Overexpression Of Superoxide Dismutase Research Articles

Superoxide dismutase 2 overexpression alleviates maternal diabetes-induced neural tube defects, restores mitochondrial function and suppresses cellular stress in diabetic embryopathy

Pregestational diabetes disrupts neurulation leading to neural tube defects (NTDs). Oxidative stress resulting from reactive oxygen species (ROS) plays a central role in the induction of NTD formation in diabetic pregnancies. We aimed to determine whether mitochondrial dysfunction increases ROS production leading to oxidative stress and diabetic embryopathy. Overexpression of the mitochondrion-specific antioxidant enzyme superoxide dismutase 2 (SOD2) in a transgenic (Tg) mouse model significantly reduced maternal diabetes-induced NTDs. SOD2 overexpression abrogated maternal diabetes-induced mitochondrial dysfunction by inhibiting mitochondrial translocation of the pro-apoptotic Bcl-2 family members, reducing the number of defective mitochondria in neuroepithelial cells, and decreasing mitochondrial membrane potential. Furthermore, SOD2 overexpression blocked maternal diabetes-increased ROS production by diminishing dihydroethidium staining signals in the developing neuroepithelium, and reducing the levels of nitrotyrosine-modified proteins and lipid hydroperoxide level in neurulation stage embryos. SOD2 overexpression also abolished maternal diabetes-induced endoplasmic reticulum stress. Finally, caspase-dependent neuroepithelial cell apoptosis enhanced by oxidative stress was significantly reduced by SOD2 overexpression. Thus, our findings support the hypothesis that mitochondrial dysfunction in the developing neuroepithelium enhances ROS production, which leads to oxidative stress and endoplasmic reticulum (ER) stress. SOD2 overexpression blocks maternal diabetes-induced oxidative stress and ER stress, and reduces the incidence of NTDs in embryos exposed to maternal diabetes.

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

Vaccination of Elk (Cervus canadensis) with Brucella abortus Strain RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes Does Not Induce Adequate Protection against Experimental Brucella abortus Challenge.

In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk.

The Influenza Virus H5N1 Infection Can Induce ROS Production for Viral Replication and Host Cell Death in A549 Cells Modulated by Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression.

Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus.

Mice Overexpressing Both Non-Mutated Human SOD1 and Mutated SOD1(G93A) Genes: A Competent Experimental Model for Studying Iron Metabolism in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by degeneration and loss of motor neurons in the spinal cord, brainstem and motor cortex. Up to 10% of ALS cases are inherited (familial, fALS) and associated with mutations, frequently in the superoxide dismutase 1 (SOD1) gene. Rodent transgenic models of ALS are often used to elucidate a complex pathogenesis of this disease. Of importance, both ALS patients and animals carrying mutated human SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation. The aim of our study was to characterize changes in iron metabolism in one of the most commonly used models of ALS – transgenic mice overexpressing human mutated SOD1G93A gene. We analyzed the expression of iron-related genes in asymptomatic, 2-month-old and symptomatic, 4-month-old SOD1G93A mice. In parallel, respective age-matched mice overexpressing human non-mutated SOD1 transgene and control mice were analyzed. We demonstrate that the overexpression of both SOD1 and SOD1G93A genes account for a substantial increase in SOD1 protein levels and activity in selected tissues and that not all the changes in iron metabolism genes expression are specific for the overexpression of the mutated form of SOD1.

Angiotensin II-Induced Hypertension Is Attenuated by Overexpressing Copper/Zinc Superoxide Dismutase in the Brain Organum Vasculosum of the Lamina Terminalis.

