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Background The extracellular matrix (ECM), which consists of a complex mixture of glycosaminoglycans, proteoglycans and glycoproteins, is of critical importance for the structure and function of the artery wall, and regulates cell activity via cell-ECM interactions and binding of cytokines and enzymes. Fibronectin, a key ECM component, possesses multiple functional domains, including cell-binding (CBF), and heparin-binding fragments (HBF). At sites of inflammation, including the artery wall during the development of atherosclerosis, activated leukocytes release myeloperoxidase (MPO), which utilises H2O2 and Cl– to generate the powerful oxidant hypochlorous acid (HOCl). Hypothesis: That HOCl and MPO/H2O2/Cl– modify fibronectin resulting in endothelial cell dysfunction. Results Exposure of human fibronectin to increasing molar ratios of HOCl, or a MPO/Cl– with increasing concentration of H2O2, resulted in a loss of antibody reactivity against the CBF and HBF domains of fibronectin (detected by silver staining, Western blotting and ELISA). Significant oxidation and cross-linking was detected on gels, with some cross-linking being reversible by DTT, consistent with disulfide bond formation. HOCl treatment resulted in a decrease in Trp and Met levels and an increase in Met sulfoxide. Oxidant treatment was accompanied by a loss of adhesion of human coronary artery endothelial cells consistent with modifications to the cell-binding domain. Analogous modifications were detected in advanced human atherosclerotic lesions by immunofluorescence. Conclusions These data support the hypothesis that fibronectin, and particularly its CBF and HBF domains are susceptible to modification by HOCl or MPO/H2O2/Cl-, with this resulting in modification to its structure and functional properties. These changes may contribute to endothelial dysfunction and rupture of atherosclerotic lesions. |
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