OBJECTIVE

Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cell (EPC). We investigated the effect of angiotensin II (Ang II) on EPC senescence, leading to the impairment of proliferative activity.

RESULTS

EPCs were isolated from peripheral blood and characterized. Both reverse transcription (RT)-polymerase chain reaction (PCR) and Western blotting were used to assess gp91phox expression. Immunofluorescence of nitrotyrosine provided evidence of peroxynitrite formation. Our data indicate that Ang II increased the expression of gp91phox mRNA in a dose-dependent manner, which was attenuated by Ang II type 1 (AT1) receptor antagonist valsartan. Similarly, Western blotting revealed that Ang II stimulated an increase in gp91phox, whereas pre-treatment with Valsartan reduced the Ang II-induced expression of gp91phox protein. Valsartan as well as superoxide dismutase (<em>SOD</em>) also inhibited Ang II-induced peroxynitrite formation. The exposure of cultured EPC to Ang II (100 nmol/l) significantly accelerated the rate of senescence compared to a control during 14 days in culture as determined by acidic beta-galactosidase staining. Ang II-induced EPC senescence was significantly inhibited by pre-treatment of either valsartan or <em>SOD</em> (P < 0.01). Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity by using PCR-enzyme-linked immunosorbent-based assay. Ang II significantly diminished telomerase activity, although the effect was significantly reduced by pre-treatment with either valsartan or <em>SOD</em> (P < 0.01). We examined whether Ang II-induced EPC senescence translates into an impairment of EPC proliferation. MTS [<em>3</em>-(4,5-dimethylthiazol-2-yl)-5-(<em>3</em>-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] assay disclosed an inhibitory effect of Ang II on EPC proliferation.

CONCLUSIONS

Ang II increases gp91phox expression in EPC, which may contribute to oxidative stress, as evidenced by peroxynitrite formation. Ang II accelerates the onset of EPC senescence via increased oxidative stress, which may be related to telomerase inactivation. In addition, Ang II-induced EPC senescence leads to the impairment of proliferative activity.

(-)-Epigallocatechin-<em>3</em>-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the phosphorylation of epidermal growth factor receptor (EGFR). We observed similar effects of EGCG in esophageal squamous cell carcinoma KYSE 150 cells and epidermoid squamous cell carcinoma A4<em>3</em>1 cells. Pretreatment of KYSE 150 cells with EGCG (20 micromol/L) for 0.5 to 24 hours in HAM's F12 and RPMI 1640 mixed medium at <em>3</em>7 degrees C, before the addition of EGF, resulted in a decreased level of phosphorylated EGFR (by <em>3</em>2-85%). Prolonged treatment with EGCG (8 or 24 hours) also decreased EGFR protein level (both by 80%). EGCG treatment for 24 hours also caused decreased signals of HER-2/neu in esophageal adenocarcinoma OE19 cells. These effects of EGCG were prevented or diminished by the addition of superoxide dismutase (<em>SOD</em>, 5 units/mL), or <em>SOD</em> plus catalase (<em>3</em>0 units/mL), to the cell culture medium. A similar phenomenon on inactivation of EGFR was observed in A4<em>3</em>1 cells as well. Under culture conditions for KYSE 150 cells, EGCG was unstable, with a half-life of approximately <em>3</em>0 minutes; EGCG dimers and other oxidative products were formed. The presence of <em>SOD</em> in the culture medium stabilized EGCG and increased its half-life to longer than 24 hours and some EGCG epimerized to (+)-gallocatechin-<em>3</em>-gallate. A mechanism of superoxide radical-mediated dimerization of EGCG and H2O2 formation is proposed. The stabilization of EGCG by <em>SOD</em> in the culture medium potentiated the activity of EGCG in inhibiting KYSE 150 cell growth. The results suggest that in cell culture conditions, the auto-oxidation of EGCG leads to EGFR inactivation, but the inhibition of cell growth is due to other mechanisms. It remains to be determined whether the presently observed auto-oxidation of EGCG occurs in vivo. In future studies of EGCG and other polyphenolic compounds in cell culture, <em>SOD</em> may be added to stabilize EGCG and to avoid possible artifacts.

