Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and apoptosis in cultured endothelial cells.
It is hypothesized that oxidative reactions of hemoglobin driven by reactive oxygen species in the vasculature lead to endothelial cell injury or death. Bovine aortic endothelial cells were incubated with diaspirin cross-linked hemoglobin (DBBF-Hb), developed as a hemoglobin-based oxygen carrier, and hydrogen peroxide (H(2)O(2)), generated by the glucose oxidase system. The low steady flux of H(2)O(2) oxidizes the ferrous form of DBBF-Hb and drives the redox cycling of ferric and ferryl DBBF-Hb. Cells underwent rounding, swelling and detachment, and accumulated in the G2/M phase of the cell cycle. G2/M arrest preceded the onset of apoptosis as determined by increases in phosphatidylserine (PS) externalization and sub-G1 events. Redox cycling of unmodified hemoglobin also led to G2/M arrest and apoptosis. The rate and extent of DBBF-Hb oxidation correlated with the onset and extent of G2/M arrest and apoptosis and induced significant decreases in soluble reduced thiols. Earlier depletion of glutathione by pretreatment with buthionine sulfoximine rendered cells more susceptible to G2/M arrest and apoptosis. The caspase inhibitor, z-VAD-fmk, had no effect on the induction of G2/M arrest but completely inhibited the subsequent increases in PS externalization and sub-G1 events. Catalase inhibited DBBF-Hb oxidation, the loss of thiols, and the onset of G2/M arrest and apoptosis. These data support a causative role for the ferric-ferryl redox cycle in the development of endothelial cell injury.
D'Agnillo
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Laboratory of Plasma Derivatives, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Alayash
A I
AI
D000818:Animals; D001011:Aorta; D017209:Apoptosis / Q000187:drug effects; D001241:Aspirin / Q000031:analogs & derivatives* / Q000378:metabolism*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D061945:Caspase Inhibitors; D002374:Catalase / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D016195:G2 Phase; D005949:Glucose Oxidase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008938:Mitosis; D010084:Oxidation-Reduction; D010718:Phosphatidylserines / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006454:Hemoglobins; D010718:Phosphatidylserines; D013438:Sulfhydryl Compounds; D019328:Buthionine Sulfoximine; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D002374:Catalase; C083478:diaspirin-cross-linked hemoglobin; D005978:Glutathione; D001241:Aspirin