RESUMO
The extracellular matrix (ECM) is critical to biological architecture and determines cellular properties, function and activity. In many situations it is highly abundant, with collagens and elastin being some of the most abundant proteins in mammals. The ECM comprises of multiple different protein species and sugar polymers, with both different isoforms and post-translational modifications (PTMs) providing a large variety of microenvironments that play a key role in determining tissue structure and health. A number of the PTMs (e.g. cross-links) present in the ECM are critical to integrity and function, whereas others are deleterious to both ECM structure and associated cells. Modifications induced by reactive oxidants and electrophiles have been reported to accumulate in some ECM with increasing age. This accumulation can be exacerbated by disease, and in particular those associated with acute or chronic inflammation, obesity and diabetes. This is likely to be due to higher fluxes of modifying agents in these conditions. In this focused review, the role and effects of oxidants and other electrophiles on ECM are discussed, with a particular focus on the artery wall and atherosclerotic cardiovascular disease. Modifications generated on ECM components are reviewed, together with the effects of these species on cellular properties including adhesion, proliferation, migration, viability, metabolic activity, gene expression and phenotype. Increasing data indicates that ECM modifications are both prevalent in human and mammalian tissues and play an important role in disease development and progression.
Assuntos
Proteínas da Matriz Extracelular , Oxidantes , Processamento de Proteína Pós-Traducional , Humanos , Proteínas da Matriz Extracelular/metabolismo , Animais , Oxidantes/metabolismo , Matriz Extracelular/metabolismoRESUMO
Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.e. leucine-rich repeat domain (LRR), immunoglobulin domains, peroxidase domain, von Willebrand factor type C domain] of PXDN interact with the polymeric networks of the extracellular matrix (ECM), and how these interactions integrate and regulate the enzyme's cross-linking activity, without imparting oxidative damage to the ECM. In this study, we probed the interactions of four PXDN constructs with different domain compositions with components of a basement membrane extract by immunoprecipitation. Strong binding of the LRR-containing construct was detected with the major ECM protein laminin. Analysis of these interactions by surface plasmon resonance spectroscopy revealed similar kinetics and affinities of binding of the LRR-containing construct to human and murine laminin-111, with calculated dissociation constants of 1.0 and 1.5 µM, respectively. The findings are discussed with respect to the recently published in-solution structures of the PXDN constructs and the proposed biological role of this peroxidase.
Assuntos
Membrana Basal/metabolismo , Laminina/metabolismo , Peroxidases/metabolismo , Animais , Células HEK293 , Humanos , Leucina/química , Leucina/metabolismo , Camundongos , Peroxidases/química , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismoRESUMO
ECM (extracellular matrix) materials, such as laminin, perlecan, type IV collagen and fibronectin, play a key role in determining the structure of the arterial wall and the properties of cells that interact with the ECM. The aim of the present study was to investigate the effect of peroxynitrous acid, an oxidant generated by activated macrophages, on the structure and function of the ECM laid down by HCAECs (human coronary artery endothelial cells) in vitro and in vivo. We show that exposure of HCAEC-derived native matrix components to peroxynitrous acid (but not decomposed oxidant) at concentrations >1 µM results in a loss of antibody recognition of perlecan, collagen IV, and cell-binding sites on laminin and fibronectin. Loss of recognition was accompanied by decreased HCAEC adhesion. Real-time PCR showed up-regulation of inflammation-associated genes, including MMP7 (matrix metalloproteinase 7) and MMP13, as well as down-regulation of the laminin α2 chain, in HCAECs cultured on peroxynitrous acid-treated matrix compared with native matrix. Immunohistochemical studies provided evidence of co-localization of laminin with 3-nitrotyrosine, a biomarker of peroxynitrous acid damage, in type II-III/IV human atherosclerotic lesions, consistent with matrix damage occurring during disease development in vivo. The results of the present study suggest a mechanism through which peroxynitrous acid modifies endothelial cell-derived native ECM proteins of the arterial basement membrane in atherosclerotic lesions. These changes to ECM and particularly perlecan and laminin may be important in inducing cellular dysfunction and contribute to atherogenesis.
