Abstract 780: Atorvastatin Activates A Novel Nbs1-dependent Mechanism Of Accelerating Dna Repair In Atherosclerosis

Abstract

There is increasing evidence that reactive oxidant species (ROS) and DNA damage promote the development and complications of atherosclerosis. Although statin therapy reduces both ROS and DNA damage in atherosclerosis, the mechanism of this effect is unknown. We first examined expression of DNA damage and repair markers in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaques. With increasing disease severity, there was increased VSMC expression of the DNA repair markers P-ATM/ATR substrate and P-H2AX from 2.7%±2.2 and 0.5±0.71 [mean±SEM] (AHA Grade I/II), to 21%±3.5 and 36.5±2.1 (Grade III) lesions, and 86.5%±0.7 and 69.3±7.6 (Grade IV/V). Cultured plaque VSMCs also showed a 1.5 fold increased oxidant stress; a 4.4 fold increased double-stranded DNA breaks, and expression of P-H2AX by Western blots. ROS analogues induced a robust DNA damage response in VSMCs, characterised by lengthening of tails on COMET assay, and activation of ATM and P-H2AX, with completion of repair by 6 hours. Atorvastatin pre-treatment accelerated DNA repair by approximately 2 hours without inhibiting ROS induction or DNA damage, and markedly accelerated the kinetics of nibrin (NBS-1) and P-H2AX activation, both proteins recruited to sites of DNA damage, by preventing degradation of NBS-1. Atorvastatin induced phosphorylation of HDM2, an E3 ligase and putative regulator of NBS-1 stability, and siRNA knockdown of HDM2 replicated the effect of atorvastatin on NBS-1. The ability of atorvastatin to accelerate repair was completely dependent upon NBS-1, as atorvastatin was ineffective in cells either null or expressing constitutively active NBS-I. In summary, we have demonstrated a novel NBS-1-dependent mechanism by which statins accelerate DNA repair in atherosclerosis, through HDM2 phosphorylation and stabilisation of NBS-1. We believe that both NBS-1 and HDM2 are critical to DNA repair in atherosclerosis.