Lipids and inflammation 761 P4158 | BENCH CD40 ligand promotes angiotensin-II induced vascular inflammation, thrombin sensitivity, oxidative stress and endothelial dysfunction M. Hausding 1 , K. Jurk 2 , C. Becker 3 , S. Daub 1 , S. Kroeller-Schoen 1 , M. Oelze 1 , P. Wenzel 4 , S. Grabbe 3 , T. Muenzel 4 , A. Daiber 1 . 1 University Medical Center, Department of Molecular Cardiology, Mainz, Germany; 2 University Medical Center, Center for Thrombosis and Hemostasis, Mainz, Germany; 3 University Medical Center, Department of Dermatology, Mainz, Germany; 4 University Medical Center Mainz, II. Med. Clinic, Mainz, Germany Purpose: CD40 ligand (CD40L) is involved in the vascular infiltration of immune cells, development of atherosclerotic lesions which was shown to be attenuated in CD40L-/- mice. T cell mediated CD40L release during thrombosis points to a role of CD40L in the intravascular inflammatory process, thrombin sensitivity, oxidative stress and vascular dysfunction in angiotensin (AT)-II-mediated hypertension. Methods: AT-II (1mg/kg/d for 7days) was infused in C57/Bl6 control and CD40L-/- mice by using osmotic minipumps. Vascular function was recorded by isometric tension studies, reactive oxygen species (ROS) by enhanced chemiluminescence and protein expression by ELISA and Western blot. Thrombin sensitivity was as- sessed by thrombin release analysis and monocyte-thrombocyte aggregates by flow cytometry. Leukocyte adhesion to cultured endothelial cells was investigated as well. Results: CD40L-/- mice showed less pronounced adverse effects on endothelial function and oxidative stress upon AT-II infusion compared to control mice. AT-II in- fused CD40L-/- mice displayed significantly decreased levels of TH1 cytokines as a surrogate of decreased activation of CD4+ T cells. We found lower levels of ac- tivated F4/80+ and CD68+ monocytes in aortic tissue from AT-II treated CD40L-/- mice indicating a strong anti-inflammatory phenotype. This observation was ac- companied by a lowered NOX-2 and P-selectin expression in aorta during AT-II infusion of CD40L-/- mice. T-bet and interleukin-12 receptor as markers for a TH1 effector immune response activated by innate immune cells decreased in AT-II infused CD40L-/- versus wild type mice. Control mice showed truncated bleeding when treated with AT-II, which was not observed in the absence of CD40L indi- cating that CD40L-/- mice had an impaired coagulation cascade. Thrombocyte activation was increased in PRP from AT-II treated control mice but significantly inhibited in CD40L-/- mice as assessed by tissue factor-mediated thrombin burst and formation of thrombocyte-monocyte aggregates. Adhesion of primary leuko- cytes to cultured endothelial cells was increased when isolated from whole blood of AT-II treated control mice but this increase was absent in CD40L-/- mice. Conclusions: In summary, our results demonstrate that CD40L-/- deficiency im- proves angiotensin-II induced vascular inflammation, dysfunction and oxidative stress as well as thrombin sensitivity supporting an essential role of immune cells and inflammatory pathways for AT-II induced arterial hypertension. CD40 ligand represents a molecule which provides a link between atherothrombotic events and the immune system. P4159 | BENCH Endothelial Overexpression of LOX-1 increases stroke size in vivo A. Akhmedov 1 , R.D. Spescha 1 , F. Paneni 1 , G.G. Camici 1 , T.F. Luescher 2 . 1 University of Zurich, Cardiology & Cardiovascular Research, Zurich, Switzerland; 2 University Hospital Zurich, Zurich, Switzerland Background: Stroke is one of the most common causes of death and long term disability worldwide primarily affecting the elderly population. Lectin-like oxidized LDL receptor 1 (LOX-1) is the receptor for oxidized LDL identified in endothelial cells. Binding of OxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO. Accumulating evidence suggests that LOX-1 is involved in endothelial dysfunction, inflammation, atherogenesis, myocardial infarction, and intimal thickening after balloon catheter injury. Interestingly, a re- cent study demonstrated that acetylsalicylic acid (aspirin), which could prevent is- chemic stroke, inhibited Ox-LDL-mediated LOX-1 expression in human coronary endothelial cells. The expression of LOX-1 was increased at a transient ischemic core site in the rat middle cerebral artery occlusion model. These data suggest that LOX-1 expression induces atherosclerosis in the brain and is the precipitating cause of ischemic stroke. Therefore, the goal of the present study was to investi- gate the role of endothelial LOX-1 in stroke using experimental mouse model. Methods and results: 12-week-old male LOX-1TG generated recently in our group and wild-type (WT) mice were applied for a transient middle cerebral artery occlusion (MCAO) model to induce ischemia/reperfusion (I/R) brain injury after 45 min of ischemia followed by 24 h of reperfusion. LOX-1TG mice developed 24 h post-MCAO significantly larger infarcts in the brain compared to WT (81.51±8.84 vs. 46.41±10.13, n=7, p < 0.05) as assessed morphologically using Triphenylte- trazolium chloride (TTC) staining. Moreover, LOX-1TG showed higher neurologi- cal deficit in RotaRod (35.57±8.92 vs. 66.14±10.63, n=7, p < 0.05) and Beder- son tests (2.22±0.14 vs. 1.25±0.30, n=9-12, p < 0.05) – two experimental phys- iological tests for neurological function. Conclusions: Thus, our data suggest that LOX-1 plays a critical role in the is- chemic stroke when expressed at unphysiological levels. Such LOX-1-associated phenotype could be due to the endothelial dysfunction. Therefore, LOX-1 may represent novel therapeutic targets for preventing ischemic stroke. P4160 | BENCH Adipose tissue as an endocrine organ: production of serum amyloid a in response to inflammatory cytokines by human adipocytes P. Calabro, L. Riegler, V. Maddaloni, F. Fimiani, G. Limongelli, F. Martone, R. D’Alessandro, B. Ziello, P. Golino, R. Calabro’. Division of Cardiology Second University of Naples Monaldi Hospital, Naples, Italy Background: Serum amyloid A (SAA) is one of the major acute-phase proteins in humans, and elevated plasma levels of this protein represent a risk factor for the development of cardiovascular disease. At first SAA was thought to be produced by hepatocytes in response to inflammatory stimuli, but several stud- ies have shown that adipose tissue can secrete a number of biologically active molecules, including several proinflammatory factors. Therefore, we tested the hypothesis that cells in adipose tissue can synthesize SAA in response to inflam- matory stimuli. Methods and results: Adipocytes and preadipocytes were isolated from abdomi- nal adipose tissue and incubated with interleukin-1 and -6, tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), or resistin, or a combination of these proteins, at different concentrations. After 48 hours, the supernatants were analyzed by using ELISAs specific for human SAA. Incubation with TNF-α led to a significant increase in SAA production, with a peak after incubation with either LPS or re- sistin (∼3 times greater than that in control adipocytes). In addition, the biggest in- crease in SAA occurred in cells exposed to all stimuli combined (∼5 times greater than that in control cells). Subsequently we investigated whether treatment with some drugs could modulate SAA production, and we observed that treatment with fluvastatin led to a significant, but not complete, inhibition of SAA release from adipocytes, whereas a large, but still not complete, modulation of SAA re- lease from adipocytes was observed after treatment with either troglitazone or aspirin. Conclusions: These results show for the first time that human adipocytes can produce SAA in response to inflammatory cytokines, thereby suggesting a new link between obesity and vascular inflammation. P4161 | BENCH Increased IGF-1 bioavailability preserves cardiac function and protects against diet-induced increases of blood pressure in high-fat fed mice J. Allen, P. Crossey. University College Dublin, Dublin, Ireland Purpose: Obesity is an established risk factor for cardiovascular disease, hyper- tension and left ventricular hypertrophy and is associated with a decline in plasma free IGF-I levels. This study evaluates the effect of high-fat feeding on cardiac and vascular function in mice with increased IGF-I bioavailability (IGFBP-1 knockout mice, BP1KO). Methods: Blood pressure was measured non-invasively after 8 and 16 weeks of feeding a 45% high fat diet. Cardiac structure and function was examined by echo- cardiography. Left ventricular and septum wall size was measured in systole and diastole. Doppler echocardiography measured mitral valve function. Mice were sacrificed and the heart and the left ventricle weighed. Results: After 8 and 16 weeks of high-fat feeding, BP1KO mice were protected against obesity-related hypertension (117.7 vs. 127 mmHg and 129.3 vs 143 mmHg BP1KO and WT respectively). At 16 weeks the left ventricular wall in sys- tole was larger in the KO cohort, but stroke volume was preserved. At 24 weeks there was no difference in ventricular or septal wall measurements. Left ventri- cle to heart weight ratio was higher in the WT than the BP1KO mice (73.38% vs 69.20%). Mitral Valve Measurements Measurement 16w HFD WT 16w HFD KO 24w HFD WT 24w HFD KO E’/A’ ratio 1.23±0.04 1.39±0.05* 1.204±0.031 1.486±0.068** IVRT 12.22±0.21 12.43±0.33 13.85±0.059 11.38±0.35** IVCT 14.3±0.53 12.92±0.47 13.81±0.044 12.48±0.89 IVRT/RR 0.096±0.001 0.009±0.0002** 0.109±0.004 0.09±0.002** IVCT/RR 0.112±0.0005 0.094±0.003* 0.378±0.008 0.343±0.015* TEI 0.652±0.024 0.577±0.017* 0.577±0.029 0.547±0.022 Values expressed as mean±SEM. *p<0.05, **p<0.01. Conclusions: Increased IGF-1 bioavailability protected against the rise in blood pressure observed in obesity. Although there was no difference in ventricular wall Downloaded from https://academic.oup.com/eurheartj/article-abstract/34/suppl_1/P4158/2862296 by guest on 31 May 2020