Angiotensin II (AngII) can access the brain via circumventricular organs (CVOs), including the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), to modulate blood pressure. Previous studies have demonstrated a role for both the SFO and OVLT in the hypertensive response to chronic AngII, yet it is unclear which intracellular signaling pathways are involved in this response. Overexpression of copper/zinc superoxide dismutase (CuZnSOD) in the SFO has been shown to attenuate the chronic hypertensive effects of AngII. Presently, we tested the hypothesis that elevated levels of superoxide (O2 ∙−) in the OVLT contribute to the hypertensive effects of AngII. To facilitate overexpression of superoxide dismutase, adenoviral vectors encoding human CuZnSOD or control adenovirus (AdEmpty) were injected directly into the OVLT of rats. Following 3 days of control saline infusion, rats were intravenously infused with AngII (10 ng/kg/min) for ten days. Blood pressure increased 33 ± 8 mmHg in AdEmpty rats (n = 6), while rats overexpressing CuZnSOD (n = 8) in the OVLT demonstrated a blood pressure increase of only 18 ± 5 mmHg after 10 days of AngII infusion. These results support the hypothesis that overproduction of O2 ∙− in the OVLT plays an important role in the development of chronic AngII-dependent hypertension.

Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation.

Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs.

Superoxide dismutase overexpression protects against glucocorticoid-induced depressive-like behavioral phenotypes in mice

In the stress response, activation of the hypothalamic–pituitary–adrenal axis, and particularly the release of glucocorticoids, plays a critical role. However, dysregulation of this system and sustained high plasma levels of glucocorticoids can result in depression. Recent studies have suggested the involvement of reactive oxygen species (ROS), such as superoxide anion, in depression. However, direct evidence for a role of ROS in the pathogenesis of this disorder is lacking. In this study, using transgenic mice expressing human Cu/Zn-superoxide dismutase (SOD1), an enzyme that catalyzes the dismutation of superoxide anions, we examined the effect of SOD1 overexpression on depressive-like behavioral phenotypes in mice. Depressive-like behaviors were induced by daily subcutaneous administration of the glucocorticoid corticosterone for 4 weeks, and was monitored with the social interaction test, the sucrose preference test and the forced swim test. These tests revealed that transgenic mice overexpressing SOD1 are more resistant to glucocorticoid-induced depressive-like behavioral disorders than wild-type animals. Furthermore, compared with wild-type mice, transgenic mice showed a reduction in the number of 8-hydroxy-2′-deoxyguanosine (a marker of oxidative stress)-positive cells in the hippocampal CA3 region following corticosterone administration. These results suggest that overexpression of SOD1 protects mice against glucocorticoid-induced depressive-like behaviors by decreasing cellular ROS levels.

High Glucose-Repressed CITED2 Expression Through miR-200b Triggers the Unfolded Protein Response and Endoplasmic Reticulum Stress.

High glucose in vivo and in vitro induces neural tube defects (NTDs). CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) is essential for neural tube closure. We explored the regulatory mechanism underlying CITED2 expression and its relationship with miRNA and endoplasmic reticulum (ER) stress. miR-200b levels were increased by maternal diabetes or high glucose in vitro, and this increase was abrogated by transgenic overexpression of superoxide dismutase 1 (SOD1) or an SOD1 mimetic. CITED2 was the target of miR-200b and was downregulated by high glucose. Two miR-200b binding sites in the 3′-untranslated region of the CITED2 mRNA were required for inhibiting CITED2 expression. The miR-200b mimic and a CITED2 knockdown mimicked the stimulative effect of high glucose on unfolded protein response (UPR) and ER stress, whereas the miR-200b inhibitor and CITED2 overexpression abolished high glucose–induced UPR signaling, ER stress, and apoptosis. The ER stress inhibitor, 4-phenylbutyrate, blocked CITED2 knockdown–induced apoptosis. Furthermore, the miR-200b inhibitor reversed high glucose–induced CITED2 downregulation, ER stress, and NTDs in cultured embryos. Thus, we showed a novel function of miR-200b and CITED2 in high glucose–induced UPR and ER stress, suggesting that miR-200b and CITED2 are critical for ER homeostasis and NTD formation in the developing embryo.

