Hydrogen Peroxide Contributes to the Relaxation Induced by C-Type Natriuretic Peptide in 2K-1C Hypertensive Rat Aorta

inflammation and antioxidant response element protein induction. Post-operative PCF (4 hr) stimulated manganese superoxide dismutase (MnSOD) within 4 hr and heme-oxygenase-1 (HO-1) within 16 hr in Thp1 cells. in addition, the 4 hr PCF sample increased thiol oxidation and caused elongation of Thp1 cells following 16 hr exposure. in summary, our data suggest that the quality of post-operative PCF adversely affects Thp1 monocytic cells and this may contribute to the development of post-surgery complications in the clinical setting. These preliminary studies may lead to the development of a successful preventative therapy to improve post-surgical outcome and patient recovery. This work was supported by T32 HL007457 (TM).



regulation of superoxide in VSMC represents a novel target for future molecular therapies to prevent neointimal hyperplasia.

 doi: 10.1016/j.freeradbiomed.2013.10.581

   Cannabidiol Attenuates Cardiac Dysfunction, Oxidative Stress, and Cell Death in Doxorubicin Induced Cardiomyopathy Partha Mukhopadhyay1, Enkui Hao1, Zongxian Cao1, Eileen Holovac1, Katalin Erdelyi1, and Pal Pacher1 1 National Institutes of Health, United States

doi: 10.1016/j.freeradbiomed.2013.10.580

   Nitric Oxide Regulates Superoxide Levels in Vascular Smooth Muscle Cells via Regulation of Superoxide Dismutase-1 Edward S Moreira1, Nadiezhda Cantú-Medellín2, Eric C Kelley2, and Melina R Kibbe1 1 Northwestern University, United States, 2University of Pittsburgh, United States Background: Reactive oxygen species (ROS) are important signaling molecules that stimulate cellular proliferation following arterial injury. This leads to the development of neointimal hyperplasia which ultimately causes vascular interventions to fail. Superoxide (O2x) derived from NADPH oxidases is a key mediator of this process. We have previously demonstrated that x NO inhibits neointimal hyperplasia in association with differential regulation of SOD-1 levels throughout the arterial wall in vivo and cell type-specific regulation in vitro. Specifically, we found that x NO increases SOD-1 in vascular smooth muscle cells (VSMC), but has no effect on SOD-1 levels in endothelial cells (EC) and adventitial fibroblasts (AF). Thus, we hypothesize that xNO differentially regulates O2x levels in vascular cells via modulation of SOD-1. Methods: Primary VSMC were isolated and propagated from male Sprague Dawley rats and used at passage 4-9. Cells were exposed to the xNO donor DETA/NO (0.5 ± 1.0 mM) for 6 to 24 h. O2x levels were measured by incubating the cells with the fluorogenic probe dihydroethidium (DHE) after thorough washing of the xNO donor, followed by quantification of the specific reaction product, 2-hydroxyethidium (2-OH-E+) via HPLC with electrochemical detection. ROS were also quantified by electron paramagnetic resonance (EPR) using the cell-permeable spin probe, 1-hydroxy-3-methoxy-carbonyl-2,2,5,5tetramethylpyrrolidine (CMH). sod1 gene expression was assayed by qPCR, SOD-1 protein levels were detected by Western blot analysis, and SOD activity assessed by a colorimetric assay. Results: to further explore the increase in SOD-1 protein and gene expression observed in VSMC after xNO treatment we evaluated O2x levels by HPLC and EPR. Treatment with DETA/NO reduced the CMH EPR signal in VSMC but not in AF or EC. HPLC detection of 2-OH-E+ revealed that pre-treatment with DETA/NO for 24 h reduced O2x levels in VSMC but not in AF or EC. in agreement with a decrease in O2x levels due to an increase in SOD-1, VSMC extracts treated with DETA/NO showed a 1.6-fold increase in SOD-1 activity. Conclusion: the cell specific xNO-induced increase in SOD-1 in VSMC causes a decrease in O2x levels. This finding is unique to VSMC and does not occur in AF or EC. This cell specific

Doxorubicin (DOX) is a widely used, potent antitumor agent; however, its clinical application is limited because of its FDUGLRWR[LFLW\ 'R[RUXELFLQ¶V FDUGLRWR[LFLW\ LQYROYHV LQFUHDVHG myocardial oxidative stress, inflammation, cell death, and cardiac dysfunction. Cannabidiol is a non-psychoactive, nontoxic, cannabinoid with potent antioxidant and anti-inflammatory effects, which is well-tolerated in humans. We aimed to explore the effects of cannabidiol using a well-established in vivo mouse model of DOX-induced cardiotoxicity. DOX-induced cardiomyopathy was characterized by increased cardiac injury, oxidative/nitrative stress, myocardial inflammation, cell death, and cardiac dysfunction (decline in ejection fraction and cardiac output). Treatment with cannabidiol markedly improved DOXinduced cardiac dysfunction, oxidative/nitrative stress, inflammation and apoptosis. These data suggest that cannabidiol may represent a novel cardioprotective strategy against DOXinduced cardiotoxicity.

 doi: 10.1016/j.freeradbiomed.2013.10.582

   Hydrogen Peroxide Contributes to the Relaxation Induced by C-Type Natriuretic Peptide in 2K-1C Hypertensive Rat Aorta Laena Pernomian1 and Lusiane Maria Bendhack2 1 School of Medicine Ribeirão Preto - USP, Brazil, 2Faculty of Pharmaceutical Sciences Ribeirão Preto - USP, Brazil This study aimed to investigate the basal levels of reactive oxygen species (ROS) in the endothelial cells and their contribution to CNP-induced relaxation in isolated aorta from 2K1C rat. All the procedures were approved by the Ethical Committee of the University of São Paulo. Male rats underwent surgery for the placement of a silver clip in the renal artery (2K1C) and control rats were only submitted to laparatomy (2K). Rats were used 6 weeks after the surgeries. 2K-1C rats were considered hypertensive when systolic arterial pressure was>160mmHg. Aortas were isolated for vascular reactivity or flow cytometry studies. Endothelial cells were loaded with DHE ȝ0 and fluorescence intensity (FI) was measured in the absence (basal) or in the presence of the superoxide scavenger 7LURQ ȝ0  &RQFHQWUDWLRQ-effect curves to CNP were performed in contracted 2K and 2K-1C rat aortic rings. the protocols were performed in intact endothelium (E+) or denuded endothelium (E-), in the absence or in presence of 7LURQȝ0  $SRF\QLQ ȝ0 or PEG-catalase (250U). Pharmacological parameters analyzed were the maximum relaxing effect (ME) and potency (pD2). Basal FI of DHE was similar in 2K-1C and 2K

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aortic rings. Tiron increased FI of DHE on 2K endothelial cells, but reduced FI of DHE on 2K-1C endothelial cells. CNP-induced relaxation was similar in E+ aortas from 2K and 2K-1C. the endothelium removal similarly increased the relaxation induced by CNP in both rat aorta groups. Tiron or Apocynin had no effect, whereas PEG-catalase reduced CNP-induced relaxation in E- 2K1C rat aorta. Our results show that CNP-induced relaxation is greater in denuded than in intact endothelium 2K and 2K-1C rat aorta. O2 contributes to the basal ROS in 2K-1C aortic endothelial cells. H2O2 contributes to the CNP-induced relaxation only in denuded 2K-1C rat aorta. Supported by FAPESP and CNPq.

 doi: 10.1016/j.freeradbiomed.2013.10.583

   Nitrite Attenuates Conjugated Linoleic Acid Induced Cardiac Dysfunction in Aged Mice

Kellianne Piell1, Natia Kelm1, Megan Caroway1, Masarath Aman1, and Marsha Cole1 1 University of Louisville, United States Conjugated Linoleic Acid (cLA) is a commercially available weight loss supplement that is not currently regulated by the FDA. Healthy individuals appear to tolerate cLA supplementation well, however it is unknown how cLA may affect the aged population or those with chronic disorders. Dietary cLA has been shown to be beneficial in numerous disease states, including cancer, diabetes, atherosclerosis and immune function. Based upon these reports, it was hypothesized that administration of cLA would improve heart function in aged wild-type (WT) mice. to test this hypothesis, WT mice were aged to 10 months and treated with cLA (via osmotic mini-pump-20mg/kg/day). Echocardiography tests revealed that cardiac function was decreased in aged compared to young WT mice (68±3 vs. 35±6, p<0.05). Contrary to the hypothesis, following a 6 week treatment with cLA, percent fractional shortening (%FS) was quantitated, revealing that cLAtreated mice had increased cardiac injury compared to controls (35±6 vs. 19±9, p<0.05). Further studies demonstrated that cLA WUHDWPHQW ȝ0  VLJQLILFDQWO\ GHFUHDVHG LQWUDFHOOXODU QLWULF R[LGH levels (chemiluminescent detection of nitrite and nitrate) in human umbilical vein endothelial cells (HUVECs) compared to controls (2.7μM vs. 1.5μM, p<0.05) (10.5μM vs. 7.7μM, p<0.05), while levels in the media remain unchanged. in addition, expression of endothelial nitric oxide synthase (eNOS) was diminished in HUVEC cultures following cLA treatment (74% of control values, p<0.05). Based upon these findings, examination of eNOS expression, an important regulator of cardiac function, was assessed and found to be decreased in mouse cardiac tissue following treatment with cLA. Importantly, supplementation with nitrite (50ppm in drinking water) in cLA-treated mice induced eNOS protein expression in cardiac tissue and rescued cardiac function. Taken together, these results suggest that supplementation with cLA may exacerbate cardiac injury in aging. Moreover, treatment with nitrite restores cardiac function in this model and reiterates the clinical importance of maintaining physiological NO levels. This work was supported by NIH K99/R00HL95769 to MPC.

 doi: 10.1016/j.freeradbiomed.2013.10.584

  

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  Exogenous Matrix Metalloproteinase 2 (MMP-2) Enters the Vascular Tissue and Induces Increased Aortic Contraction Via ROS Generation

Alejandro Ferraz do Prado1,2, Laena Pernomian1, Aline de Azevedo2, Lenaldo Branco Rocha3, José Eduardo Tanus dos Santos1, Lusiane Maria Bendhack4, and Raquel Fernanda Gerlach2 1 Faculty of Medicine, Ribeirao Preto - USP, Brazil, 2Dental School of Ribeirao Preto - USP, Brazil, 3Federal University of Triangulo Mineiro, Brazil, 4Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP, Brazil Both matrix metalloproteinases (MMPs) and oxygen reactive species (ROS) are increased in in cardiovascular diseases, and so far ROS has been shown to activate MMP-2, a key protease in cardiovascular diseases. This study tested whether MMP-2´s effect on vascular dysfunction was mediated by ROS. the cDNA of the catalytic domain of the human MMP-2 was cloned in fusion with GFP, and the activity of the hMMP-2cat/GFP was assayed. the rhMMP-2/GFP protein was catalytically active, but had a decreased activity and was more prone to autolysis in comparison to the rhMMP-2 (both were expressed and purified from E coli). Both proteins were injected into the lumen of New Zealand rabbit aortas, and filled vessels (MMP-2-FA) were incubated for 30 min. Concentration used: 1.2 μg/ml or 16 mmol/L, 4x the level of MMP2 found in healthy humans. Both proteins crossed the aortic laminas, but no signs of destruction of cells or the matrix were noticed by SEM at the ultrastructural level. the exogenous MMP-2 increase in aortic wall was tracked by direct observation of GFP, immunostaining and gelatinolytic activity. This last parameter doubled in the MMP-2-FA, as well as in MMP-2-FA+PMSF (a serine-proteinase inhibitor), but displayed normal values in MMP2-FA+ Doxycycline (an MMP inhibitor). ROS activity was increased by 85% (±4%) in MMP-2-FA, while there was only a slight increase in the MMP-2-FA co-infused with the MMP inhibitor Doxycycline (15 ±8%). Apocinin and PEG Catalase decreased the levels of ROS in MMP-2-FA to basal levels (P<0.001), while Tiron only reduced ROS levels by 17%(±8%)(p<0.05). We assessed the contractile response to phenylephrine in the MMP-2-FA, and observed a 25% increase in the Emax of endothelium-denuded MMP-2-FA in comparison to aortas filled with vehicle or to endothelium-intact MMP-2-FA (P<0.05). This effect was completely abrogated by the GM6001 (MMP inhibitor) and Apocinin in MMP-2-FA. This study shows for the first time that increased levels of MMP-2 described in plasma of healthy patients are sufficient to increase ROS in vessels, leading to abnormal function.

 doi: 10.1016/j.freeradbiomed.2013.10.585

   Cys42Ser PKG IĮ Mutant Mice Are Protected from Doxorubicin-Induced Cardiomyopathy

Oleksandra Prysyazhna1, Joseph Robert Burgoyne1, and Philip Philip Eaton1 1 King's College London, United Kingdom Doxorubicin (DOX) is an anthracyclin antibiotic commonly used in the treatment of many cancers. However, its cardiotoxicity remains a clinical concern and has been a major limitation of DOX use. Reactive oxygen species, generated by the interaction of DOX with iron, can damage the cardiac myocytes to cause cell death. Recent studies have shown phosphodiesterase-5 inhibitors

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