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P419 FENOFIBRATE ATTENUATES NICOTINE-INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION IN THE RAT
78th EAS Congress
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
the aorta, the coronary sinus and the subclavian vein and analyzed for IL-1beta, IL-6, IFN-gamma, TNF-alpha and hsCRP.The study population was divided and analyzed in two ways, according to: 1. the presence or absence of CAD, documented by coronary angiography and 2. the level of EF (< or>40%), documented by left ventriculography or MUGA. Results: In 71.4% of the patients, levels of IL-1beta, IL-6, IFN-gamma, TNF-alpha and hsCRP were within normal values. There were not significant differences among the levels of all markers in relation to the site of blood sampling. Patients with CAD (47.6%) had higher levels of IL-1beta and IFNgamma in the aorta and higher levels of hsCRP in all sites compared to patients without CAD.Levels of IL-1beta, IL-6, IFN-gamma, TNF-alpha and hsCRP were also higher in all sites in patients with EF <40% (63.2%) compared to those with EF >40%. None of the observed differences was significant. Conclusion: This is the first study to report the levels of certain inflammatory markers in three different sites in patients undergoing ICD implantation for ventricular arrhythmias. Our results do not support a clear relationship between inflammation and life-threatening ventricular arrhythmias. However, in patients with CAD or low EF a trend towards higher levels of the markers was observed, suggesting an enhanced inflammatory activity in those patients. P417 ATHEROGENIC NON-UNIFORM SHEAR STRESS INDUCES ENDOTHELIAL DYSFUNCTION AND INCREASED LEUKOCYTE RECRUITMENT IN RESPONSE TO TNF-a 1 K. Urschel1 , A. Worner ¨ , K. Beronov2 , W.G. Daniel1 , C.D. Garlichs1 , I. Cicha1 . 1 Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, 2 Bavarian Academy of Sciences and Humanities, Leibniz Supercomputing Centre, Munich, Germany Objective: Atherosclerosis-prone regions are characterized by non-uniform shear stress patterns inducing endothelial activation. This process contributes to inflammatory cell recruitment in the arterial wall. We investigated the influence of circulating TNF-a on leukocyte recruitment and adhesion molecule expression in endothelial cells exposed to laminar versus non-uniform shear stress. Methods: HUVECs seeded in bifurcating flow-through cell culture slides were exposed to laminar or non-uniform shear stress either with or without an additional TNF-a stimulation. Subsequently, HUVECs were perfused with medium containing THP-1 monocytes or freshly isolated PBMCs. Adherent leukocytes were quantified by light microscopy. Adhesion molecule expression was determined by immunofluorescence. Results: Adhesion of leukocytes to unstimulated HUVECs was nearly undetectable under laminar shear stress and was slightly increased under nonuniform shear stress. Upon TNF-a stimulation, upregulation of E-selectin and VCAM-1 expression significantly increased over time (1−4 h) under non-uniform shear stress. Moreover, exposure of HUVECs to non-uniform shear stress followed by 2h stimulation with circulating TNF-a induced a 12-fold increase in THP-1 monocyte recruitment. These effects were prevented in HUVEC exposed to laminar shear stress. Essentially the same adhesion pattern was observed, when freshly isolated PMBCs were used. The significant differences in TNF-a-induced THP-1 monocyte recruitment and adhesion molecule expression between laminar and non-uniform shear stress regions were abolished in the absence of shear stress preconditioning or by HUVEC pre-treatment with a NF-úB-inhibitor. Conclusions: Non-uniform shear stress increases endothelial susceptibility to circulating TNF-a and induces leukocyte recruitment. Interference with this process may inhibit inflammatory response in atherosclerosis-prone regions. P418 CREATION OF A RAT EXPERIMENTAL MODEL OF ABDOMINAL AORTIC ANEURYSMS K.M. Mata1 , F.S. Rocha1 , P.S. Prudente1 , C.M. Prado1 , E.M. Floriano1 , J. Elias Jr.2 , E. Rizzi3 , R.F. Gerlach4 , M.A. Rossi1 , S.G. Ramos1 . 