Superoxide Dismutase 1 Regulsted the Inflammatory Remodeling of Aorta from Hypertensive Mice with Angiotensin II

demonstrated that global cell superoxide (DHE), mitochondrial superoxide (MitoSOX), as well as cell H2O2 (amplex red) production rates are significantly different among the 3 strains, as follow: LDLR-/-NntMut > B6J-NntMut> B6J-Unib-NntW. These findings suggest that all cell sources of reactive oxygen species, including mitochondria, are augmented in additive manner in single and double mutant mice. Therefore, an increased oxidative stress observed in macrophages from the double mutant hypercholesterolemic mice (LDLR-/-NntMut) can be attributed to both mutations, Nnt and LDL receptor genes, which impact on processes of generation and consumption of NADPH, respectively.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.148 109 6HSLDSWHULQ5HVWRUHVWKH0HWDEROLF3KHQRW\SH6ZLWFK ,QGXFHGE\$QWL$QJLRJHQLF)DFWRUVLQ3UHHFODPSVLD

Lissette Carolina Sanchez-Aranguren1,2, Sandra Milena SanabriaBarrera1,2, Cindy Tatiana Espinosa-Gonzalez1, Laura Maria GonzalezOrtiz1, Jeannette Vasquez-Vivar3, and Marcos Lopez1,2 1 Cardiovascular Foundation of Colombia, Santander, Colombia, 2 Universidad del Valle, Cali, Colombia, 3Medical College of Wisconsin, USA Preeclampsia (PE) is the leading cause of maternal and neonatal deaths worldwide, affecting 5-8% of all pregnancies. Currently, there are no effective ways of treatment. During normal placentation, trophoblast adapt to normal hypoxic conditions regulated by hypoxia inducible factor alpha (HIF-Į  +,)-1a orchestrates normal angiogenesis by releasing levels of soluble fms-like tyrosine kinase-1 (sFlt-1) which modulate vascular endothelial growth factor (VEGF) signaling. In PE, placenta secrete damaging levels of anti-angiogenic factors (AAFs) such as sFlt-1 and pro-inflammatory factors (IFs) like TNF-Į DQG &reactive protein inducing maternal endothelial dysfunction and hypertension. However, the underlying mechanisms that interrupt the harmony of a normal pregnancy to induce PE remain unknown. Here, we report for the first time that sFlt-1 affects mitochondrial maximal respiration and spare respiratory capacity in endothelial cells (ECs) in a dose dependent manner, leading to a metabolic phenotype switch to glycolysis. In addition, sFlt-1 exacerbate proton leak, suggesting changes in mitochondrial membrane potential that were confirmed by JC-1 fluorescence. In contrast, we found that first trimester extravillous trophoblast (HTR8/SVneo) have a basal glycolytic metabolic profile. Surprisingly, sFlt-1, even at high concentrations, did not disturb its mitochondrial metabolic phenotype. IFs caused detrimental effects on both ECs and HTR8/SVneo, ratifying their role in PE pathogenesis. Sepiapterin (SE), a precursor RI H126¶ FR-factor tetrahydrobiopterin, restored the metabolic phenotype switch induced by AAFs and IFs in ECs, and protected ECs and HTR8/SVneo from AAFs- and IFs-induced mitochondrial superoxide formation as detected by MitoSOX fluorescence. Our results demonstrate for the first time that AAFs and IFs are bioenergetic disruptors in ECs, but not in HTR8/SVneo trophoblasts which explain the damaging role of AAFs in maternal endothelium and the hallmark of hypertension. In addition, we postulate the use of SE, known to be safe during pregnancy, as a promising therapeutic approach to prevent PE.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.149

110 0LWRFKRQGULD'HULYHG2[LGDQWVDQG&HOOXODU 6HQHVFHQFH Morufat Abisola Sanusi1 and Karl E. Herbert1 1 University of Leicester, UK

Oxidants are widely implicated in vascular ageing and cardiovascular disease. For example, within atherosclerotic plaques senescent vascular smooth muscle cells (VSMC) accumulate with evidence of oxidative stress and oxidative damage to DNA. In this way, cell senescence may contribute to some of the deleterious changes in the ageing cardiovascular system. Our previous data showed that angiotensin II caused StressInduced Premature Senescence (SIPS) in primary human VSMC via oxidant generation; this may account for the beneficial antiageing effects of lifelong angiotensin inhibitor administration to rodents. Prevention of senescence with a mitochondria-targeted antioxidant, Mito-TEMPO, suggested the mechanism was dependent on mitochondrial superoxide. The current study aimed to investigate if modulation of mitochondrial reactive oxygen species signalling is a general mechanism for senescence induction in human primary VSMC. The electron transport chain inhibitors antimycin A and rotenone both stimulated SIPS in VSMC. This was accompanied by an increase in mitochondrial and cytosolic O2Ɣ- observed using flow cytometry with MitoSOX and DHE. Interestingly, antimycin A and rotenone also lead to a reduction in overall H2O2 levels suggesting a possible protective mechanism and highlighting the complexity of the signalling mechanism involving mitochondrial oxidants. Analysis using TaqMan qPCR suggested that changes in antioxidant gene expression do not account for the reduction in peroxide levels. Further studies to localize H2O2 changes following mitochondrial perturbation are necessary. As a first step data with the genetically encoded H2O2 fluorescent sensor, HyPer, in the cytosolic and mitochondrial compartments will be presented. Supported by the Medical Research Council, UK.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.150 111 6XSHUR[LGH'LVPXWDVH5HJXOVWHGWKH,QIODPPDWRU\ 5HPRGHOLQJRI$RUWDIURP+\SHUWHQVLYH0LFHZLWK $QJLRWHQVLQ,,

Atsushi Sato1, Yasushi Shiraishi1, Takayuki Namba1, Norio Ishigami1, Toyokazu Kimura1, Shunpei Horii1, Hirotaka Yada1, Takehiko Kujiraoka1, and Takeshi Adachi1 1 National Defense Medical Collage, Japan Angiotensin II (Ang II) induces aortic remodeling, which is associated with H2O2 generation by NADPH oxidase (NOX) in combination with SOD1. To investigate the role of SOD1 in Ang IIinduced aortic hypertrophy in vivo. We employed SOD1 deficient (SOD1-/-) mice and continuously infused with Ang II (3.2 mg/kg/day) for seven days. There was no difference in hypertensive responses, however, aortic wall thickening was decreased in SOD1-/- mice. Ang II infusion enhanced the phosphorylation of STAT3 in aortas in WT mice, which was blunted in SOD1-/- mice. RT-PCR revealed that the upregulations of aortic IL-6, NOX2, IL-1ȕ, and MCP-1, by AngII were also blunted in aorta from SOD1-/- mice. In cultured rat vascular smooth muscle cells, by down-regulation of SOD1 with siRNA, the

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culture fluid IL-6 was unchanged by Ang II and the phosphorylation of STAT3 was blunted either with Ang II (24 h). These results indicated the activation of STAT3 by IL-6 was involved in aortic remodeling and SOD1 promoted the local H2O2 and inflammation by Ang II, which leaded to aortic remodeling.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.151 112 &RRUGLQDWHG0LFUR51$6LOHQFLQJRI1R[1$'3+ 2[LGDVHDQG0\RFDUGLQ5HJXODWHV6PRRWK0XVFOH &HOO'HGLIIHUHQWLDWLRQLQ9DVFXODU'LVHDVH

Brandon M Schickling1 and Francis J Miller1,2 1 The University of Iowa, USA, 2VA Medical Center, Iowa City, IA, USA The transcription factor myocardin plays a critical role in the regulation of smooth muscle cell (SMC) phenotypic switching in vascular disease. The Nox4 NADPH oxidase has been implicated in SMC differentiation and its expression associated with changes in myocardin levels. However, the molecular basis of Nox4 regulation and the mechanisms by which vascular injury result in molecular reprogramming by myocardin are poorly understood. We hypothesized that microRNAs (miR)-mediate changes in Nox4 expression and regulate expression of genes implicated in 60& GLIIHUHQWLDWLRQ $QDO\VLV RI WKH ¶875 RI 1R[ LGHQWLILHG putative miR-9 and miR-25 binding sites which were confirmed with luciferase reporter assays. Treatment of human SMCs with a miR-9 or miR-25 mimic (1) silenced Nox4 mRNA and decreased ROS levels; (2) suppressed myocardin mRNA expression; (3) decreased expression of multiple differentiation genes; and (4) was sufficient to induce cell migration. Expression of miR-9 and miR-25 was increased in cultured human SMCs and human pulmonary artery segments after treatment with TNF-Į RU thrombin, and in murine carotid artery ten days after partial carotid ligation. Interestingly, despite silencing by a miR-25 mimic, the P\RFDUGLQ ¶875 ELQGV PL5-9 but not miR-25. We found that miR-25 induced the expression of miR-9 and pretreatment with a miR-9 inhibitor prevented miR-25-mediated silencing of Nox4 and myocardin. Suggesting a potential mechanism, miR-25 mimic caused demethylation of the miR-9 promoter. Finally, a miR-9 inhibitor prevented the migration of cultured SMC to thrombin and decreased neointimal formation by more than 50% to carotid partial ligation in mice. We conclude that following vascular injury (1) miR-25 induces epigenetic modifications resulting in the expression of miR-9; (2) miR-9 and mir-25 cooperate to silence Nox4; (3) Nox4 effects on SMC differentiation involve miR-9 regulation of myocardin; (4) inhibition of miR-9 blocks neointimal formation. These findings identify miR-9/Nox4 as part of a novel regulatory pathway and therapeutic target in vascular disease.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.152 113 9LWDPLQ&(QKDQFHV(QGRWKHOLQ%5HFHSWRU0HGLDWHG 9DVFXODU&RQWUDFWLOH)XQFWLRQLQ*XLQHD3LJ$UWHULHV Gry Freja Skovsted1, Pernille Tveden-Nyborg1, and Jens Lykkesfeldt1 1 University of Copenhagen, Denmark

