BACKGROUND

Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF.

RESULTS

Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)-induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased approximately equal 4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers <em>3</em>'-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (<em>SOD</em>) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase <em>3</em> knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF.

CONCLUSIONS

Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.

OBJECTIVE

The mRNA levels of antioxidant enzymes, matrix metalloproteinases, cathepsin V/L2, and tissue inhibitor of matrix metalloproteinases (TIMPs) were determined in keratoconus and normal corneas. Protein levels or enzyme activities were analyzed when RNA levels were different.

METHODS

A total of 25 physiologic (normal) and <em>3</em>2 keratoconus corneas were studied. mRNAs were analyzed by semiquantitative reverse transcription-polymerase chain reaction and Southern blot analysis. Proteins were assessed by immunohistochemistry and/or Western blot analysis. Catalase activity was measured in corneal extracts. Antioxidant enzymes examined were catalase, superoxide dismutase (<em>SOD</em>)-1, <em>SOD</em><em>3</em>, glutathione reductase, glutathione S-transferase and aldehyde dehydrogenase <em>3</em>A1. Degradative enzymes examined were cathepsin V/L2 and matrix metalloproteinase (MMP)-1, -2, -7, -9, and -14. Tissue inhibitor of matrix metalloproteinase (TIMP)-1, -2, and -<em>3</em> were also examined.

RESULTS

Keratoconus corneas exhibited a 2.2-fold increase of catalase mRNA level (P < 0.01) and 1.8-fold of enzyme activity (P < 0.0<em>3</em>); a 1.5-fold increase of cathepsin V/L2 mRNA (P < 0.0<em>3</em>) and abnormal protein distribution; and a 1.8-fold decrease of TIMP-1 mRNA (P < 0.05) and 2.8-fold decrease of protein (P < 0.0001) compared with normal (physiologic) corneas. RNA levels for other antioxidant and degradative enzymes were similar between normal and keratoconus corneas.

CONCLUSIONS

Keratoconus corneas have elevated levels of cathepsins V/L2, -B, and -G, which can stimulate hydrogen peroxide production, which, in turn, can upregulate catalase, an antioxidant enzyme. In addition, decreased TIMP-1 and increased cathepsin V/L2 levels may play a role in the matrix degradation that is a hallmark of keratoconus corneas. The findings support the hypothesis that keratoconus corneas undergo oxidative stress and tissue degradation.

(-)-Epigallocatechin-<em>3</em>-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. Nevertheless, the dose-response relationship of the inhibitory activity in vivo has not been systematically characterized. The present studies were conducted to address these issues, as well as the involvement of reactive oxygen species (ROS), in the inhibitory action of EGCG in vivo and in vitro. We characterized the inhibitory actions of EGCG against human lung cancer H1299 cells in culture and in xenograft tumors. The growth of tumors was dose dependently inhibited by EGCG at doses of 0.1, 0.<em>3</em> and 0.5% in the diet. Tumor cell apoptosis and oxidative DNA damage, assessed by the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and phosphorylated histone 2A variant X (gamma-H2AX), were dose dependently increased by EGCG treatment. However, the levels of 8-OHdG and gamma-H2AX were not changed by the EGCG treatment in host organs. In culture, the growth of viable H1299 cells was dose dependently reduced by EGCG; the estimated concentration that causes 50% inhibition (IC(50)) (20 microM) was much higher than the IC(50) (0.15 microM) observed in vivo. The action of EGCG was mostly abolished by the presence of superoxide dismutase (<em>SOD</em>) and catalase, which decompose the ROS formed in the culture medium. Treatment with EGCG also caused the generation of intracellular ROS and mitochondrial ROS. Although EGCG is generally considered to be an antioxidant, the present study demonstrates the pro-oxidative activities of EGCG in vivo and in vitro in the described experimental system.

Chronic systemic exposure of mice, rats, and Drosophila to D-galactose causes the acceleration of senescence and has been used as an aging model. The underlying mechanism is yet unclear. To investigate the mechanisms of neurodegeneration in this model, we studied cognitive function, hippocampal neuronal apoptosis and neurogenesis, and peripheral oxidative stress biomarkers, and also the protective effects of the antioxidant R-alpha-lipoic acid. Chronic systemic exposure of D-galactose (100 mg/kg, s.c., 7 weeks) to mice induced a spatial memory deficit, an increase in cell karyopyknosis, apoptosis and caspase-<em>3</em> protein levels in hippocampal neurons, a decrease in the number of new neurons in the subgranular zone in the dentate gyrus, a reduction of migration of neural progenitor cells, and an increase in death of newly formed neurons in granular cell layer. The D-galactose exposure also induced an increase in peripheral oxidative stress, including an increase in malondialdehyde, a decrease in total anti-oxidative capabilities (T-AOC), total superoxide dismutase (T-<em>SOD</em>), and glutathione peroxidase (GSH-Px) activities. A concomitant treatment with lipoic acid ameliorated cognitive dysfunction and neurodegeneration in the hippocampus, and also reduced peripheral oxidative damage by decreasing malondialdehyde and increasing T-AOC and T-<em>SOD</em>, without an effect on GSH-Px. These findings suggest that chronic D-galactose exposure induces neurodegeneration by enhancing caspase-mediated apoptosis and inhibiting neurogenesis and neuron migration, as well as increasing oxidative damage. In addition, D-galactose-induced toxicity in mice is a useful model for studying the mechanisms of neurodegeneration and neuroprotective drugs and agents.

Superoxide dismutase (<em>SOD</em>) is an enzyme that detoxifies superoxide (O2.-), a potentially toxic oxygen-derived species. Attempts to increase intracellular concentrations of <em>SOD</em> by direct application are complicated because <em>SOD</em>, being a relatively large molecule, does not readily cross cell membranes. We have identified a set of stable nitroxides that possess <em>SOD</em>-like activity, have the advantage of being low molecular weight, membrane permeable, and metal independent, and at pH 7.0 have reaction rate constants with O2.- ranging from 1.1 x 10(<em>3</em>) to 1.<em>3</em> x 10(6) M-1 s-1. These <em>SOD</em> mimics protect mammalian cells from damage induced by hypoxanthine/xanthine oxidase and H2O2, although they exhibit no catalase-like activity. In addition, the nitroxide <em>SOD</em> mimics rapidly oxidize DNA-FeII and thus may interrupt the Fenton reaction and prevent formation of deleterious OH radicals and/or higher oxidation states of metal ions. Whether by <em>SOD</em>-like activity and/or interception of an electron from redox-active metal ions they protect cells from oxidative stress and may have use in basic and applied biological studies.

Aerobic cells adjust the expression of antioxidant enzymes to maintain reactive oxygen species within tolerable levels. In addition, phosphatidylinositol <em>3</em> kinase (PI<em>3</em>K) and its downstream protein kinase effector Akt adapt cells to survive in the presence of oxidative stress. Here we provide evidence for an association between these two defense systems via transcriptional regulation of Cu/Zn-superoxide dismutase (Cu/Zn-<em>SOD</em>). PC12 pheochromocytoma cells expressing active Akt1 exhibit lower ROS levels in response to hydrogen peroxide, as determined with the superoxide-sensitive probe hydroethidine. Transfection of constitutive or 4-hydroxytamoxifen-inducible versions of Akt1 results in higher messenger RNA and protein levels of Cu/Zn-<em>SOD</em>. Luciferase reporter constructs, carrying different length fragments of the human sod1 gene promoter, have identified a region between -552 and -<em>3</em>55 that is targeted by PI<em>3</em>K and Akt and that contains a putative site of regulation by nuclear factor-kappaB (NF-kappaB). Nerve growth factor (NGF) and Akt augment the transactivating activity and produce higher nuclear levels of p65-NF-kappaB. Electrophoretic mobility shift assays indicate that the putative NF-kappaB regulatory sequence binds p65-NF-kappaB more efficiently in nuclear extracts from these cells. A dominant-negative mutant of IkappaBalpha further demonstrates that the PI<em>3</em>K/Akt axis targets the sod1 promoter at the level of the newly characterized NF-kappaB site. These results illustrate a new mechanism by which the PI<em>3</em>K/Akt pathway protects cells against oxidative stress, involving the upregulation of Cu/Zn-<em>SOD</em> gene expression, and the results identify NF-kappaB as a key mediator in the regulation of this gene.

Inhibition of either the insulin-like or target of rapamycin (TOR) pathways in the nematode Caenorhabditis elegans extends life span. Here, we demonstrate that starvation and inhibition of the C. elegans insulin receptor homolog (daf-2) elicits a daf-16-dependent up-regulation of a mitochondrial superoxide dismutase (<em>sod</em>-<em>3</em>). We also find that although heat and oxidative stress result in nuclear localization of the DAF-16 protein, these stressors do not activate a <em>SOD</em>-<em>3</em> reporter, suggesting that nuclear localization alone may not be sufficient for transcriptional activation of DAF-16. We show that inhibition of either TOR activity or key components of the cognate translational machinery (eIF-4G and EIF-2B homologs) increases life span by both daf-16-dependent and -independent mechanisms. Finally, we demonstrate that at least one nematode hexokinase is localized to the mitochondria. We propose that the increased life spans conferred by alterations in both the TOR and insulin-like pathways function by inappropriately activating food-deprivation pathways.

A freely available Windows-based program, RNSim1A, is utilized to predict metal-independent reactive nitrogen species (RNS) chemistry (oxidation, nitrosation, and nitration) under simulated biological conditions and make the following specific predictions. (1) The peak in oxidative reactions that occurs in vitro with 1:1 fluxes of (*)NO and O(2)(*)(-) does not occur under biological conditions. (2) By far, the quantitatively dominant (92-99.6%) process in vivo is oxidation, compared to nitrosation and nitration. (<em>3</em>) Only five of the many possible RNS reactions involving thiol (glutathione, GSH) and tyrosine are quantitatively important biologically. (4) Under inflammatory conditions, approximately 1% of O(2)(*)(-) reacts with (*)NO to produce ONOO(-), with the remainder reacting with <em>SOD</em>. (5) The dominant reaction of tyrosyl radical is a radical swap with GSH, producing the glutathiyl radical and regenerating tyrosine. (6) Nitrosothiol is formed virtually exclusively via radical recombination (RS(*) + (*)NO) as opposed to reaction with nitrous anhydride (N(2)O(<em>3</em>)). (7) Nitrosothiol is an intermediate, not an endproduct, and responds dynamically to changes in the immediate chemical environment. (8) The formation of a nitroso group on a particular thiol can be considered a marker of increased reactivity of that thiol, and it is likely that other modifications of that thiol (oxidation, glutathiolation) are more abundant than nitrosation and may be the functionally significant modification. (9) Specific chemical mechanisms are proposed for posttranslational protein modification via nitrosation, nitration, glutathiolation, and also dithiol/disulfide exchange, with important roles for the thiolate anion and O(2) (suggesting possible mechanisms for O(2) sensing) and variable degrees of exposure of cysteine thiol and tyrosine phenolate. (10) Patterns of reactivity are similar for low (20 nM) and high (500 nM) steady-state levels of NO. (11) The dominant reactions are those involving reactants at the highest concentrations (CO(2), thiol, O(2)). Because of the dominance of oxidative processes caused by RNS, the term nitroxidative stress is proposed, emphasizing the oxidative (as opposed to nitrosative or nitrative) stress that dominates RNS actions under biological conditions.

We have isolated and characterized over 10,000 bp of the human EC <em>SOD</em> gene (<em>SOD</em><em>3</em> or EC 1.15.1.1) and its 5'- and <em>3</em>'-flanking regions. Human genomic Southern blot analysis supports the existence of a single gene, without evidence for pseudogenes. The human EC <em>SOD</em> gene spans approximately 5900 bp. The gene can be divided into <em>3</em> exons and 2 introns. The 720-bp coding region is uninterrupted and located within exon <em>3</em>. The 560 bp 5' to the transcription start site were sequenced. No obvious TATA box was identified. A variety of conserved cis elements were identified by database searching. Exon <em>3</em> is surrounded by an Alu-J repetitive element in reverse orientation at the 5' and by an Alu-Sx repetitive element in the <em>3</em>'-flanking DNA. The relative levels of EC <em>SOD</em> tissue-specific expression were determined by RNA gel blot analysis. Adult heart, placenta, pancreas, and lung had the most expression, followed by kidney, skeletal muscle, and liver. Little EC <em>SOD</em> message was found in the brain. A second unique mRNA, approximately 4.2 kb in length, was highly expressed in skeletal muscle. When tissue enzyme activity is compared to relative mRNA levels, there is a marked disparity in the brain, pancreas, and lung, suggesting that these tissues have enhanced affinity for circulating EC <em>SOD</em> or translate the EC <em>SOD</em> message more efficiently than other tissues. These results indicate that the EC <em>SOD</em> gene contains unique transcriptional regulatory elements and that its expression may be regulated at the post-transcriptional or post-translational level. The characterization of the human EC <em>SOD</em> gene should now allow the development of further insights into its biology and provide the basis for studies of its role in human heritable disorders.

Oxidative stress damage plays a vital role in cerebral ischemia/reperfusion (I/R) pathogenesis. The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway can be activated by pharmacological and dietary means to attenuate cellular oxidative stress. Resveratrol, a plant-derived polyphenolic compound, has antioxidant property. Recent studies have demonstrated that resveratrol has protective effects against cerebral I/R injury. However, little is known about its mechanism. Hence, this study identified the neuroprotective effect of resveratrol pretreatment and elucidate the Nrf2/ARE signaling mechanism after focal cerebral I/R injury in rats. Adult male Sprague-Dawley rats were randomly assigned to sham-operated group, ischemia/reperfusion physiological saline-treated group, and ischemia/reperfusion resveratrol-pretreatmented (15 and <em>3</em>0 mg/kg) groups. Rats were pretreatmented with resveratrol or physiological saline of corresponding volume administered intraperitoneally for 7 days before surgery and <em>3</em>0 min before middle cerebral artery occlusion. At 24 h after reperfusion, neurological score, infarct volume, and brain water content were assessed. Oxidative stress was evaluated by malondialdehyde (MDA) levels and superoxide dismutase (<em>SOD</em>) activity. Pathological changes of brain tissue were observed by HE staining. RT-PCR and Western blot analysed the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). TUNEL staining detected apoptotic cells. The protein expression of Caspase-<em>3</em> were studied by immunohistochemistry. Resveratrol pretreatment significantly ameliorated neurological scores, reduced infarct volume and brain water content, decreased MDA levels, restored the <em>SOD</em> activity, upregulated the protein and mRNA expression of Nrf2 and HO-1, downregulated the protein expression of caspase-<em>3</em>. TUNEL-positive cells significantly decreased compared with the physiological saline-treated group. HE staining also showed that resveratrol significantly improved neuronal injury. These results showed that resveratrol pretreatment had neuroprotective effects on cerebral I/R injury. This neuroprotective effect is likely exerted by upregulated expression of transcription factor Nrf2 and HO-1 to ameliorate oxidative damage, decreased the protein expression of caspase-<em>3</em>. Our finding is important for understanding the neuroprotective mechanism of resveratrol and promoting its clinical therapeutic utility.

Syncytial cells in soybean (Glycine max cultivar [cv.] Peking) roots infected by incompatible and compatible populations of soybean cyst nematode (SCN [Heterodera glycines]) were collected using laser capture microdissection (LCM). Gene transcript abundance was assayed using Affymetrix soybean GeneChips, each containing <em>3</em>7,744 probe sets. Our analyses identified differentially expressed genes in syncytial cells that are not differentially expressed in the whole root analyses. Therefore, our results show that the mass of transcriptional activity occurring in the whole root is obscuring identification of transcriptional events occurring within syncytial cells. In syncytial cells from incompatible roots at three dpi, genes encoding lipoxygenase (LOX), heat shock protein (HSP) 70, superoxidase dismutase (<em>SOD</em>) were elevated almost tenfold or more, while genes encoding several transcription factors and DNA binding proteins were also elevated, albeit at lower levels. In syncytial cells formed during the compatible interaction at three dpi, genes encoding prohibitin, the epsilon chain of ATP synthase, allene oxide cyclase and annexin were more abundant. By 8 days, several genes of unknown function and genes encoding a germin-like protein, peroxidase, LOX, GAPDH, <em>3</em>-deoxy-D-arabino-heptolosonate 7-phosphate synthase, ATP synthase and a thioesterase were abundantly expressed. These observations suggest that gene expression is different in syncytial cells as compared to whole roots infected with nematodes. Our observations also show that gene expression is different between syncytial cells that were isolated from incompatible and compatible roots and that gene expression is changing over the course of syncytial cell development as it matures into a functional feeding site.

MnTBAP is often referred to as an <em>SOD</em> mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O2.- dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for <em>SOD</em> enzyme in a <em>SOD</em>-deficient Escherichia coli model, whereas MnTE-2-PyP-treated <em>SOD</em>-deficient E. coli grew as well as a wild-type strain. This <em>SOD</em>-specific system indicates that MnTBAP does not act as an <em>SOD</em> mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.<em>3</em> mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, <em>3</em>-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was <em>3</em><em>3</em>-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O2.- related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO2 adduct. The log kcat (O2.-) value for MnTBAP is estimated to be about <em>3</em>.16, which is approximately 5 and approximately 6 orders of magnitude smaller than the <em>SOD</em> activities of the potent <em>SOD</em> mimic MnTE-2-PyP and Cu,Zn-<em>SOD</em>, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the <em>SOD</em>-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only approximately 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the <em>SOD</em>-active impurities in the commercial MnTBAP sample (log k red (ONOO-) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is>> 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O2.-, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO-/CO<em>3</em>.- from O2.- pathways.

1. Free radicals may play an important role in several pathological conditions of the central nervous system (CNS) where they directly injure tissue and where their formation may also be a consequence of tissue injury. 2. Free radicals produce tissue damage through multiple mechanisms, including excito-toxicity, metabolic dysfunction, and disturbance of intracellular homeostasis of calcium. <em>3</em>. Oxidative stress can significantly worsen acute insults, such as ischemia, as well as chronic neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and Parkinson's disease. 4. For instance, recent findings suggest a causal role for chronic oxidative stress in familial ALS, as this disease is linked to missence mutations of the copper/zinc superoxide dismutase (<em>SOD</em>). 5. Thus, therapeutic approaches which limit oxidative stress may be potentially beneficial in several neurological diseases.

The C. elegans eat-<em>3</em> gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-<em>3</em> locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-<em>3</em> mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-<em>3</em> mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-<em>3</em> and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-<em>3</em> mutants. Instead, the phenotypes of eat-<em>3</em> mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-<em>3</em> mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase <em>sod</em>-2. We conclude that free radicals contribute to the pathology of C. elegans eat-<em>3</em> mutants.

Superoxide dismutase (<em>SOD</em>) rapidly scavenges superoxide (O2-) and also prolongs the vasorelaxant effects of nitric oxide (NO), thought to be the endothelium-derived relaxing factor. This prolongation has been ascribed to prevention of the reaction between O2- with NO. We report that <em>SOD</em> supports a reversible reduction of NO to NO-. When cyanamide and catalase were used to generate NO- in the presence of <em>SOD</em>, NO was measured by the conversion of HbO2 to MetHb. When <em>SOD</em>[Cu(I)] was exposed to NO anaerobically, NO- was trapped by MetHb forming nitrosylmyoglobin. When NO was generated by <em>3</em>-morpholinosydnonimine hydrochloride in the presence of <em>SOD</em>, NO- or a similar reductant was formed, which reduced catalase compound II and promoted the formation of the catalase [Fe(III)]-NO complex. It is, therefore, conceivable that <em>SOD</em> may protect NO and endothelium-derived relaxing factor by a mechanism in addition to O2- scavenging and that NO- may be a physiologically important form of endothelium-derived relaxing factor.