Cigarette smoking is associated with impaired endothelium-dependent vasodilation and reduced nitric oxide (NO) in the exhaled air of smokers. To explore the mechanism for the impairment of NO-mediated vasodilation, we studied the effect of cigarette smoke extract (CSE) on NO synthase (eNOS) activity and content in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with CSE resulted in a time- and dose-dependent decrease in eNOS activity. The inhibitory effect of CSE on eNOS activity was not reversible. Both gas-phase and particulate-phase extracts of CSE contributed to the inhibition of eNOS activity. The protein kinase c (PKC) inhibitors staurosporine and chelerythrine did not affect the CSE-induced inhibition of eNOS activity. Catalase, superoxide dismutase (<em>SOD</em>), vitamin C, vitamin E, glutathione, and dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of eNOS activity, and incubation of PAEC with <em>3</em> mM nicotine did not change the activity of eNOS. Treatment of PAEC with CSE also caused a nonreversible, time-dependent decrease in eNOS protein content detected by Western blot analysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione contents or on lactate dehydrogenase (LDH) release. These results indicate that exposure to CSE causes an irreversible inhibition of eNOS activity in PAEC, and suggest that the decreased activity is secondary to reduced eNOS protein mass and mRNA. The decrease in eNOS activity may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.

The mechanisms of action of silymarin involve different biochemical events, such as the stimulation of the synthetic rate of ribosomal RNA (rRNA) species through stimulation of polymerase I and rRNA transcription, protecting the cell membrane from radical-induced damage and blockage of the uptake of toxins such as alpha-amanitin. Studies in patients with liver disease have shown that silymarin increases superoxide dismutase (<em>SOD</em>) activity of lymphocytes and erythrocytes, as well as the expression of <em>SOD</em> in lymphocytes. Silymarin has also been shown to increase patient serum levels of glutathione and glutathione peroxidase. Silybin 20 to 48 mg/kg/day has shown promise as a clinical antidote to acute Amanita (deathcap mushroom) poisoning. Primary efficacy data from <em>3</em> trials which examined the therapeutic potential of silymarin in patients with cirrhosis, and included patient survival as an end-point, demonstrated that silymarin had no significant beneficial effect on patient mortality. However, upon subanalysis, silymarin 420 mg/day had a significantly beneficial effect on patient survival rate (compared with patients receiving placebo) in 1 randomised, double-blind trial in patients with alcoholic cirrhosis. Silymarin 420 mg/day was also shown to improve indices of liver function [AST, ALT, gamma-glutamyl transferase and bilirubin] in patients with liver disease of various aetiology, including those exposed to toxic levels of toluene or xylene; however, it was largely ineffective in patients with viral hepatitis. Reports of adverse events while receiving silymarin therapy are rare. However, there have been accounts of nausea, epigastric discomfort, arthralgia, pruritus, headache and urticaria. Silymarin has also been reported to have possibly caused a mild laxative effect.

CONCLUSIONS

The antioxidant properties of silymarin (a mixture of at least 4 closely related flavonolignans, 60 to 70% of which is a mixture of 2 diastereomers of silybin) have been demonstrated in vitro and in animal and human studies. However, studies evaluating relevant health outcomes associated with these properties are lacking. Although silymarin has low oral absorption, oral dosages of 420 mg/day have shown some therapeutic potential, with good tolerability, in the treatment of alcoholic cirrhosis. Moreover, silybin 20 to 48 mg/kg/day has shown promise as an antidote for acute mushroom poisoning by Amanita phalloides; however, further studies paying attention to the amount of ingested mushroom and time elapsed before administration of treatment are needed to clarify its role in this indication. Studies in patients with the early onset of liver disease may demonstrate the liver regeneration properties that silymarin is promoted as possessing.