While epidemiological studies have consistently found an association between vitamin C (vitC) deficiency and risk of

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cardiovascular disease, a mechanistic link has not yet been elucidated. Recent studies point to vitC as an important epigenomic regulator, most likely involved in vascular smooth muscle cell (VSMC) de-differentiation. Here, we wished to investigate the role of vitC in vascular remodeling and identify key signaling pathways in VSMCs that may be sensitive to vitC deficiency and repletion. Experiments were performed ex vivo on uterine arteries and in vitro on primary VSMCs isolated from guinea pigs. Variations in isometric tension of artery rings in response to the endothelin receptor agonists, sarafotoxin 6c (ETBRA) and endothelin-1 (ETA+ETBRA) were studied and the effect of the vasodilator carbachol. Organ culture of artery rings for 24 hrs without vitC increased sarafotoxin 6c induced ETB receptor-mediated vasocontraction (Emax=33.7±0.6) compared to fresh artery rings (Emax=10.0±0.1). Interestingly, organ culture in presence of vitC further and significantly increased the ET B receptor-mediated vasoconstriction (Emax=81.0±0.4, p<0.001), indicating that vitC augments plasticity in VSMCs. ET A receptormediated contraction and carbachol-induced vasodilation was unaffected by vitC. Furthermore, our in vitro studies revealed that VPRRWKPXVFOH60ĮSURWHLQFRQWHQWZDVLQFUHDVHGLQ960&V treated with vitC, indicating that vitC facilitates the phenotypic switch of VSMCs from the de-differentiated to the differentiated contractile form. Collectively, these data shows a possible novel role of VitC in vascular remodeling. Our data demonstrate that VitC augments vascular ETB receptor contractile function in artery ULQJV DQG VXJJHVW WKDW WKLV HIIHFW PD\ EH FRUUHODWHG WR YLW&¶V induction of VSMC phenotypic switching.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.153 114 *HQHUDWLRQDQG&KDUDFWHUL]DWLRQRI&DUGLRP\RF\WH 6SHFLILF5HGR[6HQVRU0RXVH/LQHV

Lija Swain1, Andrea Kesemeyer 1, Stefanie Meyer Roxlau 2, Ali ElArmouche2, Viacheslav Nikolaev3, and Doerthe Katschinski 1 1 2 University Medicine Göttingen, Germany, Technische Universität Dresden, Germany, 3Clinic for Cardiology, Germany Changes in the cellular redox potential can stimulate signaltransduction pathways, which are important for physio- and pathophysiology. Regarding cardiovascular diseases these include pathways involved in disease entities like ischemia or hypertrophy resulting in heart failure. Recent advances to quantitatively describe defined redox changes include the application of newly developed redox biosensors. We generated Į0+&-driven cardiomyocyte-specific Grx1-roGFP2 sensor mice, in which the biosensor is expressed either in the cytosol (cytoGrx1-roGFP2) or in the mitochondrial matrix (mito-Grx1-roGFP2). These mice now allow quantitative measurements of glutathione redox changes in both isolated intact cardiomyocytes and in the whole heart. H2O2 and diamide induced an oxidative response in isolated cardiomyocytes, which were reverted by DTT treatment demonstrating that the Grx1-roGFP2 biosensor is functionally expressed. Using the Nernst equation the calculated glutathione redox potential (EGSH) for resting cardiomyocytes in the mitochondria is -277.98 mV ± 0.66 mV and -275.98 mV ± 0.42 mV for two independent mouse lines. In the cytoplasm the calculated EGSH is -256.95 mV ± 0.79 mV and -256.31 mV ± 0.72 mV for two independent mouse lines. A similar difference in the glutathione redox potential in the mitochondria versus cytosol was observed in Langendorff perfused whole heart imaging analyses: the redox potential in the mitochondria is -275.27 ± 4.7 mV and in the cytoplasm is -254.74 ± 6.2 mV. Isoprenaline stimulation of cardiomyocytes in the cyto-Grx1-roGFP2 showed an oscillation of the glutathione potential whereas in the mitochondria isoprenaline

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