Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-alpha elicited significant increases in caspase-<em>3</em>/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment (10(-6)-10(-4) mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular H(2)O(2) levels and H(2)O(2)-mediated apoptotic cell death induced by oxidative stressors (exogenous H(2)O(2), paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of <em>SOD</em> isoforms. Resveratrol also effectively scavenged H(2)O(2) in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H(2)O(2) and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects.

Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We conducted a genome-wide RNAi screen to identify genes necessary for the extended life span of daf-2 mutants and identified approximately 200 gene inactivations that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase <em>sod</em>-<em>3</em> in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging.

BACKGROUND

Oxidative stress results in damage to cellular structures and has been linked to many diseases, including cancer. The authors sought to determine whether the expression of three major antioxidant enzymes, copper-zinc superoxide dismutase (<em>SOD</em>1), manganese superoxide dismutase (<em>SOD</em>2), and catalase, was altered in human prostate carcinoma and its likely precursor, high grade prostatic intraepithelial neoplasia (PIN). The level of reactive oxygen species damage was evaluated by measuring the expression of the DNA adduct 8-hydroxydeoxyguanosine.

METHODS

The authors evaluated the tissue expression of the antioxidant enzymes in prostate carcinoma by immunohistochemistry, immunogold electron microscopy, and enzymatic assay. The polymerase chain reaction was used to amplify and screen tissue specimens for the genes of <em>SOD</em>1, <em>SOD</em>2, and extracellular <em>SOD</em> (<em>SOD</em>3). Matched paraffin embedded tissue sections were evaluated by RNA in situ hybridization for expression of <em>SOD</em>1 and immunohistochemically for the DNA adduct 8-hydroxydeoxyguanosine.

RESULTS

All prostatic tissues, including cancer, displayed immunoreactivity for the three antioxidant enzymes in epithelial cells, with no staining of the stroma, inflammatory cells, or endothelial cells. The number of immunoreactive cells was greater in benign epithelium than in PIN and cancer for each enzyme. The mean percentage and intensity of immunoreactive cells was greatest for <em>SOD</em>2, intermediate for <em>SOD</em>1, and lower for catalase. Staining in cancer was heterogeneous. Immunogold ultrasound studies revealed strong mitochondrial labeling for <em>SOD</em>2, which was greater in benign epithelium than in cancer; <em>SOD</em>1 labeling was invariably weaker, with nuclear labeling in benign epithelium and cytoplasmic labeling in cancer cells. There was no difference in enzyme activity for the three antioxidant enzymes between benign epithelium and cancer. No mutations were found in the 5 exons of <em>SOD</em>1, 5 exons of <em>SOD</em>2, and 3 exons of <em>SOD</em>3, except for 3 of 20 cases with polymorphisms for exon 3 of <em>SOD</em>1. Intense nuclear immunoreactivity for 8-hydroxydeoxyguanosine was present in fewer than 3% of epithelial cells, with no apparent differences among benign epithelium, PIN, and cancer.

CONCLUSIONS

<em>SOD</em>1, <em>SOD</em>2, and catalase had lower expression in PIN and prostate carcinoma than in benign epithelium. The number of immunoreactive cells in PIN was similar to cancer, indicating that these are closely related. Enzyme activities were variable, with no difference between benign epithelial cells and cancer, although this lack of change in enzyme activity could have been due to the presence of contaminating benign cells within the cancer specimens. The results of reactive oxygen species damage were found only in the epithelium and not in the stroma. Expression of the DNA adduct 8-hydroxydeoxyguanosine was present in fewer than 3% of cells, with no apparent differences among benign epithelium, PIN, and cancer. These findings suggest that oxidative stress is an early event in carcinogenesis.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (<em>SOD</em>)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable <em>SOD</em>/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((<em>3</em>)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately <em>3</em>-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.

Mitochondria are a principal site for generation of reactive oxygen species (ROS) in the heart. Peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1 alpha) plays an important role in regulating mitochondrial biogenesis and myocardial metabolism, but whether PGC-1 alpha can simultaneously upregulate myocardial mitochondrial antioxidants has not been studied. In the present study, we examined the effect of PGC-1 alpha deficiency (PGC-1 alpha(-/-)) on oxidative stress and expression of a group of mitochondrial antioxidants in normal hearts and in hearts exposed to chronic systolic pressure overload produced by transverse aortic constriction (TAC). We found that PGC-1 alpha(-/-) caused moderate but significant decreases of myocardial mitochondrial antioxidant enzymes such as <em>SOD</em>2, and thioredoxin (Trx2), but had no effect on expression of myocardial oxidative stress markers and left ventricular (LV) function under basal conditions. However, in response to TAC for 6 weeks, PGC-1 alpha(-/-) mice showed greater increases of myocardial oxidative stress markers <em>3</em>'-nitrotyrosine and 4-hydroxynonenal, more severe LV hypertrophy and dilatation, pulmonary congestion, and a greater reduction of LV fractional shortening and dP/dt(max) than did wild-type hearts. <em>SOD</em> mimetic MnTMPyP treatment (6 mg/kg/day) significantly attenuated TAC-induced LV hypertrophy and dysfunction in PGC-1 alpha(-/-) mice. These data indicate that PGC-1 alpha plays an important role in regulating expression of myocardial mitochondrial antioxidants <em>SOD</em>2 and Trx2 and in protecting hearts against TAC-induced myocardial oxidative stress, hypertrophy, and dysfunction.

The bioactivity of nitric oxide (.NO) depends, in part, on its interaction with superoxide. Usually, superoxide dismutase (<em>SOD</em>) preserves .NO bioactivity by limiting the availability of superoxide. Ascorbic acid also effectively scavenges superoxide, but the extent to which this interaction is necessary for intact .NO bioactivity is not known. Therefore, the present study examined the effect of ascorbic acid on .NO bioactivity with isolated rabbit arterial segments. A steady flux of superoxide (1.15 to 2.<em>3</em> micromol . L-1 . min-1) produced either by pyrogallol autoxidation or a hypoxanthine/xanthine oxidase system inhibited endothelium-derived .NO-mediated arterial relaxation elicited by acetylcholine. This effect of superoxide was completely blocked by <em>SOD</em> (<em>3</em>00 IU/mL) and the manganese <em>SOD</em> mimic EUK-8 (<em>3</em>00 micromol/L) and partially inhibited by ascorbic acid (10 mmol/L). Lower concentrations of ascorbic acid were ineffective despite scavenging >90% of superoxide. We increased the endogenous flux of superoxide (<em>3</em>.2+/-0.<em>3</em>-fold) by inhibiting vascular copper-zinc <em>SOD</em> with diethyldithiocarbamate. This increased endogenous flux of superoxide produced an impairment of .NO-mediated arterial relaxation that was reversed by EUK-8 (<em>3</em>00 micromol/L) but not ascorbic acid (10 mmol/L) despite equivalent scavenging of the endogenous superoxide flux. We used <em>3</em>-nitrotyrosine formation (from peroxynitrite) as an indicator of .NO interaction with superoxide and found that <em>SOD</em> and EUK-8 compete more effectively with .NO for superoxide than does ascorbic acid. These data indicate that preservation of .NO bioactivity by superoxide scavengers depends not only on superoxide scavenging activity, but also on the rate of superoxide scavenging. Normal extracellular concentrations of ascorbic acid (<em>3</em>0 to 150 micromol/L) are not likely to prevent the interaction of .NO with superoxide under physiological conditions.

