Generation of Oxidants by Hypoxic Human Pulmonary and Coronary Smooth-Muscle Cells

Mehta, J.P.; Campian, Jian; Guardiola, Juan; Cabrera, Jesús A.; Weir, Ε. Kenneth; Eaton, John W.

doi:10.1378/chest.07-2984

Cited by 27 publications

(24 citation statements)

References 15 publications

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“…In this paradox, researchers supporting hypoxia-induced ROS contend that at low oxygen levels, the flow of electrons through the electron transport chain slows down, increasing the likelihood for the electrons to escape and produce free radicals. In contrast, other reports showed decreased ROS during hypoxia (23,48,63). This controversy might rely on the nature of the free radical species measured.…”

Section: Discussionmentioning

confidence: 87%

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

Díaz

Enríquez

Moraes

2012

Molecular and Cellular Biology

115

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Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O 2 ) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.

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Section: Discussionmentioning

confidence: 87%

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

Díaz

Enríquez

Moraes

2012

Molecular and Cellular Biology

115

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“…Although a number of reports demonstrated increased ROS under hypoxia from mitochondrial (24,25) or nonmitochondrial sources (26), numerous studies reported that decreased oxygen availability in hypoxia leads to lower ROS concentrations (27)(28)(29)(30)(31)(32)(33). Results in which antioxidant compounds or enzymes were used to prevent ROS-dependent effects on HIF-1␣ are equally contradictory.…”

Section: Discussionmentioning

confidence: 99%

Stabilization of Hypoxia-inducible Factor-1α Protein in Hypoxia Occurs Independently of Mitochondrial Reactive Oxygen Species Production

Chua

Dufour

Dassa

et al. 2010

Journal of Biological Chemistry

165

143

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The transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) is a master regulator of the cellular response to low oxygen. HIF-1␣ protein accumulates in hypoxia due to inhibition of prolyl hydroxylase enzymes, which under normoxic conditions use molecular oxygen to hydroxylate HIF-1␣ on two conserved proline residues (Pro 402 and Pro 564 ), thus targeting the protein for 26 S proteasome-dependent degradation. A functional mitochondrial electron transport chain is known to be necessary for HIF-1␣ stabilization in hypoxia. It has been reported that reactive oxygen species (ROS), produced under hypoxia by complex III of the mitochondrial electron transport chain, play a critical role in the stabilization of the HIF-1␣ protein, possibly by directly inhibiting prolyl hydroxylase enzymes. In contrast, we found that ROS production by complex III is not required for hypoxia-induced HIF-1␣ stabilization. Thus, reestablishing mitochondrial oxygen consumption in the presence of a complex III inhibitor by using an artificial electron donor to complex IV or by overexpressing Ciona intestinalis alternative oxidase results in HIF-1␣ protein stabilization in hypoxia. Furthermore, five inhibitors that target different sites of the mitochondrial electron transport chain have similar effects on the HIF-1␣ protein half-life in hypoxia but vary in their effects on mitochondrial ROS production. Finally, ROS do not regulate prolyl hydroxylase activity directly. We conclude that HIF-1␣ protein stabilization in hypoxia occurs independently of mitochondrial ROS production. However, mitochondria can modulate the cellular hypoxic response through altered respiratory activity, likely by regulating the cellular oxygen availability.Hypoxia induces a complex transcriptional program designed to improve cellular oxygen supply and adapt cellular metabolism to the lack of oxygen. This hypoxic response is mediated by hypoxia-inducible factor (HIF), 2 a heterodimeric transcription factor consisting of an oxygen-regulated ␣ subunit (HIF-1␣ and HIF-2␣) and a constitutively expressed ␤ subunit. The oxygen-dependent regulation of the ␣ subunit occurs at the level of its protein stability. At normal oxygen concentration, a family of prolyl hydroxylase (PHD) enzymes catalyzes the hydroxylation of two conserved proline residues in HIF-1␣ and HIF-2␣ (1, 2). This reaction is dependent on both oxygen, which is a cosubstrate, and non-heme-reduced Fe 2ϩ , bound to the catalytic center of PHD enzymes. Hydroxylation of HIF-1␣ and HIF-2␣ at either of the two proline residues creates a recognition site for binding to the Von Hippel-Lindau (pVHL) protein. Binding of HIF-1␣ and HIF-2␣ to pVHL results in polyubiquitination by the pVHL-associated E3 ubiquitin ligase and degradation by the 26 S proteasome. Under hypoxic conditions, lack of oxygen inhibits PHD activity, thus leading to HIF-1␣ and HIF-2␣ protein stabilization and accumulation in the nucleus.The mitochondrial electron transport chain (ETC), which consumes the vast majority of cellular oxygen and therefore...

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“…In the studies by Mehta et al (12), DCF, dihydroethidium, and Amplex Red were used for ROS measurement. Those tools all possess problems with lack of specificity or organelle accumulation, which may explain the discrepant results.…”

Section: Discussionmentioning

confidence: 99%

Prolonged Hypoxia Increases ROS Signaling and RhoA Activation in Pulmonary Artery Smooth Muscle and Endothelial Cells

Annie

Waypa

Mungai

et al. 2010

Antioxidants & Redox Signaling

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Phase I of the hypoxic pulmonary vasoconstriction (HPV) response begins upon transition to hypoxia and involves an increase in cytosolic calcium ([Ca 2+ ] i ). Phase II develops during prolonged hypoxia and involves increases in constriction without further increases in [Ca 2+ ] i , suggesting an increase in Ca 2+ sensitivity. Prolonged hypoxia activates RhoA and RhoA kinase, which may increase Ca 2+ sensitivity, but the mechanism is unknown. We previously found that reactive oxygen species (ROS) trigger Phase I. We therefore asked whether ROS generation during prolonged hypoxia activates RhoA in PA smooth muscle cells (PASMCs) and endothelial cells (PAECs) during Phase II. By using a cytosolic redox sensor, RoGFP, we detected increased oxidant signaling in prolonged hypoxia in PASMCs (29.8 AE 1.3% to 39.8 AE 1.4%) and PAECs (25.9 AE 2.1% to 43.7.9 AE 3.5%), which was reversed on the return to normoxia and was attenuated with EUK-134 in both cell types. RhoA activity increased in PASMCs and PAECs during prolonged hypoxia (6.4 AE 1.2-fold and 5.8 AE 1.6-fold) and with exogenous H 2 O 2 (4.1-and 2.3-fold, respectively). However, abrogation of the ROS signal in PASMCs or PAECs with EUK-134 or anoxia failed to attenuate the increased RhoA activity. Thus, the ROS signal is sustained during prolonged hypoxia in PASMCs and PAECs, and this is sufficient but not required for RhoA activation. Antioxid. Redox Signal. 12, 603-610.

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Generation of Oxidants by Hypoxic Human Pulmonary and Coronary Smooth-Muscle Cells

Cited by 27 publications

References 15 publications

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

Stabilization of Hypoxia-inducible Factor-1α Protein in Hypoxia Occurs Independently of Mitochondrial Reactive Oxygen Species Production

Prolonged Hypoxia Increases ROS Signaling and RhoA Activation in Pulmonary Artery Smooth Muscle and Endothelial Cells

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