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P397 NEUTROPHIL IL-1R AND CHEMOKINE EXPRESSION FOLLOWING IN VIVO EXTRAVASATION IN PATIENTS WITH CORONARY ARTERY DISEASE
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Atherosclerosis Supplements 11, no. 2 (2010) 17–108
a concentration ranging from 10–1.25 mg/ml) which was significantly reduced when platelets were pretreated with aspirin suggesting that it could be attributed to arachidonic acid metabolites. The binding of either LDL or HDL to PMPs significantly reduced their aggregatory effect in a dose-dependent manner. Conclusions: This is the first study showing that both plasma lipoproteins LDL and HDL bind to platelet microparticles and significantly attenuate their aggregatory effect on human platelets. The significance of the above findings concerning the pathophysiology of atherothrombosis remains to be established. P393 EFFECT OF ACUTE INFLAMMATION ON RESPONSE OF BETA-2 ADRENOCEPTORS OF RAT KNEE JOINT VESSELS DECREASED IN PRESENCE OF DIABETES S. Hajizadeh. Tarbiat Modares University, Tehran, Iran Introduction: Due to the vasodilatory effect of acute inflammation on blood vessels we aimed in this study to investigate the responsiveness of adrenoceptors of knee joint blood vessels of rats in presence of diabetes. Methods: Wistar rats weighing 200–300 gr. were used in this study. Acute inflammation was induced by intra articular injection of kaolin 4% and induction of diabetes was performed by streptozotocine (55 mg/kg). Animals divided into 4 groups as: control, diabetic, inflammation and diabetic-inflammation. Laser Doppler flowmetry (LDF) technique was used for monitoring of knee joint blood flow. Vasodilatation of articular micro vessels was measured in response to topical application of different concentrations (10−11 to 10−1) of Salbutamol. Results: Data showed that (1) knee joint diameter and perimeter due to acute inflammation were significantly lesser in diabetic than that of inflammatory rats. (2) Responsiveness of beta-2 adrenoceptors was increased in acute inflammation. (3) Diabetes attenuated the response of beta-2 adrenoceptors in acute inflammation. Conclusion: Based on the above mentioned results, we conclude that diabetes inhibits the vasodilatory effect of acute inflammation on knee joint blood vessels. P394 REACTIVE OXYGEN SPECIES, VIA MITOCHONDRIAL KATP CHANNELS, ACTIVATE PRO-SURVIVAL PATHWAY IN PRAVASTATIN-INDUCED CARDIOPROTECTION T. Saikawa1 , L. Cong Thuc1 , Y. Teshima1 , N. Takahashi1 , Y. Nagano-Torigoe1 , K. Ezaki1 , K. Yufu1 , M. Nakagawa1 , M. Hara2 . 1 Department of Laboratory Medicine and Diagnostics, 2 Department of Intenal Medicine I, Oita University, Yufu, Japan Reactive oxygen species (ROS) are important intracellular signaling molecules and are implicated in cardioprotective pathways including ischemic preconditioning. Statins have been shown to have cardioprotective effects against ischemia/reperfusion injury, however, the precise mechanisms remain to be elucidated. We hypothesized that ROS-mediated signaling cascade may be involved in pravastatin-induced cardioprotection. Cultured rat cardiomyocytes were exposed to H2 O2 for 30 min to induce cell injury. Pravastatin significantly suppressed H2 O2 -induced cell death evaluated by propidium iodide staining and the MTT assay. Incubation with pravastatin activated catalase, and prevented a ROS burst induced by H2 O2 , which preserved mitochondrial membrane potential. Protective effects were induced very rapidly within 10 min, which was concordant with the up-regulation of phosphorylated ERK1/2. L-NAME, 5HD, N-acetylcysteine (NAC) and staurosporine inhibited ERK1/2 phosphorylation and also reduced pravastatin-induced cardioprotection, suggesting NO, mitochondrial KATP (mitoKATP ) channels, ROS and PKC should be involved in the cardioprotective signaling. We also demonstrated that pravastatin moderately up-regulated ROS generation in a 5HD-inhibitable manner. In isolated perfused rat heart experiments, pravastatin administered 10 min prior to no-flow global ischemia significantly improved left ventricular functional recovery, and also reduced infarct size, which were attenuated by the treatment with NAC. Administration of pravastatin from the beginning of reperfusion also conferred cardioprotection. Pravastatin protected the cardiomyocytes against oxidative stress by preventing the ROS burst and preserving mitochondrial function. Moderately up-regulated ROS production by mitoKATP channels opening is involved in the pro-survival signaling cascade activated by pravastatin. P395 AUGMENTED ANGIOGENESIS IN ADVENTITIA PROMOTES PLAQUE FORMATION IN ABDOMINAL AORTA OF APOLIPOPROTEIN E-DEFICIENT MICE K. Tanaka1 , D. Nagata1 , Y. Hirata1 , Y. Tabata2 , R. Nagai1 , M. Sata3 . 1 Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, 2 Biomaterials, Institute for Frontier Medical Science, Kyoto University, Kyoto, 3 Cardiovascular Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Introduction: Exaggerated formation of vasa vasorum (VV) was detected along with atherosclerotic plaque progression. In advanced lesions, VV invades into plaques and supplies inflammatory cells into them. However, it remains