This article reviews the pathophysiologic concept that superoxide and hydrogen peroxide, generated by activated leukocytes, together with low-molecular-weight chelate iron derived from fecal sources and from denatured hemoglobin, amplify the inflammatory response and subsequent mucosal damage in patients with active episodes of ulcerative colitis. The putative pathogenic mechanisms reviewed are as follows: (1) Dietary iron is concentrated in fecal material owing to normally limited iron absorption. (2) Mucosal bleeding, characteristic of ulcerative colitis, as well as supplemental oral iron therapy for chronic anemia, further conspire to maintain or elevate mucosal iron concentration in colitis. (<em>3</em>) Fenton chemistry, driven especially by leukocyte-generated superoxide and hydrogen peroxide, leads to formation of hydroxyl radicals. (4) The resultant oxidative stress leads to the extension and propagation of crypt abscesses, either through direct membrane disruption by lipid peroxidation or through generation of secondary toxic oxidants such as chloramines. (5) Chemotactic products of lipid peroxidation, including 4-hydroxynonenal, provide positive feedback to accelerate this inflammatory/oxidative process, leading to acute exacerbations of the disease. (6) Other oxidized products, such as oxidized tryptophan metabolites, created by free radical mechanisms in or near the mucosa, may act as carcinogens or tumor promotors that contribute to the exceedingly high incidence of colon carcinoma in patients suffering from chronic ulcerative colitis. In this way, self-sustaining cycles of oxidant formation may amplify flare-ups of inflammation and mucosal injury in ulcerative colitis. This concept, if proved correct by subsequent research, would provide a rationale for several novel clinical approaches to the management of ulcerative colitis, including use of <em>SOD</em> mimetics, iron chelators, and chain-breaking antioxidants.

BACKGROUND

Oxidative stress is related to endothelial dysfunction (ED) and cardiovascular outcomes in patients with chronic kidney disease. Increased asymmetric dimethylarginine (ADMA) levels are among the main causes of ED. We aim to investigate any association between ED and ADMA levels, as well as levels of oxidative stress markers, in patients with chronic kidney disease.

METHODS

One hundred fifty-nine patients without diabetes with chronic kidney disease were studied. Staging was performed according to glomerular filtration rate, determined as stages 1 to 5 according to the Kidney Disease Outcomes Quality Initiative (n = 30, 33, 28, 32, and 36, respectively). The control group consisted of 30 healthy subjects. Oxidative stress markers (plasma malondialdehyde [MDA], erythrocyte superoxide dismutase [SOD], glutathione peroxidase [GSH-Px]), trace elements (erythrocyte zinc [EZn], erythrocyte copper [ECu]), plasma selenium (Se), and serum ADMA were studied. Brachial artery endothelium-dependent vasodilatation (FMD) was calculated for all.

RESULTS

FMD, SOD, GSH-Px, EZn, ECu, and Se values were lower, whereas MDA and ADMA levels were higher in patients than controls. Glomerular filtration rate correlated negatively with MDA and ADMA levels and positively with FMD, SOD, and GSH-Px values. These parameters were significantly different among patients with stages 2, 3, 4, and 5 (hemodialysis group; P < 0.001 for all). Regression analysis showed that ADMA (beta = -0.228; P < 0.01), SOD (beta = 0.405; P < 0.001), and oxidized low-density lipoprotein levels (beta = -0.428; P < 0.001) were related independently to FMD, whereas glomerular filtration rate was not involved in the model.

CONCLUSIONS

The present results imply that FMD, oxidative stress, and ADMA levels all are associated with stage of chronic kidney disease. Additionally, levels of oxidative stress markers and ADMA independently determine endothelial function.

The role of oxidative metabolism in the up-regulation/activation of stress-induciblesignaling pathways as well as induction of micronucleus formation in bystander cells was investigated. By immunoblotting and in situ immunofluorescence, active Cu-Zn superoxide dismutase (<em>SOD</em>) enzyme and active catalase enzyme were shown to inhibit the up-regulation of p21(Waf1) as well as the induction of micronucleus formation in bystander cells from confluent cultures of normal human diploid fibroblasts irradiated with 0.<em>3</em>-<em>3</em> cGy of alpha-particles. Enzyme activity assays indicated that exogenous <em>SOD</em> became significantly associated with the cells. Reactive oxygen species apparently derived from a flavin-containing oxidase enzyme [presumably an NAD(P)H-oxidase] appeared to be major contributors to the bystander-induced up-regulation of p5<em>3</em> and p21(Waf1) as well as micronucleus formation, as evidenced by the inhibition of these effects with diphenyliodonium. Rapid activation of nuclear factor kappaB, Raf-1, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p<em>3</em>8 mitogen-activated protein kinase and their downstream effectors activator protein 1, ELK-1, p90RSK, and activating transcription factor 2 was also observed in cultures exposed to very low fluences of alpha-particles. Significant attenuation in the activation of these kinases and transcription factors occurred in irradiated cultures treated with either <em>SOD</em> or catalase. Overall, these results support the hypothesis that superoxide and hydrogen peroxide produced by flavin-containing oxidase enzymes mediate the activation of several stress-inducible signaling pathways as well as micronucleus formation in bystander cells from cultures of human cells exposed to low fluences of alpha-particles.

