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 (DBBFHb), 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 ferricferryl redox cycle in the development of endothelial cell injury. IntroductionThe release of hemoglobin or myoglobin from its cellular environment during certain pathologic conditions leads to hemoproteininduced vascular damage or dysfunction. [1][2][3][4][5][6] Adverse microvascular effects of cell-free hemoglobin have also hindered the development of safe and efficacious hemoglobin-based oxygen carriers. 7 These agents are chemically stabilized forms of hemoglobin designed to act as oxygen and volume replacement therapy in clinical settings such as emergency resuscitation and elective surgery. [7][8][9] Although the molecular mechanisms of hemoprotein-mediated cytotoxicity are not completely understood, the interactions of heme (protein or nonprotein bound) with hydrogen peroxide (H 2 O 2 ), organic peroxides (ROOH), peroxynitrite (ONOO Ϫ ), and nitric oxide (NO) have been implicated. [1][2][3][4][5][6][7] The reaction of hemoglobin with H 2 O 2 or ROOH leads to a series of reduction-oxidation (redox) transitions between the ferrous, ferric, and ferryl states of the protein. The oxy and deoxy forms of ferrous hemoglobin (HbFe 2ϩ ) and ferric hemoglobin (HbFe 3ϩ ) can each react with H 2 O 2 to produce ferryl hemoglobin (HbFe 4ϩ ) (equations 1 and 2). [10][11][12] In the case of HbFe 3ϩ , this reaction also generates a proteinbased radical that can be detected by electron paramagnetic spectroscopy. 12 HbFe 4ϩ can, in turn, be reduced to HbFe 3ϩ autocatalytically or after substrate oxidation. [10][11][12] [13][14][15] HbFe 4ϩ generated from the interaction of tert-butyl hydroperoxide and hemoglo...

Redox active hemoglobin enhances lipopolysaccharide-induced injury to cultured bovine endothelial cells

“…Aliquots were frozen at Ϫ80°C until use. The amount of Gx used in these experiments was sufficient to generate 1 M H 2O2/min at 25°C in HBSS-containing reaction mixtures using the O-dianisidine-horseradish peroxidase assay (9). This enzymatic activity produced a steady-state H 2O2 concentration of ϳ10 M under cell culture conditions and produced minimal direct cytotoxicity over the course of 16 h (9, 10).…”

Salidroside Inhibits Endogenous Hydrogen Peroxide Induced Cytotoxicity of Endothelial Cells

“…1) Hence, exposing cells to steady state concentrations of H 2 O 2 , instead of bolus additions, constitutes a superior method for oxidant delivery that mimics the physiological settin.…”

“…concentration was maintained at approximately 7 to 9 mM with 10 mU GOX. 1) In order to measure the effects of salidroside on endothelial damage induced by H 2 O 2 , the MTT reduction assay was used. As show in Fig.…”

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Erythrocyte Alterations and Increased Cardiovascular Risk in Chronic Renal Failure

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