Poster Presentations
unclear whether augmented VV could promote hyperlipidemia-induced plaque formation. Methods: Slow releasing form (30 days) of basic fibroblast growth factor (bFGF) was administered focally to augment VV. bFGF (100 mg/body) incorporated in acid gelatin hydrogel microspheres (AGHMs) (bFGF+AGHM, n = 10), AGHMs alone (AGHM, n = 7), PBS (control, n = 8) was injected into periaortic areas of retroperitoneal space of 10 to 11-week-old male ApoE-deficient mice. After 2, 3, 4 and 13 weeks, the abdominal aortas were harvested with perivascular soft tissues. The adventitial micro-vessels and macrophages were visualized by staining with Lycopersicon Esculentum (Tomato) lectin and anti-Mac3 antibody. The same procedures were performed in age-matched wild-type mice (n = 4). Results: Larger lesions were observed only in the bFGF+AGHM group (bFGF+AGHM: 3.4×104 ±0.7×104 mm2 , AGHM: 0.8×104 ±0.7×104 mm2 , control 0 mm2 , p = 0.0002) at 13 weeks. Plaque formation was associated with augmented VV, although invasion of VV into plaques was not observed at time points. At early phase, proliferation of VV and accumulation of macrophages were observed prior to plaque formation. In wild-type mice, although bFGF induced VV proliferation, lesion formation was not observed. Conclusions: Under hyperlipidemic conditions, adventitial administration of bFGF induces VV development, which potentially accelerates plaque formation and progression. Augmented VV might influence plaque progression not only in advanced lesion but also in initial one via inflammatory cell recruitment in adventita. P396 ATHEROPROTECTIVE EFFECTS OF SIMVASTATIN, TELMISARTAN AND RESVERATROL: INHIBITION OF MONOCYTE ADHESION TO ENDOTHELIUM EXPOSED TO NON-UNIFORM SHEAR-STRESS AND TNF-a K. Urschel, I. Cicha, W.G. Daniel, C.D. Garlichs. Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, Germany Objective: Non-uniform shear stress-induced endothelial activation promotes atherosclerotic plaque formation at arterial bifurcations via increased recruitment of inflammatory cells. Our previous study showed that non-uniform shear stress in combination with circulating TNF-a lead to increased endothelial VCAM-1 and E-selectin expression and enhanced monocyte adhesion. Based on these results, we now hypothesized that pharmacologic substances which suppress inflammation may prevent monocyte recruitment. Methods: HUVECs seeded in bifurcating flow-through cell culture slides were exposed to shear stress overnight, followed by 2 hours stimulation with TNF-a. Subsequently, HUVECs were perfused for 1 hour with medium containing THP-1 monocytes. During flow, cells were incubated either with simvastatin − an inhibitor of HMG-CoA-reductase, telmisartan − an AT1R blocker, or resveratrol − the active compound of red wine. Adherent THP-1 monocytes were quantified by light microscopy. Endothelial adhesion molecule expression was determined by immunofluorescence. Results: Simvastatin (1 mmol/L) suppressed non-uniform shear stress- and TNF-a-induced monocytic cell adhesion by about 30% via inhibition of VCAM-1 expression. Similar effect was observed upon treatment of endothelial cells with telmisartan: VCAM-1 expression and monocytic cell recruitment were inhibited by about 50% at concentration of 1 mmol/L. E-selectin expression was not affected by either simvastatin or telmisartan. Resveratrol dose-dependently inhibited VCAM-1 and E-selectin expression in HUVECs exposed to non-uniform shear stress and TNF-a, and reduced monocytic cell recruitment by 50% at 20 mmol/L. Conclusions: Anti-atherogenic pharmaceutical and natural compounds decrease TNF-a-induced recruitment of monocytic cells and the expression of adhesion molecules in endothelial cells exposed to atherogenic shear stress in vitro. P397 NEUTROPHIL IL-1R AND CHEMOKINE EXPRESSION FOLLOWING IN VIVO EXTRAVASATION IN PATIENTS WITH CORONARY ARTERY DISEASE 2 J. Paulsson1 , A. Moshfegh1 , E. Dadfar1 , A. Bygden-Nylander ´ , C. Held3 , S. Jacobson1 , J. Lundahl1 . 1 Karolinska Institutet, 2 Karolinska Hospital, Stockholm, 3 Uppsala University Hospital, Uppsala, Sweden The aim was to study neutrophil gene expression following in vivo extravasation in relation to the inflammatory process in patients with coronary artery disease (CAD). In vivo extravasated neutrophils were collected − by use of a skin chamber method. Neutrophils from peripheral blood and the skin chamber exudate were purified by density centrifugation. Affymetrix human Gene Chip U133 plus 2.0 and the Gene Spring GX software were used to analyse gene expression. Confirmation of gene expression was performed by RT-PCR and assessment of IL-1R signalling was studied following neutrophil in vitro stimulation by IL-1. Following extravasation 400 genes were induced and 1525 genes were suppressed in neutrophils from patients with CAD compared to controls. The general induction in both CAD patients and healthy controls following neutrophil extravasation included an increased expression of IL-1R1 and NFúB together with CCL20 and CXCL2. An increased IL-1R expression on extravasated