Assuntos
Vasos Coronários/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Ácido Peroxinitroso/farmacologia , Aterosclerose/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Oxidantes/farmacologia , OxirreduçãoRESUMO
The extracellular matrix (ECM) of tissues consists of multiple proteins, proteoglycans and glycosaminoglycans that form a 3-dimensional meshwork structure. This ECM is exposed to oxidants including peroxynitrite (ONOO-/ONOOH) generated by activated leukocytes at sites of inflammation. Fibronectin, a major ECM protein targeted by peroxynitrite, self-assembles into fibrils in a cell-dependent process. Fibrillation of fibronectin can also be initiated in a cell-independent process in vitro by anastellin, a recombinant fragment of the first type-III module in fibronectin. Previous studies demonstrated that modification of anastellin by peroxynitrite impairs its fibronectin polymerization activity. We hypothesized that exposure of anastellin to peroxynitrite would also impact on the structure of ECM from cells co-incubated with anastellin, and influence interactions with cell surface receptors. Fibronectin fibrils in the ECM of primary human coronary artery smooth muscle cells exposed to native anastellin are diminished, an effect which is reversed to a significant extent by pre-incubation of anastellin with high (200-fold molar excess) concentrations of peroxynitrite. Treatment with low or moderate levels of peroxynitrite (2-20 fold molar excess) influences interactions between anastellin and heparin polysaccharides, as a model of cell-surface proteoglycan receptors, and modulates anastellin-mediated alterations in fibronectin cell adhesiveness. Based on these observations it is concluded that peroxynitrite has a dose-dependent influence on the ability of anastellin to modulate ECM structure via interactions with fibronectin and other cellular components. These observations may have pathological implications since alterations in fibronectin processing and deposition have been associated with several pathologies, including atherosclerosis.
Assuntos
Fibronectinas , Ácido Peroxinitroso , Humanos , Fibronectinas/metabolismo , Ácido Peroxinitroso/farmacologia , Ácido Peroxinitroso/metabolismo , Matriz Extracelular/metabolismo , Adesão CelularRESUMO
Peroxynitrous acid/peroxynitrite (ONOOH/ONOO-) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO-, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO-, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO- (50 µM) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO- concentrations (500 µM), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell- and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.
Assuntos
Vasos Coronários , Ácido Peroxinitroso , Humanos , Ácido Peroxinitroso/metabolismo , Vasos Coronários/metabolismo , Nitratos , Espectrometria de Massas , Tirosina/metabolismoRESUMO
Atherosclerosis is a chronic inflammatory disease and a leading cause of mortality. It is characterized by arterial wall plaques that contain high levels of cholesterol and other lipids and activated leukocytes covered by a fibrous cap of extracellular matrix (ECM). The ECM undergoes remodelling during atherogenesis, with increased expression of aggrecan, a proteoglycan that binds low-density-lipoproteins (LDL). Aggrecan levels are regulated by proteases, including a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1). Activated leukocytes release myeloperoxidase (MPO) extracellularly, where it binds to proteins and proteoglycans. Aggrecan may therefore mediate colocalization of MPO and LDL. MPO generates hypochlorous acid (HOCl) and chloramines (RNHCl species, from reaction of HOCl with amines on amino acids and proteins) that damage LDL and proteins, but effects on aggrecan have not been examined. The present study demonstrates that HOCl cleaves truncated (G1-IGD-G2) recombinant human aggrecan at specific sites within the IGD domain, with these being different from those induced by ADAMTS1 which also cleaves within this region. Irreversible protein cross-links are also formed dose-dependently. These effects are limited by the HOCl scavenger methionine. Chloramines including those formed on amino acids, proteins, and ECM materials induce similar damage. HOCl and taurine chloramines inactivate ADAMTS1 consistent with a switch from proteolytic to oxidative aggrecan fragmentation. Evidence is also presented for colocalization of aggrecan and HOCl-generated epitopes in advanced human atherosclerotic plaques. Overall, these data show that HOCl and chloramines can induce specific modifications on aggrecan, and that these effects are distinct from those of ADAMTS1.
