Enhanced Age-Dependent ENOS Dysfunction and - Uncoupling in Glutathione Peroxidase-1-Deficient Mice

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308 Increased Oxidative Stress in Humans Under Pathological Conditions Measured by Fluorescent Heme Degradation Product in Red Blood Cells and Whole Blood 1

Enika Nagababu , Charles S Hesdorffer1, and Joseph M Rifkind1 1 National Institute on Aging We have shown in our previous studies that fluorescent heme degradation products originated from the reaction of hydrogen peroxide with hemoglobin provides a sensitive measure of oxidative stress in pathological red blood cells (RBCs). To establish the physiological significance of this measure of oxidative stress in vivo, the fluorescent heme degradation product (ex.321: em. 485) in RBCs and whole blood was measured in human subjects from the Baltimore Longitudinal Study. This study includes control subjects with no diagnosed diseases and subjects with various pathological conditions. RBCs heme degradation was significantly increased for subjects with diabetes, high blood pressure, osteoarthritis and hyperlipidemia compared with control subjects. This RBCs heme degradation was positively correlated with red cell distribution width (RDW) and negatively correlated with mean corpuscular hemoglobin (MCH). The whole blood sample includes additional heme degradation found in the plasma. It is not known whether this degradation product originate from the RBC. For whole blood heme degradation, we found a significant increase relative to controls for diabetes, hypertension, cancer, osteoarthritis, anemia and vascular diseases. The whole blood heme degradation, but not the RBC heme degradation, was positively correlated with ESR levels suggesting the involvement of inflammation. There was no age (30 to 90 years) related change in heme degradation of RBCs or whole blood. These results suggest that RBC oxidative stress may contribute to a number of pathologies. The possible effect of this oxidative stress on deformability, which can impair the delivery of oxygen, is being investigated.

doi:10.1016/j.freeradbiomed.2012.10.315

309 Disruption of Nrf2 Induces Early Onset of Atrial Remodeling and Fibrillation on Aging

Sellamuthu S. Gounder1, Dinesh Devadoss1, Sankaranarayanan Kannan2, Naser Abu-Ramelah1, Kevin S Whitehead1, E. Dale Abel1, 1 Nassir F Marrouche1, and Rajasekaran Namakkal-Soorappan 1 2 University of Utah, UT-M.D. Anderson Center, Background: Heart failure is a leading health problem with over 500,000 new cases and 275,000 deaths annually in the United States. Recent reports indicate that atrial fibrillation (AF) is linked to various forms of ventricular dysfunction (VD). Over 2 million Americans have AF, which is more prevalent in people over 65 years of age. The AHA has estimated that 25% of young adults over 40 years of age will develop AF by the year 2050. However, the molecular “cause and effect” relationship between AF and VD has not been elucidated. We hypothesize that abrogation of nuclear erythroid-2 like factor-2 (Nrf2), a master antioxidant transcriptional regulator, induces atrial remodeling and fibrillation on aging. -/Methods: Age and sex matched WT and Nrf2 mice were used in this study. Atrial mass, remodeling, antioxidants and molecular redox signaling were studied at 2 and >20 months of age. Echocardiography, immunoblotting, quantitative real-time PCR and immunofluorescence (fibrosis) analyses were performed in the atrial tissue.

Results: At 2 months of age, WT and Nrf2 mice had comparable levels of ROS in the atrium. On aging, the ROS levels were -null significantly increased in atria of Nrf2 when compared to wildtype (WT) mice at 20 months of age. While decreased Nrf2antioxidant signaling in response to increased ROS generation in -null atria of Nrf2 mice was observed, ventricular function appeared to be normal at 20 months of age. However, upon endurance exercise stress (EES), hypertrophy markers including ANF, BNF, -null PLN and SERCA2A were significantly (p<0.05) altered in Nrf2 when compared to age-matched WT mice. Further, activation of -null fibrotic process was evident in the Nrf2 mouse atrium as indicated by significantly (p<0.05) increased markers of tissue remodeling (i.e. MMP2/9) on aging. These results indicating an early onset of atrial hypertrophy/remodeling due to age-induced -null oxidative stress, which cause AF in Nrf2 mice. Age-dependent decline in Nrf2 and sustained progression of AF could lead to ventricular dysfunction and heart failure. Conclusions: Our findings indicate that atria are primary targets to age-associated oxidative stress and exhibit fibrosis, which promotes atrial fibrillation and remodeling. Early onset of oxidative stress induced atrial remodeling and sustained AF might exacerbate age-associated cardiac diseases. Thus, activation of Nrf2 signaling could be a potential therapeutic target for AF.

doi:10.1016/j.freeradbiomed.2012.10.316

310 Enhanced Age-Dependent ENOS Dysfunction and Uncoupling in Glutathione Peroxidase-1-Deficient Mice 1

Matthias Oelze , Swenja Kröller-Schön1, Edtih Lubos2, Michael Hausding1, Huige Li1, Michael Torzewski1, Philip Wenzel1, Markus Bachschmid3, Karl J. Lackner1, Thomas Münzel1, and Andreas Daiber1 1 Medical Center of the Johannes Gutenberg University, Germany, 2 University Medical Center Hamburg-Eppendorf, Germany, 3 Boston University Medical Center Recently, we showed that genetic deficiency of the mitochondrial antioxidant proteins manganese superoxide dismutase (SOD2) and aldehyde dehydrogenase-2 (ALDH-2) contribute to agerelated vascular dysfunction and mitochondrial oxidative stress. In the present study we investigate, whether the genetic deficiency -/of glutathione peroxidase (GPx-1 ) enhances aging-dependent formation of reactive oxygen and nitrogen species (RONS) and uncoupling of eNOS resulting in increased vascular dysfunction. Mice on a C57/Bl6 background of 3 different age groups were used: 2, 6 and 12 months. Vascular function was assessed via isometric tension studies using isolated aortae. Formation of RONS was assessed by optical and immunoblotting-based techniques. Dysfunction/uncoupling of endothelial NO synthase (eNOS) was assessed by the phosphorylation and Sglutathionylation pattern of immunoprecipitated eNOS. Leukocyte adhesion to cultured endothelial cells was tested upon silencing of GPx-1 by siRNA. Vascular function was significantly impaired in 12 months old -/GPx-1 -mice as compared to age-matched controls. RONSformation, envisaged by 3-nitrotyrosine staining and dihydroethidine-based fluorescence microtopography, was -/-/dramatically increased in the aged GPx-1 mice. GPx-1 deficiency resulted in pronounced protein kinase C and protein tyrosine kinase-dependent phosphorylation as well as Sglutathionylation, all of which were described to cause eNOS dysfunction or uncoupling. GPx-1 deficiency also increased the adhesion of leukocytes to cultured endothelial cells. -/GPx-1 -mice displayed increased RONS-formation as compared to their wild type littermates, which could trigger redox-signaling pathways leading to eNOS dysfunction and uncoupling ultimately

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resulting in vascular dysfunction. This loss of endothelial NO formation could contribute to adhesion and infiltration of leukocytes further contributing to vascular complications.

doi:10.1016/j.freeradbiomed.2012.10.317

311 The Impact of Prx4 on Redox Signaling, Stress Resistance and Aging in Drosophila 1

Bill Orr , Svetlana Radyuk1, and Vladimir Klichko1 1 Southern Methodist University Accumulating evidence suggests that severe disruption in thiol homeostasis and redox state represent major changes that accompany aging. By virtue of their localized actions on peroxides coupled with oxidation of such key “reducing equivalents” as thioredoxin, the peroxiredoxins are considered to be important players in maintaining redox state. Consequently, we have undertaken a series of studies to explore the potential roles of the peroxiredoxin gene family in modulating life span and health span in the fly model. Orthologs for all six members of the human peroxiredoxin family are present and here we focus on the possible functional effects of the endoplasmic reticulumassociated Prx4 at the whole animal level. Reduced expression of Prx4 (up to 90%) resulted in greater sensitivity to oxidative stress, an elevated H2O2 flux and increases in lipid peroxidation, but no meaningful impact on longevity. Broad over-expression at low levels gave reduced levels of oxidative damage and a significant increase in longevity, while flies expressing Prx4 globally at high levels (>5-fold) had a dramatically reduced life span and increased apoptosis. Analysis of these high level over-expressors revealed an aberrant redistribution of the Prx4 protein from the ER to cytosol and hemolymph. In addition to the known pro-apoptotic effects of the cytosolic form of Prx4, PRx4 over-expression triggered an NF-țBmediated pro-inflammatory response, similar to that observed in cells under ER stress or when microbially-challenged. Finally, we provide the first evidence that Prx 4, upon secretion into the hemolymph, elicits a JAK/STAT-mediated response. Thus, manipulating Prx4 levels in the fly generates a broad range of effects, and implicates an important role for Prx4 in signaling as well as in antioxidant function. Whether such mis-regulation effects might accompany the normal aging process is currently under investigation.

doi:10.1016/j.freeradbiomed.2012.10.318

312 Forced Running Elicits Arthritis and Oxidative Damage in Antioxidant Impaired Mice 1

Kathryn Pate , Rebecca Oberley-Deegan1, Michael Weaver1, Fabienne Gally1, David Goldstrohm1, James Crapo1, Russell Bowler1, and Elizabeth Regan1 1 National Jewish Health Osteoarthritis (OA) is a degenerative disease that affects over 12% of the US population between ages 25-74. Mechanical stress and injury lead to the development of OA. Chondrocytes release reactive oxygen species (ROS) in response to mechanical stress, and excess ROS damage the joint if not kept at low levels by antioxidants. A major antioxidant in the joint is extracellular superoxide dismutase (EcSOD), which binds to the cartilage matrix and scavenges superoxide to protect against oxidative damage. A naturally occurring EcSOD polymorphism (R213G)

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exists that affects the heparin binding domain of the protein and reduces its ability to bind to molecules in the extracellular matrix. We hypothesized that excessive mechanical loading induced by high intensity running would increase ROS in the knee joint and that mice heterozygous (Het) for the R213G variant would have impaired ROS scavenging and show enhanced joint damage compared to wild-type (WT) animals. WT and Het mice were randomized to 8 weeks forced treadmill running or 8 weeks cage confinement. At baseline, Het animals showed a 24% decrease in knee EcSOD protein compared to WT animals. Structural damage in tibiofemoral articular cartilage was observed in Het runners and was accompanied by enhanced protein nitration, chondrocyte DNA oxidation, and apoptosis in cartilage tissue. These results indicate that EcSOD is an important antioxidant in the joint that protects against mechanical stress-induced damage. Deficiency of ECSOD may lead to accelerated cartilage degradation or increased incidence of OA in joints subject to high level mechanical loading. Patients with OA may benefit from antioxidant therapies.

doi:10.1016/j.freeradbiomed.2012.10.319

313 Apple Peel Extract Restricts the SenescenceAssociated Secretory Phenotype and Induction of Proximal Epithelial Invasion and Mesenchymal Transition 1

Nilay Patel , Donald McCarthy1, Toni Bartling1, Pauline Mary Carrico2, and Juan Andres Melendez1 1 2 University at Albany, SUNY Empire State College The senescence associated secretory phenotype (SASP) is a characteristic of senescent fibroblasts, which alters the tissue microenvironment promoting tumor initiation and invasion through the pro-inflammatory paracrine mechanisms. These mechanisms include increases in secreted factors such as Matrix Metalloproteinase-1 (MMP-1, interstitial collagenase), IL-6, and IL-8, all of which are associated with a diverse array of agerelated disease processes. We have previously demonstrated that senescence associated MMP-1 expression is induced by ROS, and that this is increase is reversed by anti-oxidant treatment. Here, we demonstrate that Apple peel extract (APE) can limit senescence associated ROS production, MMP-1, IL-6, and IL-8 expression, as well as c-jun phosphorylation. In addition, the SASP-dependent invasive capacity of MDA-MB-231 breast cancer cells is inhibited by pretreatment with APE. The decreased invasive capacity of MDA-MB-231 is accompanied by shifts in the expression of epithelial-mesenchymal transition markers E-cadherin, and ȕ catenin. Together, these studies demonstrate SASP-driven invasion is redox- mediated and amenable to antioxidant-based therapeutics.

doi:10.1016/j.freeradbiomed.2012.10.320

314 Effect of Moderate Physical Exercise on Oxidative Damage and DNA Repair Capacity in Young Adults versus Older Healthy Adults 1

Raquel Retana-Ugalde , Gie Bele García-Discua1, and Víctor Manuel Mendoza-Núñez1 1 Universidad Nacional Autónoma de México FES, Mexico Background: Intense physical exercise increases free radicals

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