Mechanisms and Biological Consequences of Damage to Arterial Endothelial Cells by Peroxynitrous Acid

Abstract

Peroxynitrous acid (ONOOH) is a potent oxidant formed by the spontaneous reaction of NO. (from nitric oxide synthase enzymes) and O2-. (from multiple sources) at sites of inflammation. Elevated levels of this oxidant have been linked with multiple human pathologies associated with chronic inflammation including atherosclerosis. In this study the effects of ONOOH (either bolus, or generated at a slow rate from SIN-1), and its adduct with CO2, ONOOCO2-, on human coronary artery endothelial cells (HCAEC) have been examined, as endothelial cell dysfunction is a critical early event in atherogenesis. It is shown that exposure of either intact cells, lysates or subcellular fractions to these oxidants induces dose-dependent thiol oxidation, 3-nitrotyrosine formation and aggregation / fragmentation of proteins. At very high doses a loss of cell viability is detected. Experiments with sub-cellular fractions indicates that membrane proteins are a significant target for bolus ONOOH, and the presence of an intact cell membrane decreases the extent of damage to intracellular thiols and proteins. LC/MS/MS studies have identified a number of proteins targeted by ONOOH including cytoskeletal, membrane, mitochondrial, chaperone, signalling and synthetic proteins. Overall these studies have provided novel data on the mechanisms and targets of ONOOH in endothelial cells.