Hypoxia induces nitric oxide synthase in rheumatoid synoviocytes: consequences on NADPH oxidase regulation

Abstract

We investigated the effects of hypoxia on inducible NO synthase (iNOS) activity and expression in rheumatoid arthritis (RA) synoviocytes. We further studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. Human cultured synoviocytes were treated for 24 hours with IL-1β, TNF-α or neither, and submitted to hypoxia or normoxia for the last 6 hours. Nitrite production and iNOS expression were increased under hypoxia conditions in RA cells in comparison to normoxia. Hypoxia did not potentate the basal and cytokine-induced superoxide productions, while NOXs’ subunit expression and p47-phox phosphorylation were increased. Nitrosylation of NOXs and p47-phox was not raised under hypoxia conditions. Finally, peroxynitrite production was significantly increased under hypoxia conditions, in comparison to normoxia. Our results provide evidence for upregulation of iNOS and NOX activities in RA synoviocytes under hypoxia conditions, associated to an increased peroxynitrite production. Synovial cell metabolism under hypoxia conditions might be different from that in normoxia.     

NOX1 abet mesangial fibrogenesis via iNOS induction in diabetes

Both NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) are the main sources of reactive oxygen species in kidney. However, their interactions in oxidative stress and contributions to kidney fibrosis during diabetic nephropathy have not been studied. Human mesangial cells were treated with normal glucose (5.6 mmol/L), high glucose (30 mmol/L) in the presence or absence of AGE (200 mg/L). Protein expressions of NOX1, NOX2, NOX4, and iNOS were examined by immunoblotting. NOX was genetically silenced with specific RNAi to study the interactions between NOX and iNOS in diabetic milieu. Superoxide (O^·?) and peroxynitrite (ONOO^·?) productions were assessed by dihydroethidium and hydroxyphenyl fluorescein, respectively. Fibrotic factors were determined by biochemistry assay. Superoxide, peroxynitrite, TGF-β, and fibronectin productions as well as the protein expressions of NOX1, NOX2, NOX4, and iNOS were increased in the diabetic milieu (high glucose 30 mmol/L plus AGE 200 mg/L). However, abolishment of iNOS induction with 1400W or iNOS RNAi would restore peroxynitrite, TGF-β, and fibronectin productions completely to basal level and attenuate superoxide production. Moreover, NOX1 inhibition not only prevented iNOS induction but also abrogated changes consequent to iNOS induction such as mesangial fibrogenesis.     

Involvement of NADPH oxidase in up-regulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts induced by oxidized LDL and in apolipoprotein E-deficient mice

The present study demonstrated that oxidized LDL (oLDL) increased the generation of superoxide and hydrogen peroxide (H(2)O(2)), the abundances of NADPH oxidase (NOX)4, NOX2, p22-phox and lectin-like oLDL receptor-1 (LOX-1) in wild-type or heat shock factor-1 (HSF1)-deficient mouse embryo fibroblasts (MEF). LOX-1 antibody inhibited LDL or oLDL-induced expression of NOX components in MEF. Abundance of HSF1 or plasminogen activator inhibitor-1 (PAI-1) was increased by oLDL in wild-type, but not in HSF1-deficient MEF. Diphenyleneiodonium or siRNA for NOX or p22-phox inhibited oLDL-induced increases of HSF1, PAI-1 and H(2)O(2) in MEF. Increased NOX4, NOX2, LOX1, HSF1 and PAI-1 were detected in aortae and hearts of apolipoprotein E-knockout (apoE-KO) mice compared to controls, which were associated with increased serum cholesterol or plasma PAI-1. The results suggest that NOX is required for oLDL-induced HSF1 or PAI-1 expression in MEF, which was supported by the up-regulation of NOX, LOX-1, HSF1 and PAI-1 in apoE-KO mice.     

Nitric oxide synthase induction,cGMP elevation,and biopterin synthesis in vascular smooth muscle cells stimulated with interleukin-1beta in hypoxia

In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.     

Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-gamma induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 +/- 14 micro M, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 +/- 1.7 micro M, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and alpha-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.     

Intestinal NADPH Oxidase 2 Activity Increases in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91^phox expression was performed. Immunofluorescence studies estimated the co-localization of p47^phox and GP91^phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91^phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91^phox and p47^phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses.     

Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses

Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.     

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47phox

We previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 μM extracellular L -Hcy), (2) incubation with 50 μM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 μM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 μM extracellular L -Hcy) with or without 10 μM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 μM, and 100 μM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.     

Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway

Our previous studies have demonstrated that the urotensin (UII) and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM), but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK) mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.     

Prostate cancer cell lines under hypoxia exhibit greater stem-like properties

Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O(2)), 7% O(2) induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O(2) induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O(2) also extended the G(0)/G(1) stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44(bright) cells. Correspondingly, the sorted CD44(bright) cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44(dim) cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44(bright) cells under normoxia formed significantly more colonies and spheres compared with the CD44(dim) cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.     

Redox control of angiogenic factors and CD31-positive vessel-like structures in mouse embryonic stem cells after direct current electrical field stimulation

The molecular mechanisms driving angiogenesis in tissues derived from embryonic stem (ES) cells are currently unknown. Herein we investigated the effects of direct current (DC) electrical field treatment on endothelial cell differentiation and angiogenesis of mouse ES cells. Treatment of ES cell-derived embryoid bodies with field strengths ranging from 250 V/m to 750 V/m, applied for 60 s, dose-dependently increased the capillary area staining positive for the endothelial-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1), indicating stimulation of endothelial cell differentiation and angiogenesis. Consequently, increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) within 24 h was observed. Electric field treatment raised reactive oxygen species (ROS) generation for at least 48 h, which was blunted by NADPH-oxidase inhibitors diphenylen iodonium chloride (DPI) as well as 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and increased the expression of NADPH-oxidase subunits p22-phox, p47-phox, p67-phox, and gp91-phox within 24 h. Electrical field treatment resulted in activation of extracellular regulated kinase 1,2 (ERK1,2), p38, as well as c-Jun NH2-terminal kinase (JNK). Pretreatment with the JNK inhibitor SP600125 resulted in a significant decrease in capillary areas under control conditions as well as under conditions of electrical field treatment, whereas the p38 inhibitor SB203580 was without effects. By contrast, the ERK1,2 antagonist UO126 inhibited electrical field-induced angiogenesis, whereas angiogenesis under control conditions was unimpaired. The increase in capillary areas and VEGF expression as well as activation of JNK and ERK1,2 was significantly inhibited in the presence of the free radical scavenger vitamin E underscoring the role of ROS in electrical field-induced angiogenesis of ES cells.     

低氧大鼠肺动脉内皮细胞VEGF变化与PKC活性关系的探讨

目的：探讨低氧培养大鼠肺动脉血管内皮细胞ＶＥＧＦ的表达变化与ＰＫＣ活性的关系。方法：培养大鼠肺动脉血管内皮细胞,观察低氧（１％Ｏ２）培养不同时间大鼠肺动脉血管内皮细胞浆、膜ＰＫＣ活性和培养液中ＶＥＧＦ水平变化;加入ＰＫＣ抑制剂（ｓｔａｕｒｏｓｐｏｒｉｎｅ）后,测定低氧、常氧培养不同时间二者的变化。结果：低氧时膜ＰＫＣ活性和培养液中ＶＥＧＦ水平明显升高（Ｐ＜０．０１）。而加入ＰＫＣ抑制剂后,常氧和低     

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.     

Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.

Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.