Oxidative and nitrosative stress in ammonia neurotoxicity

Increased ammonia accumulation in the brain due to liver dysfunction is a major contributor to the pathogenesis of hepatic encephalopathy (HE). Fatal outcome of rapidly progressing (acute) HE is mainly related to cytotoxic brain edema associated with astrocytic swelling. An increase of brain ammonia in experimental animals or treatment of cultured astrocytes with ammonia generates reactive oxygen and nitrogen species in the target tissues, leading to oxidative/nitrosative stress (ONS). In cultured astrocytes, ammonia-induced ONS is invariably associated with the increase of the astrocytic cell volume. Interrelated mechanisms underlying this response include increased nitric oxide (NO) synthesis which is partly coupled to the activation of NMDA receptors and increased generation of reactive oxygen species by NADPH oxidase. ONS and astrocytic swelling are further augmented by excessive synthesis of glutamine (Gln) which impairs mitochondrial function following its accumulation in there and degradation back to ammonia (“the Trojan horse” hypothesis). Ammonia also induces ONS in other cell types of the CNS: neurons, microglia and the brain capillary endothelial cells (BCEC). ONS in microglia contributes to the central inflammatory response, while its metabolic and pathophysiological consequences in the BCEC evolve to the vasogenic brain edema associated with HE. Ammonia-induced ONS results in the oxidation of mRNA and nitration/nitrosylation of proteins which impact intracellular metabolism and potentiate the neurotoxic effects. Simultaneously, ammonia facilitates the antioxidant response of the brain, by activating astrocytic transport and export of glutathione, in this way increasing the availability of precursors of neuronal glutathione synthesis.     

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.     

Montelukast abrogates prednisolone‐induced hepatic injury in rats: Modulation of mitochondrial dysfunction,oxidative/nitrosative stress,and apoptosis

The aim of this study was to investigate the protective effect of montelukast (MTK) against prednisolone‐induced hepatic injury in rats. Twenty‐eight male albino rats were categorized into four equal groups. Group I served as the control group; group II: rats orally received prednisolone (5 mg·kg^?1·d^?1) for 30 days; groups III and IV: rats orally received MTK at 10 and 20 mg·kg^?1·d^?1, respectively, simultaneously with prednisolone for 30 days. Serum liver enzymes, hepatic mitochondrial function, oxidative/nitrosative stress, and inflammatory and apoptotic markers were evaluated, and the results were confirmed by histopathological examination. MTK showed significant hepatic protection evidenced by alleviated histological lesion and improvement of mitochondrial function, oxidative/nitrosative stress, and inflammatory and apoptotic changes induced by prednisolone, with more profound protection in higher MTK dose (20 mg·kg^?1). In view of these findings, we can conclude that MTK may have hepatoprotective potential, beyond its therapeutic value for asthmatic patients during their course of corticosteroid therapy.     

Effect of paricalcitol and enalapril on renal inflammation/oxidative stress in atherosclerosis

AIM: To investigate the protective effect of paricalcitol and enalapril on renal inflammation and oxidative stress in Apo E-knock out mice. METHODS: Animals treated for 4 mo as group(1) Apo E-knock out plus vehicle, group(2) Apo E-knock out plus paricalcitol(200 ng thrice a week),(3) Apo Eknock out plus enalapril(30 mg/L),(4) Apo E-knock out plus paricalcitol plus enalapril and(5) normal. Blood pressure(BP) was recorded using tail cuff method. The kidneys were isolated for biochemical assays using spectrophotometer and Western blot analyses. RESULTS: Apo E-deficient mice developed high BP(127 ± 3 mm Hg) and it was ameliorated by enalapril and enalapril plus paricalcitol treatments but not with paricalcitol alone. Renal malondialdehyde concentrations, p22 phox, manganese-superoxide dismutase, inducible nitric oxide synthase(NOS), monocyte chemoattractant protein-1, tumor necrosis factor-alpha and transforming growth factor-β1 levels significantly elevated but reduced glutathione, Cu Zn-SOD and e NOS levels significantly depleted in Apo E-knock out animals compared to normal. Administration of paricalcitol, enalapril and combined together ameliorated the renal inflammation and oxidative stress in Apo E-knock out animals. CONCLUSION: Paricalcitol and enalapril combo treatment ameliorates renal inflammation as well as oxidative stress in atherosclerotic animals.     

Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin alpha(v)beta5

Background

Oxidized low density lipoprotein (oxLDL) has been shown to induce apoptosis and senescence of endothelial progenitor cells (EPC). In the present study, we hypothesized that even sub-apoptotic concentrations of oxLDL impair the angiogenic potential of EPC and investigated if this effect is mediated by affecting adhesion and incorporation.

Methods

A co-culture system of human microvascular endothelial cells and EPC was used to study the effect of sub-apoptotic concentrations of native (nLDL) and oxLDL on cell-cell interaction. The expression and the functional role of angiogenic adhesion molecules and integrins was monitored by FACS and neutralizing assay, respectively.

