Ataxia Telangiectasia Mutated (ATM)-mediated DNA Damage Response in Oxidative Stress-induced Vascular Endothelial Cell Senescence

Hong Zhan,Toru Suzuki,Kenichi Aizawa,Kiyoshi Miyagawa,Ryozo Nagai

doi:10.1074/jbc.m110.125138

Abstract

Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

Highlights

Aging is known to be a major cardiovascular risk factor [4]
ataxia telangiectasia mutated (ATM) belongs to the phosphoinositide 3-kinase (PI3-kinase)related protein kinase (PIKK) family which has been identified as the product mutated or inactivated in ataxia telangiectasia (A-T) patients
Our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Human umbilical vein endothelial cells (HUVECs) were purchased from Sanko Junyaku (Tokyo, Japan) and maintained with endothelial cell basal medium-2 containing EGM-2 supplement as purchased from Cambrex Bio Science (Rockland, MD) in humidified air with 5% CO2 at 37 °C. After washing twice with PBS and blocking for 5 min with 0.1% gelatin, slides were incubated with primary antibody (1:100) in 0.1% gelatin in PBS for 1 h in a humidified chamber at 37 °C. Cells were blocked three times with 0.1% gelatin, and samples were incubated with secondary antibody using Alexa Fluor 488 green or Alexa Fluor 635 red (Life Technologies) in 0.1% gelatin in PBS for 1 h in a humidified chamber at 37 °C. Cells were treated with 100 ␮M H2O2 for 30 min in the absence or presence of NAC (A) or caffeine (B), or KU-55933 (C), and whole cell lysates were subjected to Western blot analyses using the indicated antibodies. The phosphorylation of ATM, Akt, and p53 and up-regulation of p21 expression mediate actions of oxidative stress in endothelial cells.

RESULTS

HUVECs were pretreated with

Abrogation of ATM Blocks Effects of Oxidative Stress on Endothelial

Findings

DISCUSSION