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(Stroke. 2002;33:2471.)
© 2002 American Heart Association, Inc.

Original Contributions

ATM Gene Regulates Oxygen-Glucose Deprivation–Induced Nuclear Factor-B DNA-Binding Activity and Downstream Apoptotic Cascade in Mouse Cerebrovascular Endothelial Cells

Ke-jie Yin, MD, PhD; Shang-Der Chen, MD; Jin-Moo Lee, MD, PhD; Jan Xu, PhD Chung Y. Hsu, MD, PhD

From the Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St Louis, Mo.

Correspondence to Chung Y. Hsu, MD, PhD, Department of Neurology, Box 8111, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110. E-mail hsuc{at}neuro.wustl.edu

Background and Purpose— Cells lacking the ATM (ataxia telangectasia mutated) gene are hypersensitive to DNA damage caused by a variety of insults. ATM may regulate oxidative stress–induced signaling cascades involving nuclear factor-B (NF-B), a transcription factor that is upstream of a wide variety of stress-responsive genes. We investigated the potential interaction of ATM and NF-B after oxygen-glucose deprivation (OGD) in cerebral endothelial cells (CECs).

Methods— Primary cultures of mouse CECs were subjected to OGD in the absence or presence of ATM antisense oligonucleotides or the NF-B inhibitor SN50. ATM expression was determined with the use of reverse transcription–polymerase chain reaction and Western blot, and NF-B activity was assessed by electrophoretic mobility shift assay. Cells were assessed for mitochondrial DNA damage with the use of long polymerase chain reaction and were assessed for caspase-3 and caspase-8 activity with the use of fluorogenic substrates. Cell death was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and LDH release.

Results— OGD stimulated ATM gene expression at the mRNA and protein level in CECs as early as 1 hour after OGD initiation. ATM gene knockdown with the use of an antisense oligonucleotide suppressed OGD-induced ATM protein expression, which was accompanied by an attenuation of NF-B activation and the subsequent expression of downstream genes, including the antiapoptotic gene c-IAP2. ATM knockdown also accentuated OGD-induced mitochondrial DNA damage and the activation of caspase-3 and caspase-8, leading to enhanced CEC death. The specific NF-B inhibitor SN50 mimicked the effects of ATM knockdown.

Conclusions— We conclude that ATM may play a cytoprotective role in OGD-induced CEC death via a NF-B–dependent signaling pathway.

Key Words: ataxia telangectasia • cell death • cerebral cortex • endothelium, vascular • genetics • glucose • NF-kappa B • oxygen

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