Extracellular superoxide dismutase (EC<em>SOD</em> or <em>SOD</em><em>3</em>) is highly expressed in lungs and functions as a scavenger of O(2)(*-). ECM fragmentation, which can be triggered by oxidative stress, participates in the pathogenesis of chronic obstructive pulmonary disease (COPD) through attracting inflammatory cells into the lungs. The level of <em>SOD</em><em>3</em> is significantly decreased in lungs of patients with COPD. However, the role of endogenous <em>SOD</em><em>3</em> in the development/progression of emphysema is unknown. We hypothesized that <em>SOD</em><em>3</em> protects against emphysema by attenuating oxidative fragmentation of ECM in mice. To test this hypothesis, <em>SOD</em><em>3</em>-deficient, <em>SOD</em><em>3</em>-transgenic, and WT C57BL/6J mice were exposed to cigarette smoke (CS) for <em>3</em> d (<em>3</em>00 mg total particulate matter/m(<em>3</em>)) to 6 mo (100 mg/m(<em>3</em>) total particulate matter) or by intratracheal elastase injection. Airspace enlargement, lung inflammation, lung mechanical properties, and exercise tolerance were determined at different time points during CS exposure or after elastase administration. CS exposure and elastase administration caused airspace enlargement as well as impaired lung function and exercise capacity in <em>SOD</em><em>3</em>-null mice, which were improved in mice overexpressing <em>SOD</em><em>3</em> and by pharmacological <em>SOD</em> mimetic. These phenomena were associated with <em>SOD</em><em>3</em>-mediated protection against oxidative fragmentation of ECM, such as heparin sulfate and elastin, thereby attenuating lung inflammatory response. In conclusion, <em>SOD</em><em>3</em> attenuates emphysema and reduces oxidative fragmentation of ECM in mouse lung. Thus, pharmacological augmentation of <em>SOD</em><em>3</em> in the lung may have a therapeutic potential in the intervention of COPD/emphysema.

We have demonstrated the selective induction of manganese superoxide dismutase (Mn<em>SOD</em>) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A <em>3</em>-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and Mn<em>SOD</em> were less striking. Expression of catalase, Mn<em>SOD</em>, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc <em>SOD</em> (CuZn<em>SOD</em>) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. Mn<em>SOD</em> mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZn<em>SOD</em> mRNA remained unchanged. The <em>3</em>-fold induction of Mn<em>SOD</em> mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. Mn<em>SOD</em> mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive Mn<em>SOD</em> protein was produced in a corresponding dose- and time-dependent manner. Induction of Mn<em>SOD</em> gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.

In a previous study we demonstrated that oxalate induced free radical injury can promote calcium oxalate stone formation. In the present study, we tested whether the antioxidants vitamin E, superoxide dismutase (<em>SOD</em>), catalase and desferoxamine (DFO) can provide protection against oxalate toxicity in LLC-PK(1) cells. LLC-PK(1) cells were exposed to oxalate (1.0 mM) or oxalate+calcium oxalate monohydrate crystals (COM, 500 microg) for <em>3</em>, 6, and 9 h. Cellular injury was assessed by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) content, catalase and glutathione peroxidase activities were also measured. The effect of vitamin E (200 microM), DFO (1.0 mM), <em>SOD</em> (400 U), and catalase (400 U) on oxalate-exposed cells was tested. LLC-PK(1) cells exposed to oxalate showed a significant increase in LDH release and MDA content, which was further elevated when COM crystals were added. Cellular glutathione peroxidase and catalase activities were decreased on exposure to oxalate. The addition of vitamin E, <em>SOD</em>, catalase and DFO significantly reduced the release of LDH and restored glutathione peroxidase and catalase activities towards the control level. The increased formation of MDA on oxalate or oxalate+COM toxicity was restored towards normalization by antioxidants and antioxidant enzymes. The protection rendered by vitamin E was greater than that of <em>SOD</em>, catalase and DFO. We conclude that oxalate associated free radical injury may promote stone formation by providing cellular debris for crystal nucleation and aggregation and augment crystal attachment to other tubular cells. Antioxidant administration may prevent calcium oxalate nucleation and retention in the renal tubules by preventing oxalate mediated peroxidative injury.

