- Email: [email protected]
Homocysteine induces vascular expression of TIMP-1, PCNA and gelatinolytic activity in vivo and in vitro
Romuk of the inflammatory state than other inflammatory markers in asymptomatic carriers of the -455G/A fibrinogen polymorphism. ~-~
ATORVASTATIN MODULATES VEGF AND VEGFR-2 EXPRESSION IN APOLIPOPROTEIN E DEFICIENT MICE
M. Perez-Ilzarbe, B. Nespereira, R Fernandez, J.A. Paramo, J.A. Rodriguez.
University of Navarra, Pamplona, Navarra, Spain Vascular endothelial growth factor (VEGF) is the main responsible of anglogenesis and maintenance of endothelial integrity. In hypercholesterolemia, a well-known atherosclerosis risk factor associated with endothelial dysfunction and increased reactive oxygen species, upregulation of VEGF and VEGF receptor-2 (VEGFR-2) expression has been shown in the vessel wall. HMG-CoA reductase inhibitors (statins) have beneficial effects independent of lipid lowering. We aimed to determine the effect of atorvastatin on aortic VEGF and VEGFR-2 expression in a murine model ofhypercholesterolemia. Four groups of 3 months old C57BL/6 mice were used: wild type (WT; n-7) and apolipoprotein E deficient (apoE; n-7) receiving regular chow, and other two groups treated with atorvastatin (2.5 mg/Kg day; WT-AT, apoE-AT; n-7). Mice were sacrificed after 4 weeks of treatment. Total cholesterol, triglycerides, lipid peroxidation (TBARS) and VEGF were measured in plasma. Aortic VEGF and VEGFR-2 mRNA expression was measured by real-time quantitative PCR. ApoE, exhibiting higher levels of plasma lipid peroxidation (p<0.05), had increased levels of VEGF in plasma (p<0.01), and higher expression of VEGF and VEGFR-2 mRNA in aorta (p<0.01) than WT. Atorvastatin treatment reduced plasma cholesterol (24% reduction, p<0.05), triglycerides, VEGF levels and lipid peroxidation (p<0.05). Atorvastatin reduced VEGF (p<0.05) but upregulated VEGFR-2 (p<0.05) expression in aorta of WT and apoE mice. Increased VEGF and VEGFR-2 expression in hypercolesterolemia could be a vascular homeostatic mechanism induced by oxidative stress. Atorvastatin differential effect on VEGF and VEGFR-2 expression could be an additional mechanism for the vascular effects of statins, mostly independent of cholesterol lowering. ~
HOMOCYSTEINE INDUCES VASCULAR EXPRESSION OF TIMP-1, PCNA AND GELATINOLYTIC ACTIVITY IN VIVO
AND IN VITRO J.A. Rodriguez, R. Arias, M. Perez-Ilzarbe, B. Nespereira, P. Fernandez, J.A. Paramo. University of Navarra, Pamplona, Spain Abnormally high levels of plasma homocysteine (Hcys) have been associated with an increased risk of cardiovascular disease, although the mechanism of action is not known yet. This study aimed to assess in vivo, in an acute model of murine hiperhomocysteinemia, and in vitro, in vascular smooth muscle cells (VSMC), whether Hcys could modulate the expression of proteins involved in extracellular matrix (ECM) metabolism. Primary cultures of VSMC from porcine coronary arteries were treated with 250 uM Hcys. Three months old C57BL/6 mice were injected intraperitoneally (ip) with saline (n-5), Hcys (100 mg/Kg day, n-5) or received methionine (1 g/Kg day, n-5) in drinking water. Mice were sacrificed after 4 days of treatment. Aortic TIMP-1, MMP-2 and PCNA expression were quantified by western blotting and characterized by immunohistochemistry. MMP-2 activity was assessed using gelatin zymography. Aortas from mice treated with Hcys (ip) or receiving Met exhibited higher expression of TIMP-1 (p <0.05) and PCNA (p<0.05) than the control mice. Increased TIMP-1 and PCNA immunostaining was observed in the smooth muscle of the medial layer. Similarly, Hcys-treated VSMC exhibited increased expression ofTIMP-1 (p<0.05) and PCNA (p<0.05), and higher gelatinolytic activity (p<0.05). The induction of TIMP-1 and MMP-2 upon Hcys stimulation would indicate increased ECM metabolism, while augmented PCNA would suggest VSMC proliferation in our acute experimental model. Both Hcys-related mechanisms are relevant in the onset and progression of atherosclerosis.
~THE
NADPH-OXYDASE p22phox C242T POLYMORPHISM IS ASSOCIATED W I T H AN INCREASED RISK OF ATHEROSCLEROSIS
M. Roest 1, J. Defesche 2, J. Kastelein2, R. Voorbij 1. JResearch Laboratory
of Clinical Chemistry, 2Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands Oxygen radicals may enhance the atherosclerotic process via LDL-oxidation. The major source of superoxide anion generation in endothelial cells is NADPH-oxidase. A C242T polymorphism in the p22phox subunit of
195
NADPH-oxidase is associated with reduced superoxide anion generation and therefore an important factor for the oxydative state in the arterial wall. We have studied the contribution of the p22phox C242T polymorphism of NADPH-oxidase to atherosclerosis in 187 subjects with Familial Hypercholesterolaemia, using B-mode ultrasound measurements of the carotid arterial wall. Arterial wall thickness was expressed with intima media thickness (IMT). Subjects who were NADPH-oxidase p22phox C242 homozygous had a mean IMT of 0.79 cm, which was 6% lower than in C242T heterozygotes (mean IMT-0.84 cm; P-0.03) and 13% lower than in 242T homozygotes (mean IMT of 0.89 cm; P-0.01). Furthermore, a strong interaction was observed between the NAPDH-oxidase p22phox C242T polymorphism and total cholesterol levels. Subjects who were in the highest tertile of cholesterol levels and who were 242T homozygous had a 42% increased mean IMT value compared to subjects who were in the lowest tertile of total cholesterol levels and C242 homozygous. Our findings of a relationship between genetic predisposition to low superoxide anion production in the arterial wall (NADPH-oxidase p22phox 242T polymorphism) and increased atherosclerosis are in sharp contrast with the general believe that increased oxygen radical formation contributes to increased atherosclerosis progression. ~
CENTRAL INTRACARDIAL AND CEREBRAL HEMODYNAMICS IN HYPERTENSIVE AND NORMOTENSIVE STROKE PATIENTS
T. Romanjuk, S. Kuznetsova. Institute of Gerontology, Kiev, Ukraine The central, intracardial and cerebral hemodynamics and their relationships in normotensive and hypertensive patients with ischaemic stroke were studied. Twenty patients with mild-to-moderate hypertension (group I) and 18 normotensives (group II), both 1 0 yrs after ischaemic stroke were examined by echocardiography and Doppler sonography techniques. Mean age of pts was 64.3+2.1 yrs (group I) and 59.5+3.1 yrs (group II). Group I showed significantly increased blood pressure (BP), total peripheral vascular resistance (PVR) and total elastic resistance (Eo), linear blood flow velocity in artery cerebri media (ACM) and artery cerebri posterior (ACP). Both groups were characterized of hyperkinetic type of hemodynamics with increased cardiac index (CI), enlarged end-diastolic left ventricular (LV) volume (EDV) and decreased ejection fraction (EF) in comparison with healthy people. High frequency of LVH was characteristic of stroke pts: 67% pts had LVH with eccentric one (56%). LVH frequency didn't depend on BP level. 72% pts in group I and 40% in group II had EF < 55%. Blood flow asymmetry in intracranial vessels was more pronounced in hypertensives. Circumferential resistance index (RI) of extracranial vessels was significantly higher in normotensives, evidencing for possible stenosis of carotid arteries. Cerebral ischemic dysgemia in stroke pts are closely connected with disorders in central and intracardial hemodynamics. Hyperkinetic hemodynamics, eccentric LVH and heart contractility deterioration are common findings both in normotensive and hypertensive pts. Hypertensive stroke pts show increased intracranial blood flow asymmetry and linear flow velocities. Stenosis of carotid arteries was more pronounced in normotensive stroke pts.
~THE
SERUM TRANSFORMING GROWTH FACTOR-BETA (TGF-fi) LEVEL IN PATIENTS W I T H CORONARY HEART DISEASE
E.B. Romuk 1, B. Skrzep-Poloczek 1, C. Wojciechowska2, A.R. Tomasik2, J. Szygula2, E. Birkner 1, J. Wodniecki 2. 1Department of Biochemistry, 211
Department of Cardiology, Silesian Medical University, Zabrze, Poland
Background: There is strong epidemiological evidence that endothelium is the primary target for the development of atherosclerosis. Injury to the endothelium manifests in increased expression of many circulating cell products. TGF-[3 may have a protective role in atherosclerosis. In this study we searched changes in TGF-B level during coronary heart disease. Materials and Methods: We have studied 15 men with stable heart disease, 15 with unstable heart disease and 15 men from control group. Serum level of TGF-[3 was determined with commercially available immunoassay kit. Results: We have observed statistically significant increased in TGF-[3 level in unstable coronary heart disease and stable coronary heart disease patients comparing to the control group. Conclusion: Both forms of coronary artery disease are characterized by increased levels of TGF-[3 however we can not infer the role of TGF-[3 in unstable angina on basis of our study.
73rd EAS Congress
ATORVASTATIN MODULATES VEGF AND VEGFR-2 EXPRESSION IN APOLIPOPROTEIN E DEFICIENT MICE
M. Perez-Ilzarbe, B. Nespereira, R Fernandez, J.A. Paramo, J.A. Rodriguez.
University of Navarra, Pamplona, Navarra, Spain Vascular endothelial growth factor (VEGF) is the main responsible of anglogenesis and maintenance of endothelial integrity. In hypercholesterolemia, a well-known atherosclerosis risk factor associated with endothelial dysfunction and increased reactive oxygen species, upregulation of VEGF and VEGF receptor-2 (VEGFR-2) expression has been shown in the vessel wall. HMG-CoA reductase inhibitors (statins) have beneficial effects independent of lipid lowering. We aimed to determine the effect of atorvastatin on aortic VEGF and VEGFR-2 expression in a murine model ofhypercholesterolemia. Four groups of 3 months old C57BL/6 mice were used: wild type (WT; n-7) and apolipoprotein E deficient (apoE; n-7) receiving regular chow, and other two groups treated with atorvastatin (2.5 mg/Kg day; WT-AT, apoE-AT; n-7). Mice were sacrificed after 4 weeks of treatment. Total cholesterol, triglycerides, lipid peroxidation (TBARS) and VEGF were measured in plasma. Aortic VEGF and VEGFR-2 mRNA expression was measured by real-time quantitative PCR. ApoE, exhibiting higher levels of plasma lipid peroxidation (p<0.05), had increased levels of VEGF in plasma (p<0.01), and higher expression of VEGF and VEGFR-2 mRNA in aorta (p<0.01) than WT. Atorvastatin treatment reduced plasma cholesterol (24% reduction, p<0.05), triglycerides, VEGF levels and lipid peroxidation (p<0.05). Atorvastatin reduced VEGF (p<0.05) but upregulated VEGFR-2 (p<0.05) expression in aorta of WT and apoE mice. Increased VEGF and VEGFR-2 expression in hypercolesterolemia could be a vascular homeostatic mechanism induced by oxidative stress. Atorvastatin differential effect on VEGF and VEGFR-2 expression could be an additional mechanism for the vascular effects of statins, mostly independent of cholesterol lowering. ~
HOMOCYSTEINE INDUCES VASCULAR EXPRESSION OF TIMP-1, PCNA AND GELATINOLYTIC ACTIVITY IN VIVO
AND IN VITRO J.A. Rodriguez, R. Arias, M. Perez-Ilzarbe, B. Nespereira, P. Fernandez, J.A. Paramo. University of Navarra, Pamplona, Spain Abnormally high levels of plasma homocysteine (Hcys) have been associated with an increased risk of cardiovascular disease, although the mechanism of action is not known yet. This study aimed to assess in vivo, in an acute model of murine hiperhomocysteinemia, and in vitro, in vascular smooth muscle cells (VSMC), whether Hcys could modulate the expression of proteins involved in extracellular matrix (ECM) metabolism. Primary cultures of VSMC from porcine coronary arteries were treated with 250 uM Hcys. Three months old C57BL/6 mice were injected intraperitoneally (ip) with saline (n-5), Hcys (100 mg/Kg day, n-5) or received methionine (1 g/Kg day, n-5) in drinking water. Mice were sacrificed after 4 days of treatment. Aortic TIMP-1, MMP-2 and PCNA expression were quantified by western blotting and characterized by immunohistochemistry. MMP-2 activity was assessed using gelatin zymography. Aortas from mice treated with Hcys (ip) or receiving Met exhibited higher expression of TIMP-1 (p <0.05) and PCNA (p<0.05) than the control mice. Increased TIMP-1 and PCNA immunostaining was observed in the smooth muscle of the medial layer. Similarly, Hcys-treated VSMC exhibited increased expression ofTIMP-1 (p<0.05) and PCNA (p<0.05), and higher gelatinolytic activity (p<0.05). The induction of TIMP-1 and MMP-2 upon Hcys stimulation would indicate increased ECM metabolism, while augmented PCNA would suggest VSMC proliferation in our acute experimental model. Both Hcys-related mechanisms are relevant in the onset and progression of atherosclerosis.
~THE
NADPH-OXYDASE p22phox C242T POLYMORPHISM IS ASSOCIATED W I T H AN INCREASED RISK OF ATHEROSCLEROSIS
M. Roest 1, J. Defesche 2, J. Kastelein2, R. Voorbij 1. JResearch Laboratory
of Clinical Chemistry, 2Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands Oxygen radicals may enhance the atherosclerotic process via LDL-oxidation. The major source of superoxide anion generation in endothelial cells is NADPH-oxidase. A C242T polymorphism in the p22phox subunit of
195
NADPH-oxidase is associated with reduced superoxide anion generation and therefore an important factor for the oxydative state in the arterial wall. We have studied the contribution of the p22phox C242T polymorphism of NADPH-oxidase to atherosclerosis in 187 subjects with Familial Hypercholesterolaemia, using B-mode ultrasound measurements of the carotid arterial wall. Arterial wall thickness was expressed with intima media thickness (IMT). Subjects who were NADPH-oxidase p22phox C242 homozygous had a mean IMT of 0.79 cm, which was 6% lower than in C242T heterozygotes (mean IMT-0.84 cm; P-0.03) and 13% lower than in 242T homozygotes (mean IMT of 0.89 cm; P-0.01). Furthermore, a strong interaction was observed between the NAPDH-oxidase p22phox C242T polymorphism and total cholesterol levels. Subjects who were in the highest tertile of cholesterol levels and who were 242T homozygous had a 42% increased mean IMT value compared to subjects who were in the lowest tertile of total cholesterol levels and C242 homozygous. Our findings of a relationship between genetic predisposition to low superoxide anion production in the arterial wall (NADPH-oxidase p22phox 242T polymorphism) and increased atherosclerosis are in sharp contrast with the general believe that increased oxygen radical formation contributes to increased atherosclerosis progression. ~
CENTRAL INTRACARDIAL AND CEREBRAL HEMODYNAMICS IN HYPERTENSIVE AND NORMOTENSIVE STROKE PATIENTS
T. Romanjuk, S. Kuznetsova. Institute of Gerontology, Kiev, Ukraine The central, intracardial and cerebral hemodynamics and their relationships in normotensive and hypertensive patients with ischaemic stroke were studied. Twenty patients with mild-to-moderate hypertension (group I) and 18 normotensives (group II), both 1 0 yrs after ischaemic stroke were examined by echocardiography and Doppler sonography techniques. Mean age of pts was 64.3+2.1 yrs (group I) and 59.5+3.1 yrs (group II). Group I showed significantly increased blood pressure (BP), total peripheral vascular resistance (PVR) and total elastic resistance (Eo), linear blood flow velocity in artery cerebri media (ACM) and artery cerebri posterior (ACP). Both groups were characterized of hyperkinetic type of hemodynamics with increased cardiac index (CI), enlarged end-diastolic left ventricular (LV) volume (EDV) and decreased ejection fraction (EF) in comparison with healthy people. High frequency of LVH was characteristic of stroke pts: 67% pts had LVH with eccentric one (56%). LVH frequency didn't depend on BP level. 72% pts in group I and 40% in group II had EF < 55%. Blood flow asymmetry in intracranial vessels was more pronounced in hypertensives. Circumferential resistance index (RI) of extracranial vessels was significantly higher in normotensives, evidencing for possible stenosis of carotid arteries. Cerebral ischemic dysgemia in stroke pts are closely connected with disorders in central and intracardial hemodynamics. Hyperkinetic hemodynamics, eccentric LVH and heart contractility deterioration are common findings both in normotensive and hypertensive pts. Hypertensive stroke pts show increased intracranial blood flow asymmetry and linear flow velocities. Stenosis of carotid arteries was more pronounced in normotensive stroke pts.
~THE
SERUM TRANSFORMING GROWTH FACTOR-BETA (TGF-fi) LEVEL IN PATIENTS W I T H CORONARY HEART DISEASE
E.B. Romuk 1, B. Skrzep-Poloczek 1, C. Wojciechowska2, A.R. Tomasik2, J. Szygula2, E. Birkner 1, J. Wodniecki 2. 1Department of Biochemistry, 211
Department of Cardiology, Silesian Medical University, Zabrze, Poland
Background: There is strong epidemiological evidence that endothelium is the primary target for the development of atherosclerosis. Injury to the endothelium manifests in increased expression of many circulating cell products. TGF-[3 may have a protective role in atherosclerosis. In this study we searched changes in TGF-B level during coronary heart disease. Materials and Methods: We have studied 15 men with stable heart disease, 15 with unstable heart disease and 15 men from control group. Serum level of TGF-[3 was determined with commercially available immunoassay kit. Results: We have observed statistically significant increased in TGF-[3 level in unstable coronary heart disease and stable coronary heart disease patients comparing to the control group. Conclusion: Both forms of coronary artery disease are characterized by increased levels of TGF-[3 however we can not infer the role of TGF-[3 in unstable angina on basis of our study.
73rd EAS Congress