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W01.1 Differential gene expression in areas of instability in carotid artery plaques
Workshops iiiiiiiiiiiiiiiiiiii W1 INFLAMMATION, PLAQUE ACTIVATION, AND ACUTE CORONARY SYNDROMES
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DIFFERENTIAL GENE EXPRESSION IN AREAS OF • INSTABILITY IN CAROTID ARTERY PLAQUES
M. Papaspyridonos, A. Smith, J. Cox, R. Woollard, T. Roberts, K. Burnand, K. Suckling, L. Patel. St Thomas' Hospital, Lambeth Palace Rood, London,
GlaxoSmithKline Pharmaceuticals, Dept of Transcriptome Analysis, Stevenage, United Kingdom Atherosclerotic plaque rupture is a major cause of sta'oke and myocardial infarction. Transcriptome analysis was used to identify novel genes that could be involved in plaque instability. Human carotid endarterectomy specimens were divided into 5ram segments along theft" course. Plaque segments were classified by 3 blinded observers into three groups: no ulceration (stable), ulceration without thrombosis (unstable) or ulceration with thrombosis (highly unstable), cRNA samples fi'om 11 segments were hybridised to Affymeta'ix U133 A and B GeneChip sets. The patterns and levels of gene expression in unstable regions were compared with stable segments. 75 genes wele consistently upregulated and 95 down regulated in unstable areas (p<0.05, ANOVA). 12 genes with the greatest fold diffelence in expression were selected fi'om these. Real-time quantitative PCR was then used to confirm theft" expression in a panel of 33 segments taken fi'om a cohort of 12 independent plaques. Differences in the ratio of test gene cDNA/housekeeper genes cDNA within the three types of plaque were analysed by ANOVA. Statistically significant differential expression was detected for 6 of the 12 selected genes. Plaque MMP9 (P=0.010), LpPLA2 (P=0.011), F11R (p=0.005), CSF2RA (P=0.027), SAT (P=0.005), CTSB (P=0.013) were upregulated in unstable areas of plaque. At least 6 genes were found to be consistently upregulated in unstable areas of carotid artery plaques. The expression of a further 20 genes that include known genes not previously implicated in atherosclerosis. Whole genome inta'aplaque analysis is a novel approach that could identify new markers of plaque instability and specific therapeutic targets.
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TOLL-LIKE RECEPTOR 4 IS INVOLVED IN THE REGULATION OF VASCULAR REMODELING
P. Quax, M. de Vries, A. Vink, A. Schepers, D. Eefting, N. Pfl-es, W. Jukema, H. van Bockel, D. de Kleijn. Gaubius Laboratory TNO-PG,
Leiden; Experimental Cardiology Laboratory, UMC, Utrecht; Dept. Cardiology, and Dept. Vasc. Surgery, LUMC, Leiden, The Netherlands Toll-like receptor 4 (TLR4), a key regulator of the innate immune leponse, binds bacterial lipopolysaccharides (LPS) as well as its endogenous ligands EDA-fibronectin and HSP60. We hypothesize that activation of adventitial Toll-like receptor 4 (TLR4), ta'iggel'S the inflammatory processes leading to postinterventional vascular remodeling. Placement of a periadventitial cuff around the femoral artery in mice to induce intimal hyperplasia, resulted in an rapid upregulation of TLR4 mRNA and protein as well as its endogenous ligand EDA-fibronectin. LPS application augmented cuff-induced neointima formation (9134-4-1714 versus 2353-4-10761~m2, P<0.01). In TLR4 defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wt-mice (3859-4-904 i~m2, P=0.02). In addition we could demonstrate TLR4 involvement in outward vascular remodeling. Using the cuff model in the atherosclerotic ApoE3Leiden ta'ansgenic mouse, TLR4 activation by LPS stimulated plaque formation and subsequent outward arterial remodeling. Outward remodeling was also observed in wt-mice. In TLR4 deficient mice, however, no outward arterial lemodeling was observed, independent of neointima forrnation. Moreover, carotid artery ligation in wt-mice resulted in outward remodeling without neointima forrnation in the conta'alatel'al artery. This was associated with an increase in TLR4 expression and EDA and Hsp60 mRNA levels. In conta'ast, outward remodeling was not observed following carotid ligation in the TLR4 deficient mice.
These data indicate that TLR4, its activation and downs~eam signal pathway play an important role in regulating vascular remodeling.
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CIRCULATING STROMAL STEM CELLS AND DIAGNOSTICS OF ATHEROSCLEROSIS
E.L. Soboleva, Z.A. Gabbasov, A.A. Agapov, O.S. Saburova, V.N. Stuff-nov.
Laboratory of Stem Cells, Cardiology Research Center, Moscow, Russia Using autopsy material and clonal technique, we were able to show earlier that in human atheromatous vascular intima in addition to hematopoietic stem cells sU'omal colony-forrning units ale present. In this study we applied flow cytomeU'y to examine whether ch'culating sU'omal progenitors are present in blood of patients with ischemic heart disease. Nine coronary patients and five healthy volunteers were included into the study. Coronary angiography was can'ied out in all patients and demonsu'ated critical stenosis of at least two coronary arteries or theh" large branches. In our experiments we used antibodies to osteonectin as marker for su'omal stem cells with osteogenic potencies. No osteonectin-positive cells were found in blood of healthy donors (0 cells per 100,000 events, n=5), whereas in all coronary patients theh" number was high and varied in the interval 98-1853 per 100,000 events (n=9). Moderate expression of CD45 and the absence of CD34 antigen were characteristic for phenotype of ch'culating osteonectin-positive sU'omal cells. These findings confirmed the results on the absence of sU'omal ch'culating colony-forrning units in healthy donors and theh" appearance in blood of coronary patient obtained earlier by classical cloning technique. For the first time it was demonsU'ated that there exists close relationships between progression of atherosclerosis and the presence in blood of ch'culating su'omal cells.
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CHANGES IN LDL-CHOLESTEROL LEVELS IN DIFFERENT TIME INTERVALS AFTER ACUTE MYOCARDIAL INFARCTION
R. Ankica, L. Georgievska-Ismail, S. Dzekova-Stojkova. Biochemical Lab., Institut for Heart Disease, Institutfor Medical and Experimental Biochemistry, Faculty of Medicine, Skopje, Macedonia Numerous studies have shown that the level of the serum lipids, measured in the first 24 hours of the acute myocardial infarction (AMI) is liable to changes immediately after the event and get back to its initial (basal) value within the next 6-12 weeks. In order to confirm if there are changes in the lipid profile and what they look like, particularly the LDL cholesterol in the blood, at 230 middle aged (59.8 -4-13 years old), mostly males (66.5%) patients with AIM who were discharged fi'om hospital without statin therapy, a follow up of the LDL cholesterol level was perforrned, taken of the vein blood and determined by standard enzymatic methods in different time intervals after the actual event (24 hours, 3-7 days, 10-14 days, 30-60 days, 60-90-days). The results showed that the patients with AMI have had a higher initial LDL cholesterol level (170.3 -4- 70.97 mg/dL), which showed a tendency to decrease three days after the actual event reaching the lowest level at 10-14 days after the event and to be gradually "normalized" after 60-90 days. Condusion: We can conclude that the optimal time for determining the LDL cholesterol level at the patients with AMI are the first 24 hours of the actual event, since in the next period of time there is a unreal decrease of its level in the blood. The values of the lipid profile acqufl'ed at 24 hours of the actual event should be considered as basal.
74th EAS Congress, 17-20 April 2004, Seville, Spain
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DIFFERENTIAL GENE EXPRESSION IN AREAS OF • INSTABILITY IN CAROTID ARTERY PLAQUES
M. Papaspyridonos, A. Smith, J. Cox, R. Woollard, T. Roberts, K. Burnand, K. Suckling, L. Patel. St Thomas' Hospital, Lambeth Palace Rood, London,
GlaxoSmithKline Pharmaceuticals, Dept of Transcriptome Analysis, Stevenage, United Kingdom Atherosclerotic plaque rupture is a major cause of sta'oke and myocardial infarction. Transcriptome analysis was used to identify novel genes that could be involved in plaque instability. Human carotid endarterectomy specimens were divided into 5ram segments along theft" course. Plaque segments were classified by 3 blinded observers into three groups: no ulceration (stable), ulceration without thrombosis (unstable) or ulceration with thrombosis (highly unstable), cRNA samples fi'om 11 segments were hybridised to Affymeta'ix U133 A and B GeneChip sets. The patterns and levels of gene expression in unstable regions were compared with stable segments. 75 genes wele consistently upregulated and 95 down regulated in unstable areas (p<0.05, ANOVA). 12 genes with the greatest fold diffelence in expression were selected fi'om these. Real-time quantitative PCR was then used to confirm theft" expression in a panel of 33 segments taken fi'om a cohort of 12 independent plaques. Differences in the ratio of test gene cDNA/housekeeper genes cDNA within the three types of plaque were analysed by ANOVA. Statistically significant differential expression was detected for 6 of the 12 selected genes. Plaque MMP9 (P=0.010), LpPLA2 (P=0.011), F11R (p=0.005), CSF2RA (P=0.027), SAT (P=0.005), CTSB (P=0.013) were upregulated in unstable areas of plaque. At least 6 genes were found to be consistently upregulated in unstable areas of carotid artery plaques. The expression of a further 20 genes that include known genes not previously implicated in atherosclerosis. Whole genome inta'aplaque analysis is a novel approach that could identify new markers of plaque instability and specific therapeutic targets.
•0•]
TOLL-LIKE RECEPTOR 4 IS INVOLVED IN THE REGULATION OF VASCULAR REMODELING
P. Quax, M. de Vries, A. Vink, A. Schepers, D. Eefting, N. Pfl-es, W. Jukema, H. van Bockel, D. de Kleijn. Gaubius Laboratory TNO-PG,
Leiden; Experimental Cardiology Laboratory, UMC, Utrecht; Dept. Cardiology, and Dept. Vasc. Surgery, LUMC, Leiden, The Netherlands Toll-like receptor 4 (TLR4), a key regulator of the innate immune leponse, binds bacterial lipopolysaccharides (LPS) as well as its endogenous ligands EDA-fibronectin and HSP60. We hypothesize that activation of adventitial Toll-like receptor 4 (TLR4), ta'iggel'S the inflammatory processes leading to postinterventional vascular remodeling. Placement of a periadventitial cuff around the femoral artery in mice to induce intimal hyperplasia, resulted in an rapid upregulation of TLR4 mRNA and protein as well as its endogenous ligand EDA-fibronectin. LPS application augmented cuff-induced neointima formation (9134-4-1714 versus 2353-4-10761~m2, P<0.01). In TLR4 defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wt-mice (3859-4-904 i~m2, P=0.02). In addition we could demonstrate TLR4 involvement in outward vascular remodeling. Using the cuff model in the atherosclerotic ApoE3Leiden ta'ansgenic mouse, TLR4 activation by LPS stimulated plaque formation and subsequent outward arterial remodeling. Outward remodeling was also observed in wt-mice. In TLR4 deficient mice, however, no outward arterial lemodeling was observed, independent of neointima forrnation. Moreover, carotid artery ligation in wt-mice resulted in outward remodeling without neointima forrnation in the conta'alatel'al artery. This was associated with an increase in TLR4 expression and EDA and Hsp60 mRNA levels. In conta'ast, outward remodeling was not observed following carotid ligation in the TLR4 deficient mice.
These data indicate that TLR4, its activation and downs~eam signal pathway play an important role in regulating vascular remodeling.
•0•
CIRCULATING STROMAL STEM CELLS AND DIAGNOSTICS OF ATHEROSCLEROSIS
E.L. Soboleva, Z.A. Gabbasov, A.A. Agapov, O.S. Saburova, V.N. Stuff-nov.
Laboratory of Stem Cells, Cardiology Research Center, Moscow, Russia Using autopsy material and clonal technique, we were able to show earlier that in human atheromatous vascular intima in addition to hematopoietic stem cells sU'omal colony-forrning units ale present. In this study we applied flow cytomeU'y to examine whether ch'culating sU'omal progenitors are present in blood of patients with ischemic heart disease. Nine coronary patients and five healthy volunteers were included into the study. Coronary angiography was can'ied out in all patients and demonsu'ated critical stenosis of at least two coronary arteries or theh" large branches. In our experiments we used antibodies to osteonectin as marker for su'omal stem cells with osteogenic potencies. No osteonectin-positive cells were found in blood of healthy donors (0 cells per 100,000 events, n=5), whereas in all coronary patients theh" number was high and varied in the interval 98-1853 per 100,000 events (n=9). Moderate expression of CD45 and the absence of CD34 antigen were characteristic for phenotype of ch'culating osteonectin-positive sU'omal cells. These findings confirmed the results on the absence of sU'omal ch'culating colony-forrning units in healthy donors and theh" appearance in blood of coronary patient obtained earlier by classical cloning technique. For the first time it was demonsU'ated that there exists close relationships between progression of atherosclerosis and the presence in blood of ch'culating su'omal cells.
•0•
CHANGES IN LDL-CHOLESTEROL LEVELS IN DIFFERENT TIME INTERVALS AFTER ACUTE MYOCARDIAL INFARCTION
R. Ankica, L. Georgievska-Ismail, S. Dzekova-Stojkova. Biochemical Lab., Institut for Heart Disease, Institutfor Medical and Experimental Biochemistry, Faculty of Medicine, Skopje, Macedonia Numerous studies have shown that the level of the serum lipids, measured in the first 24 hours of the acute myocardial infarction (AMI) is liable to changes immediately after the event and get back to its initial (basal) value within the next 6-12 weeks. In order to confirm if there are changes in the lipid profile and what they look like, particularly the LDL cholesterol in the blood, at 230 middle aged (59.8 -4-13 years old), mostly males (66.5%) patients with AIM who were discharged fi'om hospital without statin therapy, a follow up of the LDL cholesterol level was perforrned, taken of the vein blood and determined by standard enzymatic methods in different time intervals after the actual event (24 hours, 3-7 days, 10-14 days, 30-60 days, 60-90-days). The results showed that the patients with AMI have had a higher initial LDL cholesterol level (170.3 -4- 70.97 mg/dL), which showed a tendency to decrease three days after the actual event reaching the lowest level at 10-14 days after the event and to be gradually "normalized" after 60-90 days. Condusion: We can conclude that the optimal time for determining the LDL cholesterol level at the patients with AMI are the first 24 hours of the actual event, since in the next period of time there is a unreal decrease of its level in the blood. The values of the lipid profile acqufl'ed at 24 hours of the actual event should be considered as basal.
74th EAS Congress, 17-20 April 2004, Seville, Spain
iiiiii:..O.iiiiiiiiiiiiiiii
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