81 - Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

Abstract

Background Basement membranes (BM) are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds avidly. Hypothesis That HOCl and MPO-derived oxidants selectively modify isolated laminin and laminin in basement membrane extracts, and that MPO-mediated damage to laminin occurs in human atherosclerotic lesions. Results Mass spectrometry with peptide mass mapping was used to detect and quantify sites of oxidation and chlorination. Increased modification was detected on isolated laminin-111, and laminin in BM extracts, with increasing oxidant exposure. Damage was detected at multiple Met, Trp and Tyr residues, with Met residues the most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidized 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were also dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with MPO/H2O2/Cl- compared to reagent HOCl. Chlorination was detected at Tyr2415 in the integrin-binding region, providing a rationale for decreased cell adhesion to HOCl-modified laminin. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. Conclusions These data indicate that laminin is extensively modified by MPO-derived oxidants, and induces structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture.