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343 3-HYDROXYKYNURENINE – A NEW FACTOR ASSOCIATED WITH CAROTID ATHEROSCLEROSIS, OXIDATIVE STRESS AND INFLAMMATION IN URAEMIA
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Atherosclerosis Supplements 12, no. 1 (2011) 13–184
activity was determined spectrophotometrically, adiponectins and ADMA levels were measured by ELISA method. Our patients have hypercholesterolaemia, high LDL and ApoB levels and parallel with improved renal function, decreased cystatinC and homocysteine levels (p < 0.001). In obese patients (BMI > 30kg/m2 , n = 14) LDL (p < 0.05) and leptin concentrations (58.06 vs. 15.16ng/ml, p < 0.01) were significantly higher than in malnourished group (n = 9). We did not find significant difference between serum adiponectin levels (16.81 vs. 21.61ug/ml) and PON1 activity (91.45 vs. 101.20U/l) in obese and malnourished renal transplant patients. HTLase activity was 438.20 vs. 385.99 U/l) in obese and malnourished transplated patients..After transplantation a significant negative correlation was found between serum PON1 activity and improved renal function (p < 0.01). Between PON1 activity and adiponectin levels there was a significant correlation (p = 0.0276) and between serum PON1 activity and ADMA levels there was a negative, non-significant correlation (p = 0.2302). Between HTLase and PON1 activity and HTLase and PON/HDL ration there was a positive linear correlation (p < 0.03). Dyslipidaemic, obese transplanted patients have high LDL, leptin levels and PON1 activity does not correlate significantly with leptin, ADMA and CRP levels. With improved renal function significant increased in PON1 activity can found. Between serum PON1 and adiponectin there was a positive correlation, between HTLase and ADMA levels we found a significant, negative correlation. 343 3-HYDROXYKYNURENINE − A NEW FACTOR ASSOCIATED WITH CAROTID ATHEROSCLEROSIS, OXIDATIVE STRESS AND INFLAMMATION IN URAEMIA D. Pawlak1 , M. Mysliwiec2 , K. Pawlak1 . 1 Monitored Pharmacotherapy, 2 Nephrology and Clinical Transplantation, Medical University, Bialystok, Poland Objective: Increased oxidative stress (SOX), inflammation and carotid atherosclerosis have been reported in chronic kidney disease (CKD) patients, but their associations with kynurenine (KYN) pathway activation remain unknown. Methods: We determined the plasma concentrations of tryptophan (TRP), KYN, 3-hydroxykynurenine (3-HKYN); SOX markers: Cu/Zn superoxide dismutase (Cu/Zn SOD), total peroxide, high sensitivity C-reactive protein (hs CRP) as a indicator of inflammation, and an early indicator of the systemic atherosclerosis − intima-media thickness (IMT) in 146 CKD patients and healthy controls. Results: The plasma concentrations of TRP was decreased by 40−60% in CKD patients compared to controls, whereas KYN and 3-HKYN levels were increased by 32−96% and 184–306%, respectively. These changes were accompanied by significant increase in the KYN/TRP ratios by 140–240%, and 3-HKYN/KYN ratios by 40–154% in uremics compared to controls. CKD patients showed a significant increase in Cu/Zn SOD, total peroxide, hs CRP and IMT values compared to controls. KYN and 3-HKYN were positively associated with inflammation, SOX markers and IMT in uraemics. Finally, multiple regression analysis identified age and 3-HKYN levels as the independent variables significantly associated with increased IMT in this population (adjusted r2 = 0.304). Conclusions: The results of the present study suggest a relationship between kynurenine pathway activation and increased oxidative stress, inflammation and progression of atherosclerosis in CKD patients. It suggests a new idea that the disturbances of the endogenous kynurenine pathway can play a role in the pathogenesis of arterial damage in this population. 344 LEPTIN TRIGGERS DISTURBED CA2+ TRANSPORT IN MONOCYTES OF OVERWEIGHT PATIENTS J. Tamas ´ Padra, I. Seres, G. Foris, ´ Z. Balogh, G. Konya, ´ Z. Karanyi, ´ G. Paragh. University of Debrecen, Debrecen, Hungary The increased [Ca2+ ]i in monocytes of overweight subjects may be the cause of many concomitant pathological symptoms. The aim of the present study was to elucidate the relationship between the leptin induced NADPH oxidase activation and [Ca2+ ]i homeostasis in human monocytes. Our results are as follows: 1. The basal level of [Ca2+ ]i in resting monocytes of overweight patients (OW monocytes) was higher than that in control cells, whereas the leptininduced peak of Ca2+ signal was lower and the return to basal level was delayed in the OW monocytes. 2. When Ca2+ signals were calculated as areas under time curves (AUC), the signals were more pronounced in OW monocytes than in control cells, as a consequence of injured Ca2+ extrusion from cells. 3. Using different inhibitors, we proved that in control cells the Ca2+ signals originated from intracellular pools, whereas in OW cells they were generated predominantly by Ca2+ -influx from medium. Finally, we found correlation between the leptin induced superoxide anion generation and Ca2+ signals (in AUC). The disturbed [Ca2+ ]i homeostasis in OW monocytes was fully restored in the presence of NADPH oxidase inhibiting fluvastatin. Therefore, statins through decrease in free radical generation have beneficial effect on elevated [Ca2+ ]i and consequently on the obesity.