The ortho, meta, and para isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-<em>3</em>-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (<em>SOD</em>) activity in vitro and in vivo. The impact of their interaction with DNA and RNA on the <em>SOD</em> activity in vivo and in vitro has also been analyzed. Differences in their behavior are due to the combined steric and electrostatic factors. In vitro catalytic activities are closely related to their redox potentials. The half-wave potentials (E1/2) are +0.220 mV, +0.052 mV, and +0.060 V versus normal hydrogen electrode, whereas the rates of dismutation (kcat) are 6.0 x 10(7), 4.1 x 10(6), and <em>3</em>.8 x 10(6) M-1 s-1 for the ortho, meta, and para isomers, respectively. However, the in vitro activity is not a sufficient predictor of in vivo efficacy. The ortho and meta isomers, although of significantly different in vitro <em>SOD</em> activities, have fairly close in vivo <em>SOD</em> efficacy due to their similarly weak interactions with DNA. In contrast, due to a higher degree of interaction with DNA, the para isomer inhibited growth of <em>SOD</em>-deficient Escherichia coli.

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO- acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (<em>SOD</em>) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO- formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO-, both c-Src and PI <em>3</em>-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI <em>3</em>-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-<em>3</em>-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and <em>SOD</em>, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and <em>SOD</em>. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

OBJECTIVE

In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the mechanisms are not completely elucidated. To elucidate the mechanisms of hepatocyte cell death induced by the ROS superoxide anions and hydrogen peroxide, primary cultures of hepatocytes were exposed to the superoxide anion donor menadione (10-50 micromol/L) or H2O2 (1-5 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors, superoxide dismutase (PEG-<em>SOD</em>) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9, -6, -<em>3</em> activation and cleaved PARP, and necrotic cell death by Sytox Green staining.

RESULTS

(1) Menadione (50 micromol/L) induces JNK phosphorylation, caspase-9, -6, -<em>3</em> activation, PARP cleavage and apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50 micromol/L) induces the phosphorylation of ERK1/2 and this attenuates cell death. (2) H2O2 increases necrotic cell death at high concentration or when H2O2 detoxification is impaired. H2O2 does not activate MAPKs signalling. (<em>3</em>) PEG-<em>SOD</em> prevents ERK1/2-, JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases menadione-induced apoptosis. (4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death.

CONCLUSIONS

In normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on JNK activity and totally abolished by superoxide scavengers.

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (<em>SOD</em>+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea <em>SOD</em> isoform (<em>SOD</em>-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but <em>SOD</em>+ plants retained higher photosynthetic rates than <em>SOD</em>- plants at all developmental stages tested. Not surprisingly, <em>SOD</em>+ plants had approximately <em>3</em>-fold higher <em>SOD</em> specific activity than <em>SOD</em>- plants. However, <em>SOD</em>+ plants also exhibited a <em>3</em>- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both <em>SOD</em>+ and <em>SOD</em>- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn <em>SOD</em> II can compensate for the increased levels of <em>SOD</em> with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in <em>SOD</em>+ plants could result not only from increased <em>SOD</em> levels but from the combined increases in <em>SOD</em> and APX activity.

We examined the effects of a variety of ligands/activators of the peroxisome proliferator-activated receptor (PPAR) on the expression of the superoxide scavenger enzyme, Cu2+,Zn2+-superoxide dismutase (CuZn-<em>SOD</em>), and the superoxide generating enzyme nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase in primary cultures of human umbilical vein endothelial cells (HUVEC) and human aorta endothelial cells (HAEC). Our data show that <em>3</em> types of PPARs, PPARalpha, PPARbeta/delta/Nuc1, and PPARgamma are expressed in endothelial cells. Bezafibrate, which is a ligand/activator for PPARalpha, increased the CuZn-<em>SOD</em> gene expression and protein levels in endothelial cells. Troglitazone and pioglitazone, which are ligands/activators for PPARgamma, also induced PPARalpha gene and protein expression and increased CuZn-<em>SOD</em> gene expression and protein levels in addition to increasing PPARgamma gene and protein expression in endothelial cells. Moreover, with treatment of monounsaturated and polyunsaturated fatty acids (PUFA), the CuZn-<em>SOD</em> mRNA levels were positively correlated with PPARalpha mRNA levels (r = .872, P < .0001) in primary endothelial cells. In addition, the phorbol myristate acetate (PMA)-stimulated or PMA-nonstimulated 22-kd a-subunit (p22phox) mRNA levels and 47-kd a-subunit (p47phox) protein levels in NADPH oxidase were decreased by treatment with PPARalpha and PPARgamma ligands/activators. These results suggest that PPARalpha and PPARgamma gene and protein expression in endothelial cells may play a physiologic role as radical scavengers, although the details of these mechanisms remain to be established.

Clinically significant elevations in the expression of manganese superoxide dismutase (<em>Sod</em>2) are associated with an increased frequency of tumor invasion and metastasis in certain cancers. The aim of this study was to examine whether increases in <em>Sod</em>2 activity modulate the migratory potential of tumor cells, contributing to their enhanced metastatic behavior. Overexpression of <em>Sod</em>2 in HT-1080 fibrosarcoma cells significantly enhanced their migration 2-fold in a wound healing assay and their invasive potential <em>3</em>-fold in a transwell invasion assay. Severity of invasion was directly correlated to <em>Sod</em>2 expression levels and this invasive phenotype was similarly observed in 25<em>3</em>J bladder tumor cells, in which <em>Sod</em> expression resulted in a <em>3</em>-fold increase in invasion compared with controls. Further, migration and invasion of the <em>Sod</em>2-expressing cells was inhibited following overexpression of catalase, indicating that the promigratory/invasive phenotype of <em>Sod</em>2-expressing cells is H(2)O(2) dependent. <em>Sod</em>2 overexpression was associated with a loss of vinculin-positive focal adhesions that were recovered in cells coexpressing catalase. Tail vein injections of <em>Sod</em>2-GFP-expressing HT-1080 cells in NCR nude mice led to the development of pulmonary metastatic nodules displaying high <em>Sod</em>2-GFP expression. Isolated tumors were shown to retain high <em>Sod</em>2 activity in culture and elevated levels of the matrix degrading protein matrix metalloproteinase-1, and a promigratory phenotype was observed in a population of cells growing out from the tumor nodule. These findings suggest that the association between increased <em>Sod</em>2 activity and poor prognosis in cancer can be attributed to alterations in their migratory and invasive capacity.

We successfully established an oxidant damage of mimetic aging model using mice induced by D-galactose, and the mimetic aging model is relative to free radical and the accumulation of waste substances in metabolism. The animals were divided into <em>3</em> groups: (1) phosphate-buffered saline (PBS); (2) 1% D-galactose; (<em>3</em>) 5% D-galactose by subcutaneous injection every day. After 45 days, mice treated with D-galactose showed a significant increase in the malondialdehyde (MDA), total antioxidant status (TAS) and a decrease in superoxide dismutase (<em>SOD</em>) in the blood compared with the PBS group. In the brain, the D-galactose treated mice exhibited a higher level MDA and a lower level <em>SOD</em> activity. In the liver, only the 5% D-galactose group indicated a significant increase in MDA. By reference to the oxidative biomarkers in blood, brain and liver, we have confirmed the reliability of the mimetic aging model.

Two antioxidative strains tentatively identified as Lactobacillus fermentum, E-<em>3</em> and E-18, were isolated from intestinal microflora of a healthy child. Survival time of these strains in the presence of reactive oxygen species (ROS), like hydrogen peroxide, superoxide anions and hydroxyl radicals, was significantly increased compared with a non-antioxidative strain, and also was quite similar to a highly ROS resistant strain of Salmonella typhimurium. E-<em>3</em> and E-8 contain a remarkable level of glutathione, express Mn-<em>SOD</em>, which is important for the prevention of lipid peroxidation, and secrete hydrogen peroxide. Their significant antimicrobial activity combined with antioxidative properties may serve as defensive principles in the intestinal microbial ecosystem and overcome exo- and endogenous oxidative stress.

