in the vascular wall and plays a key role in vascular homoeostasis via the stabilization and organization of vascular ECM, and the regulation of adhesion, differentiation and proliferation of vascular cells [2]. These functions are mediated by interactions of its protein core and heparan sulfate chains with a variety of ECM molecules, growth factors and adhesion molecules. The perlecan protein core (mass 470 kDa) consists of five distinct domains. The N-terminal domain, domain I, is the main region of GAG substitution, with a cluster of three attachment sites; an additional two potential attachment sites exist on domain V [2,5]. Endothelial-cell-derived perlecan is substituted exclusively by heparan sulfate at the domain I sites [5], whereas chondroitin sulfate and keratan sulfate variants have also been isolated from other cell sources [37]. Domains II–V display homology with the LDL (low-density lipoprotein) receptor, laminin α chain, neuronal cell adhesion molecule and epidermal growth factor [38].

Peroxynitrite-mediated damage

Peroxynitrite modifies the core protein and heparan sulfate chains. Exposure of perlecan to peroxynitrite results in a decreased recognition by multiple antibodies that recognize specific native protein epitopes, and the formation of both the specific oxidation product 3-nitrotyrosine, and a generic marker of oxidative damage, protein carbonyls [39]. These changes occur in a dose-dependent manner and are modulated by both the reaction pH and the presence of bicarbonate. The specific epitopes recognized by the core protein antibodies have been characterized [4].

Decreased recognition by antibodies that recognize specific heparan sulfate sequences have been reported [39]. The three heparan sulfate antibodies examined recognize distinct domain structures: HepSS-1 recognizes predominately N-sulfated domains, JM403 binding is dependent on N-unsubstituted glucosamine residues and 10E4 recognizes a sequence containing N-acetylated and N-sulfated glucosamine and an N-unsubstituted glucosamine residue. Selective loss of 10E4 recognition, but not that of the other two antibodies, suggests that structures recognized by 10E4 may be particularly susceptible to damage [39]. These results are consistent with previous studies that have shown that isolated GAGs are cleaved by peroxynitrite-derived HO (and CO3•− in the presence of bicarbonate) [25,26]. These results are consistent with cell culture studies in which damage to both the protein and carbohydrate components was detected [40]. These results indicate that both protein and GAG damage occurs concurrently.

Studies on globular proteins have shown that peroxynitrite damage is pH-dependent, in the absence of bicarbonate [34]. This arises from the greater reactivity of ONOOH (or HO and NO2 derived from it) over ONOO. Experimental data for the heparan sulfate chains of perlecan indicate a greater loss of recognition by antibody 10E4 at pH 6 and 6.5, than 7 or 7.4. In contrast, no pH-dependence was detected for the loss of recognition of the protein epitopes, although more 3-nitrotyrosine was detected at higher pH values. These differences may lead to a greater extent of damage to the heparan sulfate chains compared with the protein core at pH 6.0 and 6.5, and the inverse at higher pHs. The higher levels of 3-nitrotyrosine at higher pHs are consistent with the greater ease of oxidation of ionized tyrosine residues.

In the presence of bicarbonate, peroxynitrite generates higher yields of 3-nitrotyrosine on globular proteins [34]. This has been ascribed to ONOOCO2 formation from ONOO. In contrast, with perlecan the loss of antibody recognition induced by peroxynitrite was lower in the presence of bicarbonate than in its absence, and higher yields of 3-nitrotyrosine were detected in the absence of bicarbonate, compared with its presence [39]. These differences may arise from a higher reactivity of CO3•− with heparan sulfate (cf. k = 7×105 dm3·mol−1·s−1 for hyaluronan [29]) compared with most protein side chains. The presence of the sugar chains therefore appears to divert oxidant damage from the protein core; this is consistent with computational modelling [39].

Adhesion of human endothelial and VSMCs (vascular smooth muscle cells) to human perlecan is dependent on the protein core [41]. Peroxynitrite exposure induced a significant reduction in endothelial cell adhesion at pH 7.5, but not pH 6.5. Decomposed oxidant did not induce this effect, and the presence of bicarbonate at pH 7.5, did not modulate it [39]. FGF2 associates with perlecan through its heparan sulfate chains, with interaction occurring via highly sulfated regions of the heparan sulfate chains on domain I, and this may protect FGF2 from proteolytic degradation [42]. Treatment with peroxynitrite at pH 6.5 had no significant effect on the ability of the heparan sulfate to signal via the FGF receptors, whereas at pH 7.5 a significant decrease was observed [39], consistent with the observed decrease is recognition of 10E4 epitopes. The ability of perlecan to stimulate FGF2-dependent cellular proliferation not only requires a capacity to bind FGF2, but also to form a ternary complex with the cognate cell-surface receptor [43]. With FGF receptor-1 expressing Baf-32 cells, this activity was impaired. These results indicate that perlecan heparan sulfate oxidation may be functionally significant.

Formation of peroxynitrite-modified perlecan has been detected in vivo by double-immunofluorescence studies of advanced human atherosclerotic lesions, with co-localization of perlecan and 3-nitrotyrosine epitopes detected in the intimal region (Figure 1). These epitopes co-localize with CD14-positive cells, implicating tissue macrophages (or monocytes) in such damage. The localization of damaged perlecan with CD14-positive cells may be of importance, as macrophage-rich sites are often associated with ECM weakening and subsequent lesion rupture [44]. Modified perlecan has also been detected in human asthmatic tissue [45].

Double immunofluorescence staining for perlecan, 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections

Figure 1
Double immunofluorescence staining for perlecan, 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections

Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-(human perlecan) (mouse monoclonal antibody, clone 7B5, perlecan domain III), anti-(human CD14) (monocytes, macrophages), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit Cy3 (indocarbocyanine)-labelled IgG or goat anti-mouse Cy2 (carbocyanine)-labelled IgG. DAPI (4′,6-diamidino-2-phenylindole) was used to image cell nuclei. (A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (broken arrow). (B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vasa vasorum. (C) In sections where the antibodies for perlecan (CD14) and for 3-nitrotyrosine were replaced with non-immune mouse IgG and non-immune rabbit IgG, no staining in the intima was observed. The faint staining in the media results from elastic membranes located in this area. Scale as indicated in the bottom images. Reprinted from [39], with permission from Elsevier.

Figure 1
Double immunofluorescence staining for perlecan, 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections

Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-(human perlecan) (mouse monoclonal antibody, clone 7B5, perlecan domain III), anti-(human CD14) (monocytes, macrophages), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit Cy3 (indocarbocyanine)-labelled IgG or goat anti-mouse Cy2 (carbocyanine)-labelled IgG. DAPI (4′,6-diamidino-2-phenylindole) was used to image cell nuclei. (A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (broken arrow). (B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vasa vasorum. (C) In sections where the antibodies for perlecan (CD14) and for 3-nitrotyrosine were replaced with non-immune mouse IgG and non-immune rabbit IgG, no staining in the intima was observed. The faint staining in the media results from elastic membranes located in this area. Scale as indicated in the bottom images. Reprinted from [39], with permission from Elsevier.

MPO-mediated damage

HOCl has been shown to selectively damage the protein core of perlecan without detectable alteration of its heparan sulfate side chains, despite the presence of highly reactive glucosamine residues [46]. Loss of immunological recognition of native protein epitopes, and the appearance of oxidatively modified protein epitopes, induced by HOCl and HOBr, was associated with an impairment of endothelial cell adhesion; these changes occurred with doses of oxidant that may be achieved under pathological conditions (e.g. 425 nmol of oxidant/mg of protein). In contrast, the heparan sulfate chains of the modified perlecan retained their ability to bind FGF2 and collagen V, and were able to promote FGF2-dependent cellular proliferation [46].

Consequences of matrix damage

ECM damage can manifest as protein unfolding (e.g. collagen triple helix), increases in susceptibility to proteolytic cleavage, functional inactivation of ligand-binding sites and increased ECM permeability (reviewed in [3,30,31]). ECM can be solubilized by low levels of oxidants, without major overt structural alterations; this may be due to dissociation of ECM macromolecular assemblies. In some cases, GAG degradation appears to occur selectively, while in other cases the inverse appears to be true (e.g. inter-α-heavy chain protein substituents of hyaluronan and the link protein of hyaluronan/proteoglycan aggregates) [46,47]. At high oxidant doses, release of ECM fragments occurs [40,48,49], with the levels of this material used to quantify ECM damage [3,30,31,47]. Oxidants can also induce ECM cross-linking and aggregation [25,39,46], although this appears to be target- and oxidant-specific.

Oxidation can increase the permeability of epithelial/endothelial cell layers (e.g. in isolated glomeruli [50]) and can disrupt epithelial/endothelial cell adhesion [39,51]. This may arise from ECM degradation and/or modification of species involved in barrier function and/or cell adhesion (e.g. sialoglycoproteins, integrins, adhesion molecules and cell-associated glycocalyx). With activated neutrophils, MPO-derived oxidants may be the predominant mediator of damage underlying the rapid increase in glomerular permeability [50]. In contrast, tissue macrophages may modify ECM primarily via the formation of peroxynitrite. Other cells can also degrade ECM, though whether this is via oxidant production, enzymatic action or both remains to be established. Such processes are implicated in both normal turnover of ECM (e.g. epidermal hyaluronan) and multiple pathologies (reviewed in [3]).

Endothelial cells adhere and proliferate on oxidized ECM to a lesser extent than native ECM [51]. While highly polymerized hyaluronan is benign [52], hyaluronan fragments can modulate macrophage expression of chemokines, cytokines and growth factors (macrophage inflammatory proteins-1α and -1β, tumour necrosis factor α, plasminogen activator inhibitor-1 and macrophage metallo-elastase) as well as cell-surface proteins [e.g. VCAM-1 (vascular cell adhesion molecule-1)], with these processes mediated by IFNγ (interferon γ) and NF-κB (nuclear factor κB) [53]. Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1 (CXC chemokine ligand 1)/GRO1 (growth-related oncogene 1)-dependent manner [54] and stimulate endothelial recognition of injury through TLR4 (Toll-like receptor 4 [55]). Furthermore, hyaluronan fragments appear to stimulate endothelialization [56]. Other GAG oligomers modulate endothelial, fibroblast and smooth muscle cell proliferation [57], and interact in an altered manner with the major cell-surface receptor CD44 [22]. Hyaluronan fragments accumulate at sites of inflammation, particularly at macrophage-rich sites, as do other modified ECM components [5860].

ECM damage in CVD (cardiovascular disease)

The ECM is intimately involved in CVD. Lipoproteins (e.g. LDL) can be retained in the ECM via interactions between specific amino acids (e.g. lysine and arginine) on apolipoprotein B-100 and ECM GAGs. This retention can increase LDL concentrations and residence time in the sub-endothelial space, and may enhance LDL modification [61,62]. Such modified LDL is recognized by macrophage scavenger receptors, and this results in uncontrolled uptake and formation of lipid-laden (foam) cells; these are a defining feature of atherosclerotic lesions [62]. ECM oxidation may enhance lipoprotein retention and accumulation by increasing the affinity of LDL for ECM materials, via changes in charge, hydrophilicity/hydrophobicity and cross-linking between LDL and ECM components.

Increased versican levels have been reported in atherosclerotic lesions, with this believed to result in greater smooth muscle cell migration and proliferation [61]. Multiple studies have indicated that the total GAG content of intimal ECM decreases in atherosclerosis, Type 2 diabetes and abdominal aortic aneurysms, with an associated decrease in the heparan sulfate/dermatan sulfate ratio [63]. Whether this change is important is unclear, but decreased heparan sulfate levels may enhance smooth muscle cell proliferation and migration into the intima.

There is compelling evidence for the presence of oxidized materials in atherosclerotic lesions and for an association with CVD (reviewed in [62]). However, there are limited data on ECM oxidation in vivo [48]. ECM oxidation may be enhanced by reduced levels of extracellular SOD activity, which has been associated with increased vascular oxidative stress in patients with chronic heart failure [64]. Oxidant formation has also been associated with ECM remodelling and lesion rupture [62].

Both peroxynitrite and MPO have been postulated to play a key role in atherosclerosis (reviewed in [62]). MPO was been shown to be present and active in the intima of atherosclerotic lesions [65], after acute inflammatory response [66], in coronary artery disease patients [67] and at sites of lesion rupture, particularly in shoulder regions [65]. Co-localization of MPO protein and markers of damage induced by its oxidants has been detected in lesions [6769], with damage levels correlating with lesion severity [69]. Elevated levels of both 3-chlorotyrosine and 5-chlorouracil, products of HOCl reactions, have been demonstrated in lesions [68,70].

3-Nitrotyrosine, a marker of protein tyrosine oxidation by peroxynitrite, has been detected at markedly elevated levels in human atherosclerotic lesions, as well as on proteins extracted from lesions [7173]. Co-localization of MPO protein with 3-nitrotyrosine staining and fibronectin in the subendothelial layer of coronary arteries of a patient with coronary artery disease, suggest that fibronectin is a major target, with this potentially arising from MPO-mediated oxidation of nitrite [67]. Fibrinogen nitration enhances cross-link formation and clots, and this product has been detected in the blood of people with coronary artery disease [74].

Other protein oxidation products have been detected on lesion proteins [75], some of which (e.g. hydroxylated materials) are consistent with HO (or other highly reactive oxygen-derived oxidants) reactions [75]. Elastin isolated from human lesions is modified and contains the oxidation marker dityrosine, and elastin fragments and fibrin degradation products have been detected in people with coronary artery disease and atherosclerotic lesions (reviewed in [3]). Proteoglycans isolated from lesions have a lower mass than those from normal arteries, consistent with fragmentation, and modified dermatan sulfate proteoglycans have also been detected in animal models of atherosclerosis (reviewed in [3]). The elevated levels of iron and copper present in advanced human atherosclerotic lesions [19], and an observed association between iron levels and protein (but not lipid) oxidation [76], is consistent with metal-ion-mediated damage.

In addition to basement membrane modification, oxidative damage to the endothelial cell glycocalyx (a mesh-like structure comprising glycoproteins, proteoglycans and GAGs on the luminal side of the endothelium) has been associated with impaired vascular function. Oxidation may result in decreased glycocalyx volume and hyaluronan release, with this postulated to play a role in hyperglycaemia-induced vascular dysfunction and ischaemia/reperfusion injury models [77,78]. This damage can be decreased by radical-scavengers, SOD, NOS inhibitors and NO scavengers, consistent with both radical and peroxynitrite-mediated, damage [77].

Conclusions

Multiple studies now indicate that the ECM is exposed to multiple oxidants, and that antioxidant defences, repair and removal of oxidative damage are limited. As a result ECM damage is likely to accumulate over time and during disease development. ECM damage has been shown to result in altered structure and function, with this modulating cell adhesion, proliferation and behaviour, as well as altered cell signalling. Owing to the difficulties inherent in handling matrix materials, understanding of ECM damage mechanisms and processes are still in their infancy and knowledge of ECM modification is still far from complete.

Analysis of Free Radicals, Radical Modifications and Redox Signalling: A Biochemical Society Focused Meeting held at Aston University, Birmingham, U.K., 18–19 April 2011. Organized and Edited by Helen Griffiths (Aston University, U.K.), Corinne Spickett (Aston University, U.K.) and Paul Winyard (Exeter, U.K.).

Abbreviations

     
  • AGE

    advanced glycation end-product

  •  
  • CVD

    cardiovascular disease

  •  
  • ECM

    extracellular matrix

  •  
  • EPO

    eosinophil peroxidase

  •  
  • FGF2

    fibroblast growth factor 2

  •  
  • GAG

    glycosaminoglycan

  •  
  • LDL

    low-density lipoprotein

  •  
  • MMP

    matrix metalloproteinase

  •  
  • MPO

    myeloperoxidase

  •  
  • NOS

    nitric oxide synthase

  •  
  • eNOS

    endothelial NOS

  •  
  • iNOS

    inducible NOS

  •  
  • nNOS

    neuronal NOS

  •  
  • SOD

    superoxide dismutase

Funding

This work was funded by the Australian Research Council through the Discovery [grant number DP0988311] and Centers of Excellence Programmes [grant number CE0561607], the National Health and Medical Research Council [grant number 570829] and the National Heart Foundation [grant numbers G09S4313 and G08S3769].

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