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P414 EXPRESSION OF GENES AND PROTEINS IN ASCENDING THORACIC AORTIC ANEURYSMS AND SMOOTH MUSCLE CELLS ISOLATED FROM ANEURYSMAL SAMPLES
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Atherosclerosis Supplements 11, no. 2 (2010) 17–108
A mouse model that yields high numbers of pure FCM and NFM has been characterised, and the genome of the macrophages compared. Elevated levels of thrombotic genes were found in FCM. This, combined with changes in matrix production and matrix proteases, may explain how atherosclerotic plaques with high levels of FCM are vulnerable to thrombotic events. P412 PLEIOTROPIC EFFECTS OF ATORVASTATIN AND TGF-b/ENDOGLIN CASCADE IN THE VESSEL WALL OF ApoE/LDLR DOUBLE KNOCKOUT MICE Z. Strasky1 , L. Vecerova1 , M. Slanarova1 , J. Rathouska1 , E. Brcakova1 , S. Micuda2 , P. Nachtigal1 . 1 Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, 2 Charles University in Prague, Faculty of Medicine, Department of Pharmacology, Hradec Kralove, Czech Republic Endoglin is a transmembrane glycoprotein a part of the TGF-b receptor cascade and it is known as a type III TGF-b receptor. In our recent paper we demonstrated that administration of cholesterol-rich diet with atorvastatin (ATV) decrease cholesterol levels and increase the expression of endoglin/SMAD2/eNOS in the aorta. In this study, we studied the pleiotropic effects of atorvastatin on the expression of type II TGF-b receptor, endoglin, p-SMAD2, p-SMAD1 and p-eNOS in the mice aorta. Two-month-old female ApoE/LDLR double knockout mice were divided into two groups. The control group (n = 8) was fed with the chow diet whereas in the ATV group (n = 8), atorvastatin at dose 50 mg/kg per day was added to the same diet. Biochemical analysis of lipid profile, immunohistochemical and western blot analysis of above mentioned protein expressions in aortas were performed. Administration of atorvastatin resulted in a significant increase in total cholesterol and VLDL cholesterol when compared with control mice. On the contrary, the area of atherosclerotic lesion was decreased after ATV treatment. Western blot analysis revealed induced TGF-b IIR, endoglin, p-SMAD2, p-SMAD1 and p-eNOS expression in ATV treated mice when compared with control mice. In this study, we demonstrated that atorvastatin increase endoglin, p-SMADs and p-eNOS expressions in the vessel wall of ApoE/LDLR double knockout mice beyond its lipid lowering effects suggesting potential protective role of endoglin cascade in atherogenesis. This work was supported by grant from The Grant Agency of Charles University in Prague number 129208/C, grant MSM 002162082 and Research project MZO 001799060. P413 OXIDIZED-HEMOGLOBIN INDUCES INNATE IMMUNE CELL CHEMOTAXIS, MONOCYTE ARREST AND THEIR SUBINTIMAL MIGRATION INTO EARLY ATHEROSCLEROTIC TISSUE B. Buttari1 , E. Profumo1 , G. Domenici1 , M. Leopizzi2 , C. Di Cristofano2 , R. Businaro3 , G. Di Giammarco4 , R. Rigano` 1 . 1 Malattie Infettive, Parassitarie ` Rome, 2 Medicina ed Immunomediate, Istituto Superiore di Sanita, Sperimentale, 3 Anatomia Umana, Universita` di Roma, Sapienza, Roma, 4 Divisione di Cardiochirurgia, Policlinico SS Annunziata, Universita` di Chieti, G. d’Annunzio, Chieti, Italy Background and Aims: Signals that attract leukocytes to atherosclerotic plaque are partly defined. Our findings indicate that human hemoglobin (Hb) acts as a “self” antigen in patients with carotid atherosclerotic plaques. Aims of the study were to explore the ability of oxidized-Hb to act as a chemoattractant for innate immune cells and its possible role in atherogenic cell recruitment. Methods: Chemotaxis of monocytes and dendritic cells in response to Hb was studied in vitro using Transwells inserts and in vivo by establishing a mouse model of acute peritonitis. The ability of monocytes to adhere on endothelial layer (EA.hy 926) exposed for 2 hours to oxidized Hb was studied in static and flow dynamic conditions using a parallel wall flow chamber system mounted on inverted microscope (shear stress 2 dynes/cm2 for 2 min). Aortic tissue from patients with thoracic aortic aneurysm was perfused with 10 mg/ml of Hb over 4 hours and then with fluorescence-labelled monocytes. After 2 hours, nonadherent cells were washed away and fixed aortic tissue sections were analyzed by fluorescence microscope. Results: oxidized-Hb acts in vitro and in vivo as a chemoattractant for innate immune cells. The short-term incubation of endothelial monolayer with oxidizedHb results in a 2-fold increase of firm shear-resistant monocyte adhesion under flow conditions. Exposure of aortic tissues to oxidized-Hb triggered monocyte recruitment in early atherosclerotic aorta. Conclusion: Oxidized-Hb contributes to innate immune cell infiltration, monocyte arrest and their subintimal migration into early atherosclerotic tissues with consequent amplification of the inflammatory response.
Poster Presentations
P414 EXPRESSION OF GENES AND PROTEINS IN ASCENDING THORACIC AORTIC ANEURYSMS AND SMOOTH MUSCLE CELLS ISOLATED FROM ANEURYSMAL SAMPLES Z. Adıguzel ¨ 1 , M. Serhatlı1 , O. Ka¸car1 , A. Tuncer2 , E. Tuncer2 , K. Baysal1 . 1 Molecular Cell Biology, TUBITAK MRC Genetic Engineering and Biotechnology Institute, Kocaeli, 2 T.C. Ministry of Health, Kartal Ko¸suyolu, Advanced Training and Research Hospital, Istanbul, Turkey Aortic aneurysms are formed by an irreversible weakening of a certain part of the aorta, leading to localized dilatation of the vessel. The alterations in the vessel wall leading to this weakening are reported as; medial degeneration, increased expression of matrix metalloproteinases (MMPs), leading to loss of extracellular matrix proteins such as collagen and elastin. Aim: To understand the changes in smooth muscle specific genes and proteins in aneurysms of the ascending thoracic aorta, the involvement and relative roles of different MMPs in a large series of (n = 25) ascending thoracic aneurysms (ATAs). Patients and Methods: ATA samples were provided by Kartal Ko¸suyolu, Advanced Training and Research Hospital. Total RNA was isolated from aneurysmal tissue. Smooth muscle cells (SMC) were isolated from 15 ATA samples using the explant technique and immunohistochemistry (IHC) was performed to characterize SMC marker proteins; SMC actin, desmin and vimentin. The expression of certain genes in aneurysmal tissue and isolated cells SMC were determined by real time PCR. SMCs from healthy subjects and nondiseased aortic tissue (n = 4) were used as controls. Results: There are significant differences between the expression of SMC contractile genes and proteins such as actin, myosin heavy chain and calponin in tissue and cells of ATA patients. Measured MMP-2, -9 and -13 gene expressions, normalized to controls also differed significantly. Conclusion: Our results indicate that SMC in ATA tissues present with a phenotypic diversity, which persists in SMC isolated from tissue. Supported by the EU 7th Framework Program Project “Fighting Aneurysmal Diseases” (FAD, 200647). P415 ROLE OF THE COP9 SIGNALOSOME IN NF-úB SIGNALING IN ATHEROSCLEROSIS Y. Asare1,2 , H. Noels2 , H. Lue1 , A. Zernecke3 , C. Weber2 , M. deWinther4 , J. Bernhagen1 . 1 Department of Biochemistry and Molecular Cell Biology, 2 Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, 3 Rudolf Virchow Center for Experimental Biomedicine, University of Wurzburg, ¨ Wurzburg, ¨ Germany, 4 Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, Netherlands Antilles Inflammatory processes play a crucial role in all stages of atherogenesis: from early endothelial activation by modified lipids to the eventual rupture of the atherosclerotic plaque. Activation of the canonical NF-kappaB signal transduction pathway, mediated by phosphorylation and degradation of IkappaBalpha by the IkappaB-Kinase (IKK) complex, is a crucial step in proinflammatory target gene expression such as that of chemokines or adhesion molecules, which have been implicated in the initiation and development of atherosclerosis. The role of the COP9 signalosome (CSN), a multi-functional protein complex with functions in NF-kappaB and beta2-integrin signaling, in atherosclerosis