Oxyhemoglobin-induced apoptosis in cultured endothelial cells

Object. Oxyhemoglobin (OxyHb) is one of the most important spasmogens for c erebral vasospasm that follows aneurysmal subarachnoid hemorrhage. The cyto toxic effect of OxyHb has been documented in endothelial and smooth-muscle cells; however, the pattern of cell death-necrosis or apoptosis-as the fina l 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 spec ific methods: DNA fragmentation (electrophoreses), the apoptotic body (tran smission electron microscopy), and cleavage of poly (adenosine diphosphate ribose) polymerase (PARP [Western blotting]). Conclusions. Oxyhemoglobin decreased cell density in a concentration- and t ime-dependent manner. Analysis of DNA showed a pattern of internucleosomal cleavage characteristic of apoptosis (DNA ladder). Transmission electron mi croscopy 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 apopto sis in cultured endothelial cells.