RESUMO
Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO-/ONOOH. Both reagent ONOO-/ONOOH and SIN-1 (a thermal source of ONOO-/ONOOH) modified Tyr, Trp and Met residues. ONOO-/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO-/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO-/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.
Assuntos
Aterosclerose , Placa Aterosclerótica , Humanos , Oxidantes/metabolismo , Ácido Peroxinitroso/metabolismo , Versicanas/genética , Versicanas/metabolismo , Ácido Hialurônico/metabolismo , Placa Aterosclerótica/metabolismo , Matriz Extracelular/metabolismo , Aterosclerose/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inflamação/metabolismoRESUMO
Myeloperoxidase (MPO) is a neutrophil-derived enzyme that has been recently associated with tumour development. However, the mechanisms by which this enzyme exerts its functions remain unclear. In this study, we investigated whether myeloperoxidase can alter the function of A549 human lung cancer cells. We observed that MPO promoted the proliferation of cancer cells and inhibited their apoptosis. Additionally, it increased the phosphorylation of AKT and ERK. MPO was rapidly bound to and internalized by A549 cells, retaining its enzymatic activity. Furthermore, MPO partially translocated into the nucleus and was detected in the chromatin-enriched fraction. Effects of MPO on cancer cell function could be reduced when MPO uptake was blocked with heparin or upon inhibition of the enzymatic activity with the MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). Lastly, we have shown that tumour-bearing mice treated with 4-ABAH had reduced tumour burden when compared to control mice. Our results highlight the role of MPO as a neutrophil-derived enzyme that can alter the function of lung cancer cells.
RESUMO
Lubricin, also referred to as superficial zone protein, has been reported to be a proteoglycan. However, the structure of its glycosaminoglycan chain has not been well characterized, and this study was undertaken to investigate the structure of the glycosaminoglycan chain that decorated lubricin in human synovial fluid to provide insight into its biological role. Lubricin was detected as a major band at approximately 360 kDa which co-migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a chondroitin sulfate (CS)-containing proteoglycan that was detected by both monoclonal antibodies (MAb) 2-B-6 and MAb 3-B-3 after chondroitinase ABC treatment and keratan sulfate (KS) that was detected by MAb 5-D-4. Further analysis of lubricin-containing fractions that eluted from an anion exchange column indicated that the major population of lubricin could be separated from the CS and KS stubs which indicated that this fraction of lubricin was not decorated with glycosaminoglycan chain and was the glycoprotein form of lubricin. Lubricin present in fractions that also contained CS was found to be decorated with CS structures which were reactive with MAb 3-B-3 after chondroitinase ABC digestion using a sandwich enzyme-linked immunosorbent assay approach. Aggrecan was not found to form complexes with lubricin in synovial fluid which confirmed that the MAb 3-B-3 CS and MAb 5-D-4 KS structures decorated lubricin. These data demonstrate that lubricin present in human synovial fluid was a heterogeneous population with both glycoprotein and proteoglycan forms.
Assuntos
Glicoproteínas/química , Glicosaminoglicanos/química , Líquido Sinovial/química , Fracionamento Químico , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Sulfato de Queratano/química , Sulfato de Queratano/metabolismo , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Isoformas de Proteínas , Líquido Sinovial/metabolismoRESUMO
Matrix metalloproteinase-9 (MMP9, gelatinase B) plays a key role in the degradation of extracellular-matrix (ECM) proteins in both normal physiology and multiple pathologies, including those linked with inflammation. MMP9 is excreted as an inactive proform (proMMP9) by multiple cells, and particularly neutrophils. The proenzyme undergoes subsequent processing to active forms, either enzymatically (e.g., via plasmin and stromelysin-1/MMP3), or via the oxidation of a cysteine residue in the prodomain (the "cysteine-switch"). Activated leukocytes, including neutrophils, generate O2- and H2O2 and release myeloperoxidase (MPO), which catalyzes hypochlorous acid (HOCl) formation. Here, we examine the reactivity of HOCl and a range of low-molecular-mass and protein chloramines with the pro- and activated forms of MMP9. HOCl and an enzymatic MPO/H2O2/Cl- system were able to generate active MMP9, as determined by fluorescence-activity assays and gel zymography. The inactivation of active MMP9 also occurred at high HOCl concentrations. Low (nM-low µM) concentrations of chloramines formed by the reaction of HOCl with amino acids (taurine, lysine, histidine), serum albumin, ECM proteins (laminin and fibronectin) and basement membrane extracts (but not HEPES chloramines) also activate proMMP9. This activation is diminished by the competitive HOCl-reactive species, methionine. These data indicate that HOCl-mediated oxidation and MMP-mediated ECM degradation are synergistic and interdependent. As previous studies have shown that modified ECM proteins can also stimulate the cellular expression of MMP proteins, these processes may contribute to a vicious cycle of increasing ECM degradation during disease development.
RESUMO
Myeloperoxidase (MPO) mediates pathogen destruction by generating the bactericidal oxidant hypochlorous acid (HOCl). Formation of this oxidant is however associated with host tissue damage and disease. MPO also utilizes H2O2 to oxidize other substrates, and we hypothesized that mixtures of other plasma anions, including bromide (Br-), iodide (I-), thiocyanate (SCN-) and nitrite (NO2-), at normal or supplemented concentrations, might modulate MPO-mediated HOCl damage. For the (pseudo)halide anions, only SCN- significantly modulated HOCl formation (IC50 â¼33 µM), which is within the normal physiological range, as judged by damage to human plasma fibronectin or extracellular matrix preparations detected by ELISA and LC-MS. NO2- modulated HOCl-mediated damage, in a dose-dependent manner, at physiologically-attainable anion concentrations. However, this was accompanied by increased tyrosine and tryptophan nitration (detected by ELISA and LC-MS), and the overall extent of damage remained approximately constant. Increasing NO2- concentrations (0.5-20 µM) diminished HOCl-mediated modification of tyrosine and methionine, whereas tryptophan loss was enhanced. At higher NO2- concentrations, enhanced tyrosine and methionine loss was detected. These analytical data were confirmed in studies of cell adhesion and metabolic activity. Together, these data indicate that endogenous plasma levels of SCN- (but not Br- or I-) can modulate protein modification induced by MPO, including the extent of chlorination. In contrast, NO2- alters the type of modification, but does not markedly decrease its extent, with chlorination replaced by nitration. These data also indicate that MPO could be a major source of nitration in vivo, and particularly at inflammatory sites where NO2- levels are often elevated.