has been unknown. Here we silenced CSN5/JAB1, a subunit of the CSN responsible for the cullin-1 deNEDDylation activity of CSN, in endothelial cells (HUVECs) using siRNA technology. IkappaBalpha levels were decreased in CSN5 and CSN2 knockdown cells and further experiments revealed that this decrease was due to increased cullin-1 and cullin-1-NEDD8 levels, indicating that the CSN regulates the stability of cullin-1 which in turn affects the turnover of IkappaBalpha. Next, we observed that TNFalpha-dependent degradation of IkBalpha was enhanced (52%) by CSN5or CSN2-siRNA treatment (knockdown efficiency: ~40%) and an increase of NF-kB activity (80%). Furthermore, siRNA knockdown of CSN5 or CSN2 under pro-atherogenic conditions led to a prolongation of TNFalpha-induced phosphorylation of IKK. Finally, preliminary data indicate that CSN3 interacts with IKK in a TNFalpha-dependent manner. We propose that the CSN controls the activity of NF-kappaB and might play a vital role in the canonical NF-kappaB signal transduction pathway in inflammatory and atherogenic conditions. P416 INFLAMMATORY MARKERS IN PATIENTS UNDERGOING IMPLANTABLE CARDIOVERTER DEFIBRILLATOR IMPLANTATION FOR LIFE THREATENING VENTRICULAR ARRHYTHMIAS T. Papadopoulos1 , N. Katsiki2 , G. Dadous1 , I. Kanonidis1 , G. Sakadamis1 . 1 2nd Department of Cardiology, Hippokration University Hospital, Aristotle University of Thessaloniki, 2 Medical School, Aristotle University of Thessaloniki, Thessalon´ıki, Greece Aim: To investigate the role of inflammation in patients with life threatening ventricular arrhythmias. Methods: Blood samples were taken from twenty-one male patients (mean age = 64±9.27 yrs) undergoing ICD implantation for life-threatening ventricular arrhythmias. Samples were drawn simultaneously from three different sites i.e.
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
A mouse model that yields high numbers of pure FCM and NFM has been characterised, and the genome of the macrophages compared. Elevated levels of thrombotic genes were found in FCM. This, combined with changes in matrix production and matrix proteases, may explain how atherosclerotic plaques with high levels of FCM are vulnerable to thrombotic events. P412 PLEIOTROPIC EFFECTS OF ATORVASTATIN AND TGF-b/ENDOGLIN CASCADE IN THE VESSEL WALL OF ApoE/LDLR DOUBLE KNOCKOUT MICE Z. Strasky1 , L. Vecerova1 , M. Slanarova1 , J. Rathouska1 , E. Brcakova1 , S. Micuda2 , P. Nachtigal1 . 1 Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, 2 Charles University in Prague, Faculty of Medicine, Department of Pharmacology, Hradec Kralove, Czech Republic Endoglin is a transmembrane glycoprotein a part of the TGF-b receptor cascade and it is known as a type III TGF-b receptor. In our recent paper we demonstrated that administration of cholesterol-rich diet with atorvastatin (ATV) decrease cholesterol levels and increase the expression of endoglin/SMAD2/eNOS in the aorta. In this study, we studied the pleiotropic effects of atorvastatin on the expression of type II TGF-b receptor, endoglin, p-SMAD2, p-SMAD1 and p-eNOS in the mice aorta. Two-month-old female ApoE/LDLR double knockout mice were divided into two groups. The control group (n = 8) was fed with the chow diet whereas in the ATV