Erythropoiesis-stimulating agent improves flow-mediated dilation via reduction of oxidative stress in chronic kidney disease rats

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 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 patients. Many cardiovascular diseases are initiated by endothelial dysfunction. In this study, the influence of ESA on endothelial function was evaluated by measuring flow-mediated dilation (FMD) in anesthetized 5/6 nephrectomized (Nx) rats. Methods: Nx was established in male SD rats (7 weeks old). Continuous erythropoietin receptor activator (C.E.R.A., epoetin beta pegol; 0.6 μg/kg, once every 2 weeks) was subcutaneously administered from 1 week after Nx. Nine weeks later, endothelial function was evaluated by measuring FMD in the femoral artery of anesthetized rats with a high-resolution ultrasound system. FMD was calculated from the change in diameter of the femoral artery following reperfusion after 5 min occlusion. After that, the femoral artery was harvested and used for western blot analysis. Plasma levels of 8-OHdG and hydroperoxide were determined by ELISA and free radical elective evaluator, respectively. Results: C.E.R.A. normalized hemoglobin level of Nx rats (Nx, 12.5±0.5; Nx+C.E.R.A., 14.7±0.6 g/dL; mean ± SE, n=9) at 9 weeks after Nx. No changes in blood pressure or renal function were observed with C.E.R.A. treatment. In anesthetized rats, reperfusion after 5 min of hindlimb ischemia induced an instantaneous increase in femoral blood flow (i.e. reactive hyperemia). Following hyperemia, dilation of the femoral artery (i.e. FMD) was observed, with a maximum increase in dilation relative to baseline diameter occurring 0.5–1 min after reperfusion. C.E.R.A. significantly increased FMD of Nx rats (Nx, 11.8±2.3; Nx+C.E.R.A., 22.4±4.4%; n=9). Endothelium-independent vasodilation induced by nitroglycerin injection was not influenced by C.E.R.A treatment. C.E.R.A. significantly enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) in the femoral artery. Furthermore, Nox4 protein (a NADPH oxidase component) and nitrotyrosine (a marker of peroxynitrite) were significantly decreased in the femoral arteries of C.E.R.A.-treated rats. On the other hand, plasma 8-OHdG and hydroperoxide, which are other markers of oxidative stress, were not influenced by C.E.R.A. treatment. Conclusion: These results demonstrated that C.E.R.A. improved endothelial function in Nx rats, possibly through reduction of regional oxidative stress in the arteries. The endothelial protective effect of ESA might contribute to the improved prognosis in CKD patients treated with ESA. 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 export of NFκB was more rapid in the outer lining of the aortic arch, similar to the dynamics at high shear stress values in PAEC cells. The modeling study identified that nitrosylation of IKK in the NF-κB pathway was capable of explaining the change in dynamics. Conclusions: A combined experimental and theoretical study of the sheareNOS-NO and NFκB pathways show that the dependence of NFκB dynamics on shear stress level and location in the flow chamber may be explained by Snitrosylation of IκB kinase and an NO transport dependent mechanism. 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), (...truncated)