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PO9-237 IDENTIFICATION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 GENE EXPRESSION IN HUMAN CAROTID PLAQUES
Poster Sessions PO9 Inflammation and atherosclerosis endothelium. We therefore determined the transcriptional profile of human umbilical vein endothelial cells (HUVEC) transduced with Nrf2 expressing adenovirus, using Affymetrix gene array technology. Among the genes significantly down-regulated by over-expression of Nrf2, we identified thioredoxin interacting protein (TXNIP), which was down-regulated by 4.7-fold at 36 h and 3.3-fold at 72 h (p<0.05). This was further verified by real time-PCR. Inasmuch as TXNIP inhibits thioredoxin activity thus promoting inflammation in endothelial cells, we chose to study further the mechanism by which Nrf2 regulates TXNIP. Methods and results: TXNIP mRNA expression was down-regulated by increased multiplicities of infection (MOI) of AdNrf2 as well as increased concentrations of L-sulforaphane (L-sulf), a known activator of Nrf2. Maximal down-regulation was observed at 12 h at both mRNA and protein levels using 10 μM L-sulf. Importantly, blocking of Nrf2 expression by siRNA technique attenuated the down-regulation of TXNIP mRNA by L-sulf. Also the basal expression of TXNIP was significantly increased by Nrf2 siRNA. In order to study whether the down-regulation was transcriptional, we cloned the -3224 nt promoter fragment of TXNIP to a luciferase reporter plasmid and showed reduced luciferase activity on L-sulf exposure. Conclusions: We have shown that TXNIP is uniquely down-regulated at the transcriptional level by Nrf2. We postulate that reduced TXNIP expression may contribute to the anti-inflammatory effects of Nrf2. PATHOBIOLOGICAL ROLE OF ADVANCED GLYCATION ENDPRODUCTS VIA MAPK DEPENDENT PATHWAY
T.S. Kang 1 , H.M. Kwon 2 , Y.W. Yoon 2 , S. Rim 2 , B.K. Hong 2 , H.S. Kim 2 , H-S. Seo 3 . 1 Cardiology/Dankook Univ. Hospital, Chun-Ahn, South Korea; 2 Cardiology/Yonsei Univ.Hospital, Seoul, South Korea; 3 Cardiology Dept, Soochunhyang University Hospital, Seoul, South Korea Advanced glycation endproducts(AGEs) enhance the immunoinflammatory reaction in diabetic vasculopathy through receptors for AGE (RAGE). Recently, AGEs have been reported to play a role in neointimal formation and increase the rate of in-stent restenosis(ISR) in the diabetic coronary artery disease patients treated with stents. However, the potential roles and pathogenic mechanisms of AGE in vascular smooth muscle cell(VSMC) proliferation and ISR remain unclear. We sought to determine the AGEs related pathobiological mechanism of diabetic vasculopathy. Serum levels of AGEs were analyzed by fluorescent intensity method in the diabetic PCI patients. High AGEs level was independent risk factors for ISR by multivariate analysis. The RAGE expression in human atheroma was assessed by immunohistochemistry. Rat aortic smooth muscle cell(RASMC) culture was done with AGEs stimulation. Western blotting was performed to assess the activation of mitogen-associated protein kinase(MAPK) system in the cultured VSMC. AGEs stimulated VSMC proliferation and were associated with increased phosphorylation of ERK, JUN, and p-38 by time and dose dependent manner. The ERKs activation by AGEs was decreased by RNA interference for RAGE, indicating that AGEs activated ERKs via RAGE. AGEs may play a key role in VSMC proliferation and increase ISR in patients with diabetes melitus. Activation of VSMC proliferation by MAPK system and increased formation of ROS may be the possible mechanism of AGEs induced diabetic vasculopathy PO9-236
CARDIOVASCULAR PATHOLOGY IN PATIENTS WITH CHRONIC KIDNEY DISEASE IS ASSOCIATED WITH ENHANCED CYTOKINE SECRETION BY MONOCYTES
V.J.U. Galchinskaya, P.S. Semenovykh. Department of Nephrology, Institute of Therapy of AMS of Ukraine, Kharkov, Ukraine The chronic kidney disease (CKD) is strongly associated with increased risk of cardiovascular pathology. Chronic inflammation and activation of immune cells, in particular monocytes may promote the development of atherogenesis in the coronary vessel as well as progression of renal pathology. This research was conducted to investigate the production of proinflammatory cytokines - interleukin 1β (IL-1β) and interleukin 6 (IL-6) by monocytes in patients with CKD and coronary artery disease. The investigation was done on freshly isolated peripheral blood-derived monocytes from patients with CKD and coronary artery disease combination (basic group) and patients with CKD without cardiovascular pathology
(control group). IL-1β and IL-6 levels in incubation medium of monocytes was determined by ELISA. To stimulate cellular response angiotensin II (10E-7 M) was used. Our results showed the enhanced cytokine production by monocytes in patients with combination of CKD and coronary artery disease. In control group levels of IL-1β and IL-6 in the incubation media of monocytes were (375,4±38,9) pg/ml and (120±16,9) pg/ml, P<0,05, respectively. Monocytes from patients with CKD associated with coronary artery disease showed signifi-cantly more intensive production of IL-1β and IL-6 – (607,3±69,4) pg/ml and (309±29,8) pg/ml, P<0,05, respectively. These findings suggest that high levels of IL-1β and IL-6 in the isolated monocytes from patients with coronary artery disease testify to the pathogenic link between cardiovascular pathology and increased production of cytokines in CKD. Such effects of cytokines as heart remodelling, disturbance of an endothelium-dependent arterioles relaxation and intensification of cardiomyocytes apop-tosis may underlie this phenomenon. PO9-237
IDENTIFICATION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 GENE EXPRESSION IN HUMAN CAROTID PLAQUES
M. Zivkovic 1 , D.J. Radak 2 , A. Stankovic 1 , T. Djuric 1 , A. Rakovic 1 , S. Radak 2 , D. Alavantic 1 . 1 Laboratory for Radiobiology and Molecular Genetics, VINCA Institute of Nuclear Sciences, Belgrade, Serbia; 2 Vascular Surgery Clinic, Dedinje Cardiovascular Institute, Belgrade, Serbia Background and Aims: Atherosclerosis is now recognized as a disease of arterial inflammation. Monocyte chemoattractant protein-1 (MCP-1) is the key chemokine that regulates migration and infiltration of monocytes/macrophages in vascular wall. MCP-1 expression is increased in atherosclerotic plaques, although most of the results come from experimental models and cell cultures. The aim of our study was to investigate gene expression of MCP-1 in human carotid plaques and controls (normal artery and artery with kinking) and to compare it between two groups of clinically defined plaque phenotypes: stable (fibrolipid, moderately or highly echogen) and unstable (complicated with low echogenicity and ulcerations). Methods: Atherosclerotic plaque from 26 patients (12 stable and 14 unstable) undergoing carotid endaterectomy was investigated. Real-time PCR was performed using ABI Prism 7500 for relative quantification of MCP-1 gene expression. The 2-Ct method was used to analyze the results. Results: The MCP-1 expression was 3.5-fold significantly higher in plaques compared to control artery, as well as compared to kinking. We did not detect significantly different expression of MCP-1 between stable and unstable plaques. Conclusion: Our results confirm that MCP-1 overexpression is associated with human carotid atherosclerosis. According to our results there is no evidence that MCP-1 expression is significantly associated with specific plaque phenotype and vulnerability. These results should be confirmed on larger sample. PO9-238
FOLIC ACID REDUCES CHEMOKINE MCP-1 RELEASE AND EXPRESSION IN RATS WITH HYPERHOMOCYSTEINEMIA
M. Li, J. Chen, Y. Li, Y. Feng, Q. Zeng, Y. Liao. Institute of Cardiovasoloy, Union Hospital, Tongji Medical College, Huazhong Science and Technology University. No.1277, Wuhan, China Objective: To investigate effects of folate on the monocyte MCP-1 expression and release in rats with hyperhomoysteinemia. Methods and Results: Thirty male SD rats were randomly divided into 3 groups: Control, high homocysteinemia (Hhcy) and folate treatment (FA) group. They were fed with nomal diet, normal diet enriched by 1.7% Met and normal diet plus 1.7% Met and 0.006% folate for 45 days.Our study was showing: (1) A high methionine diet for 45 days is sufficient to induce hyperhomocysteinemia; folate supplementation to the rats fed the high-methionine diet prevented an elevation of Hcy levels in the blood (P<0.01). (2) Compared with control group, Hhcy group had elevated plasma levels of MCP-1, and homocysteine was sigificantly correlated with MCP-1 (P<0.05). (3) The protein and mRNA expression of MCP-1 in aorta was higher in rats of Hhcy group than in rats of Control group. (4) Most importantly, after folic acid supplementation, the lowering of homocysteine levels was accompanied by a marked reduction of MCP-1 expressed in
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
PO9-235
75
T.S. Kang 1 , H.M. Kwon 2 , Y.W. Yoon 2 , S. Rim 2 , B.K. Hong 2 , H.S. Kim 2 , H-S. Seo 3 . 1 Cardiology/Dankook Univ. Hospital, Chun-Ahn, South Korea; 2 Cardiology/Yonsei Univ.Hospital, Seoul, South Korea; 3 Cardiology Dept, Soochunhyang University Hospital, Seoul, South Korea Advanced glycation endproducts(AGEs) enhance the immunoinflammatory reaction in diabetic vasculopathy through receptors for AGE (RAGE). Recently, AGEs have been reported to play a role in neointimal formation and increase the rate of in-stent restenosis(ISR) in the diabetic coronary artery disease patients treated with stents