group (n = 8), atorvastatin at dose 50 mg/kg per day was added to the same diet. Biochemical analysis of lipid profile, immunohistochemical and western blot analysis of above mentioned protein expressions in aortas were performed. Administration of atorvastatin resulted in a significant increase in total cholesterol and VLDL cholesterol when compared with control mice. On the contrary, the area of atherosclerotic lesion was decreased after ATV treatment. Western blot analysis revealed induced TGF-b IIR, endoglin, p-SMAD2, p-SMAD1 and p-eNOS expression in ATV treated mice when compared with control mice. In this study, we demonstrated that atorvastatin increase endoglin, p-SMADs and p-eNOS expressions in the vessel wall of ApoE/LDLR double knockout mice beyond its lipid lowering effects suggesting potential protective role of endoglin cascade in atherogenesis. This work was supported by grant from The Grant Agency of Charles University in Prague number 129208/C, grant MSM 002162082 and Research project MZO 001799060. P413 OXIDIZED-HEMOGLOBIN INDUCES INNATE IMMUNE CELL CHEMOTAXIS, MONOCYTE ARREST AND THEIR SUBINTIMAL MIGRATION INTO EARLY ATHEROSCLEROTIC TISSUE B. Buttari1 , E. Profumo1 , G. Domenici1 , M. Leopizzi2 , C. Di Cristofano2 , R. Businaro3 , G. Di Giammarco4 , R. Rigano` 1 . 1 Malattie Infettive, Parassitarie ` Rome, 2 Medicina ed Immunomediate, Istituto Superiore di Sanita, Sperimentale, 3 Anatomia Umana, Universita` di Roma, Sapienza, Roma, 4 Divisione di Cardiochirurgia, Policlinico SS Annunziata, Universita` di Chieti, G. d’Annunzio, Chieti, Italy Background and Aims: Signals that attract leukocytes to atherosclerotic plaque are partly defined. Our findings indicate that human hemoglobin (Hb) acts as a “self” antigen in patients with carotid atherosclerotic plaques. Aims of the study were to explore the ability of oxidized-Hb to act as a chemoattractant for innate immune cells and its possible role in atherogenic cell recruitment. Methods: Chemotaxis of monocytes and dendritic cells in response to Hb was studied in vitro using Transwells inserts and in vivo by establishing a mouse model of acute peritonitis. The ability of monocytes to adhere on endothelial layer (EA.hy 926) exposed for 2 hours to oxidized Hb was studied in static and flow dynamic conditions using a parallel wall flow chamber system mounted on inverted microscope (shear stress 2 dynes/cm2 for 2 min). Aortic tissue from patients with thoracic aortic aneurysm was perfused with 10 mg/ml of Hb over 4 hours and then with fluorescence-labelled monocytes. After 2 hours, nonadherent cells were washed away and fixed aortic tissue sections were analyzed by fluorescence microscope. Results: oxidized-Hb acts in vitro and in vivo as a chemoattractant for innate immune cells. The short-term incubation of endothelial monolayer with oxidizedHb results in a 2-fold increase of firm shear-resistant monocyte adhesion under flow conditions. Exposure of aortic tissues to oxidized-Hb triggered monocyte recruitment in early atherosclerotic aorta. Conclusion: Oxidized-Hb contributes to innate immune cell infiltration, monocyte arrest and their subintimal migration into early atherosclerotic tissues with consequent amplification of the inflammatory response.