1 Pathology, 2 ˜ Preto/University of Radiology, 3 Pharmacology, Faculty of Medicine of Ribeirao ˜ Paulo, 4 Department of Morphology, Estomatology and Physiology, Dental Sao ˜ Preto/University of Sao ˜ Paulo, Ribeirao ˜ Preto, Brazil School of Ribeirao Introduction: Animal models of abdominal aortic aneurysm (AAA) are important tools for investigating the underlying pathophysiology. Objectives: To create of a new experimental model of AAA caused by outside traumatic vascular injury and turbulent flow in the abdominal aorta. Method: Male Wistar rats (150 g) were randomly divided into four groups: Aneurysm (A), Injury (I), Stenosis (S) and Control (C) (40/group). The (I) group received an outside traumatic injury in the aortic wall. The (S) group received an extrinsic stenosis at a corresponding location. The (A) group received both the injury and stenosis simultaneously, and the (C) group received a sham operation. Aneurysms developed in 60−70% of the animals in group (A). Animals were euthanized at days 1, 3, 7 and 15 to accompany the induction of aneurysm. Results/conclusion: Animals developed AAA by day 3 and reached a media 8 times larger than normal. The aneurysmal wall exhibited increased thickness due to damaged and fragmented elastic fibers, mesenchymal cell
105
proliferation, neoangiogenesis and an intense inflammatory infiltration seen within a dense collagenous matrix. We hypothesized that damaged to the vessel wall initiates an inflammatory response that further exacerbates vessel damage via the elaboration of metalloproteinases. Aortic stenosis causes turbulent flow, altering endothelial cells homeostasis and contributing to the development of aneurysms. Outside traumatic injury and disruption of endothelial cell homeostasis together cause AAA in rats. Our model illustrates that AAA is a dynamic remodeling process and will serve as an important tool for future studies of the pathophysiology of AAA. P419 FENOFIBRATE ATTENUATES NICOTINE-INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION IN THE RAT V. Chakkarwar, M. Singh. Cardiovascular Pharmacology Division, I.S.F. Institute of Pharmaceutical Sciences and Drug Research, Moga, India The study has been designed to investigate the effect of fenofibrate, in nicotineinduced vascular endothelial dysfunction (VED) in rats. Nicotine (2 mg/kg/day, i.p., 4 weeks) was administered to produce VED in rats. The development of VED was assessed by employing isolated aortic ring preparation and estimating serum and aortic concentration of nitrite/nitrate. Further, the integrity of vascular endothelium was assessed using the scanning electron microscopy of thoracic aorta. Moreover, the oxidative stress was assessed by estimating serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide anion generation. Furthermore, the lipid profile was assed by estimating serum cholesterol, triglycerides and high density lipoprotein. The administration of nicotine produced VED by impairing the integrity of vascular endothelium and subsequently decreasing serum and aortic nitrite/nitrate and attenuating acetylcholine-induced endothelium dependent relaxation. Further, nicotine produced oxidative stress, assessed in terms of increase in serum TBARS and aortic superoxide generation. Moreover, nicotine altered the lipid profile by increasing the serum cholesterol, triglycerides and decreasing the high density lipoprotein. However, treatment with fenofibrate (32 mg/kg, p.o.) or atorvastatin (30 mg/kg/day p.o., a standard agent) prevented nicotine-induced VED, oxidative stress and normalizes the altered lipid profile by improving integrity of vascular endothelium, increasing the serum and aortic nitrite/nitrate, enhancing the acetylcholine-induced endothelium dependent relaxation and decreasing serum TBARS and aortic superoxide anion generation. Thus, it may be concluded that fenofibrate reduces the oxidative stress, normalizes the altered lipid profile and consequently improves integrity of endothelium and enhances the generation of nitric oxide to prevent nicotine-induced experimental VED. P420 PEROXYNITRITE ACTIVATES THE TRANSCRIPTION FACTOR NRF2 IN HUMAN ENDOTHELIAL CELLS L. Mattart, D. Simon, D. Calay, V. Tevel, M. Van Steenbrugge, M. Raes. FUNDP − University of Namur, Namur, Belgium Endothelial dysfunction is an early phenomenon in cardiovascular diseases, characterized by a decrease in bioavailable nitric oxide (NO• ). This decrease further enhanced when there is a concomitant overproduction of superoxide • ions (O•− 2 ) and NO . It is observed during local inflammatory reaction where − NO• reacts with O•− 2 to form peroxynitrite (ONOO ). Recent data suggest that ONOO− activates the Nrf2 pathway. The aim of our work was to study the response of human EAhy926 endothelial cells to ONOO− and to highlight roles of the Nrf2 pathway. For this purpose, cells were exposed to SIN-1 (3-morpholinosydnonimine) an ONOO− donor. Nrf2 was activated after SIN-1 incubation and two of its well known target genes, Heme Oxygenase 1 (HO-1) and NAD(P)H:quinone Oxidoreductase-1 (NQO1), were overexpressed at the mRNA level. On the other hand, we demonstrated that SIN-1 treatment is associated with a significant increase in LC3-II formation, resulting in autophagosome formation, and a decreased in DNA fragmentation. Interestingly, the invalidation of Nrf2 or HO-1 by siRNA strongly increased SIN-1 induced LC3-II formation and DNA fragmentation. This study show that peroxynitrite have a protective effect on endothelial cells by inducing protective pathways such as Nrf2 and autophagy. Reference(s) L. Mattart and D. Calay are recipient of a FRIA fellowship − This research is supported by the IUAP program P06/30. P421 EARLY EFFECTS OF ATORVASTATIN AND CLOPIDOGREL ALONE OR COMBINED ON ENDOTHELIAL PROGENITOR CELLS AND MICROPARTICLES IN SUBJECTS WITH CORONARY DISEASE C. Fran¸ca1 , L. Pinheiro1 , M. Izar1 , M. Brunialti2 , R. Salomao ˜ 2 , F. Fonseca1 . 1 ˜ Paulo, Sao ˜ Paulo, Brazil Cardiology, 2 Infectology, Federal University of Sao Introduction: Atherosclerosis is an inflammatory disease complicated with thrombotic events related to endothelial dysfunction or apoptosis. Recently, the turnover of endothelial cells has been better understood with a crucial role of endothelial progenitor cells (EPC). In addition, increased amount of microparticles derived from endothelial apoptotic cells (EMP) has been reported in patients with uncontrolled risk factors. We aimed to examine early
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
the aorta, the coronary sinus and the subclavian vein and analyzed for IL-1beta, IL-6, IFN-gamma, TNF-alpha and hsCRP.The study population was divided and analyzed in two ways, according to: 1. the presence or absence of CAD, documented by coronary angiography and 2. the level of EF (< or>40%), documented by left ventriculography or MUGA. Results: In 71.4% of the patients, levels of IL-1beta, IL-6, IFN-gamma, TNF-alpha and hsCRP were within normal values. There were not significant differences among the levels of all markers in relation to the site of blood sampling. Patients with CAD (47.6%) had higher levels of IL-1beta and IFNgamma in the aorta and higher levels of hsCRP in all sites compared to patients without CAD.Levels of IL-1beta, IL-6, IFN-gamma, TNF-alpha and hsCRP were also higher in all sites in patients with EF <40% (63.2%) compared to those with EF >40%. None of the observed differences was significant. Conclusion: This is the first study to report the levels of certain inflammatory markers in three different sites in patients undergoing ICD implantation for ventricular arrhythmias. Our results do not support a clear relationship between inflammation and life-threatening ventricular arrhythmias. However, in patients with CAD or low EF a trend towards higher levels of the markers was observed, suggesting an enhanced inflammatory activity in those patients. P417 ATHEROGENIC NON-UNIFORM SHEAR STRESS INDUCES ENDOTHELIAL DYSFUNCTION AND INCREASED LEUKOCYTE RECRUITMENT IN RESPONSE TO TNF-a 1 K. Urschel1 , A. Worner ¨ , K. Beronov2 , W.G. Daniel1 , C.D. Garlichs1 , I. Cicha1 . 1 Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, 2 Bavarian Academy of Sciences and Humanities, Leibniz Supercomputing Centre, Munich, Germany Objective: Atherosclerosis-prone regions are characterized by non-uniform shear stress patterns inducing endothelial activation. This process contributes to inflammatory cell recruitment in the arterial wall. We investigated the influence of circulating TNF-a on leukocyte recruitment and adhesion molecule expression in endothelial cells exposed to laminar versus non-uniform shear stress. Methods: HUVECs seeded in bifurcating flow-through cell culture slides were exposed to laminar or non-uniform shear stress either with or without an additional TNF-a stimulation. Subsequently, HUVECs were perfused with medium containing THP-1 monocytes or freshly isolated PBMCs. Adherent leukocytes were quantified by light microscopy. Adhesion molecule expression was determined by immunofluorescence. Results: Adhesion of leukocytes to unstimulated HUVECs was nearly undetectable under laminar shear stress and was slightly increased under nonuniform shear stress. Upon TNF-a stimulation, upregulation of E-selectin and VCAM-1 expression significantly increased over time (1−4 h) under non-uniform shear stress. Moreover, exposure of HUVECs to non-uniform shear stress followed by 2h stimulation with circulating TNF-a induced a 12-fold increase in THP-1 monocyte recruitment. These effects were prevented in HUVEC exposed to laminar shear stress. Essentially the same adhesion pattern was observed, when freshly isolated PMBCs were used. The significant differences in TNF-a-induced THP-1 monocyte recruitment and adhesion molecule expression between laminar and non-uniform shear stress regions were abolished in the absence of shear stress preconditioning or by HUVEC pre-treatment with a NF-úB-inhibitor. Conclusions: Non-uniform shear stress increases endothelial susceptibility to circulating TNF-a and induces leukocyte recruitment. Interference with this process may inhibit inflammatory response in atherosclerosis-prone regions. P418 CREATION OF A RAT EXPERIMENTAL MODEL OF ABDOMINAL AORTIC ANEURYSMS K.M. Mata1 , F.S. Rocha1 , P.S. Prudente1 , C.M. Prado1 , E.M. Floriano1 , J. Elias Jr.2 , E. Rizzi3 , R.F. Gerlach4 , M.A. Rossi1 , S.G. Ramos1 . 1 Pathology, 2 ˜ Preto/University of Radiology, 3 Pharmacology, Faculty of Medicine of Ribeirao ˜ Paulo, 4 Department of Morphology, Estomatology and Physiology, Dental Sao ˜ Preto/University of Sao ˜ Paulo, Ribeirao ˜ Preto, Brazil School of Ribeirao Introduction: Animal models of abdominal aortic aneurysm (AAA) are important tools for investigating the underlying pathophysiology. Objectives: To create of a new experimental model of AAA caused by outside traumatic vascular injury and turbulent flow in the abdominal aorta. Method: Male Wistar rats (150 g) were randomly divided into four groups: Aneurysm (A), Injury (I), Stenosis (S) and Control (C) (40/group). The (I) group received an outside traumatic injury in the aortic wall. The (S) group received an extrinsic stenosis at a corresponding location. The (A) group received both the injury and stenosis simultaneously, and the (C) group received a sham operation. Aneurysms developed in 60−70% of the animals in group (A). Animals were euthanized at days 1, 3, 7 and 15 to accompany the induction of aneurysm. Results/conclusion: Animals developed AAA by day 3 and reached a media 8 times larger than normal. The aneurysmal wall exhibited increased thickness due to damaged and fragmented elastic fibers, mesenchymal cell
105
proliferation, neoangiogenesis and an intense inflammatory infiltration seen within a dense collagenous matrix. We hypothesized that damaged to the vessel wall initiates an inflammatory response that further exacerbates vessel damage via the elaboration of metalloproteinases. Aortic stenosis causes turbulent flow, altering endothelial cells homeostasis and contributing to the development of aneurysms. Outside traumatic injury and disruption of endothelial cell homeostasis together cause AAA in rats. Our model illustrates that AAA is a dynamic remodeling process and will serve as an important tool for future studies of the pathophysiology of AAA. P419 FENOFIBRATE ATTENUATES NICOTINE-INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION IN THE RAT V. Chakkarwar, M. Singh. Cardiovascular Pharmacology Division, I.S.F. Institute of Pharmaceutical Sciences and Drug Research, Moga, India The study has been designed to investigate the effect of fenofibrate, in nicotineinduced