78th EAS Congress
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
neutrophils was confirmed by flow-cytometry and immunofluorescent electron microscopy. Induction of chemokines in skin chamber exudate was confirmed by ELISA. The increased induction of chemokines following extravasation was more pronounced in CAD patients and RT-PCR confirmed a higher expression of CCL20 and CXCL2 in patients with CAD compared to healthy controls. Assessment of IL-1R signalling in neutrophils indicated induction of these chemokines following IL-1 stimulation. The results indicate a pro-inflammatory profile with induction of CCL20 and CXCL2 in in vivo extravasated neutrophils that is partly mediated by IL-1, especially in patients with CAD. P398 EXPRESSION OF INFLAMMATION-RELATED GENES IN HUMAN ATHEROSCLEROTIC PLAQUES M.D. Di Taranto1 , A. Morgante2,3 , U.M. Bracale4 , L. Del Guercio4 , F. Carbone4 , F.P. D’Armiento5 , M. Porcellini4 , G. Bracale4 , F. Salvatore2,3 , G. Fortunato2,3 . 1 Fondazione IRCCS SDN, Naples, 2 CEINGE Biotecnologie Avanzate S.C.a r.l, 3 Dip. Biochimica e Biotecnologie Mediche, Universita` degli Studi di Napoli Federico II, 4 Dipartimento Assistenziale di Chirurgia Generale Toracica, Vascolare e Endovascolare, 5 Dipartimento di Scienze Biomorfologiche e Funzionali, Sezione Anatomia Patologica e Citopatologica, Universita` degli Studi di Napoli Federico II, Napoli, Italy Aims: Atherosclerosis is an inflammatory disease which molecular mechanisms are not been completely investigated. Our aim is to perform a wide expression study of inflammation related genes in human atherosclerotic plaques. Methods: The Human Inflammation Array (Applied Biosystems) was used to perform the mRNA quantification of 92 inflammation-related genes in 12 atherosclerotic plaques, their respective adjacent regions (with a lower grade lesion) and 7 healthy arteries. The principle of the array is the real-time PCR amplification with a TaqMan probe specific for each gene. Data analysis was performed with SDS 2.3 software with the comparative Ct method using the gene beta-2-microglobulin as housekeeping and one of analysed samples as calibrator. Results: The mRNA levels of 42 genes result to be differently expressed: 13 genes were up-regulated in atherosclerotic plaques respect to control arteries whereas 29 were down-regulated. Their expression levels show an increasing or a decreasing trend from healthy arteries to plaque adjacent regions and to advanced plaques. These genes belong to many different functional classes. In particular, we observed the dys-regulation of genes encoding for enzymes involved in the arachidonic acid metabolism that lead to generation of prostaglandins and leukotrienes. Conclusions: This is the first study in which the expression of a wide panel of inflammation-related genes was investigated. Results clarify the mechanisms of inflammation involvement during atherosclerotic process and highlight new possible target for anti-inflammatory therapy in cardiovascular disease. P399 LONG-TERM ENDURANCE EXERCISE ATTENUATES THE AGING PROCESS IN CIRCULATING LEUKOCYTES 1 1 C. Werner1 , T. Furster ¨ , J. Scharhag2 , M. Bohm ¨ , U. Laufs1 . 1 Klinik fur ¨ Innere ¨ ¨ SportMedizin III, Universitatsklinikum des Saarlandes, Homburg, 2 Institut fur ¨ ¨ des Saarlandes, Saarbrucken, und Praventivmedizin, Universitat ¨ Germany Background: Physical activity reduces cardiovascular morbidity and mortality. Aging is the major cause of cardiovascular disease. Telomeres and telomereassociated proteins regulate aging on the cellular level. Our study examines telomere biology and senescence factors in endurance athletes and controls without physical activity. Methods: Peripheral blood leukocytes were isolated from young track & field athletes (n = 32, age 20 years, running 73 km/week), aged athletes performing regular endurance training (n = 25, age 51 years, running 80 km/week, 35 years training history) and two control groups of physically inactive healthy volunteers. Results: Telomere repeat amplification protocols revealed an activation of leukocyte telomerase in young athletes and in elderly athletes compared to controls. Western blots showed an up-regulation of the telomere-capping protein TRF2 in young as well as in aged athletes. The expression of p16 and p53 was reduced in aged athletes. Flow-FISH assays and real-time PCR measurements of leukocyte telomere length demonstrated that sedentary elder controls exhibited a significant reduction of leukocyte telomere length (FF 53%, PCR 70% vs. young controls). Importantly, there was a striking conservation of telomere length in aged athletes (FF 88%, PCR: 84% vs. young controls). Conclusions: Beneficial effects of exercise on telomere proteins and senescence markers occur in leukocytes of young track & field athletes. In elderly athletes with a long-term history of endurance exercise we found a potent activation of leukocyte telomerase and conservation of telomere length. These findings improve the molecular understanding of beneficial vascular effects of physical activity and implicate an anti-aging effect of physical exercise.
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P400 CYTOPROTECTIVE AND ANTIOXIDANT PATHWAYS IN PERIPHERAL BLOOD MONONUCLEAR CELLS ARE REGULATED BY LDL CHOLESTEROL LEVELS IN FAMILIAL HYPERCHOLESTEROLEMIA M. Puntoni1 , F. Bigazzi2 , F. Sbrana2 , B. Pennato3 , T. Sampietro2 . 1 CNR Institute of Clinical Physiology, 2 Gabriele Monasterio CNR Regione Toscana Foundation of Pisa, 3 Scuola Superiore Sant’Anna, Pisa, Italy Background: Hypercholesterolemia and oxidative stress have been implicated as major contributors in atherosclerosis. Endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) are cytoprotective and antioxidant pathways within the vasculature. Methods: We evaluated whether cholesterol affects these pathways in mononuclear cells from patients blood (8 males, mean age 56±12) with Familial Hypercholesterolemia (FH), before and two days after apoB absorption by LDL apheresis (LDL-A). Lipoprotein profile, routine blood chemistry (BC), arterial blood pressure (BP) and endothelial function monitored. The expression levels of eNOS, iNOS, HO-1 as well as the levels of phosphorylated eNOS (p-eNOS) and AKT (pAKT) in isolated mononuclear cells were determined using Western blot. Results: Following LDL-A no changes in BP or BC were observed; mean percent reductions in CH, LDL-C, HDL-C, TG, and Lp(a) were 74%, 82%, 7%, 56%, and 86%, respectively. Two days after LDL-A CH mean dropped from 319±28 to 158±12 mg/dL, sICAM1 and sELAM1 were also significantly reduced (p < 0.001); mononuclear subpopulation cells varied for a CD4 10% increase and a CD8 12% decrease. The levels of eNOS (2-fold) and HO-1 (3−4-fold) as well as p-eNOS (2−3-fold) and pAKT (50−70%) were increased after cholesterol removal. In contrast, iNOS levels were decreased by 2-fold. Conclusions: These results suggest that: (i) increase of cytoprotective and antioxidant signals contribute to the decrease in inflammatory markers and to restoration of vascular function after cholesterol removal, (ii) high levels of cholesterol impose an inhibition on cytoprotective/antioxidant circuits and an upregulation of inflammatory pathways, with consequently vascular dysfunction. P401 DISTINCT DEFECTS IN COLLAGEN MICRO-ARCHITECTURE UNDERLIE VESSEL WALL FAILURE IN GROWING AORTIC ABDOMINAL ANEURYSMS AND ANEURYSMS IN MARFAN SYNDROME J.-W.M. Beenakker1 , J.H.N. Lindeman2 , B.A. Ashcroft1 , M. van Es1 , N.B.R. Koekkoek1 , F.A. Prins3 , J.F. Tielemans2 , H. Abdul-Hussien3 , R.A. Bank4 , T.H. Oosterkamp1 . 1 Leiden Institute of Physics, Leiden University, 2 Dept. of Vascular Surgery, 3 Dept of Pathology, Leiden University Medical Center, Leiden, 4 Medical Biology Section, University Medical Center Groningen, Groningen, The Netherlands An aneurysm of the aorta is a common pathology that is part of the atherosclerotic spectrum of diseases. Remarkably, while it is accepted that vessel wall weakening is caused by an impaired collagen metabolism, a clear association has only been demonstrated for the rare syndromes such as the vascular type Ehlers Danlos syndrome. Here we show that vessel wall failure in growing aneurysms of AAA and Marfan patients is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA), and increased collagen cross-linking in the aneurysms. Using 3D-confocal imaging we show that the conditions are associated with profound defects in collagen micro-architecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing it to dilate easily, but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluation by atomic force microscopy showed that the wall has lost its ability to stretch easily but also revealed a second defect: while vascular collagen in the normal aortic wall behaves as a coherent network, AAA and Marfan tissues do not. As a result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by the (collagen) micro-architecture and that perturbations in the collagen networks may lead to mechanical failure. P402 THE CX3C CHEMOKINE FRACTALKINE AND GPIba MEDIATE PLATELET TRANSLOCATION ON VON WILLEBRAND FACTOR UNDER PHYSIOLOGIC FLOW CONDITIONS S. Meyer dos Santos, U. Klinkhardt, K. Scholich, K. Kuczka, S. Harder. Department of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, Germany The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and associated with the development of atherosclerosis. A flow-based adhesion assay was used to study the adhesion of platelets to immobilised FKN under physiologic flow conditions. Platelets adhered weakly to immobilised FKN (firmly adherent platelets±SEM: 23±2) at 150 s−1 wall
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
a concentration ranging from 10–1.25 mg/ml) which was significantly reduced when platelets were pretreated with aspirin suggesting that it could be attributed to arachidonic acid metabolites. The binding of either LDL or HDL to PMPs significantly reduced their aggregatory effect in a dose-dependent manner. Conclusions: This is the first study showing that both plasma lipoproteins LDL and HDL bind to platelet microparticles and significantly attenuate their aggregatory effect on human platelets. The significance of the above findings concerning the pathophysiology of atherothrombosis remains to be established. P393 EFFECT OF ACUTE INFLAMMATION ON RESPONSE OF BETA-2 ADRENOCEPTORS OF RAT KNEE JOINT VESSELS DECREASED IN PRESENCE OF DIABETES S. Hajizadeh. Tarbiat Modares University, Tehran, Iran Introduction: Due to the vasodilatory effect of acute inflammation on blood vessels we aimed in this study to investigate the responsiveness of adrenoceptors of knee joint blood vessels of rats in presence of diabetes. Methods: Wistar rats weighing 200–300 gr. were used in this study. Acute inflammation was induced by intra articular injection of kaolin 4% and induction of diabetes was performed by streptozotocine (55 mg/kg). Animals divided into 4 groups as: control, diabetic, inflammation and diabetic-inflammation. Laser Doppler flowmetry (LDF) technique was used for monitoring of knee joint blood flow. Vasodilatation of articular micro vessels was measured in response to topical application of different concentrations (10−11 to 10−1) of Salbutamol. Results: Data showed that (1) knee joint diameter and perimeter due to acute inflammation were significantly lesser in diabetic than that of inflammatory rats. (2) Responsiveness of beta-2 adrenoceptors was increased in acute inflammation. (3) Diabetes attenuated the response of beta-2 adrenoceptors in acute inflammation. Conclusion: Based on the above mentioned results, we conclude that diabetes inhibits the vasodilatory effect of acute inflammation on knee joint blood vessels. P394 REACTIVE OXYGEN SPECIES, VIA MITOCHONDRIAL KATP CHANNELS, ACTIVATE PRO-SURVIVAL PATHWAY IN PRAVASTATIN-INDUCED CARDIOPROTECTION T. Saikawa1 , L. Cong Thuc1 , Y. Teshima1 , N. Takahashi1 , Y. Nagano-Torigoe1 , K. Ezaki1 , K. Yufu1 , M. Nakagawa1 , M. Hara2 . 1 Department of Laboratory Medicine and Diagnostics, 2 Department of Intenal Medicine I, Oita University, Yufu, Japan Reactive oxygen species (ROS) are important intracellular signaling molecules and are implicated in cardioprotective pathways including ischemic preconditioning. Statins have been shown to have cardioprotective effects against ischemia/reperfusion injury, however, the precise mechanisms remain to be elucidated. We hypothesized that ROS-mediated signaling cascade may be involved in pravastatin-induced cardioprotection. Cultured rat cardiomyocytes were exposed to H2 O2 for 30 min to induce cell injury. Pravastatin significantly suppressed H2 O2 -induced cell death evaluated by propidium iodide staining and the MTT assay. Incubation with pravastatin activated catalase, and prevented a ROS burst induced by H2 O2 , which preserved mitochondrial membrane potential. Protective effects were induced very rapidly within 10 min, which was concordant with the up-regulation of phosphorylated ERK1/2. L-NAME, 5HD, N-acetylcysteine (NAC) and staurosporine inhibited ERK1/2 phosphorylation and also reduced pravastatin-induced cardioprotection, suggesting NO, mitochondrial KATP (mitoKATP ) channels, ROS and PKC should be involved in the cardioprotective signaling. We also demonstrated that pravastatin moderately up-regulated ROS generation in a 5HD-inhibitable manner. In isolated perfused rat heart experiments, pravastatin administered 10 min prior to no-flow global ischemia significantly improved left ventricular functional recovery, and also reduced infarct size, which were attenuated by the treatment with NAC. Administration of pravastatin from the beginning of reperfusion also conferred cardioprotection. Pravastatin protected the cardiomyocytes against oxidative stress by preventing the ROS burst and preserving mitochondrial function. Moderately up-regulated ROS production by mitoKATP channels opening is involved in the pro-survival signaling cascade activated by pravastatin. P395 AUGMENTED ANGIOGENESIS IN ADVENTITIA PROMOTES PLAQUE FORMATION IN ABDOMINAL AORTA OF APOLIPOPROTEIN E-DEFICIENT MICE K. Tanaka1 , D. Nagata1 , Y. Hirata1 , Y. Tabata2 , R. Nagai1 , M. Sata3 . 1 Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, 2 Biomaterials, Institute for Frontier Medical Science, Kyoto University, Kyoto, 3 Cardiovascular Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Introduction: Exaggerated formation of vasa vasorum (VV) was detected along with atherosclerotic plaque progression. In advanced lesions, VV invades into plaques and supplies inflammatory cells into them. However, it remains
Poster Presentations
unclear whether augmented VV could promote hyperlipidemia-induced plaque formation. Methods: Slow releasing form (30 days) of basic fibroblast growth factor (bFGF) was administered focally to augment VV. bFGF (100 mg/body) incorporated in acid gelatin hydrogel microspheres (AGHMs) (bFGF+AGHM, n = 10), AGHMs alone (AGHM, n = 7), PBS (control, n = 8) was injected into periaortic areas of retroperitoneal space of 10 to 11-week-old male ApoE-deficient mice. After 2, 3, 4 and 13 weeks, the abdominal aortas were harvested with perivascular soft tissues. The adventitial micro-vessels and macrophages were visualized by staining with Lycopersicon Esculentum (Tomato) lectin and anti-Mac3 antibody. The same procedures were performed in age-matched wild-type mice (n = 4). Results: Larger lesions were observed only in the bFGF+AGHM group (bFGF+AGHM: 3.4×104 ±0.7×104 mm2 , AGHM: 0.8×104 ±0.7×104 mm2 , control 0 mm2 , p = 0.0002) at 13 weeks. Plaque formation was associated with augmented VV, although invasion of VV into plaques was not observed at time points. At early phase, proliferation of VV and accumulation of macrophages were observed prior to plaque formation. In wild-type mice, although bFGF induced VV proliferation, lesion formation was not observed. Conclusions: Under hyperlipidemic conditions, adventitial administration of bFGF induces VV development, which potentially accelerates plaque formation and progression. Augmented VV might influence plaque progression not only in advanced lesion but also in initial one via inflammatory cell recruitment in adventita. P396 ATHEROPROTECTIVE EFFECTS OF SIMVASTATIN, TELMISARTAN AND RESVERATROL: INHIBITION OF MONOCYTE ADHESION TO ENDOTHELIUM EXPOSED TO NON-UNIFORM SHEAR-STRESS AND TNF-a K. Urschel, I. Cicha, W.G. Daniel, C.D. Garlichs. Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, Germany Objective: Non-uniform shear stress-induced endothelial activation promotes atherosclerotic plaque formation at arterial bifurcations via increased recruitment of inflammatory cells. Our previous study showed that non-uniform shear stress in combination with circulating TNF-a lead to increased endothelial VCAM-1 and E-selectin expression and enhanced monocyte adhesion. Based on these results, we now hypothesized that pharmacologic substances which suppress inflammation may prevent monocyte recruitment. Methods: HUVECs seeded in bifurcating flow-through cell culture slides were exposed to shear stress overnight, followed by 2 hours stimulation with TNF-a. Subsequently, HUVECs were perfused for 1 hour with medium containing THP-1 monocytes. During flow, cells were incubated either with simvastatin − an inhibitor of HMG-CoA-reductase, telmisartan − an AT1R blocker, or resveratrol − the active compound of red wine. Adherent THP-1 monocytes were quantified by light microscopy. Endothelial adhesion molecule expression was determined by immunofluorescence. Results: Simvastatin (1 mmol/L) suppressed non-uniform shear stress- and TNF-a-induced monocytic cell adhesion by about 30% via inhibition of VCAM-1 expression. Similar effect was observed upon treatment of endothelial cells with telmisartan: VCAM-1 expression and monocytic cell recruitment were inhibited by about 50% at concentration of 1 mmol/L. E-selectin expression was not affected by either simvastatin or telmisartan. Resveratrol dose-dependently inhibited VCAM-1 and E-selectin expression in HUVECs exposed to non-uniform shear stress and TNF-a, and reduced monocytic cell recruitment by 50% at 20 mmol/L. Conclusions: Anti-atherogenic pharmaceutical and natural compounds decrease TNF-a-induced recruitment of monocytic cells and the expression of adhesion molecules in endothelial cells exposed to atherogenic shear stress in vitro. P397 NEUTROPHIL IL-1R AND CHEMOKINE EXPRESSION FOLLOWING IN VIVO EXTRAVASATION IN PATIENTS WITH CORONARY ARTERY DISEASE 2 J. Paulsson1 , A. Moshfegh1 , E. Dadfar1 , A. Bygden-Nylander ´ , C. Held3 , S. Jacobson1 , J. Lundahl1 . 1 Karolinska Institutet, 2 Karolinska Hospital, Stockholm, 3 Uppsala University Hospital, Uppsala, Sweden The aim was to study neutrophil gene expression following in vivo extravasation in relation to the inflammatory process in patients with coronary artery disease (CAD). In vivo extravasated neutrophils were collected − by use of a skin chamber method. Neutrophils from peripheral blood and the skin chamber exudate were purified by density centrifugation. Affymetrix human Gene Chip U133 plus 2.0 and the Gene Spring GX software were used to analyse gene expression. Confirmation of gene expression was performed by RT-PCR and assessment of IL-1R signalling was studied following neutrophil in vitro stimulation by IL-1. Following extravasation 400 genes were induced and 1525 genes were suppressed in neutrophils from patients with CAD compared to controls. The general induction in both CAD patients and healthy controls following neutrophil extravasation included an increased expression of IL-1R1 and NFúB together with CCL20 and CXCL2. An increased IL-1R expression on extravasated
78th EAS Congress
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
neutrophils was confirmed by flow-cytometry and immunofluorescent electron microscopy. Induction of chemokines in skin chamber exudate was confirmed by ELISA. The increased induction of chemokines following extravasation was more pronounced in CAD patients and RT-PCR confirmed a higher expression of CCL20 and CXCL2 in patients with CAD compared to healthy controls. Assessment of IL-1R signalling in neutrophils indicated induction of these chemokines following IL-1 stimulation. The results indicate a pro-inflammatory profile with induction of CCL20 and CXCL2 in in vivo extravasated neutrophils that is partly mediated by IL-1, especially in patients with CAD. P398 EXPRESSION OF INFLAMMATION-RELATED GENES IN HUMAN ATHEROSCLEROTIC PLAQUES M.D. Di Taranto1 , A. Morgante2,3 , U.M. Bracale4 , L. Del Guercio4 , F. Carbone4 , F.P. D’Armiento5 , M. Porcellini4 , G. Bracale4 , F. Salvatore2,3 , G. Fortunato2,3 . 1 Fondazione IRCCS SDN, Naples, 2 CEINGE Biotecnologie Avanzate S.C.a r.l, 3 Dip. Biochimica e Biotecnologie Mediche, Universita` degli Studi di Napoli Federico II, 4 Dipartimento Assistenziale di Chirurgia Generale Toracica, Vascolare e Endovascolare, 5 Dipartimento di Scienze Biomorfologiche e Funzionali, Sezione Anatomia Patologica e Citopatologica, Universita` degli Studi di Napoli Federico II, Napoli, Italy Aims: Atherosclerosis is an inflammatory disease which molecular mechanisms are not been completely investigated. Our aim is to perform a wide expression study of inflammation related genes in human atherosclerotic plaques. Methods: The Human Inflammation Array (Applied Biosystems) was used to perform the mRNA quantification of 92 inflammation-related genes in 12 atherosclerotic plaques, their respective adjacent regions (with a lower grade lesion) and 7 healthy arteries. The principle of the array is the real-time PCR amplification with a TaqMan probe specific for each gene. Data analysis was performed with SDS 2.3 software with the comparative Ct method using the gene beta-2-microglobulin as housekeeping and one of analysed samples as calibrator. Results: The mRNA levels of 42 genes result to be differently expressed: 13 genes were up-regulated in atherosclerotic plaques respect to control arteries whereas 29 were down-regulated. Their expression levels show an increasing or a decreasing trend from healthy arteries to plaque adjacent regions and to advanced plaques. These genes belong to many different functional classes. In particular, we observed the dys-regulation of genes encoding for enzymes involved in the arachidonic acid metabolism that lead to generation of prostaglandins and leukotrienes. Conclusions: This is the first study in which the expression of a wide panel of inflammation-related genes was investigated. Results clarify the mechanisms of inflammation involvement during atherosclerotic process and highlight new possible target for anti-inflammatory therapy in cardiovascular disease. P399 LONG-TERM ENDURANCE EXERCISE ATTENUATES THE AGING PROCESS IN CIRCULATING LEUKOCYTES 1 1 C. Werner1 , T. Furster ¨ , J. Scharhag2 , M. Bohm ¨ , U. Laufs1 . 1 Klinik fur ¨ Innere ¨ ¨ SportMedizin III, Universitatsklinikum des Saarlandes, Homburg, 2 Institut fur ¨ ¨ des Saarlandes, Saarbrucken, und Praventivmedizin, Universitat ¨ Germany Background: Physical activity reduces cardiovascular morbidity and mortality. Aging is the major cause of cardiovascular disease. Telomeres and telomereassociated proteins regulate aging on the cellular level. Our study examines telomere biology and senescence factors in endurance athletes and controls without physical activity. Methods: Peripheral blood leukocytes were isolated from young track & field athletes (n = 32, age 20 years, running 73 km/week), aged athletes performing regular endurance training (n = 25, age 51 years, running 80 km/week, 35 years training history) and two control groups of physically inactive healthy volunteers. Results: Telomere repeat amplification protocols revealed an activation of leukocyte telomerase in young athletes and in elderly athletes compared to controls. Western blots showed an up-regulation of the telomere-capping protein TRF2 in young as well as in aged athletes. The expression of p16 and p53 was reduced in aged athletes. Flow-FISH assays and real-time PCR measurements of leukocyte telomere length demonstrated that sedentary elder controls exhibited a significant reduction of leukocyte telomere length (FF 53%, PCR 70% vs. young controls). Importantly, there was a striking conservation of telomere length in aged athletes (FF 88%, PCR: 84% vs. young controls). Conclusions: Beneficial effects of exercise on telomere proteins and senescence markers occur in leukocytes of young track & field athletes. In elderly athletes with a long-term history of endurance exercise we found a potent activation of leukocyte telomerase and conservation of telomere length. These findings improve the molecular understanding of beneficial vascular effects of physical activity and implicate an anti-aging effect of physical exercise.
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P400 CYTOPROTECTIVE AND ANTIOXIDANT PATHWAYS IN PERIPHERAL BLOOD MONONUCLEAR CELLS ARE REGULATED BY LDL CHOLESTEROL LEVELS IN FAMILIAL HYPERCHOLESTEROLEMIA M. Puntoni1 , F. Bigazzi2 , F. Sbrana2 , B. Pennato3 , T. Sampietro2 . 1 CNR Institute of Clinical Physiology, 2 Gabriele Monasterio CNR Regione Toscana Foundation of Pisa, 3 Scuola Superiore Sant’Anna, Pisa, Italy Background: Hypercholesterolemia and oxidative stress have been implicated as major contributors in atherosclerosis. Endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) are cytoprotective and antioxidant pathways within the vasculature. Methods: We evaluated whether cholesterol affects these pathways in mononuclear cells from patients blood (8 males, mean age 56±12) with Familial Hypercholesterolemia (FH), before and two days after apoB absorption by LDL apheresis (LDL-A). Lipoprotein profile, routine blood chemistry (BC), arterial blood pressure (BP) and endothelial function monitored. The expression levels of eNOS, iNOS, HO-1 as well as the levels of phosphorylated eNOS (p-eNOS) and AKT (pAKT) in isolated mononuclear cells were determined using Western blot. Results: Following LDL-A no changes in BP or BC were observed; mean percent reductions in CH, LDL-C, HDL-C, TG, and Lp(a) were 74%, 82%, 7%, 56%, and 86%, respectively. Two days after LDL-A CH mean dropped from 319±28 to 158±12 mg/dL, sICAM1 and sELAM1 were also significantly reduced (p < 0.001); mononuclear subpopulation cells varied for a CD4 10% increase and a CD8 12% decrease. The levels of eNOS (2-fold) and HO-1 (3−4-fold) as well as p-eNOS (2−3-fold) and pAKT (50−70%) were increased after cholesterol removal. In contrast, iNOS levels were decreased by 2-fold. Conclusions: These results suggest that: (i) increase of cytoprotective and antioxidant signals contribute to the decrease in inflammatory markers and to restoration of vascular function after cholesterol removal, (ii) high levels of cholesterol impose an inhibition on cytoprotective/antioxidant circuits and an upregulation of inflammatory pathways, with consequently vascular dysfunction. P401 DISTINCT DEFECTS IN COLLAGEN MICRO-ARCHITECTURE UNDERLIE VESSEL WALL FAILURE IN GROWING AORTIC ABDOMINAL ANEURYSMS AND ANEURYSMS IN MARFAN SYNDROME J.-W.M. Beenakker1 , J.H.N. Lindeman2 , B.A. Ashcroft1 , M. van Es1 , N.B.R. Koekkoek1 , F.A. Prins3 , J.F. Tielemans2 , H. Abdul-Hussien3 , R.A. Bank4 , T.H. Oosterkamp1 . 1 Leiden Institute of Physics, Leiden University, 2 Dept. of Vascular Surgery, 3 Dept of Pathology, Leiden University Medical Center, Leiden, 4 Medical Biology Section, University Medical Center Groningen, Groningen, The Netherlands An aneurysm of the aorta is a common pathology that is part of the atherosclerotic spectrum of diseases. Remarkably, while it is accepted that vessel wall weakening is caused by an impaired collagen metabolism, a clear association has only been demonstrated for the rare syndromes such as the vascular type Ehlers Danlos syndrome. Here we show that vessel wall failure in growing aneurysms of AAA and Marfan patients is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA), and increased collagen cross-linking in the aneurysms. Using 3D-confocal imaging we show that the conditions are associated with profound defects in collagen micro-architecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing it to dilate easily, but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluation by atomic force microscopy showed that the wall has lost its ability to stretch easily but also revealed a second defect: while vascular collagen in the normal aortic wall behaves as a coherent network, AAA and Marfan tissues do not. As a result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by the (collagen) micro-architecture and that perturbations in the collagen networks may lead to mechanical failure. P402 THE CX3C CHEMOKINE FRACTALKINE AND GPIba MEDIATE PLATELET TRANSLOCATION ON VON WILLEBRAND FACTOR UNDER PHYSIOLOGIC FLOW CONDITIONS S. Meyer dos Santos, U. Klinkhardt, K. Scholich, K. Kuczka, S. Harder. Department of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, Germany The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and associated with the development of atherosclerosis. A flow-based adhesion assay was used to study the adhesion of platelets to immobilised FKN under physiologic flow conditions. Platelets adhered weakly to immobilised FKN (firmly adherent platelets±SEM: 23±2) at 150 s−1 wall