Poster Presentations
P414 EXPRESSION OF GENES AND PROTEINS IN ASCENDING THORACIC AORTIC ANEURYSMS AND SMOOTH MUSCLE CELLS ISOLATED FROM ANEURYSMAL SAMPLES Z. Adıguzel ¨ 1 , M. Serhatlı1 , O. Ka¸car1 , A. Tuncer2 , E. Tuncer2 , K. Baysal1 . 1 Molecular Cell Biology, TUBITAK MRC Genetic Engineering and Biotechnology Institute, Kocaeli, 2 T.C. Ministry of Health, Kartal Ko¸suyolu, Advanced Training and Research Hospital, Istanbul, Turkey Aortic aneurysms are formed by an irreversible weakening of a certain part of the aorta, leading to localized dilatation of the vessel. The alterations in the vessel wall leading to this weakening are reported as; medial degeneration, increased expression of matrix metalloproteinases (MMPs), leading to loss of extracellular matrix proteins such as collagen and elastin. Aim: To understand the changes in smooth muscle specific genes and proteins in aneurysms of the ascending thoracic aorta, the involvement and relative roles of different MMPs in a large series of (n = 25) ascending thoracic aneurysms (ATAs). Patients and Methods: ATA samples were provided by Kartal Ko¸suyolu, Advanced Training and Research Hospital. Total RNA was isolated from aneurysmal tissue. Smooth muscle cells (SMC) were isolated from 15 ATA samples using the explant technique and immunohistochemistry (IHC) was performed to characterize SMC marker proteins; SMC actin, desmin and vimentin. The expression of certain genes in aneurysmal tissue and isolated cells SMC were determined by real time PCR. SMCs from healthy subjects and nondiseased aortic tissue (n = 4) were used as controls. Results: There are significant differences between the expression of SMC contractile genes and proteins such as actin, myosin heavy chain and calponin in tissue and cells of ATA patients. Measured MMP-2, -9 and -13 gene expressions, normalized to controls also differed significantly. Conclusion: Our results indicate that SMC in ATA tissues present with a phenotypic diversity, which persists in SMC isolated from tissue. Supported by the EU 7th Framework Program Project “Fighting Aneurysmal Diseases” (FAD, 200647). P415 ROLE OF THE COP9 SIGNALOSOME IN NF-úB SIGNALING IN ATHEROSCLEROSIS Y. Asare1,2 , H. Noels2 , H. Lue1 , A. Zernecke3 , C. Weber2 , M. deWinther4 , J. Bernhagen1 . 1 Department of Biochemistry and Molecular Cell Biology, 2 Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, 3 Rudolf Virchow Center for Experimental Biomedicine, University of Wurzburg, ¨ Wurzburg, ¨ Germany, 4 Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, Netherlands Antilles Inflammatory processes play a crucial role in all stages of atherogenesis: from early endothelial activation by modified lipids to the eventual rupture of the atherosclerotic plaque. Activation of the canonical NF-kappaB signal transduction pathway, mediated by phosphorylation and degradation of IkappaBalpha by the IkappaB-Kinase (IKK) complex, is a crucial step in proinflammatory target gene expression such as that of chemokines or adhesion molecules, which have been implicated in the initiation and development of atherosclerosis. The role of the COP9 signalosome (CSN), a multi-functional protein complex with functions in NF-kappaB and beta2-integrin signaling, in atherosclerosis has been unknown. Here we silenced CSN5/JAB1, a subunit of the CSN responsible for the cullin-1 deNEDDylation activity of CSN, in endothelial cells (HUVECs) using siRNA technology. IkappaBalpha levels were decreased in CSN5 and CSN2 knockdown cells and further experiments revealed that this decrease was due to increased cullin-1 and cullin-1-NEDD8 levels, indicating that the CSN regulates the stability of cullin-1 which in turn affects the turnover of IkappaBalpha. Next, we observed that TNFalpha-dependent degradation of IkBalpha was enhanced (52%) by CSN5or CSN2-siRNA treatment (knockdown efficiency: ~40%) and an increase of NF-kB activity (80%). Furthermore, siRNA knockdown of CSN5 or CSN2 under pro-atherogenic conditions led to a prolongation of TNFalpha-induced phosphorylation of IKK. Finally, preliminary data indicate that CSN3 interacts with IKK in a TNFalpha-dependent manner. We propose that the CSN controls the activity of NF-kappaB and might play a vital role in the canonical NF-kappaB signal transduction pathway in inflammatory and atherogenic conditions. P416 INFLAMMATORY MARKERS IN PATIENTS UNDERGOING IMPLANTABLE CARDIOVERTER DEFIBRILLATOR IMPLANTATION FOR LIFE THREATENING VENTRICULAR ARRHYTHMIAS T. Papadopoulos1 , N. Katsiki2 , G. Dadous1 , I. Kanonidis1 , G. Sakadamis1 . 1 2nd Department of Cardiology, Hippokration University Hospital, Aristotle University of Thessaloniki, 2 Medical School, Aristotle University of Thessaloniki, Thessalon´ıki, Greece Aim: To investigate the role of inflammation in patients with life threatening ventricular arrhythmias. Methods: Blood samples were taken from twenty-one male patients (mean age = 64±9.27 yrs) undergoing ICD implantation for life-threatening ventricular arrhythmias. Samples were drawn simultaneously from three different sites i.e.