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P38 MAPK dependent pathway is responsible for N-LDL and Ox-LDL Cox-2 induction in human endothelial cells
Nordestgaard
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MAPK DEPENDENT PATHWAY IS RESPONSIBLE F O R N-LDL AND OX-LDL COX-2 INDUCTION IN HUMAN ENDOTHELIAL CELLS
G.D. Norata 1, A. Pirillo 1, F. Pellegatta 1, A.L. Catapano 1'2. 1Department
of Pharmacological Sciences, 2Atherosclerosis Center, Bassini Hospital, University of Milan, Italy Low density lipoproteins (LDL) and oxidized low density lipoproteins (Ox-LDL) play a central role in atherogenesis. We investigated whether LDL or Ox-LDL modulate cyclooxygenase 1 and 2 (Cox-1 and Cox-2) expression in human endothelial cells (HUVECs). As Cox-2 is induced the activation of several mitogen-activated protein kinases (MAPKs), we studied the role of these pathways on the modulation of Cox-1 and Cox-2 by LDL or OxLDL. Cox-2 expression was strongly induced, in a dose dependent manner, after a 2h incubation of HUVECs with LDL or Ox-LDL; induction was maximal after 4 hours and sustained for at least 8 hours. The effect was specific for Cox-2, as Cox-1 expression was not affected by LDL or by Ox-LDL. LDL induced the phosphorylation of ERK1/2 to a large extent, while Ox-LDL were less effective; both LDL and Ox-LDL induced the phosphorylation of p38 MAPK. The induction of Cox-2 expression was mainly dependent on the activation of p38 MAPK as the preincubation with the p38 MAPK inhibitor SB203580 (lmM), effectively reduced the LDL or Ox-LDL effect on Cox-2 protein and mRNA as determined by RT-PCR. The MEK1 inhibitor, PD98059 (25mM) slightly affected Cox-2 expression. These data support the concept that LDL and Ox-LDL induce Cox-2 expression in HUVEC and that this effect is mediated through a MAPK dependent pathway. These findings suggest a novel level of regulation for Cox-2 expression induced by LDL and Ox-LDL. ~
FACTOR V LEIDEN AND RISK OF ARTERIAL THROMBOSIS: 3 CASE-CONTROL AND 3 PROSPECTIVE STUDIES BASED ON THE COPENHAGEN CITY HEART STUDY AND 2 META-ANALYSES
K. Juul, A. Tybjaerg-Hansen, R. Steffensen, S. Kofoed, G. Jensen, B.G. Nordest~aard. Copenhagen University Hospitals, Denmark
Background: Factor V Leiden (FVL) is associated with venous thrombosis; however, association between FVL and arterial thrombosis remains controversial. Objective: We investigated FVL as a risk factor for myocardial infarction (MI), ischemic stroke (IS), or non-MI ischemic heart disease (non-MI-IHD). Design: 3 case-control studies, 3 prospective studies with 21 years follow-up, and 2 meta-analyses. Setting: General population, Copenhagen, Denmark. Participants: Copenhagen City Heart Study 20 95 year old participants and patients from Copenhagen University Hospital with MI (n-469; n-493), IS (n-410; n-231) or non-MI-IItD (n-448; n-365), and 7.907 controls. 31.000 individuals from 35 studies for meta-analyses. Measurements: FVL genotype, major cardiovascular risk factors, MI, IS, and non-MI-IHD incidence and prevalence. Results: Prevalences of FVL heterozygotes and homozygotes in controls from the general population were 7.7% and 0.2%. Odds ratios and relative risks of MI in FVL carriers (-heterozygotes+homozygotes) were 1.24 (95%CI:0.91 1.69) and 0.83 (0.58 1.20) in case-control and prospective studies, respectively. Corresponding risks for IS were 0.92 (0.56 1.53) and 0.68(0.45 1.04), and for non-MI-IHD 1.01 (0.71 1.44) and 0.97 (0.66 1.42). Meta-analyses revealed aggregated odds ratios in FVL carriers for MI and adult IS of 1.20 (1.03 1.39) and 1.00 (0.75 1.34), and for premature MI and childhood IS of 1.54 (1.072.22) and 4.79(3.26~.03). Conclusion: Findings from The Copenhagen City Heart Study suggest that FVL is not associated with MI, IS or non-MI-IHD. Meta-analyses suggest, however, that FVL constitutes a modest risk factor for MI, a larger risk factor for premature MI, and a major risk factor for childhood IS. ~
FIBRINOGEN PREDICTS ISCHEMIC STROKE AND ADVANCED ATHEROSCLEROSIS, BUT NOT ECHOLUCENT, RUPTURE-PRONE CAROTID PLAQUES
S.C. Kofoed, H.H. Wittrup, H. Sillesen, B.G. Nordest~aard. Copenhagen
University Hospitals, Denmark Background: Whether the association between fibrinogen and cardiovascular events reflects an association with advanced atherosclerosis in general, or rupture-prone plaques in particular, is unclear. We examined whether
175
fibrinogen predicts incidence of ischemic stroke, advanced atherosclerosis (measured as carotid artery stenosis) and/or echolucent, rupture-prone plaques. Methods and Results: Study 1:8755 Copenhagen City Heart Study stroke free participants; we observed 235 ischemic strokes during six-years of follow-up. Study 2:318 carotid stenosis patients and 1584 age and gender matched controls. Study 3:159 patients with echolucent versus 159 patients with echo-rich carotid artery plaques. Fibrinogen above versus below the median value of 3 g/L predicted risk of ischemic stroke (relative risk: 1.9; 95% CI: 1.42.5). Significant risk was found in men (2.7; 1.74.2) but not in women (1.4; 0.92.0), in young (5.2; 1.126) and middle aged (2.9; 1.6-5.4) but not in the elderly (1.4; 1.02.0). Fibrinogen levels in those with and without ischemic stroke were 3.6 and 3.1 g/L (ANCOVA:p<0.0001). Likewise, in those with and without carotid artery stenosis fibrinogen levels were 4.7 and 3.1 g/L (p<0.0001); equivalent values for high-sensitive C-reactive protein were 3.6 and 1.4 mg/L (p<0.0001). Finally, neither fibrinogen nor highsensitive C-reactive protein levels differed between those with echolucent and echo-rich carotid artery plaques (p-0.61 and p-0.28). Conclusion: Elevated fibrinogen predicts future ischemic strokes, particular in men and in the young and middle aged. This is most likely a reflection of advanced atherosclerosis, rather than an association with rupture-prone plaques.
~HDL
AND OTHER FACTORS IN THE GENETICS OF ISCHEMIC HEART DISEASE
B.G. Nordestgaard 1, A. Tybjaerg-Hansen2. JHerlev University Hospital, 2Copenhagen University Hospital, Denmark Using the Copenhagen City Heart Study, we studied association between rare and common mutations in known candidate genes and risk of ischemic heart disease. The Copenhagen City Heart Study is a prospective cardiovascular population study initiated in 1996. We genotyped > 9000 participants. During 22 years follow-up, 1014 of the participants developed ischemic heart disease while 7882 were free of disease, and thus acted as controls. In addition, we also genotyped 948 independent patients with ischemic heart disease and documented coronary atherosclerosis. Genotype frequencies in these patients were compared with the 7882 controls in a case-control design. We studied mutations in candidate genes for ItDL metabolism including cholesteryl ester transfer protein, lipoprotein lipase, hepatic lipase, apolipoprotein E, and ABCA1. Other genes studied include extracellular superoxide dismutase and alpha-l-antitrypsin deficiency.
~ - ] ] TRANSVASCULAR LOW DENSITY LIPOPROTEIN TRANSPORT IN PATIENTS W I T H DIABETES MELLITUS (TYPE 2): A NON-INVASIVE IN VIVO ISOTOPE TECHNIQUE K. Kornerup, B.G. Nordestgaard, B. Feldt-Rasmussen, K. Borch-Johnsen, K.S. Jensen, J.S. Jensen. Copenhagen University Hospitals, Denmark The increased risk of atherosclerosis among diabetic individuals cannot be explained by conventional cardiovascular risk factors alone. We hypothesized that transvascular lipoprotein transport may be increased in patients with diabetes due to endothelial dysfunction, possibly explaining increased intimal lipoprotein accumulation and thus atherosclerosis. We developed an in vivo method for measurement of transvascular transport of low density lipoprotein (LDL) and applied it in 16 patients with maturity onset diabetes (type 2) and 29 age- and sex-matched controls. Autologous 131-iodinated LDL was reinj ected intravenously in addition to 125-iodinated albumin, and the 1-hour fractional escape rates were taken as indices of transvascular transport. Both parameters were normally distributed and they were tightly correlated (R2-0.69; P<0.0001). Transvascular transport of LDL was 5.4 and 4.1 percent/hour in patients with diabetes and controls (P<0.05); equivalent values for albumin were 6.5 and 5.3 percent/hour (P<0.05). This difference most likely was not caused by altered LDL receptor expression in the liver, glycosylation of LDL in diabetes, small LDL size in diabetic patients, statin use, differences in plasma insulin levels, or by nephropathy in diabetes. In conclusion, we demonstrate that transvascular transport of LDL may be increased in those with type 2 diabetes versus controls. This suggests that lipoprotein flux into the arterial wall could be increased in people with diabetes due to endothelial dysfunction, possibly explaining accelerated development of atherosclerosis in these patients.
73rd EAS Congress
•
]
P
3
8
MAPK DEPENDENT PATHWAY IS RESPONSIBLE F O R N-LDL AND OX-LDL COX-2 INDUCTION IN HUMAN ENDOTHELIAL CELLS
G.D. Norata 1, A. Pirillo 1, F. Pellegatta 1, A.L. Catapano 1'2. 1Department
of Pharmacological Sciences, 2Atherosclerosis Center, Bassini Hospital, University of Milan, Italy Low density lipoproteins (LDL) and oxidized low density lipoproteins (Ox-LDL) play a central role in atherogenesis. We investigated whether LDL or Ox-LDL modulate cyclooxygenase 1 and 2 (Cox-1 and Cox-2) expression in human endothelial cells (HUVECs). As Cox-2 is induced the activation of several mitogen-activated protein kinases (MAPKs), we studied the role of these pathways on the modulation of Cox-1 and Cox-2 by LDL or OxLDL. Cox-2 expression was strongly induced, in a dose dependent manner, after a 2h incubation of HUVECs with LDL or Ox-LDL; induction was maximal after 4 hours and sustained for at least 8 hours. The effect was specific for Cox-2, as Cox-1 expression was not affected by LDL or by Ox-LDL. LDL induced the phosphorylation of ERK1/2 to a large extent, while Ox-LDL were less effective; both LDL and Ox-LDL induced the phosphorylation of p38 MAPK. The induction of Cox-2 expression was mainly dependent on the activation of p38 MAPK as the preincubation with the p38 MAPK inhibitor SB203580 (lmM), effectively reduced the LDL or Ox-LDL effect on Cox-2 protein and mRNA as determined by RT-PCR. The MEK1 inhibitor, PD98059 (25mM) slightly affected Cox-2 expression. These data support the concept that LDL and Ox-LDL induce Cox-2 expression in HUVEC and that this effect is mediated through a MAPK dependent pathway. These findings suggest a novel level of regulation for Cox-2 expression induced by LDL and Ox-LDL. ~
FACTOR V LEIDEN AND RISK OF ARTERIAL THROMBOSIS: 3 CASE-CONTROL AND 3 PROSPECTIVE STUDIES BASED ON THE COPENHAGEN CITY HEART STUDY AND 2 META-ANALYSES
K. Juul, A. Tybjaerg-Hansen, R. Steffensen, S. Kofoed, G. Jensen, B.G. Nordest~aard. Copenhagen University Hospitals, Denmark
Background: Factor V Leiden (FVL) is associated with venous thrombosis; however, association between FVL and arterial thrombosis remains controversial. Objective: We investigated FVL as a risk factor for myocardial infarction (MI), ischemic stroke (IS), or non-MI ischemic heart disease (non-MI-IHD). Design: 3 case-control studies, 3 prospective studies with 21 years follow-up, and 2 meta-analyses. Setting: General population, Copenhagen, Denmark. Participants: Copenhagen City Heart Study 20 95 year old participants and patients from Copenhagen University Hospital with MI (n-469; n-493), IS (n-410; n-231) or non-MI-IItD (n-448; n-365), and 7.907 controls. 31.000 individuals from 35 studies for meta-analyses. Measurements: FVL genotype, major cardiovascular risk factors, MI, IS, and non-MI-IHD incidence and prevalence. Results: Prevalences of FVL heterozygotes and homozygotes in controls from the general population were 7.7% and 0.2%. Odds ratios and relative risks of MI in FVL carriers (-heterozygotes+homozygotes) were 1.24 (95%CI:0.91 1.69) and 0.83 (0.58 1.20) in case-control and prospective studies, respectively. Corresponding risks for IS were 0.92 (0.56 1.53) and 0.68(0.45 1.04), and for non-MI-IHD 1.01 (0.71 1.44) and 0.97 (0.66 1.42). Meta-analyses revealed aggregated odds ratios in FVL carriers for MI and adult IS of 1.20 (1.03 1.39) and 1.00 (0.75 1.34), and for premature MI and childhood IS of 1.54 (1.072.22) and 4.79(3.26~.03). Conclusion: Findings from The Copenhagen City Heart Study suggest that FVL is not associated with MI, IS or non-MI-IHD. Meta-analyses suggest, however, that FVL constitutes a modest risk factor for MI, a larger risk factor for premature MI, and a major risk factor for childhood IS. ~
FIBRINOGEN PREDICTS ISCHEMIC STROKE AND ADVANCED ATHEROSCLEROSIS, BUT NOT ECHOLUCENT, RUPTURE-PRONE CAROTID PLAQUES
S.C. Kofoed, H.H. Wittrup, H. Sillesen, B.G. Nordest~aard. Copenhagen
University Hospitals, Denmark Background: Whether the association between fibrinogen and cardiovascular events reflects an association with advanced atherosclerosis in general, or rupture-prone plaques in particular, is unclear. We examined whether
175
fibrinogen predicts incidence of ischemic stroke, advanced atherosclerosis (measured as carotid artery stenosis) and/or echolucent, rupture-prone plaques. Methods and Results: Study 1:8755 Copenhagen City Heart Study stroke free participants; we observed 235 ischemic strokes during six-years of follow-up. Study 2:318 carotid stenosis patients and 1584 age and gender matched controls. Study 3:159 patients with echolucent versus 159 patients with echo-rich carotid artery plaques. Fibrinogen above versus below the median value of 3 g/L predicted risk of ischemic stroke (relative risk: 1.9; 95% CI: 1.42.5). Significant risk was found in men (2.7; 1.74.2) but not in women (1.4; 0.92.0), in young (5.2; 1.126) and middle aged (2.9; 1.6-5.4) but not in the elderly (1.4; 1.02.0). Fibrinogen levels in those with and without ischemic stroke were 3.6 and 3.1 g/L (ANCOVA:p<0.0001). Likewise, in those with and without carotid artery stenosis fibrinogen levels were 4.7 and 3.1 g/L (p<0.0001); equivalent values for high-sensitive C-reactive protein were 3.6 and 1.4 mg/L (p<0.0001). Finally, neither fibrinogen nor highsensitive C-reactive protein levels differed between those with echolucent and echo-rich carotid artery plaques (p-0.61 and p-0.28). Conclusion: Elevated fibrinogen predicts future ischemic strokes, particular in men and in the young and middle aged. This is most likely a reflection of advanced atherosclerosis, rather than an association with rupture-prone plaques.
~HDL
AND OTHER FACTORS IN THE GENETICS OF ISCHEMIC HEART DISEASE
B.G. Nordestgaard 1, A. Tybjaerg-Hansen2. JHerlev University Hospital, 2Copenhagen University Hospital, Denmark Using the Copenhagen City Heart Study, we studied association between rare and common mutations in known candidate genes and risk of ischemic heart disease. The Copenhagen City Heart Study is a prospective cardiovascular population study initiated in 1996. We genotyped > 9000 participants. During 22 years follow-up, 1014 of the participants developed ischemic heart disease while 7882 were free of disease, and thus acted as controls. In addition, we also genotyped 948 independent patients with ischemic heart disease and documented coronary atherosclerosis. Genotype frequencies in these patients were compared with the 7882 controls in a case-control design. We studied mutations in candidate genes for ItDL metabolism including cholesteryl ester transfer protein, lipoprotein lipase, hepatic lipase, apolipoprotein E, and ABCA1. Other genes studied include extracellular superoxide dismutase and alpha-l-antitrypsin deficiency.
~ - ] ] TRANSVASCULAR LOW DENSITY LIPOPROTEIN TRANSPORT IN PATIENTS W I T H DIABETES MELLITUS (TYPE 2): A NON-INVASIVE IN VIVO ISOTOPE TECHNIQUE K. Kornerup, B.G. Nordestgaard, B. Feldt-Rasmussen, K. Borch-Johnsen, K.S. Jensen, J.S. Jensen. Copenhagen University Hospitals, Denmark The increased risk of atherosclerosis among diabetic individuals cannot be explained by conventional cardiovascular risk factors alone. We hypothesized that transvascular lipoprotein transport may be increased in patients with diabetes due to endothelial dysfunction, possibly explaining increased intimal lipoprotein accumulation and thus atherosclerosis. We developed an in vivo method for measurement of transvascular transport of low density lipoprotein (LDL) and applied it in 16 patients with maturity onset diabetes (type 2) and 29 age- and sex-matched controls. Autologous 131-iodinated LDL was reinj ected intravenously in addition to 125-iodinated albumin, and the 1-hour fractional escape rates were taken as indices of transvascular transport. Both parameters were normally distributed and they were tightly correlated (R2-0.69; P<0.0001). Transvascular transport of LDL was 5.4 and 4.1 percent/hour in patients with diabetes and controls (P<0.05); equivalent values for albumin were 6.5 and 5.3 percent/hour (P<0.05). This difference most likely was not caused by altered LDL receptor expression in the liver, glycosylation of LDL in diabetes, small LDL size in diabetic patients, statin use, differences in plasma insulin levels, or by nephropathy in diabetes. In conclusion, we demonstrate that transvascular transport of LDL may be increased in those with type 2 diabetes versus controls. This suggests that lipoprotein flux into the arterial wall could be increased in people with diabetes due to endothelial dysfunction, possibly explaining accelerated development of atherosclerosis in these patients.
73rd EAS Congress