286 - 20S Proteasome Gating Control: Implications on the Chronological Life Span of Yeast Cells

Nitric oxide (NO) causes S-glutathiolation of the reactive cysteine-674 in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), thus increasing SERCA activity, and inhibiting Ca2+ influx and migration of vascular smooth muscle cells (VSMC). Because increased VSMC migration contributes to accelerated neointimal growth and atherosclerosis in diabetes, the effect of culture of VSMC in high glucose (HG) was determined. Rat aortic VSMC were exposed to normal (5.5 mmol/L) or high (25 mmol/L) glucose for 3 days, and serum-induced cell migration during 6 h into a wounded cell monolayer was measured 5 min after adding the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or 24 h after interleukin-1β (IL-1β) to express inducible nitric oxide synthase (iNOS). In normal glucose, SNAP or IL-1β significantly inhibited migration in cells infected with adenovirus to express GFP or SERCA wild type (WT), but not with a C674S SERCA mutant. After HG, NO failed to inhibit migration, nor did it decrease calcium-dependent association of calmodulin with calcineurin, indicating that NO failed to decrease intracellular calcium levels via SERCA. In contrast, overexpression of SERCA WT, but not the SERCA C674S mutant, preserved the ability for NO to inhibit migration despite exposing the cells to HG. The antioxidant, Tempol, or overexpression of superoxide dismutase also prevented the effects of HG. Further studies showed that both biotinylated-iodoacetamide and NO-induced biotinylated glutathione labeling of SERCA C674 were decreased by HG, and a sequence-specific sulfonic acid antibody detected oxidation of the C674 SERCA thiol. These results indicate that failure of NO to inhibit migration in VSMC exposed to HG is due to oxidation of the SERCA reactive cysteine-674.

Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress

DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy.

Amyotrophic lateral sclerosis models derived from human embryonic stem cells with different superoxide dismutase 1 mutations exhibit differential drug responses

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative motor neuron (MN) disease. The gene encoding superoxide dismutase 1 (SOD1) is a causative element of familial ALS. Animal ALS models involving SOD1 gene mutations are widely used to study the underlying mechanisms of disease and facilitate drug discovery. Unfortunately, most drug candidates have failed in clinical trials, potentially due to species differences among rodents and humans. It is unclear, however, whether there are different responses to drugs among the causative genes of ALS or their associated mutations. In this study, to evaluate different SOD1 mutations, we generated SOD1-ALS models derived from human embryonic stem cells with identical genetic backgrounds, except for the overexpression of mutant variants of SOD1. The overexpression of mutant SOD1 did not affect pluripotency or MN differentiation. However, mutation-dependent reductions in neurite length were observed in MNs. Moreover, experiments investigating the effects of specific compounds revealed that each ALS model displayed different responses with respect to MN neurite length. These results suggest that SOD1 mutations could be classified based the response of MNs to drug treatment. This classification could be useful for the development of mutant-specific strategies for drug discovery and clinical trials.

Abstract 119: The Imbalance of Mitochondrial Superoxide Dismutase Activity and Superoxide Production in Endothelial Dysfunction and Hypertension

Clinical studies have shown that Sirt3 expression declines by 40% by age 65 paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 due to increased NADH and Acetyl-CoA. Sirt3 activates a major mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) by deacetylation of specific lysine residues. We hypothesized that loss of Sirt3 activity increases vascular oxidative stress due to SOD2 hyperacetylation and this promotes endothelial dysfunction and hypertension. Indeed, Western blot analysis showed 3-fold increase in SOD2 acetylation and 1.5-fold decrease in Sirt3 level in hypertensive human subjects. Infusion of angiotensin II (0.7 mg/kg/day) in C57Bl/6J mice increased acetylation of vascular SOD2 by 2-fold, reduced SOD2 activity to 52% and raised blood pressure to 156 mm Hg. We have tested if Sirt3 depletion exacerbates endothelial dysfunction and hypertension. Indeed, Western blot analysis of Sirt3-/- mice showed SOD2 hyperacetylation and reduced SOD2 activity, however, blood pressure and superoxide production was normal. Angiotensin II infusion in Sirt3-/- mice increased blood pressure to 182 mm Hg, increased superoxide, reduced endothelial nitric oxide and impaired acetylcholine-mediated vasodilatation compared with angiotensin II infused wild type mice. Treatment of hypertensive Sirt3-/- mice with SOD2 mimetic mitoTEMPO (1.4 mg/kg/day) after onset of angiotensin II-induced hypertension normalized blood pressure and restored endothelial dependent vasodilatation. We suggest that mitochondrial superoxide does not contribute to the basal vasodilation and blood pressure regulation however high salt, angiotensin II and inflammation increase mitochondrial superoxide. Indeed, superoxide production by complex I in submitochondrial particles was significantly increased in hypertensive mice and this was associated with complex I S-glutathionylation. Interestingly, SOD2 overexpression in transgenic mice prevents complex I S-glutathionylation, reduces superoxide production by complex I and attenuates hypertension. These data indicate that imbalance of SOD2 activity and mitochondrial superoxide production contributes to endothelial dysfunction and hypertension.

Epigenetic modifications in mouse cerebellar Purkinje cells: effects of aging, caloric restriction, and overexpression of superoxide dismutase 1 on 5-methylcytosine and 5-hydroxymethylcytosine

The aim of the present study was to assess alterations in DNA methylation and hydroxymethylation during aging in cerebellar Purkinje cells and to determine the effects of putatively preventative measures to such age-related changes. Using immunohistochemical techniques, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) immunoreactivity in cerebellar Purkinje cells of 12-month- and 24-month-old mice was interrogated. Additionally, the modulatory effects of caloric restriction (CR) and normal human Cu/Zn super oxide dismutase 1 overexpression on these changes were assessed. We show that aging is associated with an increase of 5-mC and 5-hmC immunoreactivity in mouse cerebellar Purkinje cells. These age-related increases were mitigated by CR but not super oxide dismutase 1 overexpression. Additionally, the ratio between 5-mC and 5-hmC decreased with age and CR treatment, suggesting that CR has a stronger effect on DNA methylation than DNA hydroxymethylation. These findings enforce the notion that aging is closely connected to marked epigenetic changes, affecting multiple brain regions, and that CR is an effective means to prevent or counteract deleterious age-related epigenetic alterations.

Abstract 5103: Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration

Abstract Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 (officially known as CDKN1B) has been reported, but the exact mechanism(s) whereby p27 interacts with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27+/+ mouse embryonic fibroblasts (MEFs) with genetically ablated p272/2 MEFs using wound-healing, transwell and time-lapse microscopic analyses, we provide direct evidence that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD, officially known as SOD2) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression, which was further mediated by ERK- and ATF1-dependent transactivation of the cAMP response element (CRE) within the Stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucial negative role in cell migration by inhibiting MnSOD expression in a STAT3-dependent manner. Citation Format: Jing An, Dongyun Zhang, Chuanshu Huang. Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5103. doi:10.1158/1538-7445.AM2015-5103

Superoxide Dismutase 1 In Vivo Ameliorates Maternal Diabetes Mellitus-Induced Apoptosis and Heart Defects Through Restoration of Impaired Wnt Signaling.