We have shown that intermittent interruption of immediate reflow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Postcon) is associated with a reduction in the generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload. Primary cultured neonatal rat cardiomyocytes were exposed to <em>3</em> h of hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the <em>3</em>-h index hypoxia by three cycles of 5 min of reoxygenation and 5 min of rehypoxia applied before 6 h of reoxygenation. Relative to sham control and hypoxia alone, the generation of ROS (increased lucigenin-enhanced chemiluminescence, <em>SOD</em>-inhibitable cytochrome c reduction, and generation of hydrogen peroxide) was significantly augmented after immediate reoxygenation as was the production of malondialdehyde, a product of lipid peroxidation. Concomitant with these changes, intracellular and mitochondrial Ca(2+) concentrations, which were detected by fluorescent fluo-4 AM and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide staining was decreased consistent with increased levels of lactate dehydrogenase after reoxygenation. Postcon treatment at the onset of reoxygenation reduced ROS generation and malondialdehyde concentration in media and attenuated cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase. Postcon treatment was associated with a decrease in intracellular and mitochondrial Ca(2+) concentrations. These data suggest that Postcon treatment reduces reoxygenation-induced injury in cardiomyocytes and is potentially mediated by attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca(2+) overload.

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA-induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²+](cyt)) oscillations and inward-rectifying potassium (K+(in)) channel activity in Arabidopsis. SA-induced stomatal closure was inhibited by pre-treatment with catalase (CAT) and superoxide dismutase (<em>SOD</em>), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA-induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA-induced stomatal closures. <em>3</em>,<em>3</em>'-Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂⁻ production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, <em>SOD</em> and SHAM. NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-<em>3</em>-oxide (cPTIO) suppressed the SA-induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA-induced NO production. SA failed to induce [Ca²+](cyt) oscillations in guard cells whereas K+(in) channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM-sensitive peroxidase, intracellular ROS accumulation and K+(in) channel inactivation.

Growing consumer interest in grass-fed beef products has raised a number of questions with regard to the perceived differences in nutritional quality between grass-fed and grain-fed cattle. Research spanning three decades suggests that grass-based diets can significantly improve the fatty acid (FA) composition and antioxidant content of beef, albeit with variable impacts on overall palatability. Grass-based diets have been shown to enhance total conjugated linoleic acid (CLA) (C18:2) isomers, trans vaccenic acid (TVA) (C18:1 t11), a precursor to CLA, and omega-<em>3</em> (n-<em>3</em>) FAs on a g/g fat basis. While the overall concentration of total SFAs is not different between feeding regimens, grass-finished beef tends toward a higher proportion of cholesterol neutral stearic FA (C18:0), and less cholesterol-elevating SFAs such as myristic (C14:0) and palmitic (C16:0) FAs. Several studies suggest that grass-based diets elevate precursors for Vitamin A and E, as well as cancer fighting antioxidants such as glutathione (GT) and superoxide dismutase (<em>SOD</em>) activity as compared to grain-fed contemporaries. Fat conscious consumers will also prefer the overall lower fat content of a grass-fed beef product. However, consumers should be aware that the differences in FA content will also give grass-fed beef a distinct grass flavor and unique cooking qualities that should be considered when making the transition from grain-fed beef. In addition, the fat from grass-finished beef may have a yellowish appearance from the elevated carotenoid content (precursor to Vitamin A). It is also noted that grain-fed beef consumers may achieve similar intakes of both n-<em>3</em> and CLA through the consumption of higher fat grain-fed portions.

Amyotrophic lateral sclerosis (ALS) is a lethal disease that is characterized by the relentless death of motoneurons. Mutations to Cu-Zn superoxide dismutase (<em>SOD</em>), though occurring in just 2-<em>3</em>% of individuals with ALS, remain the only proven cause of the disease. These mutations structurally weaken <em>SOD</em>, which indirectly decreases its affinity for Zn. Zn-deficient <em>SOD</em> induces apoptosis in motoneurons through a mechanism involving peroxynitrite. Importantly, Zn-deficient wild-type <em>SOD</em> is just as toxic as Zn-deficient ALS mutant <em>SOD</em>, suggesting that the loss of Zn from wild-type <em>SOD</em> could be involved in the other 98% of cases of ALS. Zn-deficient <em>SOD</em> could therefore be an important therapeutic target in all forms of ALS.

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration-dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO<em>3</em>a, p27, Bax and cleaved caspase-<em>3</em>, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with <em>SOD</em> and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p<em>3</em>8 MAPK, catalase, Mn<em>SOD</em> and Cu/Zn <em>SOD</em> protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO<em>3</em>a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.

In the present study, the ecotoxicity of silver nanoparticles (AgNPs) was investigated in Caenorhabditis elegans using survival, growth, and reproduction, as the ecotoxicological endpoints, as well as stress response gene expression. Whole genome microarray was used to screen global changes in C. elegans transcription profiles after AgNPs exposure, followed by quantitative analysis of selected genes. The integration of gene expression with organism and population level endpoints was investigated using C. elegans functional genomics tools, to test the ecotoxicological relevance of AgNPs-induced gene expression. AgNPs exerted considerable toxicity in C. elegans, most clearly as dramatically decreased reproduction potential. Increased expression of the superoxide dismutases-<em>3</em> (<em>sod</em>-<em>3</em>) and abnormal dauer formation protein (daf-12) genes with 0.1 and 0.5 mg/L of AgNPs exposures occurred concurrently with significant decreases in reproduction ability. Overall results of functional genomic studies using mutant analyses suggested that the <em>sod</em>-<em>3</em> and daf-12 gene expressions may have been related to the AgNPs-induced reproductive failure in C. elegans and that oxidative stress may have been an important mechanism in AgNPs toxicity.

Pre-eclampsia is a hypertensive disorder of human pregnancy that is a leading cause of premature delivery and fetal growth retardation. It is characterized by hypertension, reduced uteroplacental blood flow, proteinuria and oedema. Pre-eclampsia is associated with increased lipid peroxidation in the maternal circulation and in the placenta. Mitochondria are sources of oxygen radicals and are enriched with polyunsaturated fatty acids that are susceptible to peroxidation. Therefore, the mitochondria could be an important source of oxidative stress and lipid peroxidation. To study this, the level of lipid peroxidation in the mitochondrial fraction of placentae obtained from normally pregnant women (n=8) and women with pre-eclampsia (n=8) was examined. Placental tissues were homogenized and the mitochondrial fraction was isolated by ultracentrifugation. Mitochondrial lipid peroxides were estimated by malondialdehyde (MDA). NADPH and Fe++ were used to stimulate lipid peroxidation. Superoxide dismutase (<em>SOD</em>) was used to inhibit superoxide radicals and mannitol to inhibit hydroxyl radicals. The following results were found: (1) MDA levels were significantly greater in the mitochondrial fraction isolated from pre-eclamptic placentae than from normal placentae (27.4+/-<em>3</em>.0 versus 17.0+/-1.8 nmol/g tissue, mean+/-s.e., P<0.05); (2) the oxidative potential of the pre-eclamptic mitochondrial fraction was also higher than normal as evidenced by the significantly greater stimulation of lipid peroxidation by NADPH and Fe+ + (248+/-25 versus 164+/-<em>3</em>5 nmol/g, P<0.05); (<em>3</em>) superoxide dismutase, but not mannitol, attenuated the lipid peroxidation induced by NADPH and Fe+ + demonstrating that superoxide is the radical responsible for mitochondrial lipid peroxidation in this system; and (4) the amount of mitochondrial protein was 47 per cent greater and the activity of the mitochondrial enzyme, citrate synthase, was 56 per cent greater in the pre-eclamptic placentae indicating an increase in the amount of mitochondria in the pre-eclamptic placentae. It is concluded that: (1) mitochondrial lipid peroxidation is increased in pre-eclampsia; (2) the amount of placental mitochondria is increased in pre-eclampsia; (<em>3</em>) placental mitochondria contribute to the abnormal increase in lipid peroxidation that occurs in pre-eclamptic placentae by both an increase in their amount and an increase in their susceptibility to oxidation; and (4) mitochondrial generation of superoxide could be an important source of oxidative stress in pre-eclampsia.

