Testosterone-induced Leukocyte Migration Involves COX2 and Redox-Sensitive Processes in rats

10 Nox4 NADPH Oxidase Modulates the Intracellular Redox State Samuel Carrell1, Bojana Stanic1, and Francis Miller1 1 University of Iowa Unlike other Nox isoforms, activation of Nox4-based NADPH oxidase does not require association with cytosolic subunits and is constitutively active under physiologic conditions. It has been proposed that Nox4 activity is regulated by its level of expression. Although Nox4 has been implicated in maintaining a differentiated cell phenotype, the cellular role of Nox4 is poorly understood. We hypothesized that Nox4 modulates intracellular redox potential and thereby is a critical determinant of cellular function. Primary murine aortic smooth muscle cells (SMCs) were treated with adenovirus expressing either Nox4 to increase Nox4 levels, shNox4 to reduce Nox4 levels, or shGFP as control. Experiments were performed 72 hours later in resting confluent cells under normal growth conditions. Changes in Nox4 expression were confirmed by real time quantitative PCR and Western blotting. The ratio of NADP+ to NADPH was decreased by 44±7% in SMCs after AdshNox4, and increased by 141±28% after AdNox4. Activity of the rate limiting enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) was reduced by 38±4% after AdshNox4 and increased by 59±21% after AdNox4. AdshNox4 decreased the ratio of glutathione disulfide to reduced glutathione (GSSG:GSH) by 46±7%, while AdNox4 increased GSSG:GSH by 14±14%. Total cellular glutathione levels were decreased by 45±15% after AdshNox4 and increased by 57±21% after AdNox4. There was a greater amount of oxidized to reduced thioredoxin after AdNox4 as compared to AdshGFP. Finally, AdNox4 increased apoptosis in resting cells and after exposure to H2O2 as compared to AdshGFP and AdshNox4 and measured by flow cytometry. We conclude that changes in Nox4 expression provide a mechanism for cells to regulate intracellular redox potential thereby modifying the function of multiple cellular processes, such as kinase activity, ion channel function, and gene transcription. doi: 10.1016/j.freeradbiomed.2010.10.011

11 Assessment of Pro­angiogenic Activities of a Free  Radical Compound  Madhava Charyulu1, Syamantak Majumder2, Suvro Chatterjee3, and Arumugam Gnanamani1 1 2 Central Leather Research Institiute, AU-KBC Center for 3 Research, Anna University, AU-KBC Centere for Research, Anna University.  Angiogenesis, the growth of new capillary blood vessels in the body, is an important physiological process in the body used for healing and reproduction. Modulating angiogenesis either by inhibiting or stimulating has immense importance in present day drug development. The present study aims to investigate the effect of the pure bacterial secondary metabolite (UIBSM) on angiogenesis. We hypothesize that the free radical nature of the compound attributes to the modulation of angiogenesis. The secondary metabolite used in the present study has been extracted from the bacterial strain MTCC 5210, purified by HPLC (High Performance Liquid Chromatography) characterized for its functional group using FT-IR while functional activity such as free radical property and antioxidant property was measured using EPR (Electron Paramagnetic Resonance) and DPPH (2,2diphenyl-1-picrylhydrazyl) technique respectively. Finally, the compound was also tested for its effect on angiogenesis using an egg yolk angiogenesis model. Results of characterization studies of UIBSM demonstrates that UIBSM contains OH, C=C, C≡C, -N-H- as functional groups which were identified from their FT-IR spectrum. Further, EPR analysis

provided a nine line spectrum emphasizing the free radical property of the compound. Additionally, the antioxidant property was assessed using DPPH which reveals that the percentage inhibition of DPPH is increased with an increase in UIBSM concentration. UIBSM at different concentrations in sterile discs were placed on the vascular beds of 96h grown chick embryo to check its effect on angiogenesis. Results on CAM assay reveals that UIBSM attenuates angiogenesis in both dose and time dependent manners. In conclusion the present study showed that the compound has free radical and anti-oxidant properties while it also induced angiogenesis. A further study investigating the underlining mechanism of its effect on angiogenesis and also studying the efficacy of this compound in inducing in vivo angiogenesis will be important. doi:10.1016/j.freeradbiomed.2010.10.012