Oxidative stress is manifested in embryos exposed to maternal diabetes mellitus, yet specific mechanisms for diabetes mellitus-induced heart defects are not defined. Gene deletion of intermediates of Wingless-related integration (Wnt) signaling causes heart defects similar to those observed in embryos from diabetic pregnancies. We tested the hypothesis that diabetes mellitus-induced oxidative stress impairs Wnt signaling, thereby causing heart defects, and that these defects can be rescued by transgenic overexpression of the reactive oxygen species scavenger superoxide dismutase 1 (SOD1). Wild-type (WT) and SOD1-overexpressing embryos from nondiabetic WT control dams and nondiabetic/diabetic WT female mice mated with SOD1 transgenic male mice were analyzed. No heart defects were observed in WT and SOD1 embryos under nondiabetic conditions. WT embryos of diabetic dams had a 26% incidence of cardiac outlet defects that were suppressed by SOD1 overexpression. Insulin treatment reduced blood glucose levels and heart defects. Diabetes mellitus increased superoxide production, canonical Wnt antagonist expression, caspase activation, and apoptosis and suppressed cell proliferation. Diabetes mellitus suppressed Wnt signaling intermediates and Wnt target gene expression in the embryonic heart, each of which were reversed by SOD1 overexpression. Hydrogen peroxide and peroxynitrite mimicked the inhibitory effect of high glucose on Wnt signaling, which was abolished by the SOD1 mimetic, tempol. The oxidative stress of diabetes mellitus impairs Wnt signaling and causes cardiac outlet defects that are rescued by SOD1 overexpression. This suggests that targeting of components of the Wnt5a signaling pathway may be a viable strategy for suppression of congenital heart defects in fetuses of diabetic pregnancies.

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis.

Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is tightly regulated in intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow modulates autophagy with an implication in mitochondrial superoxide (mtO2(•-)) production. In the disturbed flow or oscillatory shear stress (OSS)-exposed aortic arch, we observed prominent staining of p62, a reverse marker of autophagic flux, whereas in the pulsatile shear stress (PSS)-exposed descending aorta, p62 was attenuated. OSS significantly increased (i) microtubule-associated protein light chain 3 (LC3) II to I ratios in human aortic endothelial cells, (ii) autophagosome formation as quantified by green fluorescent protein (GFP)-LC3 dots per cell, and (iii) p62 protein levels, whereas manganese superoxide dismutase (MnSOD) overexpression by recombinant adenovirus, N-acetyl cysteine treatment, or c-Jun N-terminal kinase (JNK) inhibition reduced OSS-mediated LC3-II/LC3-I ratios and mitochondrial DNA damage. Introducing bafilomycin to Earle's balanced salt solution or to OSS condition incrementally increased both LC3-II/LC3-I ratios and p62 levels, implicating impaired autophagic flux. In the OSS-exposed aortic arch, both anti-phospho-JNK and anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) staining for DNA damage were prominent, whereas in the PSS-exposed descending aorta, the staining was nearly absent. Knockdown of ATG5 with siRNA increased OSS-mediated mtO2(•-), whereas starvation or rapamycin-induced autophagy reduced OSS-mediated mtO2(•-), mitochondrial respiration, and complex II activity. Disturbed flow-mediated oxidative stress and JNK activation induce autophagy. OSS impairs autophagic flux to interfere with mitochondrial homeostasis. Antioxid. Redox Signal. 23, 1207-1219.

MWCNTs Induce ROS Generation, ERK Phosphorylation, and SOD-2 Expression in Human Mesothelial Cells.

Biological oxidative responses are involved in the toxicity of multiwall carbon nanotubes (MWCNTs), which may cause asbestos-like pathogenicity. Superoxide dismutase 2 (SOD-2) has been proposed as a biomarker of early responses to mesothelioma-inducing fibers. This study was conducted to investigate the alteration of SOD-2 expression in the human mesothelial cell lines Met-5A after exposure to nontoxic doses of MWCNTs and the potential signaling pathway. The parameters measured included the viability, morphological change, superoxide formation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and messenger RNA (mRNA)/protein levels of SOD-2. Our results showed that MWCNTs upregulated SOD-2 expression at both mRNA and protein level. Coincidently, both superoxide formation and ERK1/2 phosphorylation were observed in Met-5A cells exposed to MWCNTs and were diminished by pretreatment with the reactive oxidative species (ROS) scavenger, N-acetyl-l-(+)-cysteine (NAC). To further investigate the role of ROS/ERK1/2 in MWCNTs-induced SOD-2 overexpression, prior to MWCNTs exposure, cells were pretreated with the Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) inhibitor (U0126) or with NAC. Both pretreatments decreased the MWCNTs-induced overexpression of SOD-2. These results suggest that upregulation of SOD-2 in Met-5A cells exposed to MWCNTs is mediated by ROS formation and ERK1/2 activation.

Targeting mitochondrial reactive oxygen species to modulate hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O2•−) and hydrogen peroxide (H2O2) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O2•− (mtO2•−) and H2O2 (mtH2O2) increase Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Exposure of human pulmonary artery endothelial cells to hypoxia for 72h increased mtO2•− and mtH2O2. To assess the contribution of mtO2•− and mtH2O2 to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (TghSOD2) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O2) or hypoxia (10% O2) for three weeks. Compared with hypoxic control mice, MCAT mice developed smaller hypoxia-induced increases in RVSP, α-SMA staining, extracellular H2O2 (Amplex Red), Nox2 and Nox4 (qRT-PCR and Western blot), or cyclinD1 and PCNA (Western blot). In contrast, TghSOD2 mice experienced exacerbated responses to hypoxia. These studies demonstrate that hypoxia increases mtO2•− and mtH2O2. Targeting mtH2O2 attenuates PH pathogenesis, whereas targeting mtO2•− exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H2O2 release. These studies suggest that targeted reductions in mtH2O2 generation may be particularly effective in preventing hypoxia-induced PH.

Abstract 582: Overexpression of Mnsod Reduces Aortic Valve Calcification Through Repression of Pro-osteogenic Signaling

Increased BMP signaling and markers of osteogenic differentiation are strongly associated with regions of valvular calcification in aortic valves from humans with end-stage calcific aortic valve stenosis. While oxidative stress is also markedly elevated in these regions, it is unknown whether increasing antioxidant capacity can reduce pro-osteogenic signaling and valvular calcification. Therefore, we hypothesized that overexpression of manganese superoxide dismutase (MnSOD) can reduce pro-osteogenic signaling in the aortic valves from hypercholesterolemic mice, ultimately resulting in reduced valvular calcification and improved valvular function. We used Ldlr -/- /ApoB 100/100 (LA) mice that were wild-type for MnSOD (LA-MnSOD 0/0 ) or overexpressing MnSOD (LA-MnSOD Tg/0 ) and fed a western diet (TD88137) for six months. We first measured mRNA levels of the BMP ligands BMP2 and BMP4 (qRT-PCR), both of which were unchanged between LA-MnSOD 0/0 and LA-MNSOD Tg/0 mice. While there was a small but significant increase in Runx2 mRNA levels in LA-MnSOD Tg/0 mice compared to wild-type littermates (1.22±0.09, 1.0±0.09, qRT-PCR, p < 0.05.) gene expression levels of late stage osteoblast marker Sp7 were reduced by approximately 50% in LA-MnSOD Tg/0 compared to wild-type littermates (0.53±0.15, 1.0±0.24, qRT-PCR, p < 0.05). Sp7 protein levels (immunohistochemistry) were also significantly reduced in LA-MnSOD Tg/0 compared to wild-type littermates (164.5±12.3 RFU vs 259.6±10.4 RFU, p < 0.05). Critically, aortic valve calcification (Alizarin Red) was also significantly reduced in LA-MnSOD Tg/0 mice compared to their LA-MnSOD 0/0 littermates. Functionally, aortic valve cusp separation distance (high resolution echocardiography) was slightly improved in LA-MnSOD Tg/0 mice compared to LA-MnSOD 0/0 mice (0.98±0.04 mm vs 0.91±0.03 mm, p = n.s.). Collectively, these data suggest that increasing mitochondrial antioxidant capacity can reduce pro-osteogenic signaling and attenuate aortic valve calcification, and may be a potential therapeutic target to slow the progression of valvular calcification and dysfunction in patients with calcific aortic valve stenosis.