Epalrestat Upregulates Heme Oxygenase-1, Superoxide Dismutase, and Catalase in Cells of the Nervous System.

Heme oxygenase (HO)-1 has potent antioxidant and anti-inflammatory functions. Recent studies have shown that the upregulation of HO-1 is beneficial to counteract neuroinflammation, making HO-1 a new therapeutic target for neurological diseases. We have reported that epalrestat (EPS), which is currently used for the treatment of diabetic neuropathy, increases HO-1 levels through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in bovine aortic endothelial cells. In this study, we tested the hypothesis that EPS upregulates HO-1 via Nrf2 activation in the component cells of the nervous system, by using rat Schwann cells and human SH-SY5Y cells. Treatment of Schwann cells with EPS at near-plasma concentration led to a dramatic increase in HO-1 levels. Nrf2 knockdown by small interfering RNA (siRNA) suppressed the EPS-induced HO-1 expression. EPS did not promote the intracellular accumulation of free ferrous ion and reactive oxygen species, by increasing ferritin via Nrf2 during HO-1 induction. Moreover, EPS stimulated the expression of superoxide dismutase 1 and catalase, which also are Nrf2 target gene products. It also markedly increased HO-1 levels in SH-SY5Y cells through the activation of Nrf2. We demonstrated for the first time that EPS upregulates HO-1, superoxide dismutase, and catalase by activating Nrf2. We suggest that EPS has the potential to prevent several neurological diseases.

Glycolaldehyde induces cytotoxicity and increases glutathione and multidrug-resistance-associated protein levels in Schwann cells.

Schwann cell injury is observed in diabetic neuropathy. It is speculated that glycolaldehyde (GA), a precursor of advanced glycation end products (AGEs), contributes to the pathogenesis and development of diabetic neuropathy. Here, we demonstrated for the first time that GA at near-physiological concentration decreased the viability of rat Schwann cells. In contrast, methylglyoxal, glyoxal, and 3-deoxyglucosone, all of which are AGE precursors, had no effects on cell viability. It is well known that methylglyoxal causes oxidative damage. In the present study, however, GA failed to induce reactive oxygen species production in Schwann cells. The addition of glutathione (GSH) or N-acetyl-L-cysteine protected Schwann cells from the loss of viability induced by GA. Moreover, GA increased intracellular GSH level and γ-glutamylcysteine synthetase mRNA level. Flow cytometric analysis revealed that GA increased multidrug-resistance-associated protein 1 (MRP1) level as well. Moreover, we demonstrated that the knockdown of MRP1 with small interfering RNA (siRNA) enhanced the loss of cell viability induced by GA. Taken together, these findings suggest that MRP1, together with GSH, plays an important role in the GA-induced toxicity in Schwann cells.

Multidrug resistance associated protein 1 together with glutathione plays a protective role against 4-hydroxy-2-nonenal-induced oxidative stress in bovine aortic endothelial cells.

4-Hydroxy-2-nonenal (HNE), an aldehyde produced by lipid peroxidation, induces cytotoxicity and oxidative stress. Glutathione (GSH) protects against the cytotoxicity of HNE. However, the protective mechanism of GSH has not been fully examined. We examined the protective role played by the relationship between GSH and multidrug resistance associated protein 1 (MRP1) against the HNE-induced oxidative stress in bovine aortic endothelial cells (BAECs). HNE induced the loss of viability of BAECs. Exogenous GSH, which is membrane-impermeable, prevented the loss of viability induced by HNE by inhibiting HNE uptake in BAECs, probably due to the formation of the HNE-SG complex in the extracellular space. We demonstrated that HNE induced the expression of MRP1 protein, which can transport the HNE-SG complex. The induction of MRP1 protein expression by HNE disappeared in BAECs pretreated with L-buthionine sulfoximine, a GSH-depleting agent. This result suggests that HNE, together with intracellular GSH, contributes to the regulation of MRP1 protein expression. Moreover, we found that MK571, an MRP1 inhibitor, promoted the HNE-induced oxidative stress and cell death. Taken together, these findings suggest that MRP1, together with GSH, plays a protective role against the HNE-induced oxidative stress in BAECs.

Epalrestat suppresses cadmium-induced cytotoxicity through Nrf2 in endothelial cells.

Cadmium (Cd) is an industrial and environmental pollutant that targets the vascular endothelium. The vascular system is critically affected by Cd toxicity. Recent studies have indicated an association between Cd and vascular diseases, although the mechanisms of Cd implications in vascular diseases are not clear. The purpose of the present study was to determine whether epalrestat (EPS), which is used for the treatment of diabetic neuropathy, protects against Cd-induced cytotoxicity in bovine aortic endothelial cells (BAECs). In the present study, the effects of EPS at near-plasma concentration were examined on Cd-induced cytotoxicity in BAECs. Cd-induced cytotoxicity was suppressed by pretreatment with EPS. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that serves a role in regulating the expression of glutamate cysteine ligase, the rate-limiting enzyme in glutathione (GSH) synthesis. In a previous study, EPS was demonstrated to increase GSH levels in BAECs in association with the Nrf2 pathway. In the present study, EPS increased GSH levels in BAECs exposed to Cd. The protective ability of EPS against the Cd-induced cytotoxicity disappeared following Nrf2 small interfering RNA transfection. In addition, EPS affected the intracellular levels of Cd, Cd transporter ZIP8 and metallothionein. To the best of our knowledge, the current study demonstrated, for the first time, that EPS suppresses Cd-induced cytotoxicity in BAECs. The upregulation of GSH may be associated with the suppression of Cd-induced cytotoxicity by EPS. From these findings, it may be proposed that the regulation of GSH, ZIP8 and metallothionein by EPS is a promising therapeutic approach to prevent Cd-induced toxicity.

Buthionine sulfoximine promotes methylglyoxal-induced apoptotic cell death and oxidative stress in endothelial cells.

Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, is found at high levels in the blood of diabetic patients. MG induces oxidative stress and apoptosis. There is evidence that MG causes glutathione (GSH) depletion. However, it remains unknown whether GSH plays a protective role against the cytotoxic effect of MG. We examined the effect of DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, on the viability of bovine aortic endothelial cells (BAECs) exposed to MG. BAECs pretreated with BSO showed reduced ability to survive MG exposure. Flow cytometric analyses with annexin V and propidium iodide double staining revealed that BAECs exposed to MG after BSO pretreatment displayed features characteristic of apoptosis. Caspase-3 activation induced by MG was increased by BSO. Moreover, measurement of protein carbonyl levels showed that BSO promoted MG-induced oxidative stress. Taken together, these findings suggest that the depletion of GSH via BSO pretreatment promoted MG-induced apoptotic cell death and oxidative stress in BAECs.

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.

AIMS: Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs). MAIN METHODS: BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody. KEY FINDINGS: Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells. SIGNIFICANCE: We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.

Methylglyoxal causes dysfunction of thioredoxin and thioredoxin reductase in endothelial cells.

Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, induces oxidative stress and apoptosis. Under hyperglycemic conditions, the abnormal accumulation of MG is related to the development of diabetic complications. We examined the effects of MG on thioredoxin (Trx) and glutaredoxin (Grx) systems, two thiol-disulfide oxidoreductase systems that protect against oxidative damage of proteins, in bovine aortic endothelial cells (BAECs). The levels of protein carbonyls as markers of protein oxidation increased in BAECs exposed to MG at 5 mM, resulting in the loss of cell viability. Western blot analysis demonstrated that Trx protein level decreased when BAECs were exposed to 5 mM MG. MG also inactivated Trx reductase, which maintains Trx in the reduced/active state. Moreover, peroxiredoxin, which is dependent on Trx and Trx reductase to maintain its reduced state, was oxidized by 5 mM MG. No significant difference in the levels of Trx, Trx reductase, or peroxiredoxin was observed in BAECs exposed to MG at 1 mM; this concentration had little effect on protein carbonyl formation and cell viability. MG failed to decrease Grx activity, indicating that Trx is more susceptible to MG than Grx. Taken together, these findings suggest that MG causes dysfunction of the Trx system, including Trx and Trx reductase, in BAECs.

[methylglyoxal-induced superoxide anion production in endothelial cells].

Methylglyoxal (MG), a highly reactive dicarbonyl compound, is a metabolic by-product of glycolysis. MG is often detected at high levels in the blood of diabetic patients. We examined whether MG was capable of inducing reactive oxygen species (ROS) production in bovine aortic endothelial cells (BAECs). The viability of BAECs decreased with time on treatment with 5 mM MG, and was almost completely lost at 24 h. In contrast, MG at 1 mM had little influence on BAEC viability up to 24 h, but induced the elevation of intracellular glutathione content at 24 h. Exposure of BAECs to MG caused a dose-dependent increase in oxidized-hydroethidine fluorescence intensity, indicating ROS production. In addition, aconitase inactivation, which is an indicator of intracellular superoxide, was observed in MG-treated cells. Finally, we found that MG at 5 mM increased the fluorescence intensity of BES-So, a specific probe for superoxide. Together, the results suggest that MG induces superoxide production in endothelial cells, and that the accumulation of ROS may be linked to cytotoxic effects.

[Methylglyoxal-induced apoptosis of endothelial cells].

Diabetic patients exhibit increased blood plasma levels of methylglyoxal (MG), a metabolite of glucose. Since MG generates advanced glycation end-products (AGEs) that disrupt the functions of such biomolecules as proteins, it is responsible for the progression and complications of diabetes. A functional disorder of the vascular endothelium may also contribute to the progression and complications of diabetes. In endothelial cells, MG is the major precursor for the formation of AGEs. In this study, we examined the effects of MG on vascular endothelial cells and found that it induced the apoptosis of bovine aortic endothelial cells (BAECs). MG induced the collapse of mitochondrial membrane potential, an index of apoptosis, and the elevation of caspase-3 activity, an apoptotic execution enzyme, leading to cell death. Flow cytometric analyses with annexin-V and propidium iodide double staining revealed that cells exposed to a lethal dose of MG displayed features characteristic of apoptosis. MG induced an increase in the level of intracellular reactive oxygen species (ROS) prior to induction of apoptosis. Taken together, these findings suggest that BAECs exposed to MG die by apoptosis due to the increase of intracellular ROS level.

Low concentrations of clarithromycin upregulate cellular antioxidant enzymes and phosphorylation of extracellular signal-regulated kinase in human small airway epithelial cells.

BACKGROUND: It is well known that low-dose, long-term macrolide therapy is effective against chronic inflammatory airway diseases. Oxidative stress is considered to be a key pathogenesis factor in those diseases. However, the mechanism of action of low-dose, long-term macrolide therapy remains unclear. We have reported that clarithromycin (CAM), which is a representative macrolide antibiotic, could inhibit hydrogen peroxide (H2O2)-induced reduction of the glutathione (GSH)/glutathione disulfide (GSSG) ratio in human small airway epithelial cells (SAECs), via the maintenance of GSH levels through an effect on γ-glutamylcysteine synthetase (γ-GCS) expression. In this study, we examined the influence of CAM against H2O2-induced activities of cellular antioxidant enzymes and phosphorylated extracellular signal regulatory kinase (p-ERK) using SAECs, the main cells involved in chronic airway inflammatory diseases. METHODS: SAECs were pretreated with CAM (1, 5, and 10 µM) for 72 h, and subsequently exposed to H2O2 (100 µM) for 0.5-2 h. Levels of GSH and GSSG, and activities of glutathione peroxidase (GPx)-1, glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), heme oxygenase (HO)-1 and p-ERK were assayed. mRNA expressions of GPx-1 and HO-1 were measured using the real-time reverse transcription polymerase chain reaction (RT-PCR). Tukey's multiple comparison test was used for analysis of statistical significance. RESULTS: Pretreatment with low-dose (1 and 5 µM) CAM for 72 h inhibited H2O2-induced reductions of GPx-1, GR, SOD, CAT and HO-1 activities, and mRNA expressions of GPx-1 and HO-1, and improved the GSH/GSSG ratio. However, these alterations were not observed after pretreatment with high-dose (10 µM) CAM, which suppressed phosphorylation of cell proliferation-associated ERK to cause a significant (p < 0.01) decrease in cell viability. CONCLUSIONS: CAM is efficacious against deterioration of cellular antioxidant enzyme activity caused by oxidative stress under low-dose, long-term treatment conditions. On the other hand, pretreatment with high-dose CAM suppressed phosphorylation of cell proliferation-associated ERK and decreased cell viability. The present study may provide additional evidence as to why low-dose, long-term administration of macrolides is effective for treating chronic inflammatory airway diseases.

Paraquat-induced oxidative stress and dysfunction of cellular redox systems including antioxidative defense enzymes glutathione peroxidase and thioredoxin reductase.

We examined if paraquat-induced oxidative stress and cytotoxicity in pulmonary microvascular endothelial cells are associated with cellular redox systems such as the glutathione system and the thioredoxin system. Loss of viability, accompanied by marked decreases in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thioredoxin reductase activities, occurred 48 h after exposure to 1mM paraquat. These changes were preceded by an increased production of hydrogen peroxide after the decrease in glutathione peroxidase activity. Glutaredoxin activity was not decreased even after exposure to paraquat for 48 h, whereas thioredoxin activity was slightly decreased at 48 h. Unexpectedly, the activity of peroxiredoxin, a non-selenoenzyme, was almost completely lost at 24h. Loss of GAPDH activity and viability was notably aggravated by mercaptosuccinate. Selenium supplementation suppressed the loss of activities of glutathione peroxidase and thioredoxin reductase and alleviated paraquat-induced cytotoxicity. An in vitro experiment demonstrated that GAPDH was highly susceptible to reactive oxygen species generated in the xanthine-xanthine oxidase system, whereas thioredoxin reductase was considerably resistant. Taken together, the results suggest that the reduced regenerative ability of oxidatively damaged proteins including GAPDH due to the inactivation of thioredoxin reductase and glutathione peroxidase by paraquat may contribute to increasing oxidative stress, leading to cell death.

Long-term treatment of clarithromycin at a low concentration improves hydrogen peroxide-induced oxidant/antioxidant imbalance in human small airway epithelial cells by increasing Nrf2 mRNA expression.

BACKGROUND: Clarithromycin (CAM), a representative macrolide antibiotic, has been used widely at low doses for long-term therapy of chronic inflammatory airway diseases. Anti-inflammatory effects of macrolide antibiotics were first discovered in clinical practice. Although oxidative stress is known as a key pathogenesis factor in chronic airway inflammatory diseases, the mechanism of action of low-dose, long-term CAM therapy remains unclear. We aimed to examine the cytoprotective action of CAM against hydrogen peroxide (H2O2)-induced cell dysfunction, focusing on CAM dose and treatment duration, and using human small airway epithelial cells (SAECs), the main cells involved in chronic airway inflammatory diseases. METHODS: SAECs were pretreated with CAM (1, 5 or 10 µM) for 24, 48 or 72 h, and were subsequently exposed to H2O2 for 0.5-4 h. Levels of interleukin (IL)-8, glutathione (GSH) and glutathione disulfide (GSSG), and the activities of nuclear factor (NF)-κB and γ-glutamylcysteine synthetase (γ-GCS) were assayed using specific methods. IL-8 mRNA and NF erythroid 2-related factor 2 (Nrf2) mRNA expression were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). Tukey's multiple comparison test was used for analysis of statistical significance. RESULTS: Pretreatment with low-dose (1 or 5 µM), long-term (72 h) CAM inhibited H2O2-induced IL-8 levels, NF-κB activity, and IL-8 mRNA expression, and improved the GSH/GSSG ratio via the maintenance of γ-GCS expression levels. Similar to its enhancing effect on the GSH/GSSG ratio, pretreatment with low-dose CAM for 72 h significantly increased Nrf2 mRNA expression (p < 0.01 and p < 0.05). In contrast, these alterations were not observed after pretreatment with high-dose (10 µM) or short-term (24 and 48 h) CAM. CONCLUSIONS: CAM is efficacious against cell dysfunction caused by oxidative stress under low-dose, long-term treatment conditions. This effect depended on the suppression of NF-κB activation and improvement of the H2O2-induced oxidant/antioxidant imbalance that is achieved by increasing Nrf2 mRNA expression in SAECs. The present study may provide the first evidence of why low-dose, long-term administration of macrolides is effective for treating chronic inflammatory airway diseases.

Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis.

Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.

Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells.

Schwann cell injury is caused by diabetic neuropathy. The apoptosis of Schwann cells plays a pivotal role in diabetic nerve dysfunction. Glycolaldehyde is a precursor of advanced glycation end products that contribute to the pathogenesis of diabetic neuropathy. In this study, we examined whether glycolaldehyde induces endoplasmic reticulum (ER) stress and apoptosis in rat Schwann cells. Schwann cells treated with 500 µM glycolaldehyde showed morphological changes characteristic of apoptosis. Glycolaldehyde activated apoptotic signals, such as caspase-3 and caspase-8. Furthermore, it induced ER stress response involving RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring ER-to-nucleus signal kinase 1α (IRE1α), and eukaryotic initiation factor 2α (eIF2α). In addition, glycolaldehyde activated CCAAT/enhancer-binding homologous protein (CHOP), an ER stress response factor crucial to executing apoptosis. Knockdown of nuclear factor E2-related factor 2 (Nrf2), which is involved in the promotion of cell survival following ER stress, enhanced glycolaldehyde-induced cytotoxicity, indicating that Nrf2 plays a protective role in the cytotoxicity caused by glycolaldehyde. Taken together, these findings indicate that glycolaldehyde is capable of inducing apoptosis and ER stress in Schwann cells. The ER stress induced by glycolaldehyde may trigger the glycolaldehyde-induced apoptosis in Schwann cells. This study demonstrated for the first time that glycolaldehyde induced ER stress.

Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells.

Epalrestat (EPS) is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs), an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation.

Epalrestat (EPS), approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH), which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS), the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane) dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.

Methylglyoxal has deleterious effects on thioredoxin in human aortic endothelial cells.

Methylglyoxal (MG), a precursor of advanced glycation end products (AGEs), is elevated in diabetic patient's plasma. Some studies have demonstrated that MG induces oxidative stress and apoptosis. Thioredoxin (Trx) is a cytoprotective protein with anti-oxidative and anti-apoptosis functions. In this study, we examined the effects of MG on Trx in human aortic endothelial cells (HAECs). MG increased oxidized-hydroethidine fluorescence intensity, suggesting intracellular accumulation of reactive oxygen species. Flow cytometric analyses with annexin-V/propidium iodide double staining revealed that cells incubated with MG displayed features characteristic of apoptosis. The condensation of chromatin, the release of cytochrome c into cytosol, and the collapse of mitochondrial membrane potential by MG were observed. The exposure to MG decreased Trx protein levels through transcription regulation. MG induced the oxidative damage of peroxiredoxin, a Trx-dependent peroxidase. These results suggest that MG has deleterious effects on Trx in HAECs, which may be contribute to oxidative stress and apoptosis.

Paraquat-induced oxidative stress and dysfunction of the glutathione redox cycle in pulmonary microvascular endothelial cells.

Oxidative stress and changes in the antioxidant defense system that include the glutathione redox cycle in cultured pulmonary microvascular endothelial cells after exposure to paraquat at 0.1 and 0.5 mM were examined as a function of time. Cell viability was substantially lost 72 h after exposure to 0.5 mM paraquat, but not 0.1 mM paraquat. Viability loss was accompanied by increased glutathione-protein mixed disulfide formation, as well as a loss in glyceraldehyde-3-phosphate dehydrogenase activity, indicating a low defense potential. At 4 h after exposure to paraquat at both doses, however, a marked loss in NADPH was found, together with a decrease in aconitase activity. With 0.5 mM paraquat, increased NADP(+) accompanied by NADPH loss diminished constantly after 48 h without recovery of lost NADPH, suggesting destruction of pyridine nucleotides under oxidative stress. NAD(+) decreased 72 h after exposure to 0.5 mM paraquat, but NADH was not influenced. 3-Aminobenzamide did not protect the loss in NADP(+) or NAD(+) and cell viability. Although oxidized glutathione did not increase by exposure to paraquat at both doses through a 96-h exposure period, reduced glutathione increased at 48 to 72 h, with an increase in glutathione disulfide reductase activities. In contrast, a marked loss in glutathione peroxidase activity was produced 48 h after exposure to 0.5 mM paraquat, preceding cell injury. Mercaptosuccinate, an inhibitor of glutathione peroxidase, distinctly hastened viability loss by paraquat. These results indicate that the reduced ability of the glutathione redox cycle, leading to high oxidative stress, is closely associated with paraquat-induced cytotoxicity.

Nitric oxide mitigates peroxide-induced iron-signaling, oxidative damage, and apoptosis in endothelial cells: role of proteasomal function?

In this mini-review, oxidant-induced transferrin receptor-mediated iron-signaling and apoptosis are described in endothelial and neuronal cells exposed to a variety of oxidative stresses. The role of nitric oxide and nitration in the regulation of iron homeostasis and oxidant-induced apoptosis is described. The interrelationship between oxidative stress, iron-signaling, and nitric oxide-dependent proteasomal function provides a rational mechanism that connects both oxidative and nitrative modifications.

Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: Role of ubiquitin-proteasome pathway.

We investigated here the mechanism of cytoprotection of nitric oxide (*NO) in bovine aortic endothelial cells treated with H2O2. NONOates were used as *NO donors that released *NO slowly at a well defined rate in the extracellular and intracellular milieus. H2O2-mediated intracellular dichlorofluorescein fluorescence and apoptosis were enhanced by the transferrin receptor (TfR)-mediated iron uptake. *NO inhibited the TfR-mediated iron uptake, dichlorofluorescein fluorescence, and apoptosis in H2O2-treated cells. *NO increased the proteasomal activity and degradation of nitrated TfR via ubiquitination. Nomega-nitro-L-arginine methyl ester, a nonspecific inhibitor of endogenous *NO biosynthesis, decreased the trypsin-like activity of 26S proteasome. *NO, by activating proteolysis, mitigates TfR-dependent iron uptake, dichlorodihydrofluorescein oxidation, and apoptosis in H2O2-treated bovine aortic endothelial cells. The relevance of biological nitration on redox signaling is discussed.