Oxyhemoglobin-induced apoptosis in cultured endothelial cells.
OBJECTIVE
Oxyhemoglobin (OxyHb) is one of the most important spasmogens for cerebral vasospasm that follows aneurysmal subarachnoid hemorrhage. The cytotoxic effect of OxyHb has been documented in endothelial and smooth-muscle cells; however, the pattern of cell death--necrosis or apoptosis--as the final stage of cell damage has not been demonstrated. This study was undertaken to determine if OxyHb induces apoptotic changes in cultured bovine aortic endothelial cells.


METHODS
Confluent bovine aortic endothelial cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to culture dishes after exposure to OxyHb. To identify apoptotic changes, the investigators used three specific methods: DNA fragmentation (electrophoreses), the apoptotic body (transmission electron microscopy), and cleavage of poly (adenosine diphosphate ribose) polymerase (PARP [Western blotting]).


CONCLUSIONS
Oxyhemoglobin decreased cell density in a concentration- and time-dependent manner. Analysis of DNA showed a pattern of internucleosomal cleavage characteristic of apoptosis (DNA ladder). Transmission electron microscopy demonstrated condensation of nuclei and apoptotic bodies in OxyHb-treated endothelial cells. Western blotting with the PARP antibody revealed that the 116-kD PARP was cleaved to the 85-kD apoptosis-related fragment. These results for the first time demonstrated that the OxyHb induces apoptosis in cultured endothelial cells.
Ogihara
K
K
Department of Neurosurgery, University of Mississippi Medical Center, Jackson 39216, USA.
Zubkov
A Y
AY
Bernanke
D H
DH
Lewis
A I
AI
Parent
A D
AD
Zhang
J H
JH
D000818:Animals; D001011:Aorta; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002417:Cattle; D002452:Cell Count / Q000187:drug effects; D016923:Cell Death / Q000502:physiology; D002467:Cell Nucleus / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000503:physiopathology; D002532:Intracranial Aneurysm / Q000150:complications; D002546:Ischemic Attack, Transient / Q000209:etiology / Q000503:physiopathology; D008163:Luminescent Measurements; D008854:Microscopy, Electron; D009131:Muscle, Smooth, Vascular / Q000503:physiopathology; D009336:Necrosis; D009707:Nucleosomes / Q000187:drug effects; D010108:Oxyhemoglobins / Q000008:administration & dosage / Q000494:pharmacology* / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects; D013345:Subarachnoid Hemorrhage / Q000150:complications; D013997:Time Factors
D009707:Nucleosomes; D010108:Oxyhemoglobins; D011065:Poly(ADP-ribose) Polymerases