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WO4-OR-5 UROKINASE PLASMINOGEN ACTIVATOR (UPA) UPREGULATES MACROPHAGE PARAOXONASE 2 (PON2) EXPRESSION IN A REDOX-DEPENDENT PATHWAY
Workshops WO4 Plaque remodelling in atherosclerosis
4 WO3-OR-6
POSTPRANDIAL UCP2 EXPRESSION AND OXIDATIVE EFFECTS AFTER THREE DIET MODELS IN INSULIN-RESISTANT PATIENTS
M.E. Sanchez-Garcia 1 , J.A. Paniagua 1 , I. Romero 1 , A. Gallego de la Sacristana 1 , A. Vidal-Puig 2 , P. Pérez-Martínez 1 , F. Fuentes 1 , J.A. Ruano 1 , J. López-Miranda 1 , F. Pérez-Jiménez 1 . 1 Lipid and Atherosclerosis Unit. University Hospital Reina Sofía, Cordoba, Spain; 2 Molecular Mechanisms of Energy Balance Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK Objectives: To evaluate the effects of three isocaloric diets on UCP2 gene expression and its relationship with oxidative markers in patients with insulin resistance. Research Design and Methods: Eleven offspring obese type 2 diabetic patients with abdominal obesity were studied. The patients being insulin resistant as indicated by Matsuda ISIm <4 after OGTT maintained a HBA1c < 6.5% without treatment. All subjects underwent three diets of 28 days each in a crossover design: diet high in saturated fat (SAT), monounsaturated fat (MUFA; Mediterranean diet) and carbohydrate (CHO). At the end of each period, all subjets carried out an indirect calorimetry and received a 443 Kcal breakfast. Peripheral adipose tissue was collected 180 minutes after the intake and venous blood samples were obteined at 0, 60, 120 and 180 minutes. Results: The postprandial mRNA expression of UCP2 in peripheral adipocytes was lower after the consumption of the MUFA-rich diet (p<0.05). The intake of a CH-rich diet was associated with higher postprandial plasma concentrations of nitrotyrosine, as compared when feeding SAT and MUFA-rich diets (p<0.05, respectively). The postprandial plasma oxidized LDL (ox-LDL) concentrations were decreased after patient consumption a MUFA-rich diet (p<0.05). There was an increase in fasting lipid oxidation during the high fatty diet periods, as compared with CH-rich diet (p<0.05). Conclusions: In overweight insulin resistant patients, after consumption of a meal high-MUFA the UCP2 gene expression and postprandial ox-LDL and nitrotyrosine levels were lowered as compared when patient feeding SAT and CH diets.
WO4 PLAQUE REMODELLING IN ATHEROSCLEROSIS WO4-OR-3
SIGNALING PATHWAYS UNDERLYING CYTOKINE REGULATED EXPRESSION OF KEY GENES IN MACROPHAGES IMPLICATED IN ATHEROSCLEROSIS
N.N. Singh, P. Foka, E.J. Harvey, E. Huwait, S. Ali, N. Li, R. Salter, D.P. Ramji. Cardiff School of Biosciences, Cardiff University, Cardiff, UK Cytokines regulate the expression of key genes in macrophages that are involved in the control of cholesterol homeostasis and the inflammatory response, two crucial events in the pathogenesis of atherosclerosis. We have investigated the molecular mechanisms underlying the cytokine-mediated regulation of key genes in macrophages implicated in inflammation and the modulation of cholesterol homeostasis [e.g. lipoprotein lipase (LPL), apolipoprotein E (apoE), ATP-binding cassette transporter-A1 (ABCA1), monocyte chemoattractant protein-1 (MCP-1)], with emphasis on the action of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). We show that IFN-gamma and TGF-beta decrease the expression of LPL mRNA, protein and enzymatic activity in a concentration- and time-dependent manner. The action of IFN-gamma was mediated through a novel pathway involving the activation of casein kinase 2 (CK2) and phosphoinositide-3-kinase (PI3K) leading to a decrease in the binding of transcription factors Sp1 and Sp3 to regulatory sequences in the LPL gene. Further studies on MCP-1 along with RT-PCR and gene expression profiling of many potential atherosclerosis markers showed that CK2 and PI3K were involved in the IFN-gamma-mediated regulation of a large number of genes implicated in atherosclerosis. TGF-beta also inhibited LPL gene transcription through Sp1 and Sp3 but required the c-Jun N-terminal kinase (JNK) signalling pathway. The JNK pathway was also required for the TGF-beta-mediated induction of apoE and ABCA1 expression. These studies provide novel insights into the molecular mechanisms underlying the cytokine-mediated regulation of expression of key genes in macrophages implicated in atherosclerosis.
We thank Wellcome Trust and British Heart Foundation for financial support. WO4-OR-4
ROLE OF CATHEPSIN K IN THE AORTIC WALL REMODELING DURING PROGRESSION OF ATHEROSCLEROSIS IN APOE-DEFICIENT MICE
A. Samokhin 1 , A. Wong 1 , P. Saftig 2 , D. Bromme 1 . 1 Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada; 2 Biochemical Institute, Christian-Albrechts University, Kiel, Germany The metabolism of the vascular extracellular matrix plays an important role in the development of atherosclerosis. The stability and elasticity of blood vessels mainly depend on the presence and integrity of elastic and collagen fibers. Recent studies have shown that lysosomal cysteine proteinases exhibit potent elastinolytic and collagenolytic activities in mammalian tissues. To elucidate the role of cathepsin K which exerts both a strong collagenolytic and elastinolytic activity, we analyzed the effect of cathepsin K deficiency (ctsk-/-) in atherosclerosis-prone apoE-deficient mice on high fat diet. Ctsk-/- mice had significantly less macrophages in the tunica media while the number of smooth muscle cells was higher. Of note, ctsk-/- mice revealed a lower number of elastin fiber breaks which was also reflected by lower levels of serum desmosine. Moreover, the collagen content in plaque areas and fibrous cap thickness were significantly higher in the ctsk-/- mice and they had smaller numbers of buried fibrous caps which are considered to be a hallmark of plaque rupture. These differences resulted in smaller plaque areas in the brachiocephalic artery after 16 weeks of HFD when compared to their cathepsin K expressing littermates. Higher plaque stability in ctsk-/-mice also correlated with a decrease in cell apoptosis in atherosclerotic plaques. In conclusion, cathepsin K expression contributes to the destruction of elastin matrix of aortic wall that facilitates migration or death of smooth muscle cells. The collagenolytic activity of cathepsin K contributes to plaque destabilization by weakening of the collagen-containing fibrous cap of atherosclerotic plaques. WO4-OR-5
UROKINASE PLASMINOGEN ACTIVATOR (UPA) UPREGULATES MACROPHAGE PARAOXONASE 2 (PON2) EXPRESSION IN A REDOX-DEPENDENT PATHWAY
B. Fuhrman, J. Khateeb, M. Aviram. The Lipid Research Laboratory, Technion Faculty of Medicine and Rambam Medical Center, Haifa, Israel Background and aims: Urokinase plasminogen activator (uPA) is expressed in human atherosclerotic lesion macrophages, and contributes to the progression of atherosclerosis. The atherosclerotic plaque is dominated by macrophage-foam cells loaded with cholesterol and oxidized lipids. We have recently shown that uPA stimulates macrophage cholesterol accumulation due to increased cholesterol biosynthesis. Thus, we questioned whether uPA has an impact on macrophage oxidation status. Methods and results: uPA significantly increased oxidative stress in THP-1 human macrophages in a dose-dependent manner, by up to 2 or 3 fold, as determined by FACS using DCFH, or by the lipid peroxide assay, respectively. RT-PCR analysis revealed that uPA increased the expression of the NADPH oxidase cytosolic component, p47phox. Macrophages are protected against oxidative stress by paraoxonase2 (PON2), which is a ubiquitously expressed lactonase with antioxidant properties. A dose-dependent increase in macrophage PON2 lactonase activity was noted upon cell incubation with uPA, and this effect required the binding of uPA to its receptor. Similarly, uPA increased PON2 protein and gene expression, as measured by western blot and Q-PCR analysis. Preincubation of the macrophages with the antioxidants DPI or vitamin E abolished the uPA-mediated increase in PON2 expression, suggesting that this effect was, at least in part, related to the uPA-stimulation of cellular oxidative state. Conclusions: The present study shows, for the first time, that uPA increases macrophage oxidative stress by enhancing NADPH oxidase activation. This effect probably contributes to increased cellular PON2 expression, possibly as a compensatory protective mechanism against the uPA–induced macrophage oxidative stress.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
4 WO3-OR-6
POSTPRANDIAL UCP2 EXPRESSION AND OXIDATIVE EFFECTS AFTER THREE DIET MODELS IN INSULIN-RESISTANT PATIENTS
M.E. Sanchez-Garcia 1 , J.A. Paniagua 1 , I. Romero 1 , A. Gallego de la Sacristana 1 , A. Vidal-Puig 2 , P. Pérez-Martínez 1 , F. Fuentes 1 , J.A. Ruano 1 , J. López-Miranda 1 , F. Pérez-Jiménez 1 . 1 Lipid and Atherosclerosis Unit. University Hospital Reina Sofía, Cordoba, Spain; 2 Molecular Mechanisms of Energy Balance Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK Objectives: To evaluate the effects of three isocaloric diets on UCP2 gene expression and its relationship with oxidative markers in patients with insulin resistance. Research Design and Methods: Eleven offspring obese type 2 diabetic patients with abdominal obesity were studied. The patients being insulin resistant as indicated by Matsuda ISIm <4 after OGTT maintained a HBA1c < 6.5% without treatment. All subjects underwent three diets of 28 days each in a crossover design: diet high in saturated fat (SAT), monounsaturated fat (MUFA; Mediterranean diet) and carbohydrate (CHO). At the end of each period, all subjets carried out an indirect calorimetry and received a 443 Kcal breakfast. Peripheral adipose tissue was collected 180 minutes after the intake and venous blood samples were obteined at 0, 60, 120 and 180 minutes. Results: The postprandial mRNA expression of UCP2 in peripheral adipocytes was lower after the consumption of the MUFA-rich diet (p<0.05). The intake of a CH-rich diet was associated with higher postprandial plasma concentrations of nitrotyrosine, as compared when feeding SAT and MUFA-rich diets (p<0.05, respectively). The postprandial plasma oxidized LDL (ox-LDL) concentrations were decreased after patient consumption a MUFA-rich diet (p<0.05). There was an increase in fasting lipid oxidation during the high fatty diet periods, as compared with CH-rich diet (p<0.05). Conclusions: In overweight insulin resistant patients, after consumption of a meal high-MUFA the UCP2 gene expression and postprandial ox-LDL and nitrotyrosine levels were lowered as compared when patient feeding SAT and CH diets.
WO4 PLAQUE REMODELLING IN ATHEROSCLEROSIS WO4-OR-3
SIGNALING PATHWAYS UNDERLYING CYTOKINE REGULATED EXPRESSION OF KEY GENES IN MACROPHAGES IMPLICATED IN ATHEROSCLEROSIS
N.N. Singh, P. Foka, E.J. Harvey, E. Huwait, S. Ali, N. Li, R. Salter, D.P. Ramji. Cardiff School of Biosciences, Cardiff University, Cardiff, UK Cytokines regulate the expression of key genes in macrophages that are involved in the control of cholesterol homeostasis and the inflammatory response, two crucial events in the pathogenesis of atherosclerosis. We have investigated the molecular mechanisms underlying the cytokine-mediated regulation of key genes in macrophages implicated in inflammation and the modulation of cholesterol homeostasis [e.g. lipoprotein lipase (LPL), apolipoprotein E (apoE), ATP-binding cassette transporter-A1 (ABCA1), monocyte chemoattractant protein-1 (MCP-1)], with emphasis on the action of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). We show that IFN-gamma and TGF-beta decrease the expression of LPL mRNA, protein and enzymatic activity in a concentration- and time-dependent manner. The action of IFN-gamma was mediated through a novel pathway involving the activation of casein kinase 2 (CK2) and phosphoinositide-3-kinase (PI3K) leading to a decrease in the binding of transcription factors Sp1 and Sp3 to regulatory sequences in the LPL gene. Further studies on MCP-1 along with RT-PCR and gene expression profiling of many potential atherosclerosis markers showed that CK2 and PI3K were involved in the IFN-gamma-mediated regulation of a large number of genes implicated in atherosclerosis. TGF-beta also inhibited LPL gene transcription through Sp1 and Sp3 but required the c-Jun N-terminal kinase (JNK) signalling pathway. The JNK pathway was also required for the TGF-beta-mediated induction of apoE and ABCA1 expression. These studies provide novel insights into the molecular mechanisms underlying the cytokine-mediated regulation of expression of key genes in macrophages implicated in atherosclerosis.
We thank Wellcome Trust and British Heart Foundation for financial support. WO4-OR-4
ROLE OF CATHEPSIN K IN THE AORTIC WALL REMODELING DURING PROGRESSION OF ATHEROSCLEROSIS IN APOE-DEFICIENT MICE
A. Samokhin 1 , A. Wong 1 , P. Saftig 2 , D. Bromme 1 . 1 Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada; 2 Biochemical Institute, Christian-Albrechts University, Kiel, Germany The metabolism of the vascular extracellular matrix plays an important role in the development of atherosclerosis. The stability and elasticity of blood vessels mainly depend on the presence and integrity of elastic and collagen fibers. Recent studies have shown that lysosomal cysteine proteinases exhibit potent elastinolytic and collagenolytic activities in mammalian tissues. To elucidate the role of cathepsin K which exerts both a strong collagenolytic and elastinolytic activity, we analyzed the effect of cathepsin K deficiency (ctsk-/-) in atherosclerosis-prone apoE-deficient mice on high fat diet. Ctsk-/- mice had significantly less macrophages in the tunica media while the number of smooth muscle cells was higher. Of note, ctsk-/- mice revealed a lower number of elastin fiber breaks which was also reflected by lower levels of serum desmosine. Moreover, the collagen content in plaque areas and fibrous cap thickness were significantly higher in the ctsk-/- mice and they had smaller numbers of buried fibrous caps which are considered to be a hallmark of plaque rupture. These differences resulted in smaller plaque areas in the brachiocephalic artery after 16 weeks of HFD when compared to their cathepsin K expressing littermates. Higher plaque stability in ctsk-/-mice also correlated with a decrease in cell apoptosis in atherosclerotic plaques. In conclusion, cathepsin K expression contributes to the destruction of elastin matrix of aortic wall that facilitates migration or death of smooth muscle cells. The collagenolytic activity of cathepsin K contributes to plaque destabilization by weakening of the collagen-containing fibrous cap of atherosclerotic plaques. WO4-OR-5
UROKINASE PLASMINOGEN ACTIVATOR (UPA) UPREGULATES MACROPHAGE PARAOXONASE 2 (PON2) EXPRESSION IN A REDOX-DEPENDENT PATHWAY
B. Fuhrman, J. Khateeb, M. Aviram. The Lipid Research Laboratory, Technion Faculty of Medicine and Rambam Medical Center, Haifa, Israel Background and aims: Urokinase plasminogen activator (uPA) is expressed in human atherosclerotic lesion macrophages, and contributes to the progression of atherosclerosis. The atherosclerotic plaque is dominated by macrophage-foam cells loaded with cholesterol and oxidized lipids. We have recently shown that uPA stimulates macrophage cholesterol accumulation due to increased cholesterol biosynthesis. Thus, we questioned whether uPA has an impact on macrophage oxidation status. Methods and results: uPA significantly increased oxidative stress in THP-1 human macrophages in a dose-dependent manner, by up to 2 or 3 fold, as determined by FACS using DCFH, or by the lipid peroxide assay, respectively. RT-PCR analysis revealed that uPA increased the expression of the NADPH oxidase cytosolic component, p47phox. Macrophages are protected against oxidative stress by paraoxonase2 (PON2), which is a ubiquitously expressed lactonase with antioxidant properties. A dose-dependent increase in macrophage PON2 lactonase activity was noted upon cell incubation with uPA, and this effect required the binding of uPA to its receptor. Similarly, uPA increased PON2 protein and gene expression, as measured by western blot and Q-PCR analysis. Preincubation of the macrophages with the antioxidants DPI or vitamin E abolished the uPA-mediated increase in PON2 expression, suggesting that this effect was, at least in part, related to the uPA-stimulation of cellular oxidative state. Conclusions: The present study shows, for the first time, that uPA increases macrophage oxidative stress by enhancing NADPH oxidase activation. This effect probably contributes to increased cellular PON2 expression, possibly as a compensatory protective mechanism against the uPA–induced macrophage oxidative stress.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland