Binding of myeloperoxidase to the extracellular matrix of smooth muscle cells and subsequent matrix modification

Abstract

The extracellular matrix (ECM) of tissues is susceptible to modification by inflammation-associated oxidants. Considerable data support a role for hypochlorous acid (HOCl), generated by the leukocytederived heme-protein myeloperoxidase (MPO) in these changes. HOCl can modify isolated ECM proteins and cell-derived matrix, with this resulting in decreased cell adhesion, modulated proliferation and gene expression, and phenotypic changes. Whether this arises from free HOCl, or via site-specific reactions is unresolved. Here we examine the mechanisms of MPO-mediated changes to human coronary smooth muscle cell ECM. MPO is shown to co-localize with matrix fibronectin as detected by confocal microscopy, and bound active MPO can initiate ECM modification, as detected by decreased antibody recognition of fibronectin, versican and type IV collagen, and formation of protein carbonyls and HOCl-mediated damage. These changes are recapitulated by a glucose/glucose oxidase/MPO system where low continuous fluxes of H 2 o 2 are generated. HOCl-induced modifications enhance Mpo binding to ecM proteins as detected by eLiSA and Mpo activity measurements. these data demonstrate that MPO-generated HOCl induces ECM modification by interacting with ECM proteins in a site-specific manner, and generates alterations that increase MPO adhesion. This is proposed to give rise to an increasing cycle of alterations that contribute to tissue damage.The extracellular matrix (ECM) is essential for the homeostasis of tissues. In the case of the artery wall, the vascular basement membrane plays a key role in supporting and determining endothelial and smooth muscle cell survival and function 1 . This ECM forms a specialized and distinct microenvironment, with the biomacromolecules (e.g., collagens, laminins, fibronectin, perlecan, versican, elastin, nidogen, syndecans) that comprise this material, and a myriad of associated materials (e.g., cytokines, growth factors) dictating the shape and function of the artery wall, and the nature and behavior of the associated vascular cells 2 . Due to the lower turnover rate of many ECM materials (cf. estimated half-lives of 60-70 days and 74 years for arterial collagen and elastin, respectively 3,4 ), and the low levels of antioxidant defense systems in extracellular environments 5 , ECM components and proteins in biological fluids appear to be particularly susceptible to oxidant damage. These factors result in a gradual accumulation of damage in the ECM with both aging and disease, including within the artery wall during the development of atherosclerosis (reviewed 2,6 ). At least some of this damage appears to arise via the formation of oxidants generated by the leukocyte-derived heme enzyme myeloperoxidase (MPO) 7 .Alterations to the structure and properties of ECM has been associated with the progression of atherosclerosis 8 . Previous studies have shown that approximately 70% of the oxidation products detected on proteins extracted from human atherosclerotic lesions are present on ECM proteins 7 , consist...

Myeloperoxidase-derived damage to human plasma fibronectin: Modulation by protein binding and thiocyanate ions (SCN−)

“…Human plasma fibronectin (0.02 μM) was exposed to MPO/H 2 O 2 /Cl − and MPO/H 2 O 2 /SCN − systems using both low (0.02 μM) and high (0.1 μM) concentrations of MPO to reflect a non-diseased and diseased state. Under the former conditions the molar ratio of MPO to fibronectin is 1:1, allowing stoichiometric binding [ 19 ] with an assumed single high affinity binding site. With the higher MPO concentration, binding to the fibronectin may occur at multiple sites, or result in non-bound enzyme being present.…”

“…MPO binds to extracellular materials including low- and high-density lipoproteins [ [50] , [51] , [52] , [53] ], glycosaminoglycans, proteins, proteoglycans and glycoproteins of the ECM [ 19 , 23 , 25 , [54] , [55] , [56] ], via ionic interactions, resulting from the cationic nature of MPO (pI 9.2). The bound MPO appears (at least in some cases) to be catalytically-active [ 19 ], and capable of inducing the formation of di-Tyr [ 57 , 58 ], carbonyl compounds [ 19 , 59 ], and biological dysfunction [ 17 , 19 , 23 , 60 ]. High levels of fibronectin are present in human atherosclerotic lesions [ 26 , 27 ], and co-localization of MPO, HOCl-modified proteins, and macrophages has been observed in atherosclerotic lesions [ 61 ].…”

“…The optical absorbance of the resulting p -nitrophenolate anion was determined using a Spectra Max i3x microplate reader (Molecular Devices). The alkaline phosphatase system was used, rather than HRP/ABTS/H 2 O 2 , as both HRP and MPO can react with H 2 O 2 potentially resulting in erroneous absorbance values (cf [ 19 ]).…”

“…60–80 g protein L −1 for plasma) and have relatively long half-lives, such that they accumulate damage. Thus up to 70% of the damage detected in human artery atherosclerotic lesions has been reported to be present on ECM proteins [ 14 ], including laminins, fibronectin and perlecan [ [15] , [16] , [17] , [18] , [19] , [20] ]. Collagens (e.g.…”

“…The damage induced by HOCl and MPO on fibronectin and laminins, has been examined by LC-MS protein mass mapping, with modifications detected at multiple sites [ 17 , 18 ]. The uneven distribution of the observed modifications, the observed differences between reagent HOCl and the enzyme system, and also between proteins, has been suggested to arise from MPO binding, possibly via ionic interactions, to specific sites and proteins including fibronectin [ 19 , [23] , [24] , [25] ].…”