Assuntos
Nitritos , Peroxidase , Matriz Extracelular/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/metabolismo , Metionina , Nitritos/farmacologia , Dióxido de Nitrogênio , Oxidantes/metabolismo , Peroxidase/metabolismo , Triptofano , Tirosina/metabolismoRESUMO
Leukocytes produce oxidants at inflammatory sites, including within the artery wall during the development of atherosclerosis. Developing lesions contain high numbers of activated leukocytes that generate reactive nitrogen species, including peroxynitrite/peroxynitrous acid (ONOO-/ONOOH), as evidenced by the presence of oxidized/nitrated molecules including extracellular matrix (ECM) proteins. ECM materials are critical for arterial wall integrity, function, and determine cell phenotype, with smooth muscle cells undergoing a phenotypic switch from quiescent/contractile to proliferative/synthetic during disease development. We hypothesized that ECM modification by ONOO-/ONOOH might drive this switch, and thereby potentially contribute to atherogenesis. ECM generated by primary human coronary artery smooth muscle cells (HCASMCs) was treated with increasing ONOO-/ONOOH concentrations (1-1000 µM). This generated significant damage on laminin, fibronectin and versican, and lower levels on collagens and glycosaminoglycans, together with the increasing concentrations of the damage biomarker 3-nitrotyrosine. Adhesion of naïve HCASMC to ECM modified by 1 µM ONOO-/ONOOH was enhanced, but significantly diminished by higher ONOO-/ONOOH treatment. Cell proliferation and metabolic activity were significantly enhanced by 100 µM ONOO-/ONOOH pre-treatment. These changes were accompanied by increased expression of genes involved in mitosis (PCNA, CCNA1, CCNB1), ECM (LAMA4, LAMB1, VCAN, FN1) and inflammation (IL-1B, IL-6, VCAM-1), and corresponding protein secretion (except VCAM-1) into the medium. These changes induced by modified ECM are consistent with HCASMC switching to a synthetic/proliferative/pro-inflammatory phenotype, together with ECM remodelling. These changes model those in atherosclerosis, suggesting a link between oxidant-modified ECM and disease progression, and highlight the potential of targeting oxidant generation as a therapeutic strategy.
Assuntos
Aterosclerose , Ácido Peroxinitroso , Aterosclerose/metabolismo , Vasos Coronários/metabolismo , Matriz Extracelular/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Oxidantes/metabolismo , Oxirredução , Ácido Peroxinitroso/metabolismo , Fenótipo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
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.
Assuntos
Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Oxirredução , Estresse Oxidativo/fisiologia , Antioxidantes/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Progressão da Doença , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Oxidantes/metabolismo , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2-Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1-mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.
Assuntos
Dineínas , Canais de Potássio KCNQ , Membrana Celular , Músculo Liso Vascular , Miócitos de Músculo LisoRESUMO
Perlecan is a large multidomain proteoglycan that is essential for normal cartilage development. In this study, perlecan was localized in the pericellular matrix of hypertrophic chondrocytes in developing human cartilage rudiments. Perlecan immunopurified from medium conditioned by cultured human fetal chondrocytes was found to be substituted with heparan sulfate (HS), chondroitin sulfate (CS), and keratan sulfate (KS). Ligand and carbohydrate engagement (LACE) assays demonstrated that immunopurified chondrocyte-derived perlecan formed HS-dependent ternary complexes with fibroblast growth factor (FGF) 2 and either FGF receptors (FGFRs) 1 or 3; however, these complexes were not biologically active in the BaF32 cell system. Chondrocyte-derived perlecan also formed HS-dependent ternary complexes with FGF18 and FGFR3. The proliferation of BaF32 cells expressing FGFR3 was promoted by chondrocyte-derived perlecan in the presence of FGF18, and this activity was reduced by digestion of the HS with either heparinase III or mammalian heparanase. These data suggest that FGF2 and -18 bind to discrete structures on the HS chains attached to chondrocyte-derived perlecan which modulate the growth factor activities. The presence and activity of mammalian heparanase may be important in the turnover of HS and subsequent signaling required for the establishment and maintenance of functional osteo-chondral junctions in long bone growth.
Assuntos
Condrócitos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Transdução de Sinais/fisiologia , Desenvolvimento Ósseo , Células Cultivadas , Meios de Cultivo Condicionados/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Ligação ProteicaRESUMO
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 leukocyte-derived 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 H2O2 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.