12 The Endothelial [Ca2+]i  Increase During  Endothelial Derived Hyperpolarising factor  (EDHF) Responses is Mediated by Reactive Oxygen  Species (ROS) from Cytochrome P450  James G Chidgey1, Philip I Aaronson1, and Paul A Fraser1 1 King's College London EDHF is the dominant relaxation pathway in the microcirculation and H2O2 has been implicated in this response, but its role has yet to be elucidated. The cremaster muscle circulation of freshly killed Wistar rats was perfused with a Krebs buffer solution containing albumin and the calcium indicator Fura-PE3 AM. The preparation was placed on the modified stage of an intravital 2+ microscope to measure vessel diameter, and endothelial [Ca ]i was estimated from the 360/380 nm excitation ratio. The preparation was routinely superfused with phenylephrine (30µM), L-NAME (300µM) and indomethacin (3µM). Addition of carbachol (10µM) resulted in 72.6% ± 2.4 relaxation and the 360/380 nm ratio increased by 24.7% ± 1.5. The EDHF mediated relaxation 2+ and endothelial [Ca ]i increase in response to carbachol was substantially reduced by including a ROS scavenging combination -1 of superoxide dismutase and catalase (100U.ml each; relaxation 42.3% ± 15.4, ratio 5.5% ± 2.1). The cytochrome P450 inhibitors 6-(2-propargyloxyphenyl) hexanoic acid (10µM) and 17octadecynoic acid (50µM) reduced relaxation (to 18.8% ± 5.5 and 2+ 21.9% ± 3.2) and the endothelial [Ca ]i increase (to 9.1% ± 1.0 and 8.1% ± 2.1), as did the cytosolic phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (3µM; relaxation 17.1% ± 4.4, ratio 5.9% ± 0.4). The epoxyeicosatrienoic acids antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10µM) did not significantly affect the EDHF response (relaxation 72.7% ± 6.1, ratio 21.7% ± 3.2). These data suggest that ROS produced by arachidonic acid metabolism via cytochrome P450 play an important role in EDHF 2+ mediated relaxation by increasing endothelial [Ca ]i. Funded by the British Heart Foundation. doi: 10.1016/j.freeradbiomed.2010.10.013

13 Testosterone­induced Leukocyte Migration  Involves COX2 and Redox­Sensitive Processes in  rats  Andreia Zago Chignalia1, Maria Aparecida Oliveira2, Victor Debbas1, Rhian Touyz3, Francisco Rafael Martins Laurindo1, Maria Helena Catelli Carvalho2, Zuleica Bruno Fortes2, and Rita Tostes2 1 2 Heart Institute - USP, Institute of Biomedical Sciences - USP, 3 Kidney Research Centre - University of Ottawa Testosterone is related to cardiovascular diseases (CVD). However, the mechanisms whereby testosterone mediates

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vascular pro-inflammatory processes are unknown. This in vivo study investigated whether testosterone induces redox-sensitive leukocyte migration (LM). Wistar rats were treated with -7 testosterone (24h, 10 M, ip), in the absence and presence of pretreatment (4h) with flutamide (Flu, androgen receptor -5 antagonist, 10 M), apocynin (Apo, antioxidant/possible NADPH -4 -4 oxidase inhibitor, 10 M) or NS398 (COX2 inhibitor, 10 M). Mesenteric LM was assessed by intravital microscopy. Expression of NADPH oxidase subunits, COX2 and VCAM was assessed by immunoblot. NADPH oxidase activity was detected in isolated membrane-fraction dihydroethidium fluorescence. Testosterone increased LM (4.5±0.21 vs 1.51±0.1, n=12), an effect blocked by Flu (1.87±0,33), NS398 (0.86±0,19) and Apo (0,87±0,1). Testosterone increased expression of p22phox, NOX1 and VCAM (0.5 fold, n=5, p<0,05) and augmented NADPH oxidase activity (136% of control, p<0,05, n=6). These effects were inhibited by Flu, Apo and, of note, NS398 (p<0,05, n=4), indicating that COX2 may be upstream of NADPH oxidase. Our in vivo studies demonstrate that testosterone plays a role in vascular inflammation by enhancing LM. This effect is mediated through processes that involve COX2 and NADPH oxidase-driven ROS. Hence, testosterone may contribute to inflammatory processes in vascular oxidative stress-related pathologies, particularly CVD in males. Funding: FAPESP/CNPq. doi: 10.1016/j.freeradbiomed.2010.10.014

