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Bradykinin receptors in the crossroads of atherosclerosis and heart failure
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Lindstedt BRADYKININ RECEPTORS IN THE CROSSROADS OF ATHEROSCLEROSIS AND HEART FAILURE
A. Kuoppala 1, N. Shiota 1, J.O. Kokkonen 1.2, I. Liesmaa 1, K.M. Kostner3, M. Mayranpaa 1, ET. Kovanen 1, K.A. Lindstedt 1. 1 Wihuri Research
Institute, Helsinki; 2Division of Cardiology, Helsinki University Central Hospital, Helsinki, Finland," 3Division of Cardiology, AKtL, Vienna, Austria Recent work in experimental animals suggests that bradykinin (BK) exerts cardioprotective effects, which are mediated through specific BK-receptors (BK-R). However, nothing is known about the regulation of BK type 1 (BK-1R) and type 2 (BK-2R) receptor expression in the pathogenesis of human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n-13) and from normal hearts (n-6) unsuitable for donation. The patients had heart failure due to idiopathic dilated cardiomyopathy (IDC; n-7) or to coronary heart disease (CHD; n-6). In both IDC and CHD hearts, the level of BK-1R mRNA expression was significantly higher (2.8- and 2.1-fold, respectively) than in normal hearts. The induction of BK-1Rs was localized the endothelium of intramyocardial coronary vessels. In contrast, the levels of BK-2R mRNA and BK-2R protein expression were found to be significantly lower in IDC (30% and 45% of control) and CHD (38% and 62% of control) hearts. The down-regulation of BK-2Rs in failing hearts apparently concerned all myocardial cell types, and did not correlate with decreased cellularity, or with the expression pattern of other members of the G protein-coupled receptor superfamily. However, the reduction in BK-2Rs was associated with a decrease in eNOS in both IDC (53% of control) and CHD (43% of control) hearts. These results are the first to suggest that BK-receptors are differentially expressed in the pathogenesis of human heart failure, and that loss of cardioprotective BK-2Rs is a prominent feature in human heart failure. ~
CIRCULATING LEVELS OF OXIDIZED LDL IN TYPE 2 DIABETIC PATIENTS
P.P. Lindstedt, H.M. Bates, E. Lindgren, E. Hedlof. Mercodia AB, Research
& Development, Uppsala, Sweden Oxidized LDL, an atherogenic protein directly involved in the initiation and progression of atherosclerosis. Holvoet et al (Circulation 1998:98:1487 1494) demonstrated that untreated patients with angiographically proven coronary artery disease have significantly elevated plasma levels of oxidized LDL. Since coronary artery disease is the leading cause of death in patients with type 2 diabetes, we decided to compare oxidized LDL levels of chronic hyperglycemic, type 2 diabetes patients, with those of normoglycemic, nondiabetic patients. Oxidized LDL levels were measured with the Mercodia Oxidized LDL ELISA kit, based on the specific monoclonal antibody 4E6. We obtained EDTA-plasma samples from two groups of ambulatory patients from office-based physicians in New York City. In GROUP I (25 nondiabetics), the mean fasting glucose levels were 104 mg/dL (range: 76 134) and the hemoglobin Alc (HbAlc) levels were less than 6.1%. In GROUP II (25 type 2 diabetics), the mean fasting glucose levels were 331 mg/dL (range: 172 560) and the HbAlc levels were greater than 9.9%. The results follow: At oxidized LDL cutpoints of greater than 40 U/L, 60 U/L, 80 U/L, and 90 U/L, there were 24/25 (96%), 14/25 (56%), 5/25 (20%), and 2/25 (8%) nondiabetic patients, respectively. In contrast, at these same oxidized LDL cutpoints, there were 24/25 (96%), 20/25 (80%), 10/25 (40%), and 6/25 (24%) type 2 diabetic patients, respectively. This data clearly shows that patients with type 2 diabetes have a two- to three-fold greater frequency of elevated oxidized LDL values when compared with nondiabetics. These results suggest that elevated circulating levels of oxidized LDL may be useful in detecting type 2 diabetic patients with accelerated atherosclerosis who could benefit from drugs, such as statins, which lower plasma oxidized LDL levels. ~
LDL-RECEPTOR MUTATIONS IN NEW ZEALAND PATIENTS W I T H FAMILIAL HYPERCHOLESTEROLAEMIA
C.J. Lintott 1, C. Bunn 1, L. Henry 2, R.S. Scott 1, J. Mann 3, P. George 1.
1Christchurch Hospital, Christchurch," 2Dunedin Hospital," 3University of Otago, Dunedin, New Zealand This project aimed to determine the prevalence of specific mutations amongst hypercholesterolaemic patients attending Lipid Clinics in the South Island of New Zealand. DNA samples were collected from 181 apparently unrelated probands with fasting cholesterol levels > 8.0 mmol/1. Fifty four patients had a clinical diagnosis of definite familial hypercholesterolaemia (FH)
based on the presence of tendon xanthomata. The remaining 125 patients had a diagnosis of probable FH based on the presence of at least one of the following factors corneal arcus, xanthelasma, premature coronary disease in proband or first degree relative, and/or elevated cholesterol levels in a first degree relative. Samples were initially screened for familial defective apolipoprotein B100 and then were analysed by denaturing HPLC (DHPLC). After automated PCR setup of 7 patient samples plus controls simultaneously, all exons were amplified under one set of conditions, before DHPLC analysis. Duplicate abnormal products were further analysed by direct sequencing. A total of 27 different mutations were identified, including 8 novel mutations, in the 181 probands. Amongst probands with a diagnosis of definite FH, mutations were identified in 46% of individuals. Amongst those without tendon xanthomata, mutations were found in only 8% of patients. Of the 791 living first degree relatives of these probands, cholesterol levels had not been measured in 49% of relatives. Automated DHPLC proved an effective method for mutation analysis in the heterogeneous New Zealand population, and will also have considerable clinical application in the diagnosis of FH through family cascade screening.
~FREE RADICAL-INDUCED MODIFICATION OF HUMAN FIBRINOGEN: IMPLICATIONS F O R ATHEROSCLEROSIS B. Lipinski 1, I. Lipinska 2. IJoslin Diabetes Center, :Institute for Prevention
of Cardiovascular Disease, Harvard Medical School, Boston, MA, USA It is generally believed that all free radicals (FR) are endowed with oxidative properties. We have recently shown utilizing FR generated in a Fenton-type reaction (ascorbic acid/cupric chloride mixture) that they act as reductants rather than oxidants. This observation may thus explain the mechanism of aggregation of purified human fibrinogen (Fbg) where exposed to FR generated in the above system. Addition of human serum albumin (HSA) to the reaction mixture inhibited the FR-induced aggregation of Fbg. In this system HSA plays a role of a sacrificial protein that scavenges FR by reducing its intramolecular disulfide bonds. A characteristic feature of FRmodified Fbg is its remarkable resistance to degradation induced not only with plasminogen activators but with active proteolytic enzymes. Exposure of human plasma to reducing FR resulted in the formation of Fbg-lipoprotein aggregate similar to a macromolecular protein complex (MPC). Although MPC is not clottable with thrombin, it becomes effectively incorporated into fibrin clot thus increasing its mass and renedering it refractory to fibrinolysis. Increased levels of MPC were previously shown to be increased in diabetic patients. In conclusion, FR modification of Fbg lads to the formation of soluble and insoluble complexes that may interact hydrophobically with endothelium causing its dysfunction. Consequently, the deposited fibrinlike material, being resistant to fibrionolytic degradation, persists in the circulation thus contributing to atherosclerosis.
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NON-INVASIVE DETECTION OF LOCAL CEREBRAL ISCHEMIA IN THE HYPERLIPIDEMIC PATIENTS AND POSITIVE CEREBRAL HEMODYNAMICS AFTER SIMVASTATIN TREATMENT
B.M. Lipovetsky, G.V. Kataeva, A.D. Korotkov. IHB St. Petersburg, Russia Objective: Documenting local cerebral blood flow (CBF) disoders in the hyperlipidemic patients (HP) with chronic cerebrovascular disease (CVD) and evaluating CBF changes after the simvastatin treatment. Method: We used the positron emission tomography (PET) with [15-O]-labeled water to evaluate the CBE Our method of analysis is based on standardization of PET images to the stereotactic cerebral atlas. CBF of any cerebral area was assessed in relation to the CBF of the brain stem (in %% to the reference level). Results: The data on 60 cerebral areas (30 on each hemisphere) from 25 HP were analyzed. Among our HP were 8 with angina without signs of CVD (the group 1 for the comparison), 10 with CVD (group 2) and 7 with the ischemic stroke in the past (group 3),the average age of ItP being 55. In all groups the 1st PET study revealed both the cerebral areas with reduced CBF (hypoperfusion) and the areas with luxury perfusion (hyperperfusion). After the 3-month simvastatin treatment LDL-cholesterol decreased from 290 mg/dl to 190 mg/dl. The 2nd PET study showed the improvement of CBF in most areas with hypoperfusion revealed during the 1st PET study. Conclusion: These data make it possible to objectify the local CBF changes in patients and confirm the positive simvastatin effect on the cerebral hemodynamics. Simvastatin was sound to enhance cerebral perfusion and
73rd EAS Congress
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Lindstedt BRADYKININ RECEPTORS IN THE CROSSROADS OF ATHEROSCLEROSIS AND HEART FAILURE
A. Kuoppala 1, N. Shiota 1, J.O. Kokkonen 1.2, I. Liesmaa 1, K.M. Kostner3, M. Mayranpaa 1, ET. Kovanen 1, K.A. Lindstedt 1. 1 Wihuri Research
Institute, Helsinki; 2Division of Cardiology, Helsinki University Central Hospital, Helsinki, Finland," 3Division of Cardiology, AKtL, Vienna, Austria Recent work in experimental animals suggests that bradykinin (BK) exerts cardioprotective effects, which are mediated through specific BK-receptors (BK-R). However, nothing is known about the regulation of BK type 1 (BK-1R) and type 2 (BK-2R) receptor expression in the pathogenesis of human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n-13) and from normal hearts (n-6) unsuitable for donation. The patients had heart failure due to idiopathic dilated cardiomyopathy (IDC; n-7) or to coronary heart disease (CHD; n-6). In both IDC and CHD hearts, the level of BK-1R mRNA expression was significantly higher (2.8- and 2.1-fold, respectively) than in normal hearts. The induction of BK-1Rs was localized the endothelium of intramyocardial coronary vessels. In contrast, the levels of BK-2R mRNA and BK-2R protein expression were found to be significantly lower in IDC (30% and 45% of control) and CHD (38% and 62% of control) hearts. The down-regulation of BK-2Rs in failing hearts apparently concerned all myocardial cell types, and did not correlate with decreased cellularity, or with the expression pattern of other members of the G protein-coupled receptor superfamily. However, the reduction in BK-2Rs was associated with a decrease in eNOS in both IDC (53% of control) and CHD (43% of control) hearts. These results are the first to suggest that BK-receptors are differentially expressed in the pathogenesis of human heart failure, and that loss of cardioprotective BK-2Rs is a prominent feature in human heart failure. ~
CIRCULATING LEVELS OF OXIDIZED LDL IN TYPE 2 DIABETIC PATIENTS
P.P. Lindstedt, H.M. Bates, E. Lindgren, E. Hedlof. Mercodia AB, Research
& Development, Uppsala, Sweden Oxidized LDL, an atherogenic protein directly involved in the initiation and progression of atherosclerosis. Holvoet et al (Circulation 1998:98:1487 1494) demonstrated that untreated patients with angiographically proven coronary artery disease have significantly elevated plasma levels of oxidized LDL. Since coronary artery disease is the leading cause of death in patients with type 2 diabetes, we decided to compare oxidized LDL levels of chronic hyperglycemic, type 2 diabetes patients, with those of normoglycemic, nondiabetic patients. Oxidized LDL levels were measured with the Mercodia Oxidized LDL ELISA kit, based on the specific monoclonal antibody 4E6. We obtained EDTA-plasma samples from two groups of ambulatory patients from office-based physicians in New York City. In GROUP I (25 nondiabetics), the mean fasting glucose levels were 104 mg/dL (range: 76 134) and the hemoglobin Alc (HbAlc) levels were less than 6.1%. In GROUP II (25 type 2 diabetics), the mean fasting glucose levels were 331 mg/dL (range: 172 560) and the HbAlc levels were greater than 9.9%. The results follow: At oxidized LDL cutpoints of greater than 40 U/L, 60 U/L, 80 U/L, and 90 U/L, there were 24/25 (96%), 14/25 (56%), 5/25 (20%), and 2/25 (8%) nondiabetic patients, respectively. In contrast, at these same oxidized LDL cutpoints, there were 24/25 (96%), 20/25 (80%), 10/25 (40%), and 6/25 (24%) type 2 diabetic patients, respectively. This data clearly shows that patients with type 2 diabetes have a two- to three-fold greater frequency of elevated oxidized LDL values when compared with nondiabetics. These results suggest that elevated circulating levels of oxidized LDL may be useful in detecting type 2 diabetic patients with accelerated atherosclerosis who could benefit from drugs, such as statins, which lower plasma oxidized LDL levels. ~
LDL-RECEPTOR MUTATIONS IN NEW ZEALAND PATIENTS W I T H FAMILIAL HYPERCHOLESTEROLAEMIA
C.J. Lintott 1, C. Bunn 1, L. Henry 2, R.S. Scott 1, J. Mann 3, P. George 1.
1Christchurch Hospital, Christchurch," 2Dunedin Hospital," 3University of Otago, Dunedin, New Zealand This project aimed to determine the prevalence of specific mutations amongst hypercholesterolaemic patients attending Lipid Clinics in the South Island of New Zealand. DNA samples were collected from 181 apparently unrelated probands with fasting cholesterol levels > 8.0 mmol/1. Fifty four patients had a clinical diagnosis of definite familial hypercholesterolaemia (FH)
based on the presence of tendon xanthomata. The remaining 125 patients had a diagnosis of probable FH based on the presence of at least one of the following factors corneal arcus, xanthelasma, premature coronary disease in proband or first degree relative, and/or elevated cholesterol levels in a first degree relative. Samples were initially screened for familial defective apolipoprotein B100 and then were analysed by denaturing HPLC (DHPLC). After automated PCR setup of 7 patient samples plus controls simultaneously, all exons were amplified under one set of conditions, before DHPLC analysis. Duplicate abnormal products were further analysed by direct sequencing. A total of 27 different mutations were identified, including 8 novel mutations, in the 181 probands. Amongst probands with a diagnosis of definite FH, mutations were identified in 46% of individuals. Amongst those without tendon xanthomata, mutations were found in only 8% of patients. Of the 791 living first degree relatives of these probands, cholesterol levels had not been measured in 49% of relatives. Automated DHPLC proved an effective method for mutation analysis in the heterogeneous New Zealand population, and will also have considerable clinical application in the diagnosis of FH through family cascade screening.
~FREE RADICAL-INDUCED MODIFICATION OF HUMAN FIBRINOGEN: IMPLICATIONS F O R ATHEROSCLEROSIS B. Lipinski 1, I. Lipinska 2. IJoslin Diabetes Center, :Institute for Prevention
of Cardiovascular Disease, Harvard Medical School, Boston, MA, USA It is generally believed that all free radicals (FR) are endowed with oxidative properties. We have recently shown utilizing FR generated in a Fenton-type reaction (ascorbic acid/cupric chloride mixture) that they act as reductants rather than oxidants. This observation may thus explain the mechanism of aggregation of purified human fibrinogen (Fbg) where exposed to FR generated in the above system. Addition of human serum albumin (HSA) to the reaction mixture inhibited the FR-induced aggregation of Fbg. In this system HSA plays a role of a sacrificial protein that scavenges FR by reducing its intramolecular disulfide bonds. A characteristic feature of FRmodified Fbg is its remarkable resistance to degradation induced not only with plasminogen activators but with active proteolytic enzymes. Exposure of human plasma to reducing FR resulted in the formation of Fbg-lipoprotein aggregate similar to a macromolecular protein complex (MPC). Although MPC is not clottable with thrombin, it becomes effectively incorporated into fibrin clot thus increasing its mass and renedering it refractory to fibrinolysis. Increased levels of MPC were previously shown to be increased in diabetic patients. In conclusion, FR modification of Fbg lads to the formation of soluble and insoluble complexes that may interact hydrophobically with endothelium causing its dysfunction. Consequently, the deposited fibrinlike material, being resistant to fibrionolytic degradation, persists in the circulation thus contributing to atherosclerosis.
•
NON-INVASIVE DETECTION OF LOCAL CEREBRAL ISCHEMIA IN THE HYPERLIPIDEMIC PATIENTS AND POSITIVE CEREBRAL HEMODYNAMICS AFTER SIMVASTATIN TREATMENT
B.M. Lipovetsky, G.V. Kataeva, A.D. Korotkov. IHB St. Petersburg, Russia Objective: Documenting local cerebral blood flow (CBF) disoders in the hyperlipidemic patients (HP) with chronic cerebrovascular disease (CVD) and evaluating CBF changes after the simvastatin treatment. Method: We used the positron emission tomography (PET) with [15-O]-labeled water to evaluate the CBE Our method of analysis is based on standardization of PET images to the stereotactic cerebral atlas. CBF of any cerebral area was assessed in relation to the CBF of the brain stem (in %% to the reference level). Results: The data on 60 cerebral areas (30 on each hemisphere) from 25 HP were analyzed. Among our HP were 8 with angina without signs of CVD (the group 1 for the comparison), 10 with CVD (group 2) and 7 with the ischemic stroke in the past (group 3),the average age of ItP being 55. In all groups the 1st PET study revealed both the cerebral areas with reduced CBF (hypoperfusion) and the areas with luxury perfusion (hyperperfusion). After the 3-month simvastatin treatment LDL-cholesterol decreased from 290 mg/dl to 190 mg/dl. The 2nd PET study showed the improvement of CBF in most areas with hypoperfusion revealed during the 1st PET study. Conclusion: These data make it possible to objectify the local CBF changes in patients and confirm the positive simvastatin effect on the cerebral hemodynamics. Simvastatin was sound to enhance cerebral perfusion and
73rd EAS Congress