title: Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and apoptosis in cultured endothelial cells.
abstract: 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.
authors.affiliation: Laboratory of Plasma Derivatives, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: F
authors.initials: F
authors.lastname: D'Agnillo
authors.forename: A I
authors.initials: AI
authors.lastname: Alayash
mesh_terms: 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
chemical_list: 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