Assuntos
Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Ácido Hipocloroso/metabolismo , Miócitos de Músculo Liso/metabolismo , Peroxidase/efeitos adversos , Peroxidase/metabolismo , Formação de Anticorpos , Adesão Celular , Proliferação de Células , Células Cultivadas , Colágeno/imunologia , Matriz Extracelular/imunologia , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/imunologia , Expressão Gênica , Humanos , Ácido Hipocloroso/efeitos adversos , Miócitos de Músculo Liso/fisiologia , Ligação Proteica , Versicanas/imunologiaRESUMO
Endothelial cell dysfunction is an early event in cardiovascular disease and atherosclerosis. The origin of this dysfunction is unresolved, but accumulating evidence implicates damaging oxidants, including hypochlorous acid (HOCl), a major oxidant produced by myeloperoxidase (MPO), during chronic inflammation. MPO is released extracellularly by activated leukocytes and binds to extracellular molecules including fibronectin, a major matrix glycoprotein involved in endothelial cell binding. We hypothesized that MPO binding might influence the modifications induced on fibronectin, when compared to reagent HOCl, with this including alterations to the extent of damage to protein side-chains, modified structural integrity, changes to functional domains, and impact on naïve human coronary artery endothelial cell (HCAEC) adhesion and metabolic activity. The effect of increasing concentrations of the alternative MPO substrate thiocyanate (SCN-), which might decrease HOCl formation were also examined. Exposure of fibronectin to MPO/H2O2/Cl- is shown to result in damage to the functionally important cell-binding and heparin-binding fragments, gross structural changes to the protein, and altered HCAEC adhesion and activity. Differences were observed between stoichiometric, and above-stoichiometric MPO concentrations consistent with an effect of MPO binding to fibronectin. In contrast, MPO/H2O2/SCN- induced much less marked changes and limited protein damage. Addition of increasing SCN- concentrations to the MPO/H2O2/Cl- system provided protection, with 20 µM of this anion rescuing damage to functionally-important domains, decreasing chemical modification, and maintaining normal HCAEC behavior. Modulating MPO binding to fibronectin, or enhancing SCN- levels at sites of inflammation may therefore limit MPO-mediated damage, and be of therapeutic value.
Assuntos
Peroxidase , Tiocianatos , Fibronectinas/metabolismo , Humanos , Peróxido de Hidrogênio , Ácido Hipocloroso , Peroxidase/metabolismo , Ligação Proteica , Tiocianatos/farmacologiaRESUMO
Laminin is a major protein of the basement membrane (BM), a specialized extracellular matrix (ECM) of the artery wall. The potent oxidizing and nitrating agent peroxynitrous acid (ONOOH) is formed at sites of inflammation, and data implicate ONOOH in ECM damage and cardiovascular disease. Co-localization of 3-nitrotyrosine, a product of ONOOH-mediated tyrosine (Tyr) modification, and laminin has been reported in human atherosclerotic lesions. The sites and consequences of 3-nitrotyrosine (and related nitrated tryptophan) formation on laminin, it's self-assembly and cell interactions are poorly understood. In this study murine laminin-111 was exposed to ONOOH (1-500-fold molar excess). Nitration sites were mapped and quantified using LC-MS/MS. Mono-nitration was detected at 148 sites (126 Tyr, 22 Trp), and di-nitration at 14 sites. Label-free quantification showed enhanced nitration with increasing oxidant doses. Tyr nitration was â¼10-fold greater than at Trp. CO2 modulated damage in a site-specific manner, with most sites less extensively nitrated. 119 mono-nitration sites were identified with CO2 present, and no unique sites were detected. 23 di-nitration sites were detected, with 15 unique to the presence of CO2. Extensive modification was detected at sites involved in cell adhesion, protein-protein interactions and self-polymerization. Tyr-145 on the γ1 chain was extensively nitrated, and endothelial cells exhibited decreased adhesion to a nitrated peptide modelling this site. Modification of residues involved in self-polymerization interfered with the formation of ordered polymers as detected by scanning electron microscopy. These laminin modifications may contribute to endothelial cell dysfunction and modulate ECM structure and assembly, and thereby contribute to atherogenesis.