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and <em>3</em>.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-<em>SOD</em> and Mn-<em>SOD</em> was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase <em>3</em> activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.

BACKGROUND

Although recent studies indicate preeclampsia (PE) is associated with increased oxidative stress, the role of reactive oxygen species in the hypertension associated with PE remains unclear. We sought to test the hypothesis that placental ischemia increases oxidative stress which in turn, contributes to hypertension.

METHODS

Reduction in uterine perfusion pressure (RUPP) was induced by placing silver clips on the abdominal aorta and the ovarian arteries on day 14 of pregnancy. On day 20 of pregnancy, mean arterial pressure (MAP) was measured and oxidative stress was assessed in renal and placental tissues whereas systemic administration of tempol, a superoxide dismutase (SOD) mimetic, was used to evaluate the contribution of reactive oxygen species on RUPP-induced hypertension.

RESULTS

MAP (120 +/- 2 mm Hg vs.106 +/- 3 mm Hg), placental levels of 8-isoprostane (1.9 +/- 0.4 ng/g tissue vs. 0.8 +/- 0.1 ng/g tissue), and malondialdehyde (MDA) (6.9 +/- 0.6 micromol/g tissue vs. 3.9 +/- 0.4 micromol/g tissue) were increased, whereas renal cortical SOD activity was decreased in RUPP rats (1.2 +/- 0.1 units/mg protein vs. 1.6 +/- 0.1 units/mg protein) at day 20 of gestation (20 dG) compared to controls. Chronic treatment with tempol attenuated the hypertension (RUPP + tempol 112 +/- 2 mm Hg vs. RUPP, 120 +/- 2 mm Hg) associated with RUPP, whereas tempol had no effect on MAP (NP, 106 +/- 3 vs. NP + tempol, 108 +/- 2) in control rats.

CONCLUSIONS

The results of this study indicate that placental ischemia decreases innate antioxidant activity resulting in elevated oxidative stress which appears to play a role in mediating hypertension associated with chronic RUPP in pregnant rats.

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), but radioresistance remains a serious obstacle to successful treatment in many cases. To identify the proteins involved in this resistance and to evaluate their potential for predicting NPC response to radiotherapy, we first established a radioresistant subclone cell line (CNE2-IR) derived from NPC cell line CNE2 by treating the cells with five rounds of sublethal ionizing radiation. Proteomics was then performed to compare the protein profiles of CNE2-IR and CNE2, and a total of <em>3</em>4 differential proteins were identified. Among them, 14-<em>3</em>-<em>3</em>sigma and Maspin were downregulated and GRP78 and Mn-<em>SOD</em> were upregulated in the radioresistant CNE2-IR compared with control CNE2, which was conformed by Western blot. Immunohistochemistry was performed to detect the expression of the four validated proteins in the <em>3</em>9 radioresistant and 51 radiosensitive NPC tissues and their value for predicting NPC response to radiotherapy were evaluated by receiver operating characteristic analysis. The results showed that the downregulation of 14-<em>3</em>-<em>3</em>sigma and Maspin and the upregulation of GRP78 and Mn-<em>SOD</em> were significantly correlated with NPC radioresistance and the combination of the four proteins achieved a sensitivity of 90% and a specificity of 88% in discriminating radiosensitive from radiaoresistant NPC. Furthermore, the resistance to ionizing radiation can be partially reversed by the overexpression of 14-<em>3</em>-<em>3</em>sigma in the CNE2-IR. The data suggest that 14-<em>3</em>-<em>3</em>sigma, Maspin, GRP78, and Mn-<em>SOD</em> are potential biomarkers for predicting NPC response to radiotherapy and their dysregulation may be involved in the radioresistance of NPC.

OBJECTIVE

Glucagon-like peptide 1 (GLP-1), one of the incretin hormones, has been reported to increase positive inotropic activity in cardiac myocytes and protect against myocardial injury. However, the effects upon endothelial cells and the mechanisms involved are not fully understood. We assessed the hypothesis that GLP-1 has protective effects against inflammation and oxidative stress on human endothelial cells.

RESULTS

The effects of the GLP-1 analog liraglutide upon TNF-α-induced injury of the human umbilical vein endothelial cells (HUVECs) were evaluated. First, ROS induced by TNF-α was measured by staining with CM-H(2)DCFDA. Intracellular ROS production of HUVECs was significantly decreased in a dose-dependent manner until <em>3</em>0 nM while liraglutide inhibited the induction of gp91(phox) and p22(phox), subunit of NADPH oxidase, by TNF-α. In addition, protein levels of <em>SOD</em>-2, catalase and GPx were significantly increased by liraglutide. Second, rapid translocation of PKC-α into the membrane following TNF-α was evident. Liraglutide significantly inhibited this very rapid TNF-α-induced translocation of PKC-α into membrane at 2.5 min. Third, liraglutide significantly inhibited NF-κB activation and upregulated I-κB family while phosphorylation of IKK-α/β, which is upstream of NF-κB signaling, was also downregulated after 15 min of TNF-α treatment. Finally, liraglutide inhibited apoptosis of HUVEC and expression of Pentraxin-<em>3</em> induced by TNF-α.

CONCLUSIONS

Liraglutide exerts marked anti-oxidative and anti-inflammatory effects on endothelial cells with inhibition of PKC-α, NADPH oxidase, NF-κB signaling and upregulation of protective anti-oxidative enzymes.

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (<em>SOD</em>) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -<em>3</em> and poly (ADP-ribose) polymerase were cleaved. On the basis that <em>SOD</em> inactivation triggers cell death and low <em>SOD</em> levels occur in asthma, we tested whether <em>SOD</em> inactivation plays a role in airway epithelial cell death. <em>SOD</em> inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of Mn<em>SOD</em> were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of <em>SOD</em> activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.

We tested the hypotheses that 1) muscles of old mice are more susceptible to injury than muscles of young and adult mice, and 2) secondary or delayed onset injury results from free radical damage. Extensor digitorum longus muscles were injured in situ by lengthening contractions. Injury was assessed by measurement of maximum isometric tetanic force (Po) expressed as a percentage of the control value and by morphological damage. Mice were treated with a free radical scavenger, polyethylene glycol-superoxide dismutase (PEG-<em>SOD</em>). Three days postinjury, the Po of 44% for muscles of nontreated old mice was significantly lower than the Po of 58 and 61% for those of young and adult mice. In each group, the secondary injury at <em>3</em> days was alleviated by treatment with PEG-<em>SOD</em>. For treated muscles of young, adult, and old mice, values for Po were 88, 80, and 70%, respectively. We conclude that muscles of old mice are more susceptible to injury than muscles of young or adult mice and that free radicals contribute to the secondary or delayed onset injury.

1. This study investigates the role of nitric oxide (NO) and reactive oxygen species (ROS) on endothelial function of pulmonary arteries in a mice model of hypoxia-induced pulmonary hypertension. 2. In pulmonary arteries from control mice, the NO-synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) potentiated contraction to prostaglandin F2alpha (PGF2alpha) and completely abolished relaxation to acetylcholine. In extrapulmonary but not intrapulmonary arteries, acetylcholine-induced relaxation was slightly inhibited by polyethyleneglycol-superoxide dismutase (PEG-<em>SOD</em>) or catalase. <em>3</em>. In pulmonary arteries from hypoxic mice, ROS levels (evaluated using dihydroethidium staining) were higher than in controls. In these arteries, relaxation to acetylcholine (but not to sodium nitroprusside) was markedly diminished. L-NAME abolished relaxation to acetylcholine, but failed to potentiate PGF2-induced contraction. PEG-<em>SOD</em> or catalase blunted residual relaxation to acetylcholine in extrapulmonary arteries, but did not modify it in intrapulmonary arteries. Hydrogen peroxide elicited comparable (L-NAME-insensitive) relaxations in extra- and intrapulmonary arteries from hypoxic mice. 4. Exposure of gp91phox(-/-) mice to chronic hypoxia also decreased the relaxant effect of acetylcholine in extrapulmonary arteries. However, in intrapulmonary arteries from hypoxic gp91phox(-/-) mice, the effect of acetylcholine was similar to that obtained in mice not exposed to hypoxia. 5. Chronic hypoxia increases ROS levels and impairs endothelial NO-dependent relaxation in mice pulmonary arteries. Mechanisms underlying hypoxia-induced endothelial dysfunction differ along pulmonary arterial bed. In extrapulmonary arteries from hypoxic mice, endothelium-dependent relaxation appears to be mediated by ROS, in a gp91phox-independent manner. In intrapulmonary arteries, endothelial dysfunction depends on gp91phox, the latter being rather the trigger than the mediator of impaired endothelial NO-dependent relaxation

Intrarenal injection of radiocontrast medium (RCM) results in transient vasoconstriction and a persistent decline in glomerular filtration rate (GFR). Adenosine modulates this vasoconstrictor response and is postulated to increase oxygen free radical (OFR) generation. We hypothesized that the persistent decline in (GFR that follows RCM administration results in an increased generation of OFR. We evaluated the effects of RCM injection on renal blood flow, inulin clearance, hypoxanthine, xanthine, and malondialdehyde concentrations in four groups of non-volume-expanded, pentobarbital sodium anesthetized dogs in the presence and absence of intravenous allopurinol, 25 mg/min (group 1), intrarenal superoxide dismutase (<em>SOD</em>), 400 U/min (group 2), heat-inactivated intrarenal <em>SOD</em>, 400 U/min (group <em>3</em>), and simultaneous infusions of intrarenal <em>SOD</em>, 400 U/min, to one kidney and saline to the other (group 4). Both allopurinol and <em>SOD</em> significantly attenuated the fall in GFR after RCM administration over control. Malondialdehyde concentrations were attenuated over control in all treated groups, indicating a decrease in OFR generation. We conclude that intrarenal injection of RCM results in increased production of OFR. Inhibition of OFR production by allopurinol and increased OFR removal by <em>SOD</em> attenuates the effects of RCM on declines in GFR.

We investigated the importance of mitochondrial localization of the <em>SOD</em>2 (Mn<em>SOD</em>) transgene product for protection of <em>3</em>2D cl <em>3</em> hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/Zn<em>SOD</em>, <em>SOD</em>1) transgene, (2) the native <em>SOD</em>2 transgene, (<em>3</em>), the <em>SOD</em>2 transgene minus the mitochondrial localization leader sequence (Mn<em>SOD</em>-ML), and (4) the <em>SOD</em>2 mitochondrial leader sequence attached to the active portion of the <em>SOD</em>1 transgene (ML-Cu/Zn<em>SOD</em>) were transfected into <em>3</em>2D cl <em>3</em> cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/Zn<em>SOD</em>- and Mn<em>SOD</em>-ML-expressing clones had no increase in cellular radiation resistance (D0=0.89 +/- 0.01 and 1.08 +/- 0.02 Gy, respectively) compared to parent line <em>3</em>2D cl <em>3</em> (D0=1.15 +/- 0.11 Gy). In contrast, cell clones expressing either <em>SOD</em>2 or ML-Cu/Zn<em>SOD</em> were significantly radioresistant (D0=2.1 +/- 0.1 and 1.97 +/- 0.17 Gy, respectively). Mice injected intraesophageally with <em>SOD</em>2-plasmid/liposome (Mn<em>SOD</em>-PL) complex demonstrated significantly less esophagitis after <em>3</em>5 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/Zn<em>SOD</em>-PL or Mn<em>SOD</em>-ML-PL. Mice injected with intraesophageal ML-Cu/Zn<em>SOD</em>-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of <em>SOD</em> in the in vitro and in vivo protection of cells from radiation-induced cellular damage.