Tanshinone IIA (tan), a derivative of phenanthrenequinone, is one of the key components of Salvia miltiorrhiza Bunge. Previous reports showed that tan inhibited the apoptosis of cultured PC12 cells induced by serum withdrawal or ethanol. However, whether tan has a cardioprotective effect against apoptosis remains unknown. In this study, we investigated the effects of tan on cardiac myocyte apoptosis induced both by in vitro incubation of neonatal rat ventricular myocytes with H(2)O(2) and by in vivo occlusion followed by reperfusion of the left anterior descending coronary artery in adult rats. In vitro, as revealed by <em>3</em>-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, treatment with tan prior to H(2)O(2) exposure significantly increased cell viability. Tan also markedly inhibited H(2)O(2)-induced cardiomyocyte apoptosis, as detected by ladder-pattern fragmentation of genomic DNA, chromatin condensation, and hypodioloid DNA content. In vivo, tan significantly inhibited ischemia/reperfusion-induced cardiomyocyte apoptosis by attenuating morphological changes and reducing the percentage of terminal transferase dUTP nick end-labeling (TUNEL)-positive myocytes and caspase-<em>3</em> cleavage. These effects of tan were associated with an increased ratio of Bcl-2 to Bax protein in cardiomyocytes, an elevation of serum superoxide dismutase (<em>SOD</em>) activity and a decrease in serum malondialdehyde (MDA) level. Taken together, these data for the first time provide convincing evidence that tan protects cardiac myocytes against oxidative stress-induced apoptosis. The in vivo protection is mediated by increased scavenging of oxygen free radicals, prevention of lipid peroxidation and upregulation of the Bcl-2/Bax ratio.

Caenorhabditis elegans has recently been developed as a model for microbial pathogenesis, yet little is known about its immunological defenses. Previous work implicated insulin signaling in mediating pathogen resistance in a manner dependent on the transcriptional regulator DAF-16, but the mechanism has not been elucidated. We present evidence that C. elegans, like mammalian phagocytes, produces reactive oxygen species (ROS) in response to pathogens. Signs of oxidative stress occur in the intestine - the site of the host-pathogen interface - suggesting that ROS release is localized to this tissue. Evidence includes the accumulation of lipofuscin, a pigment resulting from oxidative damage, at this site. In addition, <em>SOD</em>-<em>3</em>, a superoxide dismutase regulated by DAF-16, is induced in intestinal tissue after exposure to pathogenic bacteria. Moreover, we show that the oxidative stress response genes <em>sod</em>-<em>3</em> and ctl-2 are required for DAF-16-mediated resistance to Enterococcus faecalis using a C. elegans killing assay. We propose a model whereby C. elegans responds to pathogens by producing ROS in the intestine while simultaneously inducing a DAF-16-dependent oxidative stress response to protect adjacent tissues. Because insulin-signaling mutants overproduce oxidative stress response enzymes, the model provides an explanation for their increased resistance to pathogens.

BACKGROUND

Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals.

RESULTS

We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 microg/100 g of body weight, intraperitoneal injection) on superoxide (O2-), extracellular superoxide dismutase (EC-<em>SOD</em>), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2- levels were elevated in diabetic rats (74.8+/-8x10(<em>3</em>) cpm/10 mg protein) compared with controls (<em>3</em>8.1+/-8x10(<em>3</em>) cpm/10 mg protein; P<0.01). D-4F decreased O2- levels to 1<em>3</em>.2<em>3</em>+/-1x10(<em>3</em>) (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50+/-6 cells/mL) than in controls (5+/-1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20+/-<em>3</em> cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0+/-2.0% relaxation). Western blot analysis showed decreased EC-<em>SOD</em> in the diabetic rats, whereas D-4F restored the EC-<em>SOD</em> level.

CONCLUSIONS

We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.

