241 - Role of chlorinated nucleosides in the promotion of vascular smooth muscle cell dysfunction

Abstract

Release of myeloperoxidase (MPO) at sites of inflammation plays a key role in the development of numerous pathologies and is strongly associated with atherosclerotic lesion development. MPO produces the highly reactive oxidant hypochlorous acid (HOCl), which modifies RNA and DNA forming different chlorinated nucleoside products: 8-chloroadenosine (8ClA), 8-chloroguanosine (8ClG), 5-chlorocytidine (5ClC) and the analogous deoxyribose analogues, 8CldA, 8CldG and 5CldC. It is well established that chlorinated nucleosides are elevated in inflammatory fluids and diseased tissue, including atherosclerotic lesions. However, whether they have a biological function, or are simply biomarkers of oxidation is not well understood. In this study, we examined the reactivity of model chlorinated nucleosides with human coronary artery smooth muscle cells (HCASMC), as these cells are dysregulated in atherosclerosis. Evidence was obtained for the time-dependent incorporation of 8ClA, 8ClG and 5ClC into the RNA, and 8CldA, 8CldG and 5CldC into the DNA of HCASMC, which was not readily repaired on removal of the exogenous source of chlorinated nucleosides. Exposure of HCASMC to chlorinated nucleosides, particularly 8ClA and 5CldC, resulted a decrease in metabolic activity, which was associated with reduced cellular proliferation, rather than cell death by apopotosis or necrosis. With 8ClA, evidence was also obtained increased mitochondrial membrane depolarisation and a loss in ATP, which was associated with the formation of 8ClATP. Treatment of HCASMC with 5CldC resulted in the activation of inflammatory pathways, which culminated in the elevated expression of matrix metalloproteinases, particularly MMP9. The increased expression of MMP9 was not associated with altered SMC phenotype, as no consistent change in the expression of genes characterising the contractile versus synthetic phenotype was observed. Given that MMP9 expression correlates strongly with lesion instability, these data provide a potential pathway by which chlorinated nucleic acid products could play a role in promoting the clinical manifestations of atherosclerosis.