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 (Fasdependent) 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 p47 phox 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 hyperoxiainduced 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 in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-X L /Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.The clinical treatment of respiratory failure often requires supplemental oxygen therapy. Prolonged exposure to an elevated oxygen tension (hyperoxia) in animal models causes acute and chronic lung injury that resembles acute respiratory distress syndrome. In rodent models, hyperoxia triggers an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema (1, 2). Lung tissue damage results from the direct action of increased intracellular reactive oxygen species (ROS) 2 or as a secondary consequence of inflammatory responses of the host (3, 4). The source(s) of intracellular ROS during oxygen exposure remain unclear but may involve increased mitochondrial generation and/or activation of NADPH oxidases (5, 6). The pathological changes in hyperoxiainjured lungs coincide with the injury or death of pulmonary capillary endothelial cells and alveolar epithelial cells (2, 4 -7). Epithelial cells maintain the integrity of the alveolar-capillary barrier and defend against oxidative injury. Compromised epithelial cell function may permit fluid and macromolecules to leak into the air...

Protein Kinase Cα and ζ Differentially Regulate Death-Inducing Signaling Complex Formation in Cigarette Smoke Extract-Induced Apoptosis

“…We have previously shown that DISC formation during hypoxia-reoxygenation-induced endothelial cell apoptosis was inhibited by expression of the endogenous caspase-8 inhibitor FLIP (42) and of Bcl-X L (20). We also reported that DISC formation during hyperoxia-induced endothelial cell apoptosis depended on reactive oxygen species generation, and could be inhibited by carbon monoxide, as well as by inhibitors of ERK1/2 MAPK or NADPH oxidase-dependent pathways (49).…”

Heme-oxygenase-1-induced protection against hypoxia/reoxygenation is dependent on biliverdin reductase and its interaction with PI3K/Akt pathway

“…The cytoprotective properties of HO-1 have traditionally been attributed to the by-products of heme degradation, namely bilirubin and carbon monoxide (CO). Indeed, within a narrow therapeutic range, these catalytic by-products exert powerful antioxidant [15,31], antiinflammatory [32] and anti-apoptotic effects [33][34][35], leading to reduced infarct size [16,17,[36][37]. However, emerging evidence, suggests that HO-1 may also exert cytoprotective effects, independent of heme breakdown [38] by interacting with survival signaling pathways such as PI3K-Akt and p38.…”

Epithelial Cell Death Is an Important Contributor to Oxidant-mediated Acute Lung Injury

“…Similar to our findings, Ilizarov and colleagues (54) observed in a mouse lung epithelial cell line that SOD2, but not SOD1 or catalase prevented hyperoxia-induced cell death. By contrast, other investigators have observed that strategies that prevent the assembly of the NAD(P)H oxidase on the plasma membrane of endothelial cells prevent hyperoxiainduced oxidant generation and cell death (55)(56)(57). Several possibilities might explain these results.…”