- hearts Plays a Critical Role in Modulating Cardiovascular Function Under Oxidative Stress

tetralinoleoyl (L4CL) to linoleoyltrioleoyl-CL (LO3CL). This remodeling is associated with decreased respiration (30% of baseline). ApAP restores normal CL composition and oxygen consumption to 80% of baseline when added to the heart before hypoxia/reoxygenation. Interestingly, ApAP still protects respiration when added after hypoxia before reoxygenation (66% of baseline). Then, we measured electron transport for each complex before and after hypoxia/reoxygenation. We found that electron entry through complex I is decreased by 61% after hypoxia/reoxygenation, whereas complex II and the ȕ-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) are not significantly affected. Importantly, ApAP restored normal electron transport by complex I. To assess whether this effect was caused by CL remodeling and not by a direct effect of ApAP on complex I, we showed that CL supplementation restored normal electron entry through complex I. In conclusion, our results show that hypoxia/reoxygenation inhibits mitochondrial respiration by decreasing electron entry through complex I. This modification of the respiratory chain activity is caused by remodeling of CL fatty acid composition and is normalized by ApAP treatment or by CL supplementation. Both of these interventions are amenable to rapid translation into human studies.

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Bronwyn Brown1,2, Roxanne Parungao1,3, and Clare Hawkins1,2 1 The Heart Research Institute, Australia, 2Sydney Medical School, University of Sydney, Australia, 3Discipline of Physiology, University of Sydney, Australia Myeloperoxidase (MPO) is a clinically significant enzyme found in atherosclerotic lesions. MPO forms the reactive oxidants hypochlorous and hypothiocyanous acids (HOCl and HOSCN), which have been linked to tissue damage and cellular dysfunction. HOCl and HOSCN also modify low-density lipoproteins (LDL), which leads to the increased uptake of these particles and lipid accumulation in macrophages. Macrophage accumulation of LDL modified by oxidation (oxLDL) is considered a key step in the development of atherosclerosis. Macrophages within atherosclerotic lesions are not a homogenous cell type, ZLWK WKH SUHVHQFH RI ERWK LQIODPPDWRU\ µ0¶ DQG DOWHrnatively DFWLYDWHG µ0¶ PDFURSKDJHV 7KH OHVLRQ PLFURHQYLURQPHQW SOD\V a role in determining the distribution of M1 and M2 cells. In this study, we examined the role of LDL modified by MPO oxidants in modulating both inflammatory and phenotypic markers in human macrophages. LDL was modified by exposure to HOCl or HOSCN for 24 h at 37°C before incubation with human macrophages for up to 48 h. Macrophage phenotype was examined by changes in gene expression (qPCR), chemokine/cytokine release (ELISA) and cell surface marker changes (flow cytometry). Exposure of macrophages to each modified LDL led to increases in the mRNA expression and secretion of MCP-1 and IL-1ȕ, which are associated with the M1 phenotype. No change in the mRNA expression of other M1 markers (IL-12, IL-6, TNFĮ) or the M2 markers IL-10 or TGFȕ1 was observed, but HOCl-modified LDL reduced the secretion TGFȕ1. Cell surface expression of CD 86 (M1) and CD206 (M2) were also unchanged. However, increases in IL-8 and heme oxygenase 1 (HO-1) mRNA expression were observed, consistent with the activation of pro-inflammatory pathways. With MCP-1 and IL-1ȕ, the increase in expression was more pronounced with HOSCN-LDL rather than HOCl-LDL. These data are consistent with a role of LDL modified by HOCl

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and HOSCN promoting inflammation rather than perturbing macrophage phenotype, which may help to drive lesion development in atherosclerosis.

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Rebecca Charles1, Bruce Freeman2, and Philip Eaton1 1 Kings College London, UK, 2University of Pittsburgh School of Medicine, USA Soluble epoxide hydrolase (sEH) is redox regulated by electrophilic lipids, such as 15-deoxy-prostaglandin J2 or isomers of nitro-oleic acid, including 10-nitro-octadec-9-enoic acid (NO2OA). These electrophiles adduct to Cys521 of sEH resulting in inhibition of its hydrolase activity. This is notable as sEH inhibition offers broad protection against cardiovascular disease. Previously NO2-OA administration was shown to inhibit the hydolase present in wild type (WT), but not littermate Cys521Ser sEH knock-in (KI), mice. Consistent with this, administration of NO 2-OA reduced MAP in angiotensin II-treated hypertensive WT mice, but was ineffective in lowering BP in littermate KIs. sEH inhibition has also previously been shown to limit ischemic damage in the heart and likewise NO2-OA has also shown to be anti-ischemic. This led us to the hypothesis that NO2-OA-dependent inhibition of sEH may limit myocardial injury during ischemia and reperfusion (I/R). We measured infarct size (using tetrazolium staining) after 30 min ischemia and 120 min reperfusion in isolated perfused hearts, comparing WT and Cys521Ser sEH KI responses. KI were more susceptible to injury with 56.5r3.3% myocardial infarction compared to 35.8r3.0% (n=5-8 group, p0.05) in the WT following the I/R protocol. This is consistent with a role for Cys521 of sEH in transducing cardioprotective responses of endogenous lipid electrophiles. NO2-OA pre-treatment (100 PM, 20 min) prior to ischemia significantly reduced infarction to 25.2r2.8% in WT (p<0.05), but failed to do so in the KI (50.2r2.2%). We conclude that sEH may be inhibited by endogenous lipid electrophiles adducting to Cys521. When this inhibition occurs epoxyeicosatrienoic acids may accumulate, exerting their cardioprotective actions to limit damage during I/R. Furthermore, NO2-OA has therapeutic potential as an anti-ischemic drug by inhibiting sEH.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.125 86 8S5HJXODWLRQRI1R[([SUHVVLRQLQ'863 KHDUWV 3OD\VD&ULWLFDO5ROHLQ0RGXODWLQJ&DUGLRYDVFXODU )XQFWLRQ8QGHU2[LGDWLYH6WUHVV Joseph Lamb1, Mark G Angelos1, and Chun-An (Andy) Chen1 1 Ohio State University, USA

Increased production of reactive oxygen species (ROS) and decreased antioxidant activity contributing to the cellular redox imbalance is the primary pathogenic factor of ischemia/reperfusion (I/R) injury in hearts. Redox imbalance leads to cellular dysfunction through protein oxidation, DNA

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damage, and lipid peroxidation. During I/R in hearts, NADPH oxidases (Noxs) are one of the major sources of ROS generation. The burst of ROS production during reperfusion activates stressresponse elements, including the p38 kinase whose overactivation is one of the detrimental contributors to cardiovascular dysfunction, especially I/R injury. Previously, we demonstrated that N-acetyl cysteine pre-treatment up-regulates DUSP4 expression in endothelial cells, regulating p38 and ERK1/2 activities, thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 on the modulation of the ROS generation. DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R-induced apoptosis (45.81% ± 5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47% ± 1.45%). Interestingly, DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff-perfused mouse hearts were subjected to global I/R injury. DUSP4 -/- hearts had significantly larger infarct size than WT. The increase in I/Rinduced infarct in DUSP4-/- mice significantly correlates with reduced functional recovery (assessed by: RPP%, LVDP%, HR%, and dP/dtmax) as well as lower CF% and a higher initial LVEDP. The level of Nox4 protein and mRNA expression is up-regulated in DUSP4-/- hearts. This data strongly suggests that DUSP4 is of critical importance in modulating myocardial function after I/R injury and the reason why DUSP4-/- hearts are more susceptible to I/R injury. Therefore, the identification of Nox4 modulation by DUSP4 provides a novel therapeutic target for oxidant-induced diseases, especially myocardial infarction.

