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PO11-296 EFFECTS OF HEPARIN ON THE PRODUCTION OF HOMOCYSTEINE-INDUCED EXTRACELLULAR MATRIX METALLOPROTEINASE-2 IN CULTURED RAT VSMC
Poster Sessions PO11 Proteinases and plaque remodelling
90
HC+FeO+Zn, HC+FeO+Cu+Zn. For this purpose, we evaluated the effects of the various dietary treatments on serum levels of malondialdehyde (MDA), as an index of lipid peroxidation, antioxidant enzyme superoxide dismutase (SOD) activity, and plasma level of vitamin E. Furthermore, Sudan IV staining method was used for the assessment of the development of atherosclerotic lesions on the lumen of thoracic aorta. The lowest activity of SOD activity was shown in rabbits given HC+FeO+Cu. Serum MDA level of rabbits treated with HC+FeO+Cu regimen was found to be significantly higher that that of rabbits supplemented with Cu and Zn, singly and together (P<0.05). None of supplementary regimens affect plasma level of standardized vitamin E. As expected, a modes increase in aortic sudanophilia was shown in rabbits given HC+FeO (P<0.001 as compared with other groups except those given HC without mineral supplementation and HC+FeO+Cu). In conclusion, our results are consistent with Fe as an inducer of atherogenesis and that single and combined supplementation of Cu and Zn in FeO rabbits can attenuate this effect.
PO11 PROTEINASES AND PLAQUE REMODELLING PO11-294
A NEW MOUSE MODEL OF DIABETIC ATHEROSCLEROSIS REVEALS INCREASED LESION COMPLEXITY AND CALCIFICATION WITHOUT WORSENING OF HYPERCHOLESTEROLEMIA
S. Koota 1 , P. Leppänen 1 , I. Kholová 1 , F. Bosch 2,3 , M. Laakso 4 , S. Ylä-Herttuala 1 . 1 Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland; 2 Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 3 Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 4 Department of Medicine, University of Kuopio, Kuopio, Finland Background and Aims: At the moment there is no available mouse model, where the induction of type 2 diabetes on atherosclerotic background could be achieved without unintentional changes in lipid levels. Our aim was to create and characterize a new mouse model and address the question if the driving force behind increased atherosclerosis in type 2 diabetes is hyperglycemia or insulin resistance. Methods: We crossbred two genetically modified mouse strains to achieve a model expressing both atherosclerotic and diabetic features. For atherosclerotic background we used a double knockout mouse (DKO) deficient of low-density lipoprotein receptor and apolipoprotein B48. Diabetic background was acquired from mice overexpressing insulin-like growth factor-II (IGF-II) in pancreatic beta cells. Thorough phenotypic characterization was carried out with 6- and 15-month-old mice on both normal and Western diet. Results: Results indicated that compared to atherosclerotic DKO controls, these IGF-II transgenic double knockout (IGF-II/DKO) mice demonstrated insulin resistance, hyperinsulinemia and mild hyperglycemia and showed diabetic morphological changes. No significant differences were seen in the actual atherosclerotic plaque burden. However, lesions of old IGF-II/DKO mice on Western diet displayed increased calcification and complexity, although their lipid levels were similar to those of the DKO controls. No significant correlation was found between glucose levels and plaque burden at any time. Conclusions: Type 2 diabetic factors other than hyperglycemia induce increased calcification and lesion complexity without lipid changes in a new mouse model of diabetic macroangiopathy. Studies on the exact mechanisms of the phenomenon are currently under way.
PO11-295
ENDOTHELIAL APOPTOSIS INDUCED THROMBOTIC PLAQUE EROSION: DIRECT EVIDENCE IN AN ATHEROSCLEROTIC RABBIT MODEL
F. Xu 1 , Y. Chen 1 , Y.N. Sun 2 , Y. Sun 1 , R. Li 1 , C. Liu 3 , Y. Zhang 3 . Department, Qilu Hospital of Shandong University, Jinan, China; 2 Cardiology Department, Shandong Province Hospital, Jinan, China; 3 Cardiology Department, Qilu Hospital of Shandong University, Jinan, China 1 Emergency
Aim: To investigate the role for endothelial apoptosis in thrombotic plaque erosion. Methods: Atherosclerotic plaques were established in 33 rabbits by post-balloon-injury high-cholesterol feeding for 3 months. A plaque-rich segment of abdominal aorta of every animal were randomly incubated with staurosporine (n=24) or saline (n=9) for 20 min. Three days later, abdominal aorta serum lipid and hs-CRP were detected, and thrombotic score was performed according to angiography (angiographic obvious thrombosis was defined as score 3–4). Serial sections of the incubated segments were conducted to count histological thrombosis. Endothelial integrity and apoptosis scores were established according to CD31 and TUNEL staining. Results: In the staurosporine-triggered plaques, fibrous caps were rich in positive α-actin staining, and caps rupture and lipid overflow under thrombi were not observed in serial sections stained with H&E, α-actin and Oil Red O, and endothelium was disrupted, which were similar to human plaque erosion. Compared to the controls, lipid and hs-CRP were similar in staurosporine-triggered rabbits, while thrombotic scores (0.11±0.33 vs. 1.88±1.33), angiographic obvious thrombosis (0% vs. 37.5%), histological thrombosis (11.1% vs. 75.0%) and apoptosis scores (0.22±0.44 vs. 1.92±1.02) were all remarkably higher and endothelial integrity scores (2.89±0.33 vs. 1.21±0.98) were lower (P<0.01, respectively). Endothelial integrity was reversely related with apoptosis (r=-0.91, P<0.01) and thrombosis was positively related with apoptosis (r=0.88, P<0.01). Moreover, logistic analysis demonstrated that endothelial apoptosis was an independent risk factor for histological thrombosis and angiographic obvious thrombosis (P<0.01, respectively). Conclusion: These findings suggest that endothelial apoptosis plays a critical role in thrombotic plaque erosion. PO11-296
EFFECTS OF HEPARIN ON THE PRODUCTION OF HOMOCYSTEINE-INDUCED EXTRACELLULAR MATRIX METALLOPROTEINASE-2 IN CULTURED RAT VSMC
P. Wang 1 , H.Y. Guo 1 , J.D. Lee 2 . 1 Department of Cardiology, Shaoxing People’s Hospital, The First Affiliated Hospital of Shaoxing University, Shaoxing, China; 2 First Department of Internal Medicine, Fukui Medical University, Fukui, Japan Objective: To study the effects of heparin on the production of homocysteine-induced extracellular matrix metalloproteinase-2 in cultured rats vascular smooth muscle cells. Methods: We examined the effects of different homocysteine levels (0-1000 mol/l) on matrix metalloproteinase-2 (MMP-2) production, and the effects of different heparin concentrations (0-100 g/ml) on homocysteineinduced MMP-2 in cultured rat vascular smooth muscle cells (VSMCs) using gelatin zymography and western blotting. We further compared the changes of MMP-2 under various treatments for 24h, 48h and 72h. Results: Homocysteine (50-1000 mol/l) increased the production of MMP-2 significantly in a dose-dependent manner. Increased production of MMP-2 induced by homocysteine was reduced by extracellularly added heparin in a dose-dependent manner. Production of MMP-2 under various treatments for 72h increased more than 24h and 48h. Conclusions: Extracellularly added heparin decreased homocysteineinduced MMP-2 secretion. Data suggested a mechanism how hyperhomocysteinemia involved in the pathogenesis of coronary artery disease, and a beneficial effect of heparin on these conditions.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
90
HC+FeO+Zn, HC+FeO+Cu+Zn. For this purpose, we evaluated the effects of the various dietary treatments on serum levels of malondialdehyde (MDA), as an index of lipid peroxidation, antioxidant enzyme superoxide dismutase (SOD) activity, and plasma level of vitamin E. Furthermore, Sudan IV staining method was used for the assessment of the development of atherosclerotic lesions on the lumen of thoracic aorta. The lowest activity of SOD activity was shown in rabbits given HC+FeO+Cu. Serum MDA level of rabbits treated with HC+FeO+Cu regimen was found to be significantly higher that that of rabbits supplemented with Cu and Zn, singly and together (P<0.05). None of supplementary regimens affect plasma level of standardized vitamin E. As expected, a modes increase in aortic sudanophilia was shown in rabbits given HC+FeO (P<0.001 as compared with other groups except those given HC without mineral supplementation and HC+FeO+Cu). In conclusion, our results are consistent with Fe as an inducer of atherogenesis and that single and combined supplementation of Cu and Zn in FeO rabbits can attenuate this effect.
PO11 PROTEINASES AND PLAQUE REMODELLING PO11-294
A NEW MOUSE MODEL OF DIABETIC ATHEROSCLEROSIS REVEALS INCREASED LESION COMPLEXITY AND CALCIFICATION WITHOUT WORSENING OF HYPERCHOLESTEROLEMIA
S. Koota 1 , P. Leppänen 1 , I. Kholová 1 , F. Bosch 2,3 , M. Laakso 4 , S. Ylä-Herttuala 1 . 1 Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland; 2 Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 3 Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 4 Department of Medicine, University of Kuopio, Kuopio, Finland Background and Aims: At the moment there is no available mouse model, where the induction of type 2 diabetes on atherosclerotic background could be achieved without unintentional changes in lipid levels. Our aim was to create and characterize a new mouse model and address the question if the driving force behind increased atherosclerosis in type 2 diabetes is hyperglycemia or insulin resistance. Methods: We crossbred two genetically modified mouse strains to achieve a model expressing both atherosclerotic and diabetic features. For atherosclerotic background we used a double knockout mouse (DKO) deficient of low-density lipoprotein receptor and apolipoprotein B48. Diabetic background was acquired from mice overexpressing insulin-like growth factor-II (IGF-II) in pancreatic beta cells. Thorough phenotypic characterization was carried out with 6- and 15-month-old mice on both normal and Western diet. Results: Results indicated that compared to atherosclerotic DKO controls, these IGF-II transgenic double knockout (IGF-II/DKO) mice demonstrated insulin resistance, hyperinsulinemia and mild hyperglycemia and showed diabetic morphological changes. No significant differences were seen in the actual atherosclerotic plaque burden. However, lesions of old IGF-II/DKO mice on Western diet displayed increased calcification and complexity, although their lipid levels were similar to those of the DKO controls. No significant correlation was found between glucose levels and plaque burden at any time. Conclusions: Type 2 diabetic factors other than hyperglycemia induce increased calcification and lesion complexity without lipid changes in a new mouse model of diabetic macroangiopathy. Studies on the exact mechanisms of the phenomenon are currently under way.
PO11-295
ENDOTHELIAL APOPTOSIS INDUCED THROMBOTIC PLAQUE EROSION: DIRECT EVIDENCE IN AN ATHEROSCLEROTIC RABBIT MODEL
F. Xu 1 , Y. Chen 1 , Y.N. Sun 2 , Y. Sun 1 , R. Li 1 , C. Liu 3 , Y. Zhang 3 . Department, Qilu Hospital of Shandong University, Jinan, China; 2 Cardiology Department, Shandong Province Hospital, Jinan, China; 3 Cardiology Department, Qilu Hospital of Shandong University, Jinan, China 1 Emergency
Aim: To investigate the role for endothelial apoptosis in thrombotic plaque erosion. Methods: Atherosclerotic plaques were established in 33 rabbits by post-balloon-injury high-cholesterol feeding for 3 months. A plaque-rich segment of abdominal aorta of every animal were randomly incubated with staurosporine (n=24) or saline (n=9) for 20 min. Three days later, abdominal aorta serum lipid and hs-CRP were detected, and thrombotic score was performed according to angiography (angiographic obvious thrombosis was defined as score 3–4). Serial sections of the incubated segments were conducted to count histological thrombosis. Endothelial integrity and apoptosis scores were established according to CD31 and TUNEL staining. Results: In the staurosporine-triggered plaques, fibrous caps were rich in positive α-actin staining, and caps rupture and lipid overflow under thrombi were not observed in serial sections stained with H&E, α-actin and Oil Red O, and endothelium was disrupted, which were similar to human plaque erosion. Compared to the controls, lipid and hs-CRP were similar in staurosporine-triggered rabbits, while thrombotic scores (0.11±0.33 vs. 1.88±1.33), angiographic obvious thrombosis (0% vs. 37.5%), histological thrombosis (11.1% vs. 75.0%) and apoptosis scores (0.22±0.44 vs. 1.92±1.02) were all remarkably higher and endothelial integrity scores (2.89±0.33 vs. 1.21±0.98) were lower (P<0.01, respectively). Endothelial integrity was reversely related with apoptosis (r=-0.91, P<0.01) and thrombosis was positively related with apoptosis (r=0.88, P<0.01). Moreover, logistic analysis demonstrated that endothelial apoptosis was an independent risk factor for histological thrombosis and angiographic obvious thrombosis (P<0.01, respectively). Conclusion: These findings suggest that endothelial apoptosis plays a critical role in thrombotic plaque erosion. PO11-296
EFFECTS OF HEPARIN ON THE PRODUCTION OF HOMOCYSTEINE-INDUCED EXTRACELLULAR MATRIX METALLOPROTEINASE-2 IN CULTURED RAT VSMC
P. Wang 1 , H.Y. Guo 1 , J.D. Lee 2 . 1 Department of Cardiology, Shaoxing People’s Hospital, The First Affiliated Hospital of Shaoxing University, Shaoxing, China; 2 First Department of Internal Medicine, Fukui Medical University, Fukui, Japan Objective: To study the effects of heparin on the production of homocysteine-induced extracellular matrix metalloproteinase-2 in cultured rats vascular smooth muscle cells. Methods: We examined the effects of different homocysteine levels (0-1000 mol/l) on matrix metalloproteinase-2 (MMP-2) production, and the effects of different heparin concentrations (0-100 g/ml) on homocysteineinduced MMP-2 in cultured rat vascular smooth muscle cells (VSMCs) using gelatin zymography and western blotting. We further compared the changes of MMP-2 under various treatments for 24h, 48h and 72h. Results: Homocysteine (50-1000 mol/l) increased the production of MMP-2 significantly in a dose-dependent manner. Increased production of MMP-2 induced by homocysteine was reduced by extracellularly added heparin in a dose-dependent manner. Production of MMP-2 under various treatments for 72h increased more than 24h and 48h. Conclusions: Extracellularly added heparin decreased homocysteineinduced MMP-2 secretion. Data suggested a mechanism how hyperhomocysteinemia involved in the pathogenesis of coronary artery disease, and a beneficial effect of heparin on these conditions.
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland