Reoxygenation after hypoxia and glucose depletion causes reactive oxygen species production by mitochondria in HUVEC

In hemorrhagic shock, local hypoxia is present and followed by reoxygenation during the therapeutic process. In endothelium, reactive oxygen species (ROS) have been identified as a cause of inflammatory reactions and tissular lesions in ischemic territory during reoxygenation. This study was designed to identify the enzymatic mechanisms of ROS formation during reoxygenation after hypoxia. Because severe shock, in vivo, can affect both O2 and nutriments, we combined hypoxia at a level close to that found in terminal vessels during shock, with glucose depletion, which induces a relevant additional stress. Human umbilical vein endothelial cells (HUVEC) underwent 2 h of hypoxia (Po2 approximately 20 mmHg) without glucose and 1 h of reoxygenation (Po2 approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. After 1 h of reoxygenation, fluorescence had risen by 143 +/- 17%. Cell death was equal to 8.6 +/- 2.4%. Antimycin A and stigmatellin, which inhibits the type III mitochondrial respiratory chain complex, reduced ROS production to values of 61 +/- 10 and 59 +/- 7%, respectively, but inhibitors of other chain complexes did not affect it. In addition, the increase in fluorescence was not affected by inhibition of NADPH oxidase, xanthine oxidase, NOS, cyclooxygenase, cytochrome P-450 monooxygenase, or monoamine oxidase. We did not observe any increase in cell death. These results show that, in HUVEC, mitochondria are responsible for ROS production after hypoxia and reoxygenation and suggest that a ROS release site is activated in the cytochrome b of the type III respiratory chain complex.     

活性氧和线粒体ATP敏感钾通道介导肿瘤坏死因子α对缺氧/复氧心肌的保护作用

在培养的乳鼠心肌细胞上,研究肿瘤坏死因子α(TNF-α)对缺氧/复氧损伤心肌的保护作用的机制。结果发现：(1)用TNF-α(10—500U／ml)预处理,缺氧/复氧后心肌细胞内锰超氧化物歧化酶(Mn-SOD)活性增高、乳酸脱氢酶(LDH)释放量减少(P＜0．05);(2)用抗氧化剂N-乙酰半既氨酸(NAC,1mmol/L)、抗霉素A(antimycin A,50μmol/L)、2-巯基丙酰氨基乙酸(2-MPG,400μmol/L)和铜／锌超氧化物歧化酸(Cu/Zn,SOD)抑制剂二乙基二硫代氨基甲酸盐(DDC,100nmol/L)预处理,可取消TNF-α的抑制缺氧/复氧心肌细胞LDH释放和诱导Mn-SOD活性增高的作用;(3)mitoKATP通道抑制剂5-羟基癸酸(5-HD)预处理可阻断TNF-α对缺氧/复氧心肌细胞的保护作用;选择性mitoKATP通道开放剂diazoxide(50μmol/L)预处理可减少复氧后心肌细胞LDH的释放(P＜0．01),其作用可被5-HD(100μmol/L)和NAC所抑制。上述结果表明,活性氧和线粒体ATP敏感钾通道参与介导TNF-α对缺氧/复氧损伤的心肌保护作用。     

Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.     

Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells

Endothelial cells are critical targets in both hypoxia-and reoxygenation-mediated lung injury. Reactive O₂ species (ROS) have been implicated in the pathogenesis of hypoxic and reoxygenation lung injury, and xanthine dehydrogenase/oxidase (XDH/XO) is a major generator of the ROS. Porcine pulmonary artery endothelial cells (PAEC) have no detectable XDH/XO. This study was undertaken to examine (1) ROS production by hypoxic porcine PAEC and their mitochondria and (2) ROS production and injury in reoxygenated PAEC lacking XDH/XO activity. Intracellular H₂O₂ generation and extracellular H₂O₂ and O/₂ release were measured after exposure to normoxia (room air-5% CO₂), hypoxia (0% O₂ -95% N-5% CO₂), or hypoxia followed by normoxia or hyperoxia (95% O₂-5% CO₂). Exposure to hypoxia results in significant reductions in intracellular H₂ O₂ formation and extracellular release of H₂ O₂ and O₂ by PAEC and mitochondria. The reductions occur with as little as a 2 h exposure and progress with continued exposure. During reoxygenation, cytotoxicity was not observed, and the production of ROS by PAEC and their mitochondria never exceeded levels observed in normoxic cells. The absence of XDH/XO may prevent porcine PAEC from developing injury and increased ROS production during reoxygenation. © 1995 Wiley-Liss, Inc.     

Transient MPK6 activation in response to oxygen deprivation and reoxygenation is mediated by mitochondria and aids seedling survival in Arabidopsis

Mitogen-activated protein kinases (MPKs) are regulated by diverse stresses with a reactive oxygen species (ROS) component. Here, we report the rapid and transient activation of MPK3, MPK4 and MPK6 upon oxygen deprivation as well as reoxygenation in seedlings of Arabidopsis thaliana. MPK activation peaked within 2 h of oxygen deprivation and again at a higher magnitude within 5 min of reoxygenation. MPK6 was the predominant kinase regulated by oxygen availability in both aerial and root tissue, except in mpk6 mutants, which displayed compensatory activation of MPK3. A universal consequence of oxygen deprivation in eukaryotes is inhibition of the terminal step of the mitochondrial electron transport chain (mETC). We demonstrate that treatment of seedlings with the mETC inhibitors antimycin A and potassium cyanide under normoxia promotes transient MPK6 and MPK3 activation. Confocal imaging of seedlings provided evidence that both oxygen deprivation and mETC inhibitors stimulate mitochondria-associated ROS production. We found that seedling survival of prolonged oxygen deprivation was improved in transgenics that ectopically overexpress MPK3, MPK4 and MPK6, but the induction of mRNAs associated with low oxygen acclimation responses were not markedly altered in MPK6 overexpression lines or mpk6 loss-of-function mutants. However, distinctions in MPK6 activation potential were correlated with other differences in mRNAs accumulation. Our findings suggest that oxygen deprivation and reoxygenation trigger mitochondrial ROS production to activate MPK signaling, which in turn regulate reversible processes that aid survival of transient oxygen deprivation.     

Angiostatin effects on endothelial cells mediated by ceramide and RhoA

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

Reactive oxygen species production in the early and later stage of chronic ventilatory hypoxia

Pulmonary hypertension resulting from chronic hypoxia is at least partly caused by the increased production of reactive oxygen species (ROS). The goal of the presented study was to investigate the dynamics and the site of production of ROS during chronic hypoxia. In our study Wistar rats were kept for 1, 4 and 21 days in an isobaric hypoxic chamber (F(iO2)=0.1), while controls stayed in normoxia. We compared NO production in expired air, plasma and perfusate drained from isolated rat lungs and measured superoxide concentration in the perfusate. We also detected the presence of superoxide products (hydrogen peroxide and peroxynitrite) and the level of ROS-induced damage expressed as the concentration of lipid peroxydation end products. We found that the production and release of ROS and NO during early phase of chronic hypoxia has specific timing and differs in various compartments, suggesting the crucial role of ROS interaction for development of hypoxic pulmonary hypertension.     

Reactive oxygen species upregulate expression of PAI-1 in endothelial cells

Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor alpha (TNFalpha?-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signaling pathway. Because TNFalpha induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFalpha. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFalpha (50 ng/ml) or H2O2 (10-200 micro M), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants. On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.     

Reactive oxygen and targeted antioxidant administration in endothelial cell mitochondria

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H(2)O(2). In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H(2)O(2) fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.     

Reactive oxygen species regulation by AIF- and complex I-depleted brain mitochondria

Apoptosis-inducing factor (AIF)-deficient harlequin (Hq) mice undergo neurodegeneration associated with a 40–50% reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H₂O₂ removal, or ROS production stimulated by complex I inhibitors rotenone or 1-methyl-4-phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by ～50% in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenone-stimulated rates of H₂O₂ release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested.     

Protective effect of nicotinamide on neuronal cells under oxygen and glucose deprivation and hypoxia/reoxygenation

Nicotinamide (vitamin B₃) reduces the infarct volume following focal cerebral ischemia in rats; however, its mechanism of action has not been reported. After cerebral ischemia and/or reperfusion, reactive oxygen species (ROS) and reactive nitrogen species may be generated by inflammatory cells through several cellular pathways, which can lead to intracellular calcium influx and cell damage. Therefore, we investigated the mechanisms of action of nicotinamide in neuroprotection under conditions of hypoxia/reoxygenation. Results showed that nicotinamide significantly protected rat primary cortical cells from hypoxia by reducing lactate dehydrogenase release with 1 h of oxygen-glucose deprivation (OGD) stress. ROS production and calcium influx in neuronal cells during OGD were dose-dependently diminished by up to 10 mM nicotinamide (p<0.01). This effect was further examined with OGD/reoxygenation (H/R). Cells were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) or antibodies against anti-microtubule-associated protein-2 and cleaved caspase-3. Apoptotic cells were studied using Western blotting of cytochrome c and cleaved caspase-3. Results showed that vitamin B₃ reduced cell injury, caspase-3 cleavage and nuclear condensation (DAPI staining) in neuronal cells under H/R. In addition, nicotinamide diminished c-fos andzif268 immediate-early gene expressions following OGD. Taken together, these results indicate that the neuroprotective effect of nicotinamide might occur through these mechanisms in this in vitro ischemia/reperfusion model.     

Toxicity oft-butylhydroperoxide in hepatocyte monolayers exposed to hypoxia and reoxygenation

Summary Inasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity oft-butyl hydroperoxide, shifting the dose-response curve oft-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O₂ hepatocyte monolayers seemed normal by three indices (release of⁵¹Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20%. O₂. In contrast, monolayers exposed to 2% O₂ for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O₂, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation. The study was supported by grant OH 00978 from the National Institutes for Occupational Safety and Health, Atlanta, Georgia.     

Reactive oxygen species generation and antioxidant systems in plant mitochondria

In living cells, reactive oxygen species (ROS) play a key role in signaling but these compounds can also damage macromolecules. As in other compartments, the mitochondrial ROS concentrations need to be tightly controlled. Plant mitochondria contain several antioxidant systems that are not only able to scavenge ROS and limit their production but also to repair damages to macromolecules and possibly to serve as redox sensors. They comprise ascorbate- and glutathione-dependent pathways as well as systems based on thioredoxin (TRX)- and glutaredoxin (GRX)-like molecules. This review describes the various mitochondrial redox pathways for ROS control in plants with special emphasis on the poorly studied GRX and TRX systems and provides perspectives for future research in this area.     

Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells (BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm2) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.     

Reactive oxygen species formation in the transition to hypoxia in skeletal muscle

Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H₂O₂ applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O₂ to 0%, 5%, 21%, or 40% O₂, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O₂ exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H₂O₂, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO₂, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O₂-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat