- Email: [email protected]
4P-1164 Effect of pravastatin on the aortic gene expression of ApoE-deficient mice fed with a hypercholesterolemic diet
330 4P-1160
Thursday October 2, 2003: Poster Session Molecules and clinical markers for atherosclerosis A mass spectrum analysis method for detection of C-reactive protein
H. Hsu 1 , Y.-R. Li 1 , Y.-C. Wang 1 , H.-K. Liao 2 , Y.-J. Chen 2 . 1 National Yang-Ming University, Institute of Biotechnology in Medicine, Taipei; 2 Institute of Chemistry, Academia Sinica, Taipei, Taiwan The circulating level of C-reactive protein (CRP) is considered as an important and independent risk factor for vascular diseases such as inflammation of vascular cells, development of atherosclerosis, coronary heart disease, etc. Considering the molecular structure, intact native CRP is a cyclic pentameric protein (pCRP) that consists of five non-covalently bound homo-monomers. Some recent findings report that pCRP could dissociate into modified or monomeric CRP (mCRP), but pCRP and mCRP possess quite different properties and functions. Using electrospray mass spectrometry (ESI-MS), we analyzed purified CRP in aqueous buffer solution at appropriate pH and calcium ion (Ca2+ ). Initially, we established methods to quantitate and identify the distinct m/z distribution (corresponding to their charge states) of the pCRP and mCRP. In addition, at certain concentration of Ca2+ , which is crucial to maintain the pentameric protein structure, we succeed to detect the non-covalently bound pCRP. After Beysian reconstruction process, distinct molecular weight of pCRP and mCRP can be identified from ESI-MS. This process would serve as a sensitive and accurate assay for analysis of various CRP species, even in the presence of mixture. Currently, modification of this method is under analyzing the different forms of CRP in human plasma, which will serve as a novel diagnostic tool for detection of inflammatory abnormality. On the other hand, a new membrane method by a specific protein affinity-directed mass spectrometry for examining the existence of low-abundant protein is also under development. 4P-1161
Elevation of bilirubin oxidative metabolites, biopyrrin, is a novel marker in the patients with acute myocardial infarction
H. Kunii 1 , K. Ishikawa 2 , T. Yamaguchi 3 , N. Komatsu 1 , H. Matsumoto 2 , M. Oikawa 1 , O. Yamaguchi 2 , Y. Shiratori 1 , H. Yamao 1 , S. Namiuchi 1 , M. Sugi 1 , M. Yui 1 , T. Ichihara 1 , Y. Maruyama 2 . 1 Iwaki Kyoritsu General Hospital; 2 Fukushima Medical University; 3 Tokyo Medical and Dental University, Japan Background: Bilirubin has been suggested to exhibit anti-oxidant properties. After the reaction with oxidants, bilirubin is oxidatively degraded into biopyrrins. We examined whether the elevation of biopyrrins is associated with the clinical features of the patients with acute myocardial infarction (AMI). Methods: Sixty consecutive patients hospitalized for AMI were analyzed. Urinary biopyrrin were measured by ELISA at the 1, 2, 3, 7 and 14 hospital days, adjusted by urinary creatinine, and referred as u(BPY/CRE). Results: The u(BPY/CRE) (Unit/gram-creatinine) were highest on the 3rd hospital day (3.6 ± 2.0) and decreased by the 14th hospital day (1.3 ± 1.2) (p<0.0001). The maximum u(BPY/CRE) was higher in death cases (24.1 ± 29.1) than that of survivors (3.9 ± 5.8) (p<0.05). Higher u(BPY/CRE) elevation was observed in the patients with large infarction (14.3 ± 7.1) than in those with small infarction (3.9 ± 0.4) (p<0.05). In addition, the patients with lower cardiac index exhibited higher u(BPY/CRE) levels (15.3 ± 23.6) than in those with higher cardiac index (3.8 ± 1.7) (p<0.05) although significant difference was not observed between two groups on the 14th hospital day. Elevation of u(BPY/CRE) tended to correlate with the elevation of brain-type natriuretic peptide (BNP) (p=0.116). Patients with higher BNP had higher urinary biopyrrin levels. Conclusion: Significant u(BPY/CRE) elevation was observed in the patients with AMI. The degree of this elevation was associated with infarction size, cardiac dysfunction and acute phase mortality after AMI. Monitoring the levels of u(BPY/CRE) can be a novel clinical marker in the patients with AMI. 4P-1162
Clinical evaluation of oxidative stress in cardiovascular disease
K. Nonaka, S. Seto, K. Yano, T. Nakayama, I. Sekine, T. Kondo. Nagasaki University School of Medicine, Japan Aim: Accelerated atherosclerosis plays an important role in the development of cardiovascular disease (CVD). There are increasing evidences concerning the linkage of oxidative damage with accelerated atherosclerosis, however, a few reports on the clinical studies relating CVD to the oxidative damage. Levels of 4-hydroxy-2-nonenal (4-HNE), an end product of lipid peroxidation, as well as carbonylated proteins have been reported as markers of oxidative
stress. Here we studied the levels of 4-HNE and carbonylated proteins in arterial wall from patients with CVD and investigated clinical importance of the intensity and the quality of oxidative stress. Materials and Methods: Small pieces of the left thoratic internal artery from patients with CVD were separated and frozen in liquid nitrogen before experiments. The clinical history and the medical records of all patients were carefully reviewed. The tissue samples were stained with HE and anti-4-HNE antibody. The levels of carbonylated proteins were estimated on immuno blot. Results and Discussion: HE stain revealed atherogenic changes in arteries of most patients. 4-HNE was found at fibromuscular cell in swollen internal layer and smooth muscle cells in midthelium. The levels of carbonylated proteins from the tissue extracts were related to those of 4-HNE, however, not to the severity of atherosclerosis. These levels of oxidative damage were rather related to clinical intensity of CVD patients. These data suggest that oxidative stress is a factor of the development of CVD and estimation of oxidative stress is important in evaluation of the degree of CVD. 4P-1163
Two novel mutations in the plasma platelet-activating factor acetylhydrolase (PAF-AH) gene identified in Japanese subjects
M. Ishihara 1 , M. Nagano 1 , T. Iwasaki 1 , T. Kujiraoka 1 , J. Ishii 2 , M. Tsuji 3 , M. Ito 1 , T. Kumagai 1 , T. Egashira 1 , H. Hattori 1 . 1 Department of Advanced Technology and Development, BML Inc.; 2 Hokkaido Hospital for Social Health Insurance; 3 Institute of Medical Science, Health Sciences University of Hokkaido, Japan Objective: The V279F mutation of PAF-AH is common in Japanese subjects. We studied PAF-AH mass and the V279F mutation in subjects with hyperlipidemia and diabetes. Among subjects with lower PAF-AH mass, we have identified two novel mutations of the PAF-AH gene. Methods: Plasma PAF-AH mass concentration was determined in 249 outpatients with hyperlipidemia and diabetes by a sandwich ELISA using monoclonal antibodies. The detection of the V279F mutation was carried out by the Invader® assay. The nucleotide sequence was determined by direct sequencing. Results: Plasma PAF-AH mass concentration in hyperlipidemia and diabetes was 1.67 ± 0.58 µg/mL for the wild type (n=169), 0.77 ± 0.30 µg/mL for heterozygotes with V279F (n=73), and undetectable mass for homozygotes with V279F (n=7). We further screened the PAF-AH gene in subjects with lower PAF-AH mass and identified two novel mutations. In a subject who was heterozygotes for the V279F mutation had undetectable PAF-AH mass, there was a substitution of T to A at 949 nt in exon 10, resulting amino acid change at codon 317 of Asn for Ile. In a subject without the V279F mutation had lower PAF-AH mass similar to those heterozygous for the V279F mutation, there was the insertion of A at 187 nt in exon 3, creating the termination at codon 63. By transient expression of mutant Asn317 PAF-AH gene in COS-7 cells, PAF-AH mass and activity in the culture medium were undetectable. Conclusions: We have identified two novel mutations, I317N and Y63X, in the PAF-AH gene in Japanese subjects with lower Plasma PAF-AH mass concentration. The expression analysis suggested that mutant PAF-AH protein might not be secreted to the circulation. 4P-1164
Effect of pravastatin on the aortic gene expression of ApoE-deficient mice fed with a hypercholesterolemic diet
G.-Y. Shi, S.-L. Liu, Y.-W. Chen, C.-S. Shi, M.-H. Wu, H.-L. Wu. National Cheng Kung University, Taiwan 3-Hydroxy-3-methyl-gutaryl coenzyme (HMG-CoA) reductase inhibitors or statins, constituting the most powerful class of lipid-lowering drugs, have been applied in clinics to reduce the risk of acute coronary events. However, the beneficial effects of statins cannot be fully explained by their lipid-lowing potential. In this study, we intend to identify genes in aortas that altered in statin treatment. To establish the effects of statin on the progression of atherosclerosis in hypercholesterolemic mice and to test the hypothesis that treatment with statin may result in alteration of gene expression, the apoEdeficient mice were fed with diet supplemented with 0.15% cholesterol and treated with pravastatin. The related genes whose expressions were affected by pravastatin were identified using oilgonucleotide microarray technology. ApoE-deficient mice were fed with a high cholesterol chow diet started at 8 weeks of age for a total of 16 weeks. For 6 weeks before euthanasia, the test groups were treated with pravastatin (80 mg/kg) dissolved in water by daily oral inoculation. The total cholesterol, LDL and lesion/total aortic size between the treated with pravastatin and control groups were significantly
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Thursday October 2, 2003: Poster Session Molecules and clinical markers for atherosclerosis different. Microarray analysis of the expression of 20281 murine genes in the aortas between the two groups by Agilent gene chips suggested that 93 genes were significantly regulated in apoE-deficient mice. Among these genes, 44 were up-regulated and 49 were down-regulated. 4P-1165
Increased levels of pregnancy-associated plasma protein-A in patients with hypercholesterolemia and diabetes: the effect of lipid lowering treatment
T. Stulc 1 , J. Skrha 1 , I. Malbohan 2 , L. Fialova 2 , J. Malik 1 , R. Ceska 1 . 1 3rd Department of Internal Medicine, 2 Institute of Clinical Chemistry, 1st School of Medicine, Charles University, Czech Republic Objective: Pregnancy-associated plasma protein A (PAPP-A) is a metalloproteinase produced by unstable plaques; serum PAPP-A is increased in acute coronary syndromes. The relationship between PAPP-A and coronary risk factors has not been studied to date. We have therefore investigated whether PAPP-A levels are increased in hypercholesterolemia and diabetes and whether they are affected by statin treatment. Methods: We examined 32 subjects with severe hypercholesterolemia (HLP group), 20 type 2 diabetic patients with mixed hyperlipidemia (DM group) and 26 controls. All subjects were free of vascular disease. Patients were examined before and after 12 weeks of treatment with atorvastatin 20mg/d (HLP group) or simvastatin 20mg/d (DM group). Results: In both patient groups, baseline PAPP-A levels were significantly higher than in controls (Table 1; p<0.01); moreover, there was a trend towards higher values in DM group. However, PAPP-A levels were not influenced by either treatment. Controls PAPP/A [mU/l] TC [mmol/l] TG [mmol/l]
6.32±2.59 4.99±0.60 1.11±0.62
HLP
DM
before
after
before
after
8.02±1.86 8.59±1.60 1.67±0.63
7.67±1.89 5.87±1.12 1.33±0.37
8.98±2.53 6.62±0.83 3.61±2.09
8.35±2.50 5.19±0.75 3.25±1.98
Conclusion: We have demonstrated for the first time that PAPP-A levels are elevated in hyperlipidemic subjects without clinical signs of atherosclerosis. PAPP-A may therefore not only reflect the plaque instability, but also the total atheroclerotic burden in asymptomatic subjects with hyperlipidemia. 4P-1166
Structure and expression of apoE, apoC1 and apoCII from tree shrew- a kind of in-susceptible to atherosclerosis animal
B.S. Chen 1 , X.-Y. Lu 2 , W.-w. Zeng 2 . 1 Chinese Academy of Medical Sciences; 2 Wu Gang Chinese Academy of Medical Sciences, Peking Union Medical School, Beijing, China
Quatitative analysis of apolipoprotein E secretion by human monocyte-derived macrophages in culture
K. Yamato 1 , N. Tamasawa 1 , H. Murakami 1 , J.-Z. Guan 2 , J. Tanabe 1 , J. Matsui 1 , T. Suda 1 , M. Yasujima 3 . 1 Third Department of Internal Medicine, Hirosaki, Aomori, Japan; 2 Third Department Of Internal Medicine, China; 3 Laboratory Medicine, Japan Objective: Apolipoprotein E (apo E) has an impact on lipid metabolism and its production by macrophages is considered to play a protective role against atherosclerosis. Apo A-I stimulates secretion of apo E from macrophages, and the interaction of exogenous apoA-I and macrophage-secreted apoE may inhibit atherogenesis in the vessel wall in vivo. We developed a new method to evaluate the ability of human monocyte-derived macrophages to secrete apo E, and the effects of factors such as apo A-I were examined. Methods: Monocytes separated from peripheral venous blood were cultured. The levels of apo E in macrophage-conditioned medium were quantified by immunoblotting with an anti-human apo E antiserum conjugated with alkaline phosphatase. Basic characterization including specificity, precision and reproducibility of the method was performed, and the adequacy of the method was checked by verifying the enhanced apo E secretion in response to apo A-I or recombinant human macrophage colony stimulating factor. The basal levels of apo E secretion and the response to exogenous apo A-I in macrophages from 10 healthy volunteers were measured. Results: Sufficient accuracy and sensitivity were confirmed and coefficient of variation of the method was 18±11% (n=10). The average apo E concentration in the conditioned medium of macrophages from 10 healthy subjects was 309±147 ng/mg cell protein. After the addition of apoA-I, the average apoE concentration increased, by about 60%, to 494±297 ng/ml (p<0.05). There was a positive correlation between the apo A-I-induced increase and plasma cholesterol levels (r=+0.54, p<0.05). Conclusions: The method is simple and accurate, and is effective to evaluate macrophage function in the real time. It may be applicable to the studies for the interaction of apo E and apo A-I, which is an important initial step in cholesterol efflux. 4P-1168
CRP induces PAI-1 gene expression through tyrosine kinase and NFkB pathways in vascular endothelial cells
D. Kawanami, K. Maemura, N. Takeda, T. Harada, T. Nojiri, Y. Imai, R. Nagai. Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Objectives: Recent evidences have shown that C-reactive protein (CRP) is an independent risk factor for cardiovascular diseases. However, it still remains to be elucidated whether CRP is just an inflammation marker or CRP itself functions as a proatherogenic factor in vessel walls. The purpose of this study is to examine whether CRP induces the gene expressions that promote atherosclerosis in vascular endothelial cells. Methods and Results: Northern blot analysis demonstrated that CRP induced VCAM-1, ICAM-1 and plasminogen activator inhibitor 1 (PAI-1) mRNA expressions in bovine aortic endothelial cells (BAECs) in a dose dependent manner. The PAI-1 mRNA increased from 2 hours and reached maximum at 8 hours after CRP stimulation. Treatment of BAECs with actinomycinD revealed that CRP did not affect PAI-1 mRNA stability, suggesting that CRP induces PAI-1 mRNA expression at the transcriptional level. Cycloheximide did not affect PAI-1 induction, suggesting that PAI-1 induction is not mediated by de novo protein synthesis. Next, we examined the effects of various protein kinase inhibitors on PAI-1 induction by CRP. Genistein and HerbimycinA, tyrosine kinase inhibitors, remarkably inhibited PAI-1 induction. Furthermore, parthenolide, a specific NFkB inhibitor also inhibited PAI-1 induction. Finally, we transfected the luciferase reporter gene containing NFkB binding sites as an enhancer into BAECs. Luciferase activity was increased by CRP stimulation. Conclusions: In this study, we demonstrated that CRP induced PAI-1 mRNA induction through tyrosine kinase and NFkB pathways in vascular endothelial cells. These data suggest that CRP is not only a marker for inflammation but also directly affects the gene expressions that may contribute to the development of thrombosis and atherosclerosis in vessel walls.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
THURSDAY
Purpose: Tree shrew is a kind of in-susceptible animal to atherosclerosis (AS). We discovered that both Tree Shrew (TS) and Beijing ducks (BD) have high level of HDL-C in plasma on feeding a diet supplemented with richcholesterol and fat for four and eight months respectively, no typical plaques of AS formed. However, for deeply understanding the anti-atherosclerosis mechanism, we cloned and characterized of apoE, apoC1 and apoCII genes from TS and BD Methods: The first strand cDNA of apoE/C1/CII were prepared by reverse transcription using the mRNA isolated and purified from the liver tissue of TS or BD. The apoE/C1/CII cDNA sequences were obtained with SMART RACE-PCR, and the amino acid (AA) sequences were deduced. The primary and secondary structures of the proteins were determined and predicted by using PCCGENE software. Results: The apoE cDNA sequence of TS consists of 1138 nucleotides, including 64bp 5’ untranslated region, 135bp 3’ untranslated region and 939 bp open reading frame, and is identical by 86% with that of human. The deduced sequence of AA was a 313 AA residue of apoE precursor which consists of an 18 AA signal peptide and a 295 AA mature protein which was identical by 78% with that of human beings, but TS apoE was 4 AA less than that of human, while 2 AA more than that of rabbit. Predicted by the method of Garnier, the secondary structure of TS apoE which may contain helical conformation 69.6%, extended 16.6%, turn 6.0%, coid 7.6%, was similar to that of human apoE. ApoC1 and apoCII cDNA were cloned and compared with that of human, rabbit, monkey and others. The expression of apoE/C1/CII mRNA in several tisues were detected by northern blot. In the present communication, we present the structure of apoA1/C1/CII and the distribution of their mRNA among different tissuses. The informations are important to our understanding of lipoprotein metabolism in the TS model.
4P-1167
331
Thursday October 2, 2003: Poster Session Molecules and clinical markers for atherosclerosis A mass spectrum analysis method for detection of C-reactive protein
H. Hsu 1 , Y.-R. Li 1 , Y.-C. Wang 1 , H.-K. Liao 2 , Y.-J. Chen 2 . 1 National Yang-Ming University, Institute of Biotechnology in Medicine, Taipei; 2 Institute of Chemistry, Academia Sinica, Taipei, Taiwan The circulating level of C-reactive protein (CRP) is considered as an important and independent risk factor for vascular diseases such as inflammation of vascular cells, development of atherosclerosis, coronary heart disease, etc. Considering the molecular structure, intact native CRP is a cyclic pentameric protein (pCRP) that consists of five non-covalently bound homo-monomers. Some recent findings report that pCRP could dissociate into modified or monomeric CRP (mCRP), but pCRP and mCRP possess quite different properties and functions. Using electrospray mass spectrometry (ESI-MS), we analyzed purified CRP in aqueous buffer solution at appropriate pH and calcium ion (Ca2+ ). Initially, we established methods to quantitate and identify the distinct m/z distribution (corresponding to their charge states) of the pCRP and mCRP. In addition, at certain concentration of Ca2+ , which is crucial to maintain the pentameric protein structure, we succeed to detect the non-covalently bound pCRP. After Beysian reconstruction process, distinct molecular weight of pCRP and mCRP can be identified from ESI-MS. This process would serve as a sensitive and accurate assay for analysis of various CRP species, even in the presence of mixture. Currently, modification of this method is under analyzing the different forms of CRP in human plasma, which will serve as a novel diagnostic tool for detection of inflammatory abnormality. On the other hand, a new membrane method by a specific protein affinity-directed mass spectrometry for examining the existence of low-abundant protein is also under development. 4P-1161
Elevation of bilirubin oxidative metabolites, biopyrrin, is a novel marker in the patients with acute myocardial infarction
H. Kunii 1 , K. Ishikawa 2 , T. Yamaguchi 3 , N. Komatsu 1 , H. Matsumoto 2 , M. Oikawa 1 , O. Yamaguchi 2 , Y. Shiratori 1 , H. Yamao 1 , S. Namiuchi 1 , M. Sugi 1 , M. Yui 1 , T. Ichihara 1 , Y. Maruyama 2 . 1 Iwaki Kyoritsu General Hospital; 2 Fukushima Medical University; 3 Tokyo Medical and Dental University, Japan Background: Bilirubin has been suggested to exhibit anti-oxidant properties. After the reaction with oxidants, bilirubin is oxidatively degraded into biopyrrins. We examined whether the elevation of biopyrrins is associated with the clinical features of the patients with acute myocardial infarction (AMI). Methods: Sixty consecutive patients hospitalized for AMI were analyzed. Urinary biopyrrin were measured by ELISA at the 1, 2, 3, 7 and 14 hospital days, adjusted by urinary creatinine, and referred as u(BPY/CRE). Results: The u(BPY/CRE) (Unit/gram-creatinine) were highest on the 3rd hospital day (3.6 ± 2.0) and decreased by the 14th hospital day (1.3 ± 1.2) (p<0.0001). The maximum u(BPY/CRE) was higher in death cases (24.1 ± 29.1) than that of survivors (3.9 ± 5.8) (p<0.05). Higher u(BPY/CRE) elevation was observed in the patients with large infarction (14.3 ± 7.1) than in those with small infarction (3.9 ± 0.4) (p<0.05). In addition, the patients with lower cardiac index exhibited higher u(BPY/CRE) levels (15.3 ± 23.6) than in those with higher cardiac index (3.8 ± 1.7) (p<0.05) although significant difference was not observed between two groups on the 14th hospital day. Elevation of u(BPY/CRE) tended to correlate with the elevation of brain-type natriuretic peptide (BNP) (p=0.116). Patients with higher BNP had higher urinary biopyrrin levels. Conclusion: Significant u(BPY/CRE) elevation was observed in the patients with AMI. The degree of this elevation was associated with infarction size, cardiac dysfunction and acute phase mortality after AMI. Monitoring the levels of u(BPY/CRE) can be a novel clinical marker in the patients with AMI. 4P-1162
Clinical evaluation of oxidative stress in cardiovascular disease
K. Nonaka, S. Seto, K. Yano, T. Nakayama, I. Sekine, T. Kondo. Nagasaki University School of Medicine, Japan Aim: Accelerated atherosclerosis plays an important role in the development of cardiovascular disease (CVD). There are increasing evidences concerning the linkage of oxidative damage with accelerated atherosclerosis, however, a few reports on the clinical studies relating CVD to the oxidative damage. Levels of 4-hydroxy-2-nonenal (4-HNE), an end product of lipid peroxidation, as well as carbonylated proteins have been reported as markers of oxidative
stress. Here we studied the levels of 4-HNE and carbonylated proteins in arterial wall from patients with CVD and investigated clinical importance of the intensity and the quality of oxidative stress. Materials and Methods: Small pieces of the left thoratic internal artery from patients with CVD were separated and frozen in liquid nitrogen before experiments. The clinical history and the medical records of all patients were carefully reviewed. The tissue samples were stained with HE and anti-4-HNE antibody. The levels of carbonylated proteins were estimated on immuno blot. Results and Discussion: HE stain revealed atherogenic changes in arteries of most patients. 4-HNE was found at fibromuscular cell in swollen internal layer and smooth muscle cells in midthelium. The levels of carbonylated proteins from the tissue extracts were related to those of 4-HNE, however, not to the severity of atherosclerosis. These levels of oxidative damage were rather related to clinical intensity of CVD patients. These data suggest that oxidative stress is a factor of the development of CVD and estimation of oxidative stress is important in evaluation of the degree of CVD. 4P-1163
Two novel mutations in the plasma platelet-activating factor acetylhydrolase (PAF-AH) gene identified in Japanese subjects
M. Ishihara 1 , M. Nagano 1 , T. Iwasaki 1 , T. Kujiraoka 1 , J. Ishii 2 , M. Tsuji 3 , M. Ito 1 , T. Kumagai 1 , T. Egashira 1 , H. Hattori 1 . 1 Department of Advanced Technology and Development, BML Inc.; 2 Hokkaido Hospital for Social Health Insurance; 3 Institute of Medical Science, Health Sciences University of Hokkaido, Japan Objective: The V279F mutation of PAF-AH is common in Japanese subjects. We studied PAF-AH mass and the V279F mutation in subjects with hyperlipidemia and diabetes. Among subjects with lower PAF-AH mass, we have identified two novel mutations of the PAF-AH gene. Methods: Plasma PAF-AH mass concentration was determined in 249 outpatients with hyperlipidemia and diabetes by a sandwich ELISA using monoclonal antibodies. The detection of the V279F mutation was carried out by the Invader® assay. The nucleotide sequence was determined by direct sequencing. Results: Plasma PAF-AH mass concentration in hyperlipidemia and diabetes was 1.67 ± 0.58 µg/mL for the wild type (n=169), 0.77 ± 0.30 µg/mL for heterozygotes with V279F (n=73), and undetectable mass for homozygotes with V279F (n=7). We further screened the PAF-AH gene in subjects with lower PAF-AH mass and identified two novel mutations. In a subject who was heterozygotes for the V279F mutation had undetectable PAF-AH mass, there was a substitution of T to A at 949 nt in exon 10, resulting amino acid change at codon 317 of Asn for Ile. In a subject without the V279F mutation had lower PAF-AH mass similar to those heterozygous for the V279F mutation, there was the insertion of A at 187 nt in exon 3, creating the termination at codon 63. By transient expression of mutant Asn317 PAF-AH gene in COS-7 cells, PAF-AH mass and activity in the culture medium were undetectable. Conclusions: We have identified two novel mutations, I317N and Y63X, in the PAF-AH gene in Japanese subjects with lower Plasma PAF-AH mass concentration. The expression analysis suggested that mutant PAF-AH protein might not be secreted to the circulation. 4P-1164
Effect of pravastatin on the aortic gene expression of ApoE-deficient mice fed with a hypercholesterolemic diet
G.-Y. Shi, S.-L. Liu, Y.-W. Chen, C.-S. Shi, M.-H. Wu, H.-L. Wu. National Cheng Kung University, Taiwan 3-Hydroxy-3-methyl-gutaryl coenzyme (HMG-CoA) reductase inhibitors or statins, constituting the most powerful class of lipid-lowering drugs, have been applied in clinics to reduce the risk of acute coronary events. However, the beneficial effects of statins cannot be fully explained by their lipid-lowing potential. In this study, we intend to identify genes in aortas that altered in statin treatment. To establish the effects of statin on the progression of atherosclerosis in hypercholesterolemic mice and to test the hypothesis that treatment with statin may result in alteration of gene expression, the apoEdeficient mice were fed with diet supplemented with 0.15% cholesterol and treated with pravastatin. The related genes whose expressions were affected by pravastatin were identified using oilgonucleotide microarray technology. ApoE-deficient mice were fed with a high cholesterol chow diet started at 8 weeks of age for a total of 16 weeks. For 6 weeks before euthanasia, the test groups were treated with pravastatin (80 mg/kg) dissolved in water by daily oral inoculation. The total cholesterol, LDL and lesion/total aortic size between the treated with pravastatin and control groups were significantly
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Thursday October 2, 2003: Poster Session Molecules and clinical markers for atherosclerosis different. Microarray analysis of the expression of 20281 murine genes in the aortas between the two groups by Agilent gene chips suggested that 93 genes were significantly regulated in apoE-deficient mice. Among these genes, 44 were up-regulated and 49 were down-regulated. 4P-1165
Increased levels of pregnancy-associated plasma protein-A in patients with hypercholesterolemia and diabetes: the effect of lipid lowering treatment
T. Stulc 1 , J. Skrha 1 , I. Malbohan 2 , L. Fialova 2 , J. Malik 1 , R. Ceska 1 . 1 3rd Department of Internal Medicine, 2 Institute of Clinical Chemistry, 1st School of Medicine, Charles University, Czech Republic Objective: Pregnancy-associated plasma protein A (PAPP-A) is a metalloproteinase produced by unstable plaques; serum PAPP-A is increased in acute coronary syndromes. The relationship between PAPP-A and coronary risk factors has not been studied to date. We have therefore investigated whether PAPP-A levels are increased in hypercholesterolemia and diabetes and whether they are affected by statin treatment. Methods: We examined 32 subjects with severe hypercholesterolemia (HLP group), 20 type 2 diabetic patients with mixed hyperlipidemia (DM group) and 26 controls. All subjects were free of vascular disease. Patients were examined before and after 12 weeks of treatment with atorvastatin 20mg/d (HLP group) or simvastatin 20mg/d (DM group). Results: In both patient groups, baseline PAPP-A levels were significantly higher than in controls (Table 1; p<0.01); moreover, there was a trend towards higher values in DM group. However, PAPP-A levels were not influenced by either treatment. Controls PAPP/A [mU/l] TC [mmol/l] TG [mmol/l]
6.32±2.59 4.99±0.60 1.11±0.62
HLP
DM
before
after
before
after
8.02±1.86 8.59±1.60 1.67±0.63
7.67±1.89 5.87±1.12 1.33±0.37
8.98±2.53 6.62±0.83 3.61±2.09
8.35±2.50 5.19±0.75 3.25±1.98
Conclusion: We have demonstrated for the first time that PAPP-A levels are elevated in hyperlipidemic subjects without clinical signs of atherosclerosis. PAPP-A may therefore not only reflect the plaque instability, but also the total atheroclerotic burden in asymptomatic subjects with hyperlipidemia. 4P-1166
Structure and expression of apoE, apoC1 and apoCII from tree shrew- a kind of in-susceptible to atherosclerosis animal
B.S. Chen 1 , X.-Y. Lu 2 , W.-w. Zeng 2 . 1 Chinese Academy of Medical Sciences; 2 Wu Gang Chinese Academy of Medical Sciences, Peking Union Medical School, Beijing, China
Quatitative analysis of apolipoprotein E secretion by human monocyte-derived macrophages in culture
K. Yamato 1 , N. Tamasawa 1 , H. Murakami 1 , J.-Z. Guan 2 , J. Tanabe 1 , J. Matsui 1 , T. Suda 1 , M. Yasujima 3 . 1 Third Department of Internal Medicine, Hirosaki, Aomori, Japan; 2 Third Department Of Internal Medicine, China; 3 Laboratory Medicine, Japan Objective: Apolipoprotein E (apo E) has an impact on lipid metabolism and its production by macrophages is considered to play a protective role against atherosclerosis. Apo A-I stimulates secretion of apo E from macrophages, and the interaction of exogenous apoA-I and macrophage-secreted apoE may inhibit atherogenesis in the vessel wall in vivo. We developed a new method to evaluate the ability of human monocyte-derived macrophages to secrete apo E, and the effects of factors such as apo A-I were examined. Methods: Monocytes separated from peripheral venous blood were cultured. The levels of apo E in macrophage-conditioned medium were quantified by immunoblotting with an anti-human apo E antiserum conjugated with alkaline phosphatase. Basic characterization including specificity, precision and reproducibility of the method was performed, and the adequacy of the method was checked by verifying the enhanced apo E secretion in response to apo A-I or recombinant human macrophage colony stimulating factor. The basal levels of apo E secretion and the response to exogenous apo A-I in macrophages from 10 healthy volunteers were measured. Results: Sufficient accuracy and sensitivity were confirmed and coefficient of variation of the method was 18±11% (n=10). The average apo E concentration in the conditioned medium of macrophages from 10 healthy subjects was 309±147 ng/mg cell protein. After the addition of apoA-I, the average apoE concentration increased, by about 60%, to 494±297 ng/ml (p<0.05). There was a positive correlation between the apo A-I-induced increase and plasma cholesterol levels (r=+0.54, p<0.05). Conclusions: The method is simple and accurate, and is effective to evaluate macrophage function in the real time. It may be applicable to the studies for the interaction of apo E and apo A-I, which is an important initial step in cholesterol efflux. 4P-1168
CRP induces PAI-1 gene expression through tyrosine kinase and NFkB pathways in vascular endothelial cells
D. Kawanami, K. Maemura, N. Takeda, T. Harada, T. Nojiri, Y. Imai, R. Nagai. Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Objectives: Recent evidences have shown that C-reactive protein (CRP) is an independent risk factor for cardiovascular diseases. However, it still remains to be elucidated whether CRP is just an inflammation marker or CRP itself functions as a proatherogenic factor in vessel walls. The purpose of this study is to examine whether CRP induces the gene expressions that promote atherosclerosis in vascular endothelial cells. Methods and Results: Northern blot analysis demonstrated that CRP induced VCAM-1, ICAM-1 and plasminogen activator inhibitor 1 (PAI-1) mRNA expressions in bovine aortic endothelial cells (BAECs) in a dose dependent manner. The PAI-1 mRNA increased from 2 hours and reached maximum at 8 hours after CRP stimulation. Treatment of BAECs with actinomycinD revealed that CRP did not affect PAI-1 mRNA stability, suggesting that CRP induces PAI-1 mRNA expression at the transcriptional level. Cycloheximide did not affect PAI-1 induction, suggesting that PAI-1 induction is not mediated by de novo protein synthesis. Next, we examined the effects of various protein kinase inhibitors on PAI-1 induction by CRP. Genistein and HerbimycinA, tyrosine kinase inhibitors, remarkably inhibited PAI-1 induction. Furthermore, parthenolide, a specific NFkB inhibitor also inhibited PAI-1 induction. Finally, we transfected the luciferase reporter gene containing NFkB binding sites as an enhancer into BAECs. Luciferase activity was increased by CRP stimulation. Conclusions: In this study, we demonstrated that CRP induced PAI-1 mRNA induction through tyrosine kinase and NFkB pathways in vascular endothelial cells. These data suggest that CRP is not only a marker for inflammation but also directly affects the gene expressions that may contribute to the development of thrombosis and atherosclerosis in vessel walls.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
THURSDAY
Purpose: Tree shrew is a kind of in-susceptible animal to atherosclerosis (AS). We discovered that both Tree Shrew (TS) and Beijing ducks (BD) have high level of HDL-C in plasma on feeding a diet supplemented with richcholesterol and fat for four and eight months respectively, no typical plaques of AS formed. However, for deeply understanding the anti-atherosclerosis mechanism, we cloned and characterized of apoE, apoC1 and apoCII genes from TS and BD Methods: The first strand cDNA of apoE/C1/CII were prepared by reverse transcription using the mRNA isolated and purified from the liver tissue of TS or BD. The apoE/C1/CII cDNA sequences were obtained with SMART RACE-PCR, and the amino acid (AA) sequences were deduced. The primary and secondary structures of the proteins were determined and predicted by using PCCGENE software. Results: The apoE cDNA sequence of TS consists of 1138 nucleotides, including 64bp 5’ untranslated region, 135bp 3’ untranslated region and 939 bp open reading frame, and is identical by 86% with that of human. The deduced sequence of AA was a 313 AA residue of apoE precursor which consists of an 18 AA signal peptide and a 295 AA mature protein which was identical by 78% with that of human beings, but TS apoE was 4 AA less than that of human, while 2 AA more than that of rabbit. Predicted by the method of Garnier, the secondary structure of TS apoE which may contain helical conformation 69.6%, extended 16.6%, turn 6.0%, coid 7.6%, was similar to that of human apoE. ApoC1 and apoCII cDNA were cloned and compared with that of human, rabbit, monkey and others. The expression of apoE/C1/CII mRNA in several tisues were detected by northern blot. In the present communication, we present the structure of apoA1/C1/CII and the distribution of their mRNA among different tissuses. The informations are important to our understanding of lipoprotein metabolism in the TS model.
4P-1167
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