Results

We observed an inhibition of tube formation and impairment of EPC integration into the vascular network of mature endothelial cells by oxLDL. In contrast, nLDL did not affect angiogenic properties of EPC. Incubation of EPC with sub-apoptotic oxLDL concentrations significantly decreased E-selectin and integrin α_vβ₅ expression (37.6% positive events vs. 71.5% and 24.3% vs. 49.9% compared to control culture media without oxLDL). Interestingly, expression of α_vβ₃, VE-cadherin and CD31 remained unchanged. Blocking of E-selectin and integrin α_vβ₅ by neutralizing antibody effectively inhibited adhesion of EPC to differentiated endothelial cells (56.5% and 41.9% of control; p < 0.001).

Conclusion

In conclusion, oxidative alteration of LDL impairs angiogenic properties of EPC at sub-apoptotic levels by downregulation of E-selectin and integrin α_vβ₅, both substantial mediators of EPC-endothelial cell interaction.     

Changes in proteomics profile during maturation of marrow-derived dendritic cells treated with oxidized low-density lipoprotein

Increasing evidence suggests that dendritic cells (DCs) and oxidized low-density lipoprotein (ox-LDL) participate in atherosclerosis. However, few data on the molecular mechanisms of this process are available. To address this question, we used iTRAQ labeling followed by LC-MS/MS analysis to identify many proteins that changed markedly during the maturation of dendritic cells stimulated with ox-LDL. Among a total of 781 identified proteins, 93 were upregulated and 100 were downregulated. The major and significant changes in upregulated proteins were that ox-LDL not only affected the levels of intracellular cathepsins G, Z, D and S, but also significantly enhanced cathepsin S secretion by the treated cells. Our results may provide clues for a more comprehensive understanding the pathogenesis of atherosclerosis.     

Recognition and detection of 8-oxo-rG in RNA using the DNA/OMeRNA chimera probes containing fluorescent adenosine-diazaphenoxazine analog

Recent studies indicate that oxidative damage to RNA results in dysfunction of translation and eventual pathogenesis. A representative oxidized base in RNA is 8-hydroxyguanosine (8-oxo-rG), however, unlike its DNA counterpart (8-oxo-dG), its role in pathogenesis has not attracted much attention until recently. The 2′-deoxyadenosine derivative with a diazaphenoxazine skeleton at the 6-amino group (Adap) was shown to be selective for 8-oxo-dG in DNA. In this study, the 2′-O-methoxy derivative of Adap (2′-OMeAdap) was designed as a selective molecule for 8-oxo-rG in RNA. 8-Oxo-rG in the homopurine RNA was selectively recognized by the ODN probe incorporating Adap. In contrast, although it was not possible by the Adap-containing ODN prove due to the instability of the corresponding duplex, 8-oxo-rG in homopyrimidine RNA was selectively detected by the 2′-OMeRNA probe incorporating 2′-OMeAdap.     

Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Interleukin-10 down-regulates oxLDL induced expression of scavenger receptor A and Bak-1 in macrophages derived from THP-1 cells

Here, we investigated the therapeutic potential of IL-10 by testing its effects on oxLDL-induced lipoprotein uptake and apoptosis by flow cytometry in THP-1-derived macrophages. The mRNA and protein expressions of lipid scavenger receptors (SR-A, CD36) and apoptosis-related proteins (Bcl-2, Bak-1) were also detected. Co-incubation of oxLDL with IL-10 reduced DiI-oxLDL uptake by 16.1 ± 3.8%, 35.2 ± 3.8% and 28.9 ± 1.8% at 6, 12 and 24 h of treatment, respectively. Furthermore, treatment with oxLDL for 24 h enhanced the SR-A mRNA and protein expressions by 89.3 ± 17.1% and 70.1 ± 17.6%, respectively. IL-10 abrogated the oxLDL-induced SR-A mRNA expression by 50.2 ± 3.9% and its protein by 45.6 ± 1.9%. Meanwhile IL-10 had no effect on the oxLDL-induced increase of CD36 expression. IL-10 inhibited the oxLDL-induced cell apoptosis in a time-dependent manner by 17.3 ± 3.3%, 36.4 ± 2.8% and 31.0 ± 4.3% at 6, 12 and 24 h, respectively. OxLDL increased Bak-1 mRNA and protein expressions by 38.4 ± 13.3% and 36.9 ± 12.1%, respectively. However co-stimulation of oxLDL with IL-10 for 24 h inhibited Bak-1 expression to 28.4 ± 7.2% (mRNA) and 25.7 ± 6.3% (protein). Meanwhile, IL-10 had no effect on the oxLDL-induced decrease of Bcl-2 expression. Our findings suggested that IL-10 reduced the oxLDL-induced lipoprotein uptake and apoptosis partly via down-regulating the oxLDL-induced expression of SR-A and Bak-1 in THP-1-derived macrophages.     

Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes

It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFalpha and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2', 7'-dichlorofluorescin (DCFH) to 2', 7'-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (p<0,001) while aqueous extract (50 microg/ml) by 43% (p<0,01). The ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production in monocytes by 76% (p<0,01). Effective concentrations (25-100 microg/ml) were well below the cytotoxic levels of the extracts which started at 1 mg/ml as assessed by LDH leakage and trypan blue exclusion. Penetration of some active substances into the cells was necessary for inhibition to take place as juged from the effect of preincubation time. These results demonstrate that artichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes.     

Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS

Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.     

氧化型低密度脂蛋白诱导血管平滑肌细胞凋亡的机理研究

近年来的研究发现,氧化型低密度脂蛋白(oxi-dizedlowdensitylipoprotein,OX-LDL)是导致动脉粥样硬化发生的重要因素[1].OX-LDL具有双重效应,既有强烈的促细胞生长效应,又可诱导细胞发生凋亡.这主要根据过氧化物量的变化而定,少量的OX-LDL可促进增殖,而长时间大量的OX-LDL作用于平滑肌细胞则可导致其凋亡[2].OX-LDL诱导的平滑肌细胞凋亡有助于氧化脂质的生成,导致动脉粥样硬化形成.在动脉粥样硬化晚期,由于斑块中的平滑肌细胞凋亡,细胞外基质分泌减少,使斑块极不稳定而易于破裂,诱发急性临床事件如心肌梗塞、猝死等的发生[3].OX-…     

Detection by whole genome microarrays of a spontaneous 126-gene deletion during construction of a ytfE mutant: confirmation that a ytfE mutation results in loss of repair of iron-sulfur centres in proteins damaged by oxidative or nitrosative stress

We show that genomic hybridization allows detection of a spontaneous secondary deletion of 126 genes that occurred during construction of an Escherichia coli ytfE mutant, LMS4209, explaining some of its unexpected growth defects. We confirm that YtfE is required to repair damage to iron-sulfur centres and for hydrogen peroxide resistance.     

Direct observation of the distribution of fluorescent probes in phosphatidylcholine/cholesterol vesicles using flow microfluorometry

The technique of flow microfluorometry has been extended to the study of small lipid complexes to assess either the lipid (hydrophobic) or aqueous (hydrophilic) compartments of selected natural or model membrane systems. sn-1-Palmitoyl-sn-2-oleoyl-phosphatidylcholine/cholesterol unilamellar vesicles, averaging 268 nm in diameter and containing varying concentrations of the synthetic lipophile probe, sn-1-palmitoyl-sn-2-12-[N-4-nitrobenzo-2-oxa-1,3-diazole]-aminocaproyl-phosphatidylcholine (NBD-PC), were analyzed using an Ortho Series 50-H Cytofluorograf and an Ortho 2150 computer system. NBD-labeled vesicles were analyzed for green fluorescence and the intensity of scattered light, the later being analyzed both at low angle (2–5°) and at 90° to the incident beam. At the high amplification required for vesicle detection, background signals from the sheath buffer, nonspecific laser light, and electronic noise were observed. However, this background noise signal was removed by appropriately setting a discriminator window. Profiles of signals falling within this region were then constructed. For the settings selected, more than 98% of data recorded could be attributed to observations on vesicles. Size information from the intensity of scattered light was obtained by comparison of the sample with fluorescent microspheres after correcting for the particle-scattering function difference between hollow and solid spheres and for refractive index differences. Additionally, cytograms and profiles were constructed for vesicles containing 5 m 6-carboxyfluorescein, 3′,6′-dihydroxy-3-oxospiro(isobenzofuran-1 (3H),9′-(9H)xanthen)-6-carboxylic acid, trapped in the aqueous core. Thus, the utility of flow microfluorometry has been extended to much smaller particle populations than studied previously by this technique. It has significant potential for studying several important properties of selected populations of vesicles and lipoproteins including (i) the size and fluorescence distribution of particles, (ii) the equilibrium distribution of probes among different size populations and among different domains within populations, (iii) the time dependence of probe transfer from a specific labeled population to a specific unlabeled population, (iv) the time dependence of vesicle fusion (combining aqueous compartments), and (v) sorting particles which are labeled differently.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

Characterization of the lipid and protein organization in HBsAg viral particles by steady-state and time-resolved fluorescence spectroscopy

Hepatitis B surface antigen (HBsAg) particles, produced in the yeast Hansenula polymorpha, are 20 nm particles, composed of S surface viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing, we further characterized them by fluorescence techniques. Fluorescence correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 S proteins per particle. The S proteins were characterized through the intrinsic fluorescence of their thirteen Trp residues. Fluorescence quenching and time-resolved fluorescence experiments suggest the presence of both low emissive embedded Trp residues and more emissive Trp residues at the surface of the HBsAg particles. The low emission of the embedded Trp residues is consistent with their close proximity in alpha-helices. Furthermore, S proteins exhibit restricted movement, as expected from their tight association with lipids. The lipid organization of the particles was studied using viscosity-sensitive DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg particles were found to be composed of an ordered rigid lipid interface, probably organized as a phospholipid monolayer, and a more hydrophobic and fluid inner core, likely composed of triglycerides and free fatty acids. However, the lipid core of HBsAg particles was substantially more polar than the LDL one, probably due to its larger content in proteins and its lower content in sterols. Based on our data, we propose a structural model for HBsAg particles where the S proteins deeply penetrate into the lipid core.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.