323 - Peroxynitrite Modifies Components of the Extracellular Matrix of Human Atherosclerotic Lesions

Abstract

Background The extracellular matrix (ECM) is critical to the functional and mechanical properties of arteries, with matrix proteins interacting with growth factors and enzymes to regulate endothelial and smooth muscle cell function. These interactions are perturbed in atherosclerosis, where activated leukocytes generate oxidants that may induce endothelial cell dysfunction, alter ECM composition, and give rise to ECM remodelling, arterial stiffening and plaque formation. Hypothesis: that peroxynitrous acid (ONOOH) modifies isolated ECM proteins, ECM synthesised by human coronary artery endothelial cells (HCAEC), and ECM in human atherosclerotic lesions. Results Perlecan, fibronectin and laminins were detected in native HCAEC-ECM in vitro, and in human atherosclerotic lesions. A dose-dependent loss of antibody reactivity against perlecan protein and the cell-binding epitopes of fibronectin and laminin was detected on exposure to ONOOH. This was accompanied by a dose-dependent generation of the ONOOH biomarkers 3-nitrotyrosine, 6-nitrotryptophan and dityrosine. Tyr and Trp modification was quantified by loss of parent amino acids, and product formation. LC-MS peptide mass mapping indicates that changes occur in critical functional regions including the cell binding domain, consistent with decreased HCAEC adhesion and spreading on ONOOH-modified ECM proteins compared to controls in vitro. Studies on advanced human atherosclerotic lesions have provided strong evidence for co-localisation of perlecan, fibronectin and laminins with 3-nitrotyrosine epitopes. Conclusions ONOOH induces structural and functional changes in ECM proteins, and particularly to fibronectin and laminins, critical to endothelial cell function. These data suggest a mechanism through which ONOOH can modify arterial ECM basement membrane in atherosclerotic lesions and give rise to ECM remodelling.