Original ContributionPeroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin
Graphical abstract
Introduction
Peroxynitrous acid (ONOOH) is a powerful oxidizing and nitrating agent formed under inflammatory conditions by the diffusion-controlled reaction of nitric oxide (●NO) and superoxide (O2-●) radicals (k2 6.7×109 M−1 s−1 [1]). ●NO is produced by nitric oxide synthases (NOS), with the inducible NOS protein expressed in activated macrophages being a major source. This isoform generates micromolar concentrations of ●NO [2]. O2-● is generated via multiple enzymatic and cellular sources, including NADPH oxidases (NOx, particularly of phagocytic cells) and electron transport chains of endothelial and smooth muscle cells amongst others [3]. NOx enzymes are particularly effective sources, generating fluxes in the nano-micromolar range as part of the immune response [4]. ONOOH is more reactive than its precursors radicals (●NO and O2-●) and can induce both direct damage, via two-electron oxidation reactions (e.g. with metal-ion centres, thiols and thioethers, with rate constants in the range 103-106 M−1 s−1 [5], [6]), and by one-electron reactions of radicals (nitrogen dioxide, ●NO2, and hydroxyl radicals, HO●) arising from homolysis of the -O-O- bond [7]. The peroxynitrite anion (ONOO−, pKa 6.5) reacts with CO2 to a give the short-lived nitrosoperoxocarbonate anion (ONOOCO2−), which may undergo homolysis to give carbonate radical anions (CO3-●) and nitrogen dioxide radical (●NO2) [8]. ONOOH is known to damage multiple biological molecules [5], [6], and this oxidation is associated with multiple inflammatory conditions including cardiovascular disease [9], [10], [11], [12]. Previous research on ONOOH-mediated damage has been focused primarily on cells, apolipoproteins and antioxidants, with relatively little attention paid to damage to the extracellular matrix (ECM).
The ECM is a major component of all tissue types, with this typically forming a complex framework that provides a 3-dimensional structure and support to tissues. There is growing evidence that the ECM has multiple functions including: a) providing mechanical strength and elasticity; b) acting as a scaffold for cell adhesion and growth; c) controlling cell migration and proliferation; d) binding signalling molecules, pro-enzymes, enzyme inhibitors and cytokines; and e) modulating the activity of these materials [13], [14]. Glycosaminoglycans can form gels of varying pore size and charge density thereby acting as a selective sieve that regulates molecular traffic. Proteoglycans (proteins with covalently attached large glycosaminoglycan chains) bind secreted signalling molecules (e.g. growth factors) and can enhance or inhibit their activity via multiple mechanisms including: (1) immobilization thereby restricting range of action; (2) steric blocking of active sites; (3) providing a reservoir for delayed release; (4) protection from proteolytic degradation thereby prolonging action; (5) via alteration or concentration of the protein for presentation to cell-surface receptors. Alterations to this highly organized network are therefore likely to modulate both cell behaviour (e.g. loss of adhesion and enhanced proliferation) and tissue structure and stability.
Recent data indicate that the ECM is highly susceptible to oxidative damage due to its abundance, its low rate of turnover (which results in greater accumulation of damage) and the relatively low levels of extracellular repair and turnover systems [15], [16]. A number of recent studies have demonstrated that ONOOH can induce structural and functional changes to extracellular proteins and proteoglycans (proteins with covalently attached glycosaminoglycans), with such damage detected in a number of biological samples including human atherosclerotic lesions [17], [18], [19], [20]. Atherosclerosis is characterized by chronic inflammation, large numbers of activated leukocytes, lipid accumulation, endothelial cell dysfunction, smooth muscle cell migration and proliferation, loss of elasticity, calcification and in some cases lesion rupture and thrombus formation (reviewed [21]). Convincing data has been presented for the presence of elevated levels of oxidation products at both extra- and intra-cellular sites within plaques (e.g. [22], [23], [24]). The majority of this protein damage appears to be present on ECM materials [25].
The basement membrane (BM) is a specialized ECM structure that underlies endothelial cells in the artery wall, and prevents smooth muscle cell infiltration in to the intimal space. It is rich in laminins (together with type IV collagen, nidogen, entactin, perlecan and other species [26]), which plays a crucial role in the architecture of basement membranes, as its hetero-trimeric structure of one α−, one β−, and one γ−chain, allowing cross-links to be formed between the N-termini to form laminin sheets. The C-terminus of the longest of the three chains (the α-chain) binds to integrins, α-dystroglycan, heparan sulfates and sulfated glycolipids on cell surfaces, and therefore plays a critical role in cell adhesion. Binding sites present on the laminin chains for agrin, and nidogen function as a link between type-IV collagen networks and other ECM molecules [27]. The structure and morphology of ECM components is known to change during the development of atherosclerosis [12], [13], and particularly within the fibrotic cap that stabilizes lesions and prevents plaque rupture.
As ONOOH is likely to be generated continually within the inflamed artery wall, and in close proximity to ECM proteins, we hypothesized that ONOOH would induce significant chemical and structural changes to laminin, and that this would modulate the composition, structure and function of this protein and particularly its capacity to bind endothelial cells. We have therefore examined damage to isolated laminin-111 (used as model protein), and basement membrane extracts by ONOOH, as well as the possible presence of nitrated materials on laminin in human atherosclerotic lesions.
Section snippets
Materials
All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Milli-Q grade water (Millipore Advantage A10; Merck-Millipore, Billerica, MA, USA) was used for HPLC buffers; all aqueous reaction mixtures were treated with Chelex-100 resin (Bio-Rad, Hercules, CA, USA) to remove trace metal ions. ONOO- was synthesized in a two-phase system using isoamyl nitrite and H2O2, with unreacted H2O2 eliminated using MnO2 [28]. The resulting stocks were stored at −80 °C and used immediately after
Effect of ONOOH on epitopes present on isolated laminin-111, and laminin within BME
Surface-bound isolated laminin-111, or the mixture of proteins present in the BME (both 1 nM protein), were exposed to ONOOH (1 nM–100 μM, molar calculations based on complete protein absorption) and probed for loss of laminin epitopes with a polyclonal antibody raised against EHS tumour-derived laminin-111 (Fig. 1). A significant loss of antibody epitope recognition of isolated laminin-111 was observed with 10 and 100 μM ONOOH when compared to the untreated laminin-111 (Fig. 1A). A significant
Discussion
ONOOH is a powerful oxidizing and nitrating agent formed at sites of inflammation as a result of the formation and subsequent cross-reaction of ●NO and O2−●[1]. Particularly high levels of this species are believed to be formed by activated macrophage cells due to the high activity of the inducible NOS isoform present in these cells and their capacity to generate high levels of O2−● via the oxidative burst and NOx isoforms; fluxes of ●NO and O2−● have been reported to be in the high
Acknowledgements
We thank Mr. Pat Pisansarakit for tissue culture support and Dr. G. Hoefler (Institute of Pathology, Center for Applied Biomedicine, Medical University of Graz, Graz, Austria) for providing the human tissues samples. We are grateful to the Australian Research Council (through the Centres of Excellence Scheme, CE0561607, and Discovery Programs DP0988311), and the Novo Nordisk Foundation (Laureate Research Grant NNF13OC0004294 to MJD) for financial support.
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