OP28 - Laminin, a key component of tissue extracellular matrix, is a major target for peroxynitrous acid

Abstract

Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to laminin was characterized with purified murine laminin-111, and murine BM extracts containing multiple matrix components. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. This damage was modulated by bicarbonate, a known modifier of ONOOH reactions. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Significant damage was detected with equimolar or greater concentrations of ONOOH. Endothelial cell adhesion to isolated laminin-111 was significantly decreased (~25%) compared to controls, on exposure to 10 μM or higher levels of ONOOH. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture.