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PO9-243 EXERCISE TRAINING AND THE INFLAMMATORY RESPONSE IN ZDF (TYPE 2) DIABETIC RATS
Poster Sessions PO9 Inflammation and atherosclerosis antibodies to carbamylated-LDL (r=0.23, P=0.037) and carbamylated-MSA (r=0.308, P=0.005). Conclusions: The data reveals increased IgG antibody levels to carbamylated proteins and apoptotic cells in uremia. Antibodies to modified LDL and apoptotic cells may influence their macrophage uptake and may potentially direct atherosclerosis in uremic patients. PO9-243
EXERCISE TRAINING AND THE INFLAMMATORY RESPONSE IN ZDF (TYPE 2) DIABETIC RATS
F. Teixeira 1 , E. Teixeira de Lemos 1,2 , F. Reis 1 , S. Baptista 1 , R. Pinto 3 , B. Sepodes 3 , H. Vala 2 , P. Rocha-Pereira 4 , A. Santos-Silva 5 . 1 Therapeutics Unit, Institute of Pharmacology and Experimental Therapeutics, Medicine Faculty, Coimbra University, Coimbra, Portugal; 2 Polytechnic Institute of Viseu, Viseu, Portugal; 3 Pharmacology and Pharmacotoxicology Unit, Pharmacy School, University of Lisbon, Lisbon, Portugal; 4 Department of Chemistry, University of Beira Interior, Covilhã, Portugal; 5 Department of Biochemistry, Faculty of Pharmacy and Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
PO9-244
UPTAKE OF 11C-PK11195, A MARKER OF INFLAMMATORY CELLS, INTO ATHEROSCLEROTIC PLAQUES IN MICE
I. Laitinen 1 , P. Marjamäki 1 , M. Haaparanta 1 , K. Någren 1 , V.J.O. Laine 2 , P. Leppänen 3 , S. Ylä-Herttuala 3 , A. Roivainen 1 , J. Knuuti 1 . 1 Turku PET Centre, Turku University Hospital, Turku, Finland; 2 Department of Pathology, Turku University Hospital, Turku, Finland; 3 A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland Background and Aims: Inflammation provokes atherosclerosis and dictates the outcome of coronary artery disease. 11C-PK11195 has previously been used for PET (positron emission tomography) imaging of multiple sclerosis suggesting suitability also for the imaging of atherosclerosis-associated inflammation. 11C-PK11195 is a high-affinity ligand for mitochondrial benzodiatzepine receptors found in many peripheral tissues and in leukocytes. We hypothesised that the uptake of 11C-PK11195 would be more prominent in macrophage-rich atherosclerotic plaques compared to silent plaques. Methods: LDL receptor- and ApoB48 -deficient mice (Ldlr -//Apob100/100) (n=5) were kept on Western-type diet. Normally fed C57BL/6 mice (n=3) were used as controls. An intravenous bolus of 11C-PK11195 (23 MBq) was given and the animals were sacrificed after 20 minutes. The weight and radioactivity of plasma and tissue samples was measured. The dissected, frozen aorta was sectioned longitudinally and analysed with digital autoradiography (BAS-5000 Fuji Analyzer). Adjacent sections were immunostained for macrophage markers and all sections
were HE-stained. The autoradiographs were analysed for count densities in regions of interest. Results: Ldlr -/- /Apob100/100 mice developed both silent and cellrich atherosclerotic plaques with moderate inflammation. Autoradiographs revealed diffuse 11C-PK11195 uptake in the tissue, but not specifically in the cell-rich plaques. The uptake of 11C-PK11195 was not increased in the aorta of atherosclerotic mice compared to the control mice. Conclusions: Our results indicate that the uptake of 11C-PK11195 to the atherosclerotic vessels is not specific to inflamed plaques. This may limit the use of 11C-PK11195 in PET imaging of atherosclerotic plaques. PO9-245
REGULATORY ROLE OF MATURING HUMAN MAST CELLS IN INFLAMMATION
J.A. Trosien 1 , K.A. Lindstedt 1 , M. Laine 2 , P.T. Kovanen 1 . 1 Wihuri Research Institute, Helsinki, Finland; 2 Department of Medicine, Helsinki University Central Hospital and Minerva Institute for Medical Research, Helsinki, Finland, Helsinki, Finland Objective: Mast cells (MCs) are abundant in the shoulder region of atherosclerotic plaques, where also T cells and macrophages are present. Since MCs infiltrate tissues as hematopoietic progenitor cells, we here investigate the contribution of maturing human mast cells to inflammatory processes in vitro. Methods and Results: Conditioned media from unstimulated human cord blood-derived MCs were screened for cytokines at different stages of their maturation. High concentrations of MCP-1 (up to 11 ng/ml), MDC (up to 2.5 ng/ml), and IL-8 (up to 2.4 ng/ml) were detected. Moreover, leptin (up to 0.6 ng/ml), IL-6, IL-10 and NAP-2 were also present in the conditioned media. Real-time PCR revealed that leptin expression depended on the degree of MC maturation. Pro-inflammatory molecules, such as PGE2 and TNF-α, increased the leptin-secretion in a dose-dependent manner, whereas IgE-dependent MC activation was without effect. T cells cultured in MC conditioned media showed a suppressed proliferative response to CD3CD28 stimulus, and the level of suppression depended on the degree of MC maturation. IL-10 (up to 0.8 ng/ml), a known T cell inhibitor, was present in the conditioned media, and neutralizing it with an antibody restored T cell proliferation. Conclusions: Maturing human mast cells secrete pro-inflammatory cytokines and chemokines, such as leptin, MDC, MCP-1, and IL-8. However, they also secrete IL-10, capable of suppressing T cell proliferation in vitro. Thus, maturing mast cells are not innocent bystanders but actively participate in tissue homeostasis by secreting both pro- and anti-inflammatory cytokines. PO9-246
HUMAN PARAOXONASE 2: AN ENDOGENOUS ENZYME AS DEFENSE SYSTEM AGAINST VASCULAR OXIDATIVE STRESS AND ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS
S. Horke 1 , I. Witte 1 , P. Wilgenbus 1 , M. Krueger 1 , D. Strand 2 , U. Foerstermann 1 . 1 Institute of Pharmacology, Johannes Gutenberg University of Mainz, Mainz, Germany; 2 Internal Medicine I, Johannes Gutenberg University of Mainz, Mainz, Germany Background: In the vascular system, oxidative stress, i.e. elevated levels of reactive oxygen species (ROS) predispose to the development of atherosclerosis. Because ROS are constantly formed in cells and removed by antioxidant defenses, it is mandatory to understand the regulatory systems involved. The family of paraoxonases (PONs) consists of three proteins, PON1, PON2 and PON3, which share high levels of identity. PON1 and/or PON3 have been reported to protect against oxidation of plasma lipoproteins and coronary heart disease, whereas much less information is available on PON2. Methods and Results: By qRT-PCR and Western blot we show that PON2 is equally expressed in major vascular cell types. In order to evaluate the importance of PON2 as an antioxidant defense, we established HUVEC-derived EA.hy 926 endothelial cells stably overexpressing PON2 and used RNA interference for its temporal knockdown. Overexpression of PON2 markedly reduced ROS levels, whereas its temporal knockdown increased ROS levels significantly. Investigating its subcellular localization by various approaches, we found PON2 to be localized predominantly in the nuclear membrane and the endoplasmic reticulum (ER). The ER is the site of lipid-induced cytotoxicity in atherogenesis. Interestingly, reporter gene
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
Background and Aims: Sub clinical chronic inflammation could be a unifying factor linking type 2 diabetes (T2D) and atherosclerosis. The beneficial effects of physical activity in reducing the risk of CHD could at least in part be mediated by improving the inflammatory markers. We examined the putative effects of exercise training on insulin levels, hs-CRP, uric acid and pro-inflammatory markers IL-6 and TNF-alpha, in the T2D model Zucker diabetic fatty (ZDF) rats. Methods: Male ZDF (Gmi fa/fa) rats and their littermates (Gmi +/+), aged 8 weeks, were randomly assigned in two groups: an exercise swimming trained and a sedentary one. Training was conducted 1h/day, 3 days/week, for 12 weeks. The rats were sacrificed 48 hours after the last bout exercise. Results: Such 12 weeks swimming training regimen has significantly reduced the plasma glucose (-11.3%), HbA1c (-6.6%), total cholesterol (-17.3%) and triglycerides (- 32.1%) concentrations. Nevertheless, there was not body weight loss, neither in the diabetic nor in the control trained groups. The ZDF (fa/fa) rats have presented hyperinsulinaemia, which was significantly prevented (p<0.05) by exercise. Serum uric acid levels were reverted (- 37.3%) by swimming, as well as hs-CRP (-26.0%). In the ZDF (fa/fa) group that underwent exercise, IL-6 and TNF-alpha values have significantly improved (-20.0% and -13.0%, respectively). Conclusions: The present results indicate that a 12 week swimming training exercise is effective in improving insulin and chronic inflammation, being also beneficial for other metabolic abnormalities associated with T2D. Acknowledgements for the grant of Merck Sharp & Dohme Foundation, Portugal.
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EXERCISE TRAINING AND THE INFLAMMATORY RESPONSE IN ZDF (TYPE 2) DIABETIC RATS
F. Teixeira 1 , E. Teixeira de Lemos 1,2 , F. Reis 1 , S. Baptista 1 , R. Pinto 3 , B. Sepodes 3 , H. Vala 2 , P. Rocha-Pereira 4 , A. Santos-Silva 5 . 1 Therapeutics Unit, Institute of Pharmacology and Experimental Therapeutics, Medicine Faculty, Coimbra University, Coimbra, Portugal; 2 Polytechnic Institute of Viseu, Viseu, Portugal; 3 Pharmacology and Pharmacotoxicology Unit, Pharmacy School, University of Lisbon, Lisbon, Portugal; 4 Department of Chemistry, University of Beira Interior, Covilhã, Portugal; 5 Department of Biochemistry, Faculty of Pharmacy and Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
PO9-244
UPTAKE OF 11C-PK11195, A MARKER OF INFLAMMATORY CELLS, INTO ATHEROSCLEROTIC PLAQUES IN MICE
I. Laitinen 1 , P. Marjamäki 1 , M. Haaparanta 1 , K. Någren 1 , V.J.O. Laine 2 , P. Leppänen 3 , S. Ylä-Herttuala 3 , A. Roivainen 1 , J. Knuuti 1 . 1 Turku PET Centre, Turku University Hospital, Turku, Finland; 2 Department of Pathology, Turku University Hospital, Turku, Finland; 3 A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland Background and Aims: Inflammation provokes atherosclerosis and dictates the outcome of coronary artery disease. 11C-PK11195 has previously been used for PET (positron emission tomography) imaging of multiple sclerosis suggesting suitability also for the imaging of atherosclerosis-associated inflammation. 11C-PK11195 is a high-affinity ligand for mitochondrial benzodiatzepine receptors found in many peripheral tissues and in leukocytes. We hypothesised that the uptake of 11C-PK11195 would be more prominent in macrophage-rich atherosclerotic plaques compared to silent plaques. Methods: LDL receptor- and ApoB48 -deficient mice (Ldlr -//Apob100/100) (n=5) were kept on Western-type diet. Normally fed C57BL/6 mice (n=3) were used as controls. An intravenous bolus of 11C-PK11195 (23 MBq) was given and the animals were sacrificed after 20 minutes. The weight and radioactivity of plasma and tissue samples was measured. The dissected, frozen aorta was sectioned longitudinally and analysed with digital autoradiography (BAS-5000 Fuji Analyzer). Adjacent sections were immunostained for macrophage markers and all sections
were HE-stained. The autoradiographs were analysed for count densities in regions of interest. Results: Ldlr -/- /Apob100/100 mice developed both silent and cellrich atherosclerotic plaques with moderate inflammation. Autoradiographs revealed diffuse 11C-PK11195 uptake in the tissue, but not specifically in the cell-rich plaques. The uptake of 11C-PK11195 was not increased in the aorta of atherosclerotic mice compared to the control mice. Conclusions: Our results indicate that the uptake of 11C-PK11195 to the atherosclerotic vessels is not specific to inflamed plaques. This may limit the use of 11C-PK11195 in PET imaging of atherosclerotic plaques. PO9-245
REGULATORY ROLE OF MATURING HUMAN MAST CELLS IN INFLAMMATION
J.A. Trosien 1 , K.A. Lindstedt 1 , M. Laine 2 , P.T. Kovanen 1 . 1 Wihuri Research Institute, Helsinki, Finland; 2 Department of Medicine, Helsinki University Central Hospital and Minerva Institute for Medical Research, Helsinki, Finland, Helsinki, Finland Objective: Mast cells (MCs) are abundant in the shoulder region of atherosclerotic plaques, where also T cells and macrophages are present. Since MCs infiltrate tissues as hematopoietic progenitor cells, we here investigate the contribution of maturing human mast cells to inflammatory processes in vitro. Methods and Results: Conditioned media from unstimulated human cord blood-derived MCs were screened for cytokines at different stages of their maturation. High concentrations of MCP-1 (up to 11 ng/ml), MDC (up to 2.5 ng/ml), and IL-8 (up to 2.4 ng/ml) were detected. Moreover, leptin (up to 0.6 ng/ml), IL-6, IL-10 and NAP-2 were also present in the conditioned media. Real-time PCR revealed that leptin expression depended on the degree of MC maturation. Pro-inflammatory molecules, such as PGE2 and TNF-α, increased the leptin-secretion in a dose-dependent manner, whereas IgE-dependent MC activation was without effect. T cells cultured in MC conditioned media showed a suppressed proliferative response to CD3CD28 stimulus, and the level of suppression depended on the degree of MC maturation. IL-10 (up to 0.8 ng/ml), a known T cell inhibitor, was present in the conditioned media, and neutralizing it with an antibody restored T cell proliferation. Conclusions: Maturing human mast cells secrete pro-inflammatory cytokines and chemokines, such as leptin, MDC, MCP-1, and IL-8. However, they also secrete IL-10, capable of suppressing T cell proliferation in vitro. Thus, maturing mast cells are not innocent bystanders but actively participate in tissue homeostasis by secreting both pro- and anti-inflammatory cytokines. PO9-246
HUMAN PARAOXONASE 2: AN ENDOGENOUS ENZYME AS DEFENSE SYSTEM AGAINST VASCULAR OXIDATIVE STRESS AND ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS
S. Horke 1 , I. Witte 1 , P. Wilgenbus 1 , M. Krueger 1 , D. Strand 2 , U. Foerstermann 1 . 1 Institute of Pharmacology, Johannes Gutenberg University of Mainz, Mainz, Germany; 2 Internal Medicine I, Johannes Gutenberg University of Mainz, Mainz, Germany Background: In the vascular system, oxidative stress, i.e. elevated levels of reactive oxygen species (ROS) predispose to the development of atherosclerosis. Because ROS are constantly formed in cells and removed by antioxidant defenses, it is mandatory to understand the regulatory systems involved. The family of paraoxonases (PONs) consists of three proteins, PON1, PON2 and PON3, which share high levels of identity. PON1 and/or PON3 have been reported to protect against oxidation of plasma lipoproteins and coronary heart disease, whereas much less information is available on PON2. Methods and Results: By qRT-PCR and Western blot we show that PON2 is equally expressed in major vascular cell types. In order to evaluate the importance of PON2 as an antioxidant defense, we established HUVEC-derived EA.hy 926 endothelial cells stably overexpressing PON2 and used RNA interference for its temporal knockdown. Overexpression of PON2 markedly reduced ROS levels, whereas its temporal knockdown increased ROS levels significantly. Investigating its subcellular localization by various approaches, we found PON2 to be localized predominantly in the nuclear membrane and the endoplasmic reticulum (ER). The ER is the site of lipid-induced cytotoxicity in atherogenesis. Interestingly, reporter gene
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
Background and Aims: Sub clinical chronic inflammation could be a unifying factor linking type 2 diabetes (T2D) and atherosclerosis. The beneficial effects of physical activity in reducing the risk of CHD could at least in part be mediated by improving the inflammatory markers. We examined the putative effects of exercise training on insulin levels, hs-CRP, uric acid and pro-inflammatory markers IL-6 and TNF-alpha, in the T2D model Zucker diabetic fatty (ZDF) rats. Methods: Male ZDF (Gmi fa/fa) rats and their littermates (Gmi +/+), aged 8 weeks, were randomly assigned in two groups: an exercise swimming trained and a sedentary one. Training was conducted 1h/day, 3 days/week, for 12 weeks. The rats were sacrificed 48 hours after the last bout exercise. Results: Such 12 weeks swimming training regimen has significantly reduced the plasma glucose (-11.3%), HbA1c (-6.6%), total cholesterol (-17.3%) and triglycerides (- 32.1%) concentrations. Nevertheless, there was not body weight loss, neither in the diabetic nor in the control trained groups. The ZDF (fa/fa) rats have presented hyperinsulinaemia, which was significantly prevented (p<0.05) by exercise. Serum uric acid levels were reverted (- 37.3%) by swimming, as well as hs-CRP (-26.0%). In the ZDF (fa/fa) group that underwent exercise, IL-6 and TNF-alpha values have significantly improved (-20.0% and -13.0%, respectively). Conclusions: The present results indicate that a 12 week swimming training exercise is effective in improving insulin and chronic inflammation, being also beneficial for other metabolic abnormalities associated with T2D. Acknowledgements for the grant of Merck Sharp & Dohme Foundation, Portugal.
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