vascular endothelial dysfunction (VED) in rats. Nicotine (2 mg/kg/day, i.p., 4 weeks) was administered to produce VED in rats. The development of VED was assessed by employing isolated aortic ring preparation and estimating serum and aortic concentration of nitrite/nitrate. Further, the integrity of vascular endothelium was assessed using the scanning electron microscopy of thoracic aorta. Moreover, the oxidative stress was assessed by estimating serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide anion generation. Furthermore, the lipid profile was assed by estimating serum cholesterol, triglycerides and high density lipoprotein. The administration of nicotine produced VED by impairing the integrity of vascular endothelium and subsequently decreasing serum and aortic nitrite/nitrate and attenuating acetylcholine-induced endothelium dependent relaxation. Further, nicotine produced oxidative stress, assessed in terms of increase in serum TBARS and aortic superoxide generation. Moreover, nicotine altered the lipid profile by increasing the serum cholesterol, triglycerides and decreasing the high density lipoprotein. However, treatment with fenofibrate (32 mg/kg, p.o.) or atorvastatin (30 mg/kg/day p.o., a standard agent) prevented nicotine-induced VED, oxidative stress and normalizes the altered lipid profile by improving integrity of vascular endothelium, increasing the serum and aortic nitrite/nitrate, enhancing the acetylcholine-induced endothelium dependent relaxation and decreasing serum TBARS and aortic superoxide anion generation. Thus, it may be concluded that fenofibrate reduces the oxidative stress, normalizes the altered lipid profile and consequently improves integrity of endothelium and enhances the generation of nitric oxide to prevent nicotine-induced experimental VED. P420 PEROXYNITRITE ACTIVATES THE TRANSCRIPTION FACTOR NRF2 IN HUMAN ENDOTHELIAL CELLS L. Mattart, D. Simon, D. Calay, V. Tevel, M. Van Steenbrugge, M. Raes. FUNDP − University of Namur, Namur, Belgium Endothelial dysfunction is an early phenomenon in cardiovascular diseases, characterized by a decrease in bioavailable nitric oxide (NO• ). This decrease further enhanced when there is a concomitant overproduction of superoxide • ions (O•− 2 ) and NO . It is observed during local inflammatory reaction where − NO• reacts with O•− 2 to form peroxynitrite (ONOO ). Recent data suggest that ONOO− activates the Nrf2 pathway. The aim of our work was to study the response of human EAhy926 endothelial cells to ONOO− and to highlight roles of the Nrf2 pathway. For this purpose, cells were exposed to SIN-1 (3-morpholinosydnonimine) an ONOO− donor. Nrf2 was activated after SIN-1 incubation and two of its well known target genes, Heme Oxygenase 1 (HO-1) and NAD(P)H:quinone Oxidoreductase-1 (NQO1), were overexpressed at the mRNA level. On the other hand, we demonstrated that SIN-1 treatment is associated with a significant increase in LC3-II formation, resulting in autophagosome formation, and a decreased in DNA fragmentation. Interestingly, the invalidation of Nrf2 or HO-1 by siRNA strongly increased SIN-1 induced LC3-II formation and DNA fragmentation. This study show that peroxynitrite have a protective effect on endothelial cells by inducing protective pathways such as Nrf2 and autophagy. Reference(s) L. Mattart and D. Calay are recipient of a FRIA fellowship − This research is supported by the IUAP program P06/30. P421 EARLY EFFECTS OF ATORVASTATIN AND CLOPIDOGREL ALONE OR COMBINED ON ENDOTHELIAL PROGENITOR CELLS AND MICROPARTICLES IN SUBJECTS WITH CORONARY DISEASE C. Fran¸ca1 , L. Pinheiro1 , M. Izar1 , M. Brunialti2 , R. Salomao ˜ 2 , F. Fonseca1 . 1 ˜ Paulo, Sao ˜ Paulo, Brazil Cardiology, 2 Infectology, Federal University of Sao Introduction: Atherosclerosis is an inflammatory disease complicated with thrombotic events related to endothelial dysfunction or apoptosis. Recently, the turnover of endothelial cells has been better understood with a crucial role of endothelial progenitor cells (EPC). In addition, increased amount of microparticles derived from endothelial apoptotic cells (EMP) has been reported in patients with uncontrolled risk factors. We aimed to examine early