Assuntos
Laminina/química , Laminina/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Dióxido de Carbono/química , Cromatografia Líquida , Biologia Computacional/métodos , Matriz Extracelular/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Nitratos/química , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Espectrometria de Massas em TandemRESUMO
The extracellular matrix (ECM) influences the structure and function of the arterial wall and modulates the behavior of vascular cells through ECM-cell interactions. Alterations to the ECM have been implicated in multiple pathological processes, including atherosclerosis which is characterized by low-grade chronic inflammation and the infiltration and proliferation of smooth muscle cells during disease development. Considerable evidence has been presented for a role for inflammation-derived oxidation in atherogenesis, with enzymatically-active myeloperoxidase (MPO), elevated levels of 3-chlorotyrosine (a biomarker of MPO-catalyzed damage) and oxidized ECM materials detected in advanced human atherosclerotic lesions. Whether oxidant-modified ECM contributes to the altered behavior of smooth muscle cells is however unclear. This study therefore investigated the effects of hypochlorous acid (HOCl), a major MPO-derived oxidant, on the structure of the native ECM synthesized by human coronary artery smooth muscle cells (HCAMSCs) and whether modified ECM proteins affected HCASMC adhesion, proliferation and gene expression. Exposure of native HCASMC-derived ECM to reagent HOCl or a MPO-Cl--H2O2 system resulted in extensive ECM modifications as evidenced by the loss of antibody recognition of epitopes on type IV collagen, laminin, versican and fibronectin. Oxidation of HCASMC ECM markedly reduced HCASMC adhesion to matrix components, but facilitated subsequent proliferation in vitro. Multiple genes were upregulated in HCASMCs in response to HOCl-modified HCASMC-ECM including interleukin-6 (IL-6), fibronectin (FN1) and matrix-metalloproteinases (MMPs). These data reveal a mechanism through which inflammation-induced ECM-modification may contribute to the behavioral switching of HCASMCs, a key process in plaque formation during the development of atherosclerosis.
Assuntos
Adesão Celular , Proliferação de Células , Vasos Coronários/citologia , Matriz Extracelular/química , Ácido Hipocloroso/farmacologia , Miócitos de Músculo Liso/citologia , Células Cultivadas , Vasos Coronários/metabolismo , Matriz Extracelular/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Metaloproteinases da Matriz/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxidantes/farmacologia , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
Dysfunction of endothelial cells of the artery wall is an early event in cardiovascular disease and atherosclerosis. The cause(s) of this dysfunction are unresolved, but accumulating evidence suggests that oxidants arising from chronic low-grade inflammation are contributory agents, with increasing data implicating myeloperoxidase (MPO, released by activated leukocytes), and the oxidants it generates (e.g. HOCl and HOSCN). As these are formed extracellularly and react rapidly with proteins, we hypothesized that MPO-mediated damage to the matrix glycoprotein fibronectin (FN) would modulate FN structure and function, and its interactions with human coronary artery endothelial cells (HCAEC). Exposure of human plasma FN to HOCl resulted in modifications to FN and its functional epitopes. A dose-dependent loss of methionine and tryptophan residues, together with increasing concentrations of methionine sulfoxide, and modification of the cell-binding fragment (CBF) and heparin-binding fragment (HBF) domains was detected with HOCl, but not HOSCN. FN modification resulted in a loss of HCAEC adhesion, impaired cell spreading and reduced cell proliferation. Exposure to HCAEC to HOCl-treated FN altered the expression of HCAEC genes associated with extracellular matrix (ECM) synthesis and adhesion. Modifications were detected on HCAEC-derived ECM pre-treated with HOCl, but not HOSCN, with a loss of antibody recognition of the CBF, HBF and extra-domain A. Co-localization of epitopes arising from MPO-generated HOCl and cell-derived FN was detected in human atherosclerotic lesions. Damage was also detected on FN extracted from lesions. These data support the hypothesis that HOCl, but not HOSCN, targets and modifies FN resulting in arterial wall endothelial cell dysfunction.