OBJECTIVE

Peroxisome proliferator-activated receptors (PPAR) alpha and beta/delta are essential transcriptional regulators of fatty acid oxidation in the heart. However, little is known about the roles of PPARgamma in the heart. The present study is to investigate in vivo role(s) of PPARgamma in the heart.

METHODS

A Cre-loxP mediated cardiomyocyte-restricted PPARgamma knockout line was investigated. In these mice, exon 1 and 2 of PPARgamma were targeted to eliminate PPARgamma from cardiomyocytes.

RESULTS

PPARgamma null mice exhibited pathological changes around <em>3</em> months of age, featuring progressive cardiac hypertrophy with mitochondrial oxidative damage. Most mice died from dilated cardiomyopathy. Cardiac expression of Sod2 (encoding manganese superoxide dismutase; Mn<em>SOD</em>), a mitochondrial antioxidant enzyme was downregulated both in transcript and protein levels in cardiac samples in PPARgamma knockout mice independent of pathological changes. Promoter analyses revealed that Sod2 is a target gene of PPARgamma. Consequently, myocardial superoxide content in PPARgamma knockout mice was increased, leading to extensive oxidative damage. Treatment with a <em>SOD</em> mimetic compound, MnTBAP, prevented superoxide-induced cardiac pathological changes in PPARgamma knockout mice.

CONCLUSIONS

The present study demonstrates that PPARgamma is critical to myocardial redox homeostasis. These findings should provide new insights into understanding the roles of PPARgamma in the heart.

Endothelium-derived microparticles have recently been described as a new marker of endothelial cell dysfunction. Increased levels of circulating microparticles have been documented in inflammatory disorders, diabetes mellitus, and many cardiovascular diseases. Perturbations of angiogenesis play an important role in the pathogenesis of these disorders. We demonstrated previously that isolated endothelial microparticles (EMPs) impair endothelial function in vitro, diminishing acetylcholine-induced vasorelaxation and nitric oxide production by rat aortic rings and simultaneously increasing superoxide production. Herein, using the Matrigel assay of angiogenesis in vitro and a topological analysis of the capillary-like network by human umbilical vein endothelial cells (HUVECs), we investigated the effects of EMPs on formation of the vascular network. All parameters of angiogenesis were affected by treatment for 48 h with isolated EMPs in a concentration of 10(5) but not 10(<em>3</em>) or 10(4) EMPs/ml. The effects included decreases in total capillary length (24%), number of meshes (45%), and branching points (<em>3</em>6%) and an increase in mesh area (<em>3</em>8%). The positional and topological order indicated that EMPs affect angiogenic parameters uniformly over the capillary network. Treatment with the cell-permeable <em>SOD</em> mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (Mn-TBAP) partially or completely restored all parameters of angiogenesis affected by EMPs. EMPs reduced cell proliferation rate and increased apoptosis rate in time- and dose-dependent manners, and this phenomenon was also prevented by Mn-TBAP treatment. Our data demonstrate that EMPs have considerable impact on angiogenesis in vitro and may be an important contributor to the pathogenesis of diseases that are accompanied by impaired angiogenesis.

Peroxisome proliferator-activated receptor (PPAR)-gamma is a ligand-activated transcription factor of nuclear hormone receptor superfamily. Thiazolidinedione rosiglitazone is a potent agonist of PPARgamma which was shown to induce neuroprotection in animal models of focal ischemia and spinal cord injury. We currently evaluated the therapeutic potential of rosiglitazone (6 mg/kg at 5 min, 6 h and 24 h; i.p.) following controlled cortical impact (CCI)-induced traumatic brain injury (TBI) in adult mice. CCI injury increased the cortical PPARgamma mRNA levels which were further elevated by rosiglitazone treatment. In addition, rosiglitazone treatment significantly decreased the cortical lesion volume measured at 7 days compared to vehicle treatment (by 56+/-7%; p<0.05; n=6/group). Following TBI, the spared cortex of the rosiglitazone group showed significantly less numbers of GSI-B4(+) activated microglia/macrophages and ICAM1(+) capillaries, and curtailed induction of pro-inflammatory genes IL6, MCP1 and ICAM1 compared to vehicle group. Rosiglitazone-treated mice also showed significantly less number of TUNEL(+) apoptotic neurons and curtailed induction of caspase-<em>3</em> and Bax, compared to vehicle control. In addition, rosiglitazone significantly enhanced the post-TBI expression of the neuroprotective chaperones HSP27, HSP70 and HSP<em>3</em>2/HO1, and the anti-oxidant enzymes catalase, Cu/Zn-<em>SOD</em> and Mn-<em>SOD</em>, compared to vehicle. Treatment with GW9662 (a specific PPARgamma antagonist) prevented all the above PPARgamma-mediated actions. Thus, PPARgamma activation confers neuroprotection after TBI by anti-inflammatory, anti-apoptotic and anti-oxidative mechanisms.

