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  • Author or Editor: Andrew D. Parent x
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Kotaro Ogihara, Alexander Y. Zubkov, David H. Bernanke, Adam I. Lewis, Andrew D. Parent and John H. Zhang

Object. 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.

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Alexander Y. Zubkov, Kotaro Ogihara, Phani Tumu, Anita Patlolla, Adam I. Lewis, Andrew D. Parent and John Zhang

Object. Mitogen-activated protein kinase (MAPK) is an important signaling factor in vascular proliferation and contraction, which are the two features of cerebral vasospasm that follow subarachnoid hemorrhage. The authors studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery (BA).

Methods. Isometric tension was used to record the contractile response of rabbit BA to hemolysate, and Western blots were obtained using antibodies for MAPK.

The following results are reported. 1) Hemolysate produced a concentration-dependent contraction of rabbit BA; however, preincubation of arteries with the MAPK kinase (MEK) inhibitor PD-98059 markedly reduced this contraction. The administration of PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by 10% hemolysate. 2) The Janus tyrosine kinase 2 inhibitor AG-490, preincubated with arterial rings, reduced the contractile response to hemolysate but failed to relax the sustained contraction induced by this agent. The Src-tyrosine kinase inhibitor damnacanthal and the phosphatidylinositol 3—kinase inhibitor wortmannin failed to reduce hemolysate-induced contraction. 3) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity as seen on Western blots of rabbit BA. The MAPK was enhanced 1 minute after hemolysate exposure and the effect reached maximum levels at 5 minutes. The immunoreactivity of MAPK decayed slowly over time, but the level of this kinase was still higher than the basal level, even at 2 hours after exposure to hemolysate. Preincubation of arteries with the MEK inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity.

Conclusions. Hemolysate produced contraction of rabbit BA, possibly by activation of MAPK, and therefore MAPK inhibitors may be useful in the treatment of cerebral vasospasm.