Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation

Abstract

Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47phox subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved both intrinsic and extrinsic Carbon monoxide inhibited the activation of and the and of and We also show that carbon monoxide of heme oxygenase-1 with mechanisms underlying the of carbon monoxide Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47phox subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved both intrinsic and extrinsic Carbon monoxide inhibited the activation of and the and of and We also show that carbon monoxide of heme oxygenase-1 with mechanisms underlying the of carbon monoxide of oxygen to oxygen causes and lung injury that hyperoxia inflammatory the lung that the to and and from the of reactive oxygen species reactive oxygen signal heme lung endothelial reactive oxygen signal heme lung endothelial or as of inflammatory of the of oxygen remain activation of NADPH with the injury or death of endothelial and the of the and cell and to the and death mechanisms underlying lung injury and cell death and apoptosis, necrosis, or mixed cell death signaling hyperoxia-induced lung cell of the apoptosis or by and the cell death intrinsic (mitochondria-dependent) pathway and extrinsic the of by the of and by with of the of proteins as of the intrinsic of this as to the membrane of the as and to the with the membrane and the release of cytochrome c and from the hyperoxia also to membrane to the cytochrome c complex with downstream the of apoptosis the the extrinsic pathway death as the with cell death-inducing signal complex of and the of death and caspase-8. from the to the activation injury and the underlying mechanisms remain as of the of also and as of heme heme to and the of to by of and as and as and at low of as well as of injury and injury mechanisms of of signaling activation of and the we that prevented hyperoxia-induced apoptosis by of both extrinsic and intrinsic by and activation and cytochrome c release from the inhibited plasma membrane formation by attenuating and trafficking from the Golgi We the the of with that by and that and and from was from was from oxygenase-1 was from was from and from and from with and to from the and and by the and of of and from and of endothelial as and of of lung endothelial by and cell and with with endothelial cell at to and with to and with to the and the with low by endothelial and and to by cell and and of endothelial cell at the to and to of with or and to of hyperoxia the to to and to and the or of release was of was at to the with and to to was by at the of cell death was by of by formation of was by the with at the of the the was and the the of of the to hyperoxia the or of the of to the on and the cell at at was and the cell was with on with at of of to cell of cell of formation of was on at of formation was was as at to of and cell at and the to and at at and the plasma membrane the of the to by the of of and at the of was of of both and at at the of and was and and Golgi complex was as with the and with was at and the was was and the was to of was the of and with and at from the and with by at and the Golgi was by and as and proteins from and of or was to of at and with of at and with of and was at the and the and on by to membrane was with and with the at the with the was and the was with and and from cell was to the and as of the as the was by and of was and to hyperoxia and hyperoxia-induced by the Hyperoxia caused cell death as by and release at of and Hyperoxia caused the release of cytochrome c from the and cytochrome c of by with inhibited hyperoxia-induced cell as by cell and release and to cytochrome c release from the and the of the of by the of and we also the of on hyperoxia-induced cell of at of the inhibited hyperoxia-induced cell death and release and of inhibited cytochrome c release from the and the We also the of on the of inhibited activation inhibited the activation of of hyperoxia-induced cell or the or of the was by by release or from as to to cytochrome c or also of as the the of the the that the of the of the extrinsic to hyperoxia of the or of with by with to of to hyperoxia formation associated with the of to the of and the of of the hyperoxia the formation and the of We also signaling of formation to and the and of by as plasma membrane trafficking and formation extrinsic by or the or of the and to with by to also to to the or of of or NADPH or the to with by as to and or also to to of as the with and to hyperoxia was by the of as the of from or or the to with by to and with of the hyperoxia inhibited and the of with We that hyperoxia-induced extrinsic with NADPH or to to the of NADPH with the as by the with attenuated also inhibited hyperoxia-induced with genetic from the p47phox of NADPH oxidase. studies show that p47phox endothelial to as and Hyperoxia the of and hyperoxia formation that NADPH the activation of the extrinsic pathway to hyperoxia and by the activation of this that formation at the plasma membrane by from the Golgi the of hyperoxia on of the cell from to hyperoxia Golgi complex and plasma membrane formation was by with by with caspase-8. 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Recent studies show that apoptosis involved the and activation of and with this of was required the trafficking of to the and the of the extrinsic pathway with this we that hyperoxia and with with activation and on because by NADPH and also inhibited by we also that inhibited trafficking of to by NADPH of activation hyperoxia and the of by by extrinsic to or of or of hyperoxia-induced lung with inflammatory cell the and the of formation associated with and with injury and inhibited apoptosis injury associated with the activation of the to by of by and attenuated lung and lung cell apoptosis hyperoxia of low lung the pathway by cell with we that prevented hyperoxia-induced cytochrome c release from the also inhibited hyperoxia-induced membrane as by release also cell we the that of with the of this although we that this the of to membrane and cytochrome c release we cell death to hyperoxia and found that hyperoxia caused apoptosis, involving both extrinsic and intrinsic inhibited cell death by inhibiting and formation the plasma inhibiting activation and also by and of with to hyperoxia-induced the