A simple microtiter plate based colorimetric assay for superoxide dismutase is described. The method, involves generation of superoxide by pyrogallol autoxidation and the inhibition of superoxide dependent reduction of the tetrazolium dye MTT [<em>3</em>-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] to its formazan, measured at 570 nm. The reaction was terminated by the addition of dimethyl sulfoxide (DMSO) which also helps to solubilize the formazan formed and the colour evolved was stable for many hours. The method was compared with other known methods to measure the activity of purified erythrocyte Cu,Zn<em>SOD</em> and superoxide dismutase activity from various rat tissues. This procedure involves inexpensive reagents, allows a rapid and sensitive measurement of <em>SOD</em> activity and the microtiter plate assay is suitable for use with large number of samples.

Myocardial ischemia and reperfusion have been shown to impair coronary vasorelaxation to endothelium-dependent vasodilators. To examine the time course of this dysfunction, occlusion of the left anterior descending (LAD) coronary artery (90 minutes) was followed by reperfusion for 0, 2.5, 5, 20, 180, or 270 minutes. Coronary arterial rings from the ischemic LAD and control left circumflex (LCx) arteries were tested for responsiveness to the endothelium-dependent receptor-mediated vasodilator, acetylcholine (ACh), and the endothelium-dependent nonreceptor-mediated vasodilator, A2<em>3</em>187, as well as the endothelium-independent vasodilator, NaNO2. ACh relaxation was not impaired after 90 minutes of ischemia without reperfusion. However, 2.5 minutes of reperfusion resulted in depressed ACh responses (<em>3</em>6 +/- 10% of control) that was further reduced to 16 +/- 6% at 20 minutes, and remained comparably depressed at every time thereafter. A2<em>3</em>187 vasodilator responses were also attenuated after reperfusion, although the reduced response occurred later (that is, at 20 minutes). There was no significant decrease in response to NaNO2 in the LAD at any time or to any vasodilator in LCx control rings. Treatment with recombinant human superoxide dismutase (h<em>SOD</em>, 5 mg/kg/hr, that is, 15,545 <em>SOD</em> units/kg/hr), starting 10 minutes before reperfusion, preserved the vasodilator response to ACh (82 +/- 6%) and A2<em>3</em>187, but treatment with the hydroxyl ion scavenger N-(2-mercapto proprionyl)-glycine (MPG) (8 mg/kg/hr) only protected the A2<em>3</em>187 response. No damage to the surface of the endothelium was observed by scanning electron microscopy at any time point. Myocardial cell damage increased with time of reperfusion as assessed by increasing plasma CK activities and amounts of necrotic tissue indexed to area at risk. Significant myocardial injury occurred at <em>3</em> hours after reperfusion. These findings suggest that endothelial dysfunction resulting in reduced endothelium-derived relaxing factor release occurs before the development of myocardial cell necrosis and may be due to oxygen-derived free radicals produced rapidly on reperfusion.

