Shear stress and nitric oxide transport affect NFkB dynamics in endothelial cells

110 Endothelial function and vascular remodelling Conclusion: Vascular-specific eNOS rescue in eNOS-KO resulted in restoration of endothelial eNOS-activity in conductance and resistance arteries and normal physiological response to shear but did not lower BP. These data demonstrate an obligatory role of extra-vascular eNOS for regulation of blood pressure. P581 | BENCH Erythropoiesis-stimulating agent improves flow-mediated dilation via reduction of oxidative stress in chronic kidney disease rats K. Serizawa, K. Yogo, Y. Tashiro, M. Hirata, K. Endo. Chugai pharmceutical Co.Ltd, Gotemba Shizuoka, Japan P582 | BENCH Shear stress and nitric oxide transport affect NFkB dynamics in endothelial cells S.M. Bovens 1 , N. Foin 1 , N. O’Clery 2 , K. Van Der Heiden 3 , S. Cuhlmann 4 , H. Carlsen 5 , M. Barahona 2 , P.C. Evans 6 , R. Krams 1 . 1 Imperial College London, Department of Bioengineering, London, United Kingdom; 2 Imperial College London, London, United Kingdom; 3 Erasmus Medical Center, Rotterdam, Netherlands; 4 Imperial College London, National Heart and Lung Institute (NHLI), London, United Kingdom; 5 University of Oslo, Department of Nutrition, Oslo, Norway; 6 University of Sheffield, Department of Cardiovascular Science, Sheffield, United Kingdom Introduction: Shear stress is known to regulate the pro-inflammatory transcription factor nuclear factor kappaB (NFκB) and nitric oxide (NO) synthesis. Both signalling pathways influence each other, yet the exact nature of the interaction between shear stress, endothelial NO and NFκB regulation is unclear. Here we present a combined experimental and theoretical study of the dynamics of NFκB pathway in response to shear stress and NO transport. Methods: Porcine aortic endothelial cells (PAEC) were studied in parallel plate flow chambers at two shear stress levels (2 and 10 dyne/cm2 ) over 12 time points (0-330 minutes) at four different locations along the length of a flow chamber. Additionally, the inner (low shear stress and NO-transport) and outer linings (high shear stress and rapid NO-transport) of the aortic arch of C57/Bl6 mice after treatment with LPS were studied via immunohistochemical en face staining of p65 (NFκB). Nuclear and total NFκB were measured. A coupled ODE model describing the dynamics of the eNOS-NO and the NFκB pathway was used to interpret the nature of the coupling between both pathways Results: In cultured PAEC cells, exposure to low shear stress resulted in a more prolonged nuclear NFκB translocation while high shear stress resulted in oscillatory dynamics. Addition of L-NAME, an inhibitor of NO synthesis, increased the period of NFκB activation in cells exposed to high shear, suggesting that sheardependent NO synthesis modulates the dynamics of NFκB activation. In the aortic arch of C57/Bl6 mice, LPS-induced dynamics of the NFκB pathway was dependent on location, as immediately after LPS (30-120 minutes) accumulation of NFκB was increased in the inner lining of the aortic arch, and nuclear P583 | BENCH Hydroxyamine chloridrate reduces oxidative-stress damage subsequent to balloon-injury rat model S. Muscoli 1 , M. Macrini 1 , D. Della Rocca 1 , V. Cammalleri 1 , C. Muscoli 2 , V. Mollace 2 , F. Romeo 1 . 1 Tor Vergata Polyclinic, Rome, Italy; 2 Magna Graecia University of Catanzaro, Catanzaro, Italy Scientific evidence showing that generation of reactive oxygen species play a relevant role in the proliferation of vascular smooth muscle cells (smc) subsequent to balloon injury (BI) in rat model. Data suggests that superoxide dismutase mimetics, exert protective effects against vascular injury in rats, although the molecular mechanism is still unclear. We have investigated on the protective effects of the peroxinitrite decomposition catalyst, Hydroxyamine Chloridrate (IAC), on smc proliferation generated in response to BI of common carotid artery in rats. The rats were treated with Hydroxyamine Chloridrate (10-20-40 mg/kg/day, i.p.) for 14 consecutive days (Fig. 1). Animals were housed and cared for in accordance with National Institutes of Health guidelines on laboratory animal welfare. Immunohistochemical analisys (Fig. 2) were employed to identify the presence of nitrotyrosine (NT), the footprint of peroxynitrite. NT staining was observed using a confocal microscope to also identify the inner localization of free radicals within the cells. The in situ visualization of superoxide was also employed by using the hydroethidine histochemistry (Fig. 3). In animals undergoing BI a significant restenosis and neointima formation occurred. This effect was associated to an elevated production of peroxynitrite, reactive free radicals, and to significant modulation of tissue cNOS and eNOS (as a measure of NO activation), nitrotyrosine and malondialdehyde levels (the marker of peroxidative processes). Treatment with IAC, dose-dependently reduced postinjury neointima formation, an effect accompanied by decreased peroxynitrite generation and MDA accumulation. These results suggest that novel peroxynitrite antagonist may reduce post-injury neointima formation via inhibition of ROS intracellular pathway. P584 | BENCH Perfusion and microvascular adaptation in a rabbit model of developing heart failure assessed with a 3D imaging cryomicrotome M.G.J.T.B. Van Lier 1 , N. Hakimzadeh 1 , R. Coronel 2 , J.A.E. Spaan 1 , M. Siebes 1 , J.P.H.M. Van Den Wijngaard 1 . 1 Academic Medical Center, Department of Biomedical Engineering & Physics, Amsterdam, Netherlands; 2 Center for Heart Failure Research, Academic Medical Center, Department of Experimental Cardiology, Amsterdam, Netherlands Purpose: Vascular remodeling during chronic ischemia can ameliorate the development of heart failure. In this study, we aimed to quantify changes in myocardial perfusion (perf.) and neovascularization induced by gradual coronary occlusion. Methods: Regional ischemia was induced in rabbits by an ameroid constrictor on a side branch of the LAD. Sham-operated animals served as control (CTRL). After 8 weeks, different-colored 15 μm fluorescent microspheres (μsph) were infused into the left ventricle (LV) at rest and during adenosine or regadenoson induced hyperemia (HYP) to determine myocardial perf. Renal flow and μsph served as reference for perf. quantification. After sacrifice, the coronary arteries were filled with fluorescent cast. The heart was frozen, alternately cut and block-face imaged at 14 μm slice thickness using an imaging cryomicrotome. The reconstructed high-resolution 3D vascular anatomy (figure) and co-localized μsph deposition (shown in red) were quantified and compared between ischemic (IS) and remote (RM) myocardial regions. Results: Thus far, μsph were analyzed in three IS and one CTRL heart. The Purpose: Anemia in chronic kidney disease (CKD) patients is an important risk factor for cardiovascular disease, and treatment of anemia by erythropoiesisstimulating agent (ESA) has been reported to improved QOL in CKD (...truncated)