14 The Effects of Exposure Regimen and Age on  Chronic Ozone Exposure Induction of  Atherosclerosis  Gin C. Chuang1,2, Edward M. Postlethwait2,3, and Scott W. Ballinger1,2 1 2 Dept of Pathology, University of Alabama at Birmingham, Center for Free Radical Biology, University of Alabama at Birmingham, 3 Dept of Environmental Health Sciences, University of Alabama at Birmingham Ozone (O3) is a common air pollutant known to induce pulmonary oxidative stress, as well as exacerbate respiratory diseases such as asthma. Recent studies indicate that O3 inhalation may also confer increased risks of cardiovascular morbidity or mortality. We recently reported that 8 weeks of O3 exposure (5 O3 days/week; 40 total O3 days) significantly increased aortic intimal -/lesion size in the apoE mouse model of atherosclerosis, and that vascular oxidative stress markers increased significantly in C57Bl/6J mice after only 5 days of O3 exposure. Since the typical human exposure to O3 is intermittent, a 2 and 4 cycle exposure model was used in this follow-up study for increased physiological relevance; each cycle was 5 days of O3 followed by 9 days of -/filtered air. We hypothesized that apoE mice exposed to increased cycles of O3 will exhibit increased aortic atherosclerosis and oxidative stress, and the latter should normalize if the animals are allowed 5 days to recover without further exposures. Aortas of 4-cycle O3 exposed mice showed decreased aconitase activity and protein nitration, as well as increased SOD2 activity, but these alterations were not detected in the animals after 5 additional recovery days. These findings are consistent with our hypothesis that O3 exposure-induced vascular oxidative stress is transient and resolves after the exposure. We further observed that increased atherosclerosis induced by either 2 or 4 cycles of O3 exposure (10 and 20 total O3 days, respectively) was not statistically significant relative to controls, suggesting that the O3-/free days in each exposure cycle were protective. Lastly, apoE mice exposed to 3 weeks of O3 (5 O3 days/week; 15 total O3 days) starting at 5 days of age exhibited significantly increased aortic lesion size at 13 weeks of age versus controls, suggesting that young age at the time of O3 exposure may confer susceptibility. Conclusions: The nature of the O3 exposure regimen is critical in determining its atherogenic potential; transient vascular oxidative stress may play a role in O3-induced atherogenesis; and newborns may be more susceptible to the

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pro-atherogenic effects of O3 inhalation. doi: 10.1016/j.freeradbiomed.2010.10.015

15 Sirt1 Deficiency and Increased Oxidative Stress are  Associated to Accelerated Cardiovascular  Remodeling in Hypertensive Mice Submitted to a  Western Diet  Nicolas Clavreul1, Patricia Sansilvestri-morel1, Jérôme Paysant1, Olympe Beugné1, Christine Mauclair1, Tony J. Verbeuren1, and Alain Rupin1 1 Servier Research Institute To better understand the mechanism by which a Western diet can accelerate cardiovascular complications associated to hypertension, we set up a model of hypertensive mice receiving a high fat and high sucrose (HFHS) diet during 13 weeks. Double transgenic mice overexpressing human angiotensinogen and human renin (AGT/REN) have a moderate increase of their systolic blood pressure (+10 mmHg) compared to C57BL6. The HFHS diet slightly enhanced this difference (+15 mmHg). Fasting blood glucose concentration was higher in hypertensive mice under the HFHS diet compared to C57BL6 with the same diet (230 vs 188 mg/dl) and oral glucose tolerance tests confirmed that AGT/REN mice became more glucose intolerant. Although they reached the same body weight under the HFHS diet (21 g to 33 g), only the hypertensive mice showed a significant increase of heart weight that was correlated with an up-regulation of collagen I and alpha actin mRNA expression. In the aorta of AGT/REN mice under the HFHS diet, we found an increased TGFβ, Nox2 and CSF1-R mRNA expression, suggesting a macrophage infiltration, that was absent in the C57BL6 mice. Superoxide anion production in the aorta and nitrosyl hemoglobin in blood, a biomarker of NO synthesis, both measured by electron spin resonance, were consistent with an increased vascular oxidative stress with the HFHS diet. Interestingly, Sirt1, an important regulator of oxidative stress and glucose metabolism, was significantly reduced in the heart and the aorta of the animals under the HFHS diet. This study suggests that the diet-induced worsening of glucose tolerance, vascular inflammation and cardiac hypertrophy in hypertensive mice may be related to Sirt1 deficiency and increased oxidative stress. doi: 10.1016/j.freeradbiomed.2010.10.016

16 A Comparative Analysis of Diesel Exhaust Particulates with Varying Physicochemical Properties on Viability and Stress Response in Murine Endothelial Cells David P. Cox1,2, Bertram E. Drury3, and Terrance J. Kavanagh1,2 1 2 University of Washington, Center for Ecogenetics & 3 Environmental Health, University of Missouri Human exposure to traffic-related air pollution, specifically ultrafine ambient particulate matter (PM), has been associated with increased risk for cardiovascular dysfunction. Epidemiologic studies have further increased the complexity of PM-related cardiovascular dysfunction by identifying numerous variables such as proximity to traffic density in addition to particle source, size, and physicochemical properties. Diesel Exhaust particles (DEP) are a major component of urban-generated PM and DEP exposure has been associated with increased reactive oxygen species (ROS), which, depending on the levels can influence cellular function and/or toxicity. Endothelial cells are sensitive to increased ROS and their dysfunction has been implicated in several pathologies including hypertension and atherosclerosis.

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