Poster presentations
345 CHLOROGENIC ACID REDUCE PLASMA LIPIDS AND INFLAMMATORY-RELATED GENE EXPRESSION IN HYPERLIPIDEMIC RABBITS Y.-M. Yu1 , W.-C. Chang2 . 1 Nutrition and Health Sciences, 2 Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan City, Taiwan R.O.C. Atherosclerosis is a major cause of coronary heart disease. Chlorogenic acid (CA) is a phenolic compound present in fruits, vegetables and some drinks, including apples, pears, berries, black tea and coffee. It has been found to have antioxidant, antimutagenic, and immunomodulatory properties. In this study, we investigated the effects of chlorogenic acid on oxidative stress and antiinflammatory gene expression in hyperlipidemic rabbits. Twenty-four NZW male rabbits were assigned randomly into four dietary groups and were sacrificed after eight weeks. The normal group was fed regular rabbit chow and the control group was fed a high fat and cholesterol diet. The probucol group and the CA group were fed the same diet as the control group plus 0.1% (w/w) probucol or chlorogenic acid. The result indicated that the serum levels of total cholesterol, LDL-cholesterol, triglyceride, MDA, luminol-CL and intimal thickening of thoracic aorta were significantly increased in the control group compared with the normal group, and decreased in the probucol and the CA group. The lag time of LDL oxidation in the control group was shorter than the normal group, and longer in the probucol and the CA group. The mRNA expression of VCAM-1 (vascular cell adhesion molecule-1) and MCP-1 (monocyte chemotactic protein-1) were significantly increased in the control group compared with the normal group, and decreased in the probucol and the CA group. In conclusion, chlorogenic acid suppresses the progression of atherosclerosis by lowering serum lipid, lipid oxidation and inflammatory-related gene expression including VCAM-1and MCP-1 in hyperlipidemic rabbits. 346 IMPACT OF CIGARETTE SMOKE ON THE TRANSMIGRATION OF MONOCYTES THROUGH AN ENDOTHELIAL CELL LAYER I. Gallitz1 , R. Janssens1 , K. Stolle1 , Y. Steffen1 , S. Wagner1 , S. Lebrun2 , M. Lietz1 . 1 PHILIP MORRIS Research Laboratories GmbH, Koeln, Germany, 2 PHILIP MORRIS International, Neuchatel, Switzerland The transmigration of monocytes through the vascular endothelium is one of the key steps in the development of atherosclerosis. Cigarette smoking is a known risk factor for the development of atherosclerosis, but the mechanisms involved are not completely understood. To investigate the impact of cigarette smoke on the transmigration of monocytes through an endothelial cell layer, we developed a transmigration assay. Human coronary artery endothelial cells (HCAEC) were grown on transwell filter inserts and treated either directly, i.e., with cigarette smoke bubbled through phosphate-buffered saline (sbPBS) or total particulate matter (TPM), or indirectly, i.e., with the supernatant of sbPBS- or TPMexposed monocytes. The transmigration rate of THP-1 cells (human acute monocytic leukemia cell line) was determined by flow cytometry. Blocking antibodies against surface molecules (e.g., E-selectin, ICAM-1, VCAM-1) were used to investigate the molecular mechanisms involved in the cigarette-smokeenhanced transmigration of THP-1 cells through the endothelial cell layer. Direct treatment showed no effect. Indirect treatment showed a cigarette-smokeenhanced transmigration rate which was higher for TPM than for sbPBS. 347 INVOLVEMENT OF PROTEIN TYROSINE PHOSPHATASE HYPERHOMOCYSTEINEMIA AND HYPERCHOLESTEROLEMIA INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION S. Sharma, M. Singh, P.L. Sharma. Cardiovascular Division, Department of Pharmacology, ISF College of Pharmacy, Moga, India Introduction: Protein tyrosine phosphatase (PTPase) regulates processes involved in pathological vessel wall functions viz. inhibition of survival pathways and reduce the phosphorylation status of enzymes. Aim/Objective: The Present study is designed to investigate involvement of PTPase in vascular endothelium dysfunction (VED) associated with Hyperhomocysteinemia (Hhcy) and hypercholesterolemia (Hch). Furthermore possible downstream mechanisms inhibited by PTPase were also elaborated in both the conditions. Methods and Purpose: Hhcy was induced in a group of male wistar rats by administration of L-Methionine (1.7% w/w., p.o.) while Hch was produced by feeding high cholesterol diet. After 4 weeks of Methionine and Cholesterol rich diet administration, Hhcy was confirmed by estimating serum Hhcy conc. (Hhcy > 12 mm/l) and Hch was confirmed by increase in serum conc. of cholesterol, TG-c and decrease in HDL-c level. VED was assessed, in terms of attenuation of endothelium-dependent relaxation (EDR), a decrease in serum nitrate/nitrite (N/N) level, as well as mRNA expression of eNOS (rtPCR) and disruption of integrity of vascular endothelium (Electron microscopy). Results: Sodium orthovanadate (SOV), (8 mg/kg, p.o., 16 mg/kg, p.o and 24 mg/kg, p.o) and atorvastatin (30 mg/kg, p.o) (positive control) treatment significantly improved EDR, Serum N/N, mRNA expression of eNOS and integrity of vascular endothelium. However, this ameliorative effect of SOV was
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
activity was determined spectrophotometrically, adiponectins and ADMA levels were measured by ELISA method. Our patients have hypercholesterolaemia, high LDL and ApoB levels and parallel with improved renal function, decreased cystatinC and homocysteine levels (p < 0.001). In obese patients (BMI > 30kg/m2 , n = 14) LDL (p < 0.05) and leptin concentrations (58.06 vs. 15.16ng/ml, p < 0.01) were significantly higher than in malnourished group (n = 9). We did not find significant difference between serum adiponectin levels (16.81 vs. 21.61ug/ml) and PON1 activity (91.45 vs. 101.20U/l) in obese and malnourished renal transplant patients. HTLase activity was 438.20 vs. 385.99 U/l) in obese and malnourished transplated patients..After transplantation a significant negative correlation was found between serum PON1 activity and improved renal function (p < 0.01). Between PON1 activity and adiponectin levels there was a significant correlation (p = 0.0276) and between serum PON1 activity and ADMA levels there was a negative, non-significant correlation (p = 0.2302). Between HTLase and PON1 activity and HTLase and PON/HDL ration there was a positive linear correlation (p < 0.03). Dyslipidaemic, obese transplanted patients have high LDL, leptin levels and PON1 activity does not correlate significantly with leptin, ADMA and CRP levels. With improved renal function significant increased in PON1 activity can found. Between serum PON1 and adiponectin there was a positive correlation, between HTLase and ADMA levels we found a significant, negative correlation. 343 3-HYDROXYKYNURENINE − A NEW FACTOR ASSOCIATED WITH CAROTID ATHEROSCLEROSIS, OXIDATIVE STRESS AND INFLAMMATION IN URAEMIA D. Pawlak1 , M. Mysliwiec2 , K. Pawlak1 . 1 Monitored Pharmacotherapy, 2 Nephrology and Clinical Transplantation, Medical University, Bialystok, Poland Objective: Increased oxidative stress (SOX), inflammation and carotid atherosclerosis have been reported in chronic kidney disease (CKD) patients, but their associations with kynurenine (KYN) pathway activation remain unknown. Methods: We determined the plasma concentrations of tryptophan (TRP), KYN, 3-hydroxykynurenine (3-HKYN); SOX markers: Cu/Zn superoxide dismutase (Cu/Zn SOD), total peroxide, high sensitivity C-reactive protein (hs CRP) as a indicator of inflammation, and an early indicator of the systemic atherosclerosis − intima-media thickness (IMT) in 146 CKD patients and healthy controls. Results: The plasma concentrations of TRP was decreased by 40−60% in CKD patients compared to controls, whereas KYN and 3-HKYN levels were increased by 32−96% and 184–306%, respectively. These changes were accompanied by significant increase in the KYN/TRP ratios by 140–240%, and 3-HKYN/KYN ratios by 40–154% in uremics compared to controls. CKD patients showed a significant increase in Cu/Zn SOD, total peroxide, hs CRP and IMT values compared to controls. KYN and 3-HKYN were positively associated with inflammation, SOX markers and IMT in uraemics. Finally, multiple regression analysis identified age and 3-HKYN levels as the independent variables significantly associated with increased IMT in this population (adjusted r2 = 0.304). Conclusions: The results of the present study suggest a relationship between kynurenine pathway activation and increased oxidative stress, inflammation and progression of atherosclerosis in CKD patients. It suggests a new idea that the disturbances of the endogenous kynurenine pathway can play a role in the pathogenesis of arterial damage in this population. 344 LEPTIN TRIGGERS DISTURBED CA2+ TRANSPORT IN MONOCYTES OF OVERWEIGHT PATIENTS J. Tamas ´ Padra, I. Seres, G. Foris, ´ Z. Balogh, G. Konya, ´ Z. Karanyi, ´ G. Paragh. University of Debrecen, Debrecen, Hungary The increased [Ca2+ ]i in monocytes of overweight subjects may be the cause of many concomitant pathological symptoms. The aim of the present study was to elucidate the relationship between the leptin induced NADPH oxidase activation and [Ca2+ ]i homeostasis in human monocytes. Our results are as follows: 1. The basal level of [Ca2+ ]i in resting monocytes of overweight patients (OW monocytes) was higher than that in control cells, whereas the leptininduced peak of Ca2+ signal was lower and the return to basal level was delayed in the OW monocytes. 2. When Ca2+ signals were calculated as areas under time curves (AUC), the signals were more pronounced in OW monocytes than in control cells, as a consequence of injured Ca2+ extrusion from cells. 3. Using different inhibitors, we proved that in control cells the Ca2+ signals originated from intracellular pools, whereas in OW cells they were generated predominantly by Ca2+ -influx from medium. Finally, we found correlation between the leptin induced superoxide anion generation and Ca2+ signals (in AUC). The disturbed [Ca2+ ]i homeostasis in OW monocytes was fully restored in the presence of NADPH oxidase inhibiting fluvastatin. Therefore, statins through decrease in free radical generation have beneficial effect on elevated [Ca2+ ]i and consequently on the obesity.
Poster presentations
345 CHLOROGENIC ACID REDUCE PLASMA LIPIDS AND INFLAMMATORY-RELATED GENE EXPRESSION IN HYPERLIPIDEMIC RABBITS Y.-M. Yu1 , W.-C. Chang2 . 1 Nutrition and Health Sciences, 2 Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan City, Taiwan R.O.C. Atherosclerosis is a major cause of coronary heart disease. Chlorogenic acid (CA) is a phenolic compound present in fruits, vegetables and some drinks, including apples, pears, berries, black tea and coffee. It has been found to have antioxidant, antimutagenic, and immunomodulatory properties. In this study, we investigated the effects of chlorogenic acid on oxidative stress and antiinflammatory gene expression in hyperlipidemic rabbits. Twenty-four NZW male rabbits were assigned randomly into four dietary groups and were sacrificed after eight weeks. The normal group was fed regular rabbit chow and the control group was fed a high fat and cholesterol diet. The probucol group and the CA group were fed the same diet as the control group plus 0.1% (w/w) probucol or chlorogenic acid. The result indicated that the serum levels of total cholesterol, LDL-cholesterol, triglyceride, MDA, luminol-CL and intimal thickening of thoracic aorta were significantly increased in the control group compared with the normal group, and decreased in the probucol and the CA group. The lag time of LDL oxidation in the control group was shorter than the normal group, and longer in the probucol and the CA group. The mRNA expression of VCAM-1 (vascular cell adhesion molecule-1) and MCP-1 (monocyte chemotactic protein-1) were significantly increased in the control group compared with the normal group, and decreased in the probucol and the CA group. In conclusion, chlorogenic acid suppresses the progression of atherosclerosis by lowering serum lipid, lipid oxidation and inflammatory-related gene expression including VCAM-1and MCP-1 in hyperlipidemic rabbits. 346 IMPACT OF CIGARETTE SMOKE ON THE TRANSMIGRATION OF MONOCYTES THROUGH AN ENDOTHELIAL CELL LAYER I. Gallitz1 , R. Janssens1 , K. Stolle1 , Y. Steffen1 , S. Wagner1 , S. Lebrun2 , M. Lietz1 . 1 PHILIP MORRIS Research Laboratories GmbH, Koeln, Germany, 2 PHILIP MORRIS International, Neuchatel, Switzerland The transmigration of monocytes through the vascular endothelium is one of the key steps in the development of atherosclerosis. Cigarette smoking is a known risk factor for the development of atherosclerosis, but the mechanisms involved are not completely understood. To investigate the impact of cigarette smoke on the transmigration of monocytes through an endothelial cell layer, we developed a transmigration assay. Human coronary artery endothelial cells (HCAEC) were grown on transwell filter inserts and treated either directly, i.e., with cigarette smoke bubbled through phosphate-buffered saline (sbPBS) or total particulate matter (TPM), or indirectly, i.e., with the supernatant of sbPBS- or TPMexposed monocytes. The transmigration rate of THP-1 cells (human acute monocytic leukemia cell line) was determined by flow cytometry. Blocking antibodies against surface molecules (e.g., E-selectin, ICAM-1, VCAM-1) were used to investigate the molecular mechanisms involved in the cigarette-smokeenhanced transmigration of THP-1 cells through the endothelial cell layer. Direct treatment showed no effect. Indirect treatment showed a cigarette-smokeenhanced transmigration rate which was higher for TPM than for sbPBS. 347 INVOLVEMENT OF PROTEIN TYROSINE PHOSPHATASE HYPERHOMOCYSTEINEMIA AND HYPERCHOLESTEROLEMIA INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION S. Sharma, M. Singh, P.L. Sharma. Cardiovascular Division, Department of Pharmacology, ISF College of Pharmacy, Moga, India Introduction: Protein tyrosine phosphatase (PTPase) regulates processes involved in pathological vessel wall functions viz. inhibition of survival pathways and reduce the phosphorylation status of enzymes. Aim/Objective: The Present study is designed to investigate involvement of PTPase in vascular endothelium dysfunction (VED) associated with Hyperhomocysteinemia (Hhcy) and hypercholesterolemia (Hch). Furthermore possible downstream mechanisms inhibited by PTPase were also elaborated in both the conditions. Methods and Purpose: Hhcy was induced in a group of male wistar rats by administration of L-Methionine (1.7% w/w., p.o.) while Hch was produced by feeding high cholesterol diet. After 4 weeks of Methionine and Cholesterol rich diet administration, Hhcy was confirmed by estimating serum Hhcy conc. (Hhcy > 12 mm/l) and Hch was confirmed by increase in serum conc. of cholesterol, TG-c and decrease in HDL-c level. VED was assessed, in terms of attenuation of endothelium-dependent relaxation (EDR), a decrease in serum nitrate/nitrite (N/N) level, as well as mRNA expression of eNOS (rtPCR) and disruption of integrity of vascular endothelium (Electron microscopy). Results: Sodium orthovanadate (SOV), (8 mg/kg, p.o., 16 mg/kg, p.o and 24 mg/kg, p.o) and atorvastatin (30 mg/kg, p.o) (positive control) treatment significantly improved EDR, Serum N/N, mRNA expression of eNOS and integrity of vascular endothelium. However, this ameliorative effect of SOV was