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Non_invasive analysis of oxidative stress in myocardial remodeling and failure in mice using in vivo electron spin resonance (ESR) spectroscopy
ABSTRACTS / Journal of Molecular and Cellular Cardiology 41 (2006) 1039 – 1079
Y-A-3. Catheter-based adenovirus-mediated anti-monocyte chemoattractant gene therapy attenuates in-stent neointima formation in non-human primates Kaku Nakano, Kensuke Egashira, Kisho Ohtani, Gang Zhao, Kouta Funakoshi, Yoshiko Ihara, Kenji Sunagawa. Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyushu University Background: We have previously demonstrated great benefit from anti-monocyte chemoattractant protein-1 (MCP1) gene therapy by ‘‘systemic’’ transfer of an N-terminal deletion mutant of human MCP-1 (called 7ND) gene into skeletal muscle for treatment of restenosis and atherosclerosis. However, recent evidence suggests that ’’local’’ gene transfer may be a clinically relevant approach. We therefore tested the hypothesis that catheter-based adenovirus-mediated anti-MCP-1 gene therapy attenuates stent-associated neointima formation. Methods and Results: Bare metal stents were implanted in iliac arteries of cynomolgus monkeys fed a high cholesterol diet. Immediately after the stenting procedure, normal saline or recombinant adenoviral vector containing LacZ or the 7ND gene was administered locally into the stenting site through a REMEDY channel-delivery catheter. Compared to saline infusion or LacZ gene transfer, 7ND gene transfer markedly reduced inflammatory changes at an early stage and attenuated neointima formation after 4 weeks. This strategy also reduced the increased production of pro-inflammatory and growthpromoting factors such platelet-derived growth factor. No systemic adverse effects of 7ND gene transfer were detected. There were no significant differences in serum cholesterol levels among the three groups. Conclusion: These data suggest that catheter-based adenovirus-mediated anti-MCP-1 gene therapy may be a clinically relevant and feasible strategy for treatment of in-stent restenosis. Keywords: Restenosis; Gene therapy; MCP-1 doi:10.1016/j.yjmcc.2006.08.035
Y-A-4. Non-invasive analysis of oxidative stress in myocardial remodeling and failure in mice using in vivo electron spin resonance (ESR) spectroscopy Tomomi Ide, Hideo Utsumi, Hiroyuki Tsutsui, Kenji Sunagawa. Kyushu University Department of Cardiovascular Medicine Oxidative stress plays an important role in the development and progress of myocardial remodeling and failure. We demonstrated the generation of reactive oxygen species (ROS) is enhanced in the tissue homogenates obtained from the failing hearts in vitro by electron spin resonance (ESR) spectroscopy. However, there is no direct evidence of ROS generation in the hearts in vivo. The aim of this study was to assess the time-dependent changes of ROS generation in the hearts after myocardial infarction(MI) using a technique of in
1045
vivo ESR measurement that has been developed for direct and non-invasive analysis of ROS generation in living animals. We created MI in mice by ligating the left coronary artery and performed in vivo ESR analysis at day 2, 7, 14, and 28 after MI. The in vivo generation of ROS was analyzed by the ESR signal decay of methoxy-PROXYL, injected intravenously. Left ventricular (LV) function was assessed by echocardiography. The ESR signal decay measured at the chest of mice was not changed at day 2, but was increased in MI compared to sham-operated mice at day 7 (Sham vs MI; 0.122 T 0.005 vs 0.142 T 0.007/min; p<0.05)and further increased at day 14 (0.12 T 0.01 vs 0.16 T 0.01/min, p<0.01) and 28 (0.12 T 0.01 vs 0.16 T 0.01/min, p<0.01), associated with the progression of LV dilatation and dysfunction. The increased ESR signal decay in MI was completely suppressed by the administration of antioxidant, Tiron. It was further confirmed by the increased ESR signal decay also in the cardiac homogenates obtained from MI mice. The present study provided the direct evidence in the post-MI living mice that ROS were generated within the hearts during the progression of remodeling and failure. Keywords: Heart failure; Free radicals; Remodeling doi:10.1016/j.yjmcc.2006.08.036
Y-A-5. Adipogenesis in obesity is coupled with angiogenesis Satoshi Nishimura 1, Ryozo Nagai 1, Seiryo Sugiura 2. 1 Department of Cardiovascular Medicine, The University of Tokyo. 2 Graduate School of Frontier Sciences. The University of Tokyo Ample evidences suggest that angiogenesis plays a pivotal role in adipogenesis in obesity, but little is known about the detailed mechanism. We have developed a visualization technique based on laser confocal microscopy that allows us to analyze angiogenesis and adipogenesis in intact living adipose tissue three-dimensionally. We found that there were close spatial and temporal interrelationships between blood vessel formation and adipogenesis in obese adipose tissue. Sprouting of new blood vessels from preexisting vasculature was coupled with differentiation of adipocytes, which took place in clusters of cells consisting of differentiating small adipocytes and perivascular cells that had surface phenotypes of CD34+, CD68+ and lectin+. The perivascular cells could be clearly distinguished from macrophages that were scattered in stroma and showed CD34 , CD68+, and lectin . Anti-VEGF treatment inhibited not only angiogenesis but also the formation of adipo-/angiogenic cell clusters, indicating that the coupled adipo-/angiogenesis is essential for differentiation of adipocytes in obesity, and that VEGF is a key mediator of that process. Interestingly, a thiazolinedione, pioglitazone, induced adipogenesis that was decoupled from angiogenesis. In conclusion, our visualization provides novel evidence for the dynamic interactions between differentiating adipocytes, stromal cells and angiogenesis in living adipose tissue in obesity.
Y-A-3. Catheter-based adenovirus-mediated anti-monocyte chemoattractant gene therapy attenuates in-stent neointima formation in non-human primates Kaku Nakano, Kensuke Egashira, Kisho Ohtani, Gang Zhao, Kouta Funakoshi, Yoshiko Ihara, Kenji Sunagawa. Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyushu University Background: We have previously demonstrated great benefit from anti-monocyte chemoattractant protein-1 (MCP1) gene therapy by ‘‘systemic’’ transfer of an N-terminal deletion mutant of human MCP-1 (called 7ND) gene into skeletal muscle for treatment of restenosis and atherosclerosis. However, recent evidence suggests that ’’local’’ gene transfer may be a clinically relevant approach. We therefore tested the hypothesis that catheter-based adenovirus-mediated anti-MCP-1 gene therapy attenuates stent-associated neointima formation. Methods and Results: Bare metal stents were implanted in iliac arteries of cynomolgus monkeys fed a high cholesterol diet. Immediately after the stenting procedure, normal saline or recombinant adenoviral vector containing LacZ or the 7ND gene was administered locally into the stenting site through a REMEDY channel-delivery catheter. Compared to saline infusion or LacZ gene transfer, 7ND gene transfer markedly reduced inflammatory changes at an early stage and attenuated neointima formation after 4 weeks. This strategy also reduced the increased production of pro-inflammatory and growthpromoting factors such platelet-derived growth factor. No systemic adverse effects of 7ND gene transfer were detected. There were no significant differences in serum cholesterol levels among the three groups. Conclusion: These data suggest that catheter-based adenovirus-mediated anti-MCP-1 gene therapy may be a clinically relevant and feasible strategy for treatment of in-stent restenosis. Keywords: Restenosis; Gene therapy; MCP-1 doi:10.1016/j.yjmcc.2006.08.035
Y-A-4. Non-invasive analysis of oxidative stress in myocardial remodeling and failure in mice using in vivo electron spin resonance (ESR) spectroscopy Tomomi Ide, Hideo Utsumi, Hiroyuki Tsutsui, Kenji Sunagawa. Kyushu University Department of Cardiovascular Medicine Oxidative stress plays an important role in the development and progress of myocardial remodeling and failure. We demonstrated the generation of reactive oxygen species (ROS) is enhanced in the tissue homogenates obtained from the failing hearts in vitro by electron spin resonance (ESR) spectroscopy. However, there is no direct evidence of ROS generation in the hearts in vivo. The aim of this study was to assess the time-dependent changes of ROS generation in the hearts after myocardial infarction(MI) using a technique of in
1045
vivo ESR measurement that has been developed for direct and non-invasive analysis of ROS generation in living animals. We created MI in mice by ligating the left coronary artery and performed in vivo ESR analysis at day 2, 7, 14, and 28 after MI. The in vivo generation of ROS was analyzed by the ESR signal decay of methoxy-PROXYL, injected intravenously. Left ventricular (LV) function was assessed by echocardiography. The ESR signal decay measured at the chest of mice was not changed at day 2, but was increased in MI compared to sham-operated mice at day 7 (Sham vs MI; 0.122 T 0.005 vs 0.142 T 0.007/min; p<0.05)and further increased at day 14 (0.12 T 0.01 vs 0.16 T 0.01/min, p<0.01) and 28 (0.12 T 0.01 vs 0.16 T 0.01/min, p<0.01), associated with the progression of LV dilatation and dysfunction. The increased ESR signal decay in MI was completely suppressed by the administration of antioxidant, Tiron. It was further confirmed by the increased ESR signal decay also in the cardiac homogenates obtained from MI mice. The present study provided the direct evidence in the post-MI living mice that ROS were generated within the hearts during the progression of remodeling and failure. Keywords: Heart failure; Free radicals; Remodeling doi:10.1016/j.yjmcc.2006.08.036
Y-A-5. Adipogenesis in obesity is coupled with angiogenesis Satoshi Nishimura 1, Ryozo Nagai 1, Seiryo Sugiura 2. 1 Department of Cardiovascular Medicine, The University of Tokyo. 2 Graduate School of Frontier Sciences. The University of Tokyo Ample evidences suggest that angiogenesis plays a pivotal role in adipogenesis in obesity, but little is known about the detailed mechanism. We have developed a visualization technique based on laser confocal microscopy that allows us to analyze angiogenesis and adipogenesis in intact living adipose tissue three-dimensionally. We found that there were close spatial and temporal interrelationships between blood vessel formation and adipogenesis in obese adipose tissue. Sprouting of new blood vessels from preexisting vasculature was coupled with differentiation of adipocytes, which took place in clusters of cells consisting of differentiating small adipocytes and perivascular cells that had surface phenotypes of CD34+, CD68+ and lectin+. The perivascular cells could be clearly distinguished from macrophages that were scattered in stroma and showed CD34 , CD68+, and lectin . Anti-VEGF treatment inhibited not only angiogenesis but also the formation of adipo-/angiogenic cell clusters, indicating that the coupled adipo-/angiogenesis is essential for differentiation of adipocytes in obesity, and that VEGF is a key mediator of that process. Interestingly, a thiazolinedione, pioglitazone, induced adipogenesis that was decoupled from angiogenesis. In conclusion, our visualization provides novel evidence for the dynamic interactions between differentiating adipocytes, stromal cells and angiogenesis in living adipose tissue in obesity.