OBJECTIVE

We investigated whether NADPH oxidase-dependent production of superoxide contributes to activation of NF-kappaB in endothelial cells by the saturated free fatty acid palmitate.

RESULTS

After incubation of human endothelial cells with palmitate at a concentration known to induce cellular inflammation (100 mumol/L), we measured superoxide levels by using electron spin resonance spectroscopy and the spin trap 1-hydroxy-<em>3</em>-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Palmitate exposure induced a >2-fold increase in superoxide levels, an effect associated with activation of NF-kappaB signaling as measured by phospho-IkappaBalpha, NF-kappaB activity, IL-6, and ICAM expression. Reduction in superoxide levels by each of <em>3</em> different interventions-pretreatment with superoxide dismutase (<em>SOD</em>), diphenylene iodinium (DPI), or knockdown of NADPH oxidase 4 (NOX4) by siRNA-attenuated palmitate-mediated NF-kappaB signaling. Inhibition of toll like receptor-4 (TLR4) signaling also suppressed palmitate-mediated superoxide production and associated inflammation, whereas palmitate-mediated superoxide production was not affected by overexpression of a phosphorylation mutant IkappaBalpha (NF-kappaB super repressor) that blocks cellular inflammation downstream of IKKbeta/NF-kappaB. Finally, high-fat feeding increased expression of NOX4 and an upstream activator, bone morphogenic protein (BMP4), in thoracic aortic tissue from C57BL/6 mice, but not in TLR4(-/-) mice, compared to low-fat fed controls.

CONCLUSIONS

These results suggest that NADPH oxidase-dependent superoxide production links palmitate-stimulated TLR4 activation to NF-kappaB signaling in endothelial cells.

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (<em>SOD</em>), the anion channel blocker DIDS, and selective silencing of the chloride channel-<em>3</em> (ClC-<em>3</em>) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-<em>3</em> channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.

Ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) have antioxidant properties that could improve redox-sensitive vascular changes associated with hypertension. We determined whether vitamins C and E influence vascular function and structure in hypertension by modulating activity of NADPH oxidase and superoxide dismutase (<em>SOD</em>). Adult stroke-prone spontaneously hypertensive rats (SHRSP) were divided into <em>3</em> groups: control (C; n=6), vitamin C-treated (vit C, 1000 mg/day; n=7), and vitamin E-treated (vit E, 1000 IU/day; n=8). All rats were fed 4% NaCl. Blood pressure was measured weekly. After 6 weeks of treatment, the rats were killed, and mesenteric arteries were mounted as pressurized preparations. Vascular O(2)(-) generation and NADPH oxidase activity were measured by chemiluminescence. Vascular <em>SOD</em> activity and plasma total antioxidant status (TAS) were determined spectrophotometrically. Blood pressure increased from 212+/-7 to 265+/-6 mm Hg in controls. Treatment prevented progression of hypertension (vit C, 222+/-6 to 2<em>3</em>4+/-14 mm Hg; vit E, 220+/-9 to 227+/-10 mm Hg). Acetylcholine-induced vasodilation was improved (P<0.05), and media-to-lumen ratio was reduced (P<0.05) in the treated rats. O(2)(-) was lower in vitamin-treated groups compared with controls (vit C, 10+/-4 nmol. min(-1). g(-1) dry tissue weight; vit E, 9.6+/-<em>3</em>.5 nmol. min(-1). g(-1) dry tissue weight; C, 21+/-9 nmol. min(-1). g(-1) dry tissue weight; P<0.05). Both vitamin-treated groups showed significant improvement (P<0.01) in TAS. These effects were associated with decreased activation of vascular NADPH oxidase (vit C, 46+/-10; vit E, 50+/-9; C, 70+/-16 nmol. min(-1). g(-1) dry tissue weight, P<0.05) and increased activation of <em>SOD</em> (vit C, 12+/-2; vit E, 8+/-1; C, 4.6+/-1 U/mg; P<0.05). Our results demonstrate that vitamins C and E reduce oxidative stress, improve vascular function and structure, and prevent progression of hypertension in SHRSP. These effects may be mediated via modulation of enzyme systems that generate free radicals.