Recent studies suggest that superoxide dismutase 1 (<em>SOD</em>1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human <em>SOD</em>1, G85R, fused to YFP, in a line of transgenic G85R <em>SOD</em>1-YFP mice. These mice, but not wild-type human <em>SOD</em>1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R <em>SOD</em>-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R <em>SOD</em>-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R <em>SOD</em>-YFP, but not WT <em>SOD</em>-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, <em>3</em> Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R <em>SOD</em>-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Reactive oxygen species, such as superoxide radicals, are thought to underlie the pathogenesis of various diseases. Almost <em>3</em> to 10% of the oxygen utilized by tissues is converted to its reactive intermediates, which impair the functioning of cells and tissues. Superoxide dismutase (<em>SOD</em>) catalyzes the conversion of single electron reduced species of molecular oxygen to hydrogen peroxide and oxygen. There are several classes of <em>SOD</em> that differ in their metal binding ability, distribution in different cell compartments, and sensitivity to various reagents. Among these, Cu, Zn superoxide dismutase (<em>SOD</em>1) is widely distributed and comprises 90% of the total <em>SOD</em>. This ubiquitous enzyme, which requires Cu and Zn for its activity, has great physiological significance and therapeutic potential. The present review describes the role of <em>SODs</em>, especially Cu, Zn <em>SOD</em>, in several diseases, such as familial amyotrophic lateral sclerosis (FALS), Parkinson's disease, Alzheimer's disease, dengue fever, cancer, Down's syndrome, cataract, and several neurological disorders. Mutations in the <em>SOD</em>1 gene cause a familial form of amyotrophic lateral sclerosis. The mechanism by which mutant <em>SOD</em>1 causes the degeneration of motor neurons is not well understood. Transgenic mice expressing multiple copies of FALS-mutant <em>SOD</em>1s develop an ALS-like motor neuron disease. Vacuolar degeneration of mitochondria has been identified as the main pathological feature associated with motor neuron death and paralysis in several lines of FALS-<em>SOD</em>1 mice. Various observations and conclusions linking mutant <em>SOD</em>1 and FALS are discussed in this review in detail.

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26-28 month) male C57BL6 control mice, and <em>3</em> weeks of sodium nitrite (50 mg L(-1) in drinking water) restored nitrite levels to or above young (4-6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 9<em>3</em>.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (<em>SOD</em>) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. <em>3</em><em>3</em>2 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (<em>3</em>84 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100-<em>3</em>00% greater and <em>SOD</em> activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Cytoplasmic superoxide dismutase (<em>SOD</em>-1; EC 1.15.1.1) is encoded by human chromosome 21. The <em>SOD</em>-1 gene locus is located at chromosomal region 21q22, which is involved in Down syndrome. cDNA clones containing sequences of human <em>SOD</em>-1 were previously isolated. In the present study the nucleotide sequence of one clone, designated pS61-10, was determined. It contains 459 nucleotides representing the entire coding region and 95 nucleotides of the <em>3</em>' untranslated region. In human cells two poly(A)-containing <em>SOD</em>-1 RNAs of 0.7 and 0.5 kilobases were detected. These two species are also present in monkey cells, whereas mouse cells contain only a 0.5-kilobase RNA. In a mouse/human hybrid line that contains chromosome 21 as the only human chromosome, the two human <em>SOD</em>-1 RNAs were detected, indicating that both are encoded by this chromosome. These RNAs were found in poly(A)-containing polysomal RNA and were translated in vitro to <em>SOD</em>-1 polypeptide; they are therefore functional mRNAs. In normal human fibroblasts 0.002-0.006% of the poly(A)-containing RNA was <em>SOD</em>-1 RNA. The level in monosomic 21 cells was 70% of this value and the level in fibroblasts from Down syndrome patients was about 2 times higher than normal.

To determine if short-term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = <em>3</em>0) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately <em>3</em>0%) for 8 weeks. Ex vivo carotid artery endothelium-dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 +/- 5 vs. 95 +/- 2% of maximum dilation, P < 0.05), whereas old calorie-restricted (OCR) and YCR did not differ (96 +/- 1 vs. 94 +/- <em>3</em>%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (<em>SOD</em>) mimetic that restored EDD in OAL (to 97 +/- 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese <em>SOD</em> activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium-independent dilation did not differ among groups. Short-term CR initiated in old age reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase-mediated superoxide production and stimulation of anti-oxidant enzyme activity), and upregulation of sirtuin-1.