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The atheroprotective activity of Ganoderma and its action mechanism in mice
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Abstracts / Atherosclerosis 252 (2016) e197ee235
EAS16-0523, EXPERIMENTAL CADIOVASCULAR MEDICINE II. CONSEQUENCES OF LYSOSOMAL ACID LIPASE DEFICIENCY MACROPHAGES
IN
M.C. Duta-Mare 1, B. Radovic 1, S. Schlager 1, M. Wegscheider 1, D. KolbLenz 2, T. Madl 1, S. Stryeck 1, D. Kratky 1. 1 Medical University of Graz, Institute of Molecular Biology and Biochemistry, Graz, Austria; 2 Medical University of Graz, Center for Medical Research/Institute of Cell BiologyHistology and Embryology, Graz, Austria Objectives: Lysosomal acid lipase (LAL) is highly expressed in mouse peritoneal macrophages (MPM) and hydrolyzes exogenous (via lipoproteins) or endogenous (via autophagy) originated triacylglycerols (TG) and cholesteryl esters (CE). Human LAL deficiency causes severe atherosclerotic lesions and macrophages play a central role in the pathology evolution, becoming hyperplasic and hypertrophic with increasing lysosomal lipid accumulation. We used macrophages from LAL deficient (Lal-/-) mice to investigate the role of the enzyme in lipid metabolism. Methods: We determined the lipid profile of Lal-/- macrophages by GCMS, analyzed cell morphology by electron microscopy, and quantified metabolites by NMR spectroscopy. Using qPCR, flow cytometry, and western blotting analyses we studied the apoptotic phenotype and autophagy in macrophages. Results: Macrophages isolated from Lal-/- mice accumulate lysosomal TG and CE at the expense of phospholipids. Interestingly, a pro-apoptotic profile of Lal-/- macrophages was confirmed both by mRNA and flow cytometry analyses, a feature which is opposite to tissue-resident (e.g. liver) macrophages. In addition, decreased alanine, increased lactate and glycine levels but similar glycerol and glycogen concentrations in Lal-/compared to wildtype MPM contrasted the results observed in Lal-/livers. Conclusions: LAL deficiency causes an intra-lysosomal entrapment of lipids and increases overall inflammation. Together with alterations in plasma lipoprotein profiles, this results in accelerated atherosclerosis. Despite defective lysosomal lipid degradation, NMR spectroscopy confirmed still a functional metabolic machinery in macrophages. Due to non-lipid nutrient-rich culture conditions, which compensate the cellular needs towards utilizing glucose as energy source, Lal-/- macrophages maintain their metabolic homeostasis in controlled culture conditions.
EAS16-0246, EXPERIMENTAL CADIOVASCULAR MEDICINE II. PEROXIREDOXIN 1 HAVE PROTECTIVE ROLE IN THE VASCULAR DISEASE BY REGULATING MACROPHAGES S. Kim, S.J. Jeong, G.T. Oh. Immune and Vascular Cell Network Research Center-National Creative Initiatives, Department of Life Sciences-Ewha Womans University, Seoul, Republic of Korea Objectives: Peroxiredoxin1 (Prx1), an anti-oxidant enzyme, was first identified by a novel stress-inducible protein, MSP23 (macrophage 23-kDa stress protein). However, it has remained unclear what is the major cell type related Prx1 deficient induced atherosclerosis. The aim of this study is elucidating the role of Prx1 in immune responses related to vascular disease. Methods: Immune cell population changes were detected by analysis of baseline Prx1 WT and KO mice using FACS. ApoE-Prx1 DKO BM transplanted ApoE KO mice and ApoE KO BM transplanted ApoE KO mice were used to analyze the plaque size and macrophage cholesterol efflux pathways. Results: Immune cell population had no difference between Prx1 WT and KO mice. However, the expression of Prx1 were remarkably higher in the peritoneal macrophages. Interestingly, ApoE-Prx1 DKO BM transplanted ApoE KO mice showed increased plaque size more than ApoE KO BM transplanted ApoE KO mice. These results suggested that Prx1 may have pivotal role in macrophage lineage cells as atherogenic condition. Then we checked the macrophage in atherosclerotic plaque. As we expected, macrophage infiltration and foam cell formation were significantly increased under the Prx1 deficient mice.
Conclusions: Prx1 deficiency is associated with increase the foam cell formation and decrease the macrophage cholesterol efflux. In conclusion, our data suggest that Prx1 in macrophages play protective role in atherosclerosis.
EAS16-0517, EXPERIMENTAL CADIOVASCULAR MEDICINE II. THE ATHEROPROTECTIVE ACTIVITY OF GANODERMA AND ITS ACTION MECHANISM IN MICE F.E. Mo 1, 2, P.L. Hsu 1, 2, Y.C. Lin 1. 1 National Cheng Kung University, Department of Cell Biology and Anatomy, Tainan, Taiwan; 2 National Cheng Kung University, Institute of Basic Medical Sciences, Tainan, Taiwan Objectives: Disturbed flow and low wall shear stress induce atherosclerosis through regulating the mechanosensors on endothelial cells and inducing a series of atherogenic gene expression. Ganoderma, or lingzhi in Chinese, is a traditional Chinese medicine with a variety of medicinal activities. We previously demonstrated that Ganoderma triterpenoids prevent myocardial injury in a stressed heart by alleviating oxidative stress. Because oxidative stress is also critically involved in atherogenesis, we intented to assess the atheroprotective potential of Ganoderma triterpenoids. Methods: We used a mouse model of neointima formation induced by carotid artery ligation. Neointima formation is a feature of atherosclerosis. Blood recirculation and flow turbulence was created in the ligated carotid artery. To test the effect of triterpenoids, mice were subcutaneously injected with purified triterpenoids (300 mg/kg/day) immediately after ligation. Carotid arteries were collected 2 weeks after ligation for histological analysis. Results: Neointima formation-induced by carotid artery ligation was inhibited in mice treated with triterpenoids. The oxidative injury and proliferation of endothelial cells were reduced by triterpenoids. The proliferation of vascular smooth muscle cells in intima was reduced in triterpenoids-treated mice as well. The levels of endothelial MCP-1 were downregulated after treating triterpenoids for 3 days. Conclusions: Our results demonstrated that Ganoderma triterpenoids prevent neointima formation through their antioxidant activities to alleviate the oxidative stress-induced by disturbed flow. We are currently investing the molecular mechanism underlying the atheroprotective activity of Ganoderma triterpenoids.
EAS16-0793, EXPERIMENTAL CADIOVASCULAR MEDICINE II. THE APOE*3LEIDEN.GK+/- MOUSE AS NOVEL TRANSLATIONAL MODEL FOR DYSLIPIDEMIA, TYPE 2 DIABETES AND MACROVASCULAR COMPLICATIONS asson 2, E. M. Pouwer 1, S. Heinonen 2, M. Behrendt 2, A.C. Andre € nssonPieterman 1, A. van den Hoek 1, W. Jukema 3, B. Leighton 2, A.C. Jo Rylander 2, H. Princen 1. 1 TNO, Metabolic Health Research, Leiden, Netherlands; 2 AstraZeneca R&D, Cardiovascular & Metabolic Disease€lndal, Sweden; 3 Leiden University Medical Centre, Innovative Medicines, Mo Cardiology, Leiden, Netherlands Objectives: There is a lack of predictive preclinical animal models combining atherosclerosis and diabetes. The objective of this pilot study was to evaluate the response of APOE*3-Leiden. Glucokinase+/- (E3L.GK+/- ) mice to fat and cholesterol containing diets with respect to changes in plasma lipids and glucose levels, and development of diabetic macrovascular complications. E3L mice are a well-established model for dietinduced hyperlipidemia and atherosclerosis, whereas GK+/- mice are a translational disease model for glucose control in type 2 diabetes. Methods: 10-23 week old female GK+/- , E3L and E3L.GK+/- mice (n¼6-10/ sex/genotype) were fed a Western diet+0.15% cholesterol (8 weeks), enriched with glucose in the last 2 weeks, followed by a 29-week period on a Western diet+1% cholesterol. Plasma parameters were analyzed and histopathological analysis for atherosclerosis, liver steatosis, kidney injury and pancreatic changes was performed.
Abstracts / Atherosclerosis 252 (2016) e197ee235
EAS16-0523, EXPERIMENTAL CADIOVASCULAR MEDICINE II. CONSEQUENCES OF LYSOSOMAL ACID LIPASE DEFICIENCY MACROPHAGES
IN
M.C. Duta-Mare 1, B. Radovic 1, S. Schlager 1, M. Wegscheider 1, D. KolbLenz 2, T. Madl 1, S. Stryeck 1, D. Kratky 1. 1 Medical University of Graz, Institute of Molecular Biology and Biochemistry, Graz, Austria; 2 Medical University of Graz, Center for Medical Research/Institute of Cell BiologyHistology and Embryology, Graz, Austria Objectives: Lysosomal acid lipase (LAL) is highly expressed in mouse peritoneal macrophages (MPM) and hydrolyzes exogenous (via lipoproteins) or endogenous (via autophagy) originated triacylglycerols (TG) and cholesteryl esters (CE). Human LAL deficiency causes severe atherosclerotic lesions and macrophages play a central role in the pathology evolution, becoming hyperplasic and hypertrophic with increasing lysosomal lipid accumulation. We used macrophages from LAL deficient (Lal-/-) mice to investigate the role of the enzyme in lipid metabolism. Methods: We determined the lipid profile of Lal-/- macrophages by GCMS, analyzed cell morphology by electron microscopy, and quantified metabolites by NMR spectroscopy. Using qPCR, flow cytometry, and western blotting analyses we studied the apoptotic phenotype and autophagy in macrophages. Results: Macrophages isolated from Lal-/- mice accumulate lysosomal TG and CE at the expense of phospholipids. Interestingly, a pro-apoptotic profile of Lal-/- macrophages was confirmed both by mRNA and flow cytometry analyses, a feature which is opposite to tissue-resident (e.g. liver) macrophages. In addition, decreased alanine, increased lactate and glycine levels but similar glycerol and glycogen concentrations in Lal-/compared to wildtype MPM contrasted the results observed in Lal-/livers. Conclusions: LAL deficiency causes an intra-lysosomal entrapment of lipids and increases overall inflammation. Together with alterations in plasma lipoprotein profiles, this results in accelerated atherosclerosis. Despite defective lysosomal lipid degradation, NMR spectroscopy confirmed still a functional metabolic machinery in macrophages. Due to non-lipid nutrient-rich culture conditions, which compensate the cellular needs towards utilizing glucose as energy source, Lal-/- macrophages maintain their metabolic homeostasis in controlled culture conditions.
EAS16-0246, EXPERIMENTAL CADIOVASCULAR MEDICINE II. PEROXIREDOXIN 1 HAVE PROTECTIVE ROLE IN THE VASCULAR DISEASE BY REGULATING MACROPHAGES S. Kim, S.J. Jeong, G.T. Oh. Immune and Vascular Cell Network Research Center-National Creative Initiatives, Department of Life Sciences-Ewha Womans University, Seoul, Republic of Korea Objectives: Peroxiredoxin1 (Prx1), an anti-oxidant enzyme, was first identified by a novel stress-inducible protein, MSP23 (macrophage 23-kDa stress protein). However, it has remained unclear what is the major cell type related Prx1 deficient induced atherosclerosis. The aim of this study is elucidating the role of Prx1 in immune responses related to vascular disease. Methods: Immune cell population changes were detected by analysis of baseline Prx1 WT and KO mice using FACS. ApoE-Prx1 DKO BM transplanted ApoE KO mice and ApoE KO BM transplanted ApoE KO mice were used to analyze the plaque size and macrophage cholesterol efflux pathways. Results: Immune cell population had no difference between Prx1 WT and KO mice. However, the expression of Prx1 were remarkably higher in the peritoneal macrophages. Interestingly, ApoE-Prx1 DKO BM transplanted ApoE KO mice showed increased plaque size more than ApoE KO BM transplanted ApoE KO mice. These results suggested that Prx1 may have pivotal role in macrophage lineage cells as atherogenic condition. Then we checked the macrophage in atherosclerotic plaque. As we expected, macrophage infiltration and foam cell formation were significantly increased under the Prx1 deficient mice.
Conclusions: Prx1 deficiency is associated with increase the foam cell formation and decrease the macrophage cholesterol efflux. In conclusion, our data suggest that Prx1 in macrophages play protective role in atherosclerosis.
EAS16-0517, EXPERIMENTAL CADIOVASCULAR MEDICINE II. THE ATHEROPROTECTIVE ACTIVITY OF GANODERMA AND ITS ACTION MECHANISM IN MICE F.E. Mo 1, 2, P.L. Hsu 1, 2, Y.C. Lin 1. 1 National Cheng Kung University, Department of Cell Biology and Anatomy, Tainan, Taiwan; 2 National Cheng Kung University, Institute of Basic Medical Sciences, Tainan, Taiwan Objectives: Disturbed flow and low wall shear stress induce atherosclerosis through regulating the mechanosensors on endothelial cells and inducing a series of atherogenic gene expression. Ganoderma, or lingzhi in Chinese, is a traditional Chinese medicine with a variety of medicinal activities. We previously demonstrated that Ganoderma triterpenoids prevent myocardial injury in a stressed heart by alleviating oxidative stress. Because oxidative stress is also critically involved in atherogenesis, we intented to assess the atheroprotective potential of Ganoderma triterpenoids. Methods: We used a mouse model of neointima formation induced by carotid artery ligation. Neointima formation is a feature of atherosclerosis. Blood recirculation and flow turbulence was created in the ligated carotid artery. To test the effect of triterpenoids, mice were subcutaneously injected with purified triterpenoids (300 mg/kg/day) immediately after ligation. Carotid arteries were collected 2 weeks after ligation for histological analysis. Results: Neointima formation-induced by carotid artery ligation was inhibited in mice treated with triterpenoids. The oxidative injury and proliferation of endothelial cells were reduced by triterpenoids. The proliferation of vascular smooth muscle cells in intima was reduced in triterpenoids-treated mice as well. The levels of endothelial MCP-1 were downregulated after treating triterpenoids for 3 days. Conclusions: Our results demonstrated that Ganoderma triterpenoids prevent neointima formation through their antioxidant activities to alleviate the oxidative stress-induced by disturbed flow. We are currently investing the molecular mechanism underlying the atheroprotective activity of Ganoderma triterpenoids.
EAS16-0793, EXPERIMENTAL CADIOVASCULAR MEDICINE II. THE APOE*3LEIDEN.GK+/- MOUSE AS NOVEL TRANSLATIONAL MODEL FOR DYSLIPIDEMIA, TYPE 2 DIABETES AND MACROVASCULAR COMPLICATIONS asson 2, E. M. Pouwer 1, S. Heinonen 2, M. Behrendt 2, A.C. Andre € nssonPieterman 1, A. van den Hoek 1, W. Jukema 3, B. Leighton 2, A.C. Jo Rylander 2, H. Princen 1. 1 TNO, Metabolic Health Research, Leiden, Netherlands; 2 AstraZeneca R&D, Cardiovascular & Metabolic Disease€lndal, Sweden; 3 Leiden University Medical Centre, Innovative Medicines, Mo Cardiology, Leiden, Netherlands Objectives: There is a lack of predictive preclinical animal models combining atherosclerosis and diabetes. The objective of this pilot study was to evaluate the response of APOE*3-Leiden. Glucokinase+/- (E3L.GK+/- ) mice to fat and cholesterol containing diets with respect to changes in plasma lipids and glucose levels, and development of diabetic macrovascular complications. E3L mice are a well-established model for dietinduced hyperlipidemia and atherosclerosis, whereas GK+/- mice are a translational disease model for glucose control in type 2 diabetes. Methods: 10-23 week old female GK+/- , E3L and E3L.GK+/- mice (n¼6-10/ sex/genotype) were fed a Western diet+0.15% cholesterol (8 weeks), enriched with glucose in the last 2 weeks, followed by a 29-week period on a Western diet+1% cholesterol. Plasma parameters were analyzed and histopathological analysis for atherosclerosis, liver steatosis, kidney injury and pancreatic changes was performed.