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Bruke Tedla1, Adam Bush1, Thomas D Coates2, Henry Forman1, John Wood2, and Jon A Detterich2 1 University of Southern California, USA, 2Children's Hospital Los Angeles, USA Recent evidence suggests that sickle cell trait (SCT, heterozygotes for HbS) subjects who are exposed to intense training conditions are prone to sudden death. Glutamine is known to elevate NADPH/NADP+. The aim of this study was to examine the effect of oxidative stress secondary to muscle exertion, and assess its impact on red blood cell (RBC) glutamine, GSH/GSSG and NADPH/NADP+ balance, RBC deformability and whole blood viscosity. Methods: SCT and control (CTRL) underwent handgrip exercise. Blood was collected pre and post handgrip. RBC deformability and whole blood viscosity at native hematocrit were measured using laser ektacytometry and a tube viscometer. RBC glutamine, GSH/GSSG and RBC NADPH/NADP+ were also collected and analyzed. Results: No significant differences in deformability or viscosity were observed between CTRL and SCT. In SCT, low/mid shear viscosity increased post handgrip, whereas high shear viscosity decreased in CTRL post handgrip. There was no significant difference in GSH/GSSG ratio after handgrip between SCT and CTRL, but there was a trend toward higher pre-handgrip NADPH/NADP and a statistically significant drop in NADPH/NADP in SCT compared to control. In contrast, when

controlled for the pre handgrip NADPH/NADP levels, there was no difference in the percent change of NADPH/NADP; however, the absolute change was associated with the pre-handgrip NADPH/NADP level in SCT subjects but not CTRL. Conclusions: There is a subpopulation in our cohort of SCT subjects with an elevated RBC NADPH/NADP pre-handgrip compared to CTRL but the percent change in that ratio after handgrip is not different from CTRL. This suggests that there is differential modulation of NADPH/NADP in SCT subjects other than GSH recycling.

doi: 10.1016/j.freeradbiomed.2015.10.127 doi: xxxxx

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Anna E Dikalova1, Hanna A Itani1, Rafal R Nazarewicz1, William G McMaster1, Joshua P Fessel1, Jorge L Gamboa1, David G Harrison 1, and Sergey Dikalov1 1 Vanderbilt University Medical Center, USA Clinical studies have shown that Sirt3 expression declines by 40% by age 65 paralleling the increased incidence of hypertension. Metabolic syndrome, hyperlipidemia and diabetes further inactivate Sirt3 due to increased NADH and Acetyl-CoA levels. 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 in hypertensive human subjects showed 3-fold increase in SOD2 acetylation and 1.5-fold decrease in Sirt3 while SOD2 expression was not affected. Infusion of angiotensin II (0.7mg/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 H2O2 sensing by Sirt3 induces mitochondrial superoxide and this contributes to hypertension. Indeed, expression of mitochondria targeted catalase in mCAT mice prevents Sirt3 S-glutathionylation, preserves SOD2 activity and attenuates hypertension. We found that Sirt3 depletion (Sirt3-/-) exacerbates angiotensin II induced hypertension (182 mm Hg), increases vascular superoxide, reduces endothelial nitric oxide and impairs acetylcholine-mediated vasodilatation compared with angiotensin II infused wild type mice. Treatment of Sirt3-/- mice with SOD2 mimetic mitoTEMPO (1.4 mg/kg/day) after onset of angiotensin II-induced hypertension normalized blood pressure and improved endothelial dependent vasodilatation. We suggest that reduced SOD2 activity promote mitochondrial superoxide production. Indeed, superoxide production by complex I in submitochondrial particles was significantly increased in hypertensive mice and this was associated with complex I Sglutathionylation. Interestingly, SOD2 overexpression in transgenic mice prevents complex I S-glutathionylation, attenuates superoxide production by complex I and reduces hypertension. These data indicate that Sirt3 redox impairment lead to imbalance of SOD2 activity and mitochondrial superoxide production contributing to endothelial dysfunction and

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