1. ¹Department of Neurosurgery, Washington University School of Medicine, St Louis, Missouri, USA
2. ²Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri, USA
3. ³Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, Missouri, USA
4. ⁴Department of Cell Biology & Physiology, Washington University School of Medicine, St Louis, Missouri, USA

Correspondence: Professor JM Gidday, Department of Neurosurgery, Box 8057, Washington University School of Medicine, St Louis, MO 63110, USA. E-mail: gidday@nsurg.wustl.edu

Received 7 July 2004; Revised 7 October 2004; Accepted 11 October 2004; Published online 23 February 2005.

Abstract

Cerebral ischemia–reperfusion leads to vascular dysfunction characterized by endothelial cell injury or death. In the present study, we used an in vitro model to elucidate mechanisms of human brain microvascular endothelial cell (HBMEC) injury after episodic ischemia–reperfusion. Near-confluent HBMEC cultures were exposed to intermittent hypoxia–reoxygenation (HX/RO) and, at different recovery time points, cell viability was assessed by the MTT assay, apoptotic death by fluorescence microscopy of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-positive cells, and nuclear translocation of apoptosis-inducing factor (AIF) and cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) by immunoblotting of subcellular fractions. Reductions in HBMEC viability were proportional to the number of HX/RO cycles, and not the total duration of hypoxia. Using four cycles of 1-h HX with 1 h of intervening normoxic RO, cell viability was reduced 30% to 40% between 12 and 48 h. Treatment with the PARP-1 inhibitors 3-aminobenzamide or 4-amino-1,8-naphthalimide during the insult improved HBMEC viability at 24 h after insult, and resulted in dose-dependent reductions in TUNEL-positivity at 16 h after insult, but not if these treatments were delayed by 4 h. HX/RO-induced increases in nuclear AIF translocation, as well as PARP-1 cleavage, were also reduced dose-dependently at 4 h after insult by the inhibitors. The caspase inhibitor z-VAD-fmk blocked PARP-1 cleavage, but did not affect AIF translocation and was only modestly cytoprotective. These findings indicate that PARP-1 activation and a PARP-1-dependent, caspase-independent, nuclear translocation of AIF contribute to apoptotic cerebral endothelial cell death after ischemia–reperfusion, underscoring the potential for ischemic microvascular protection by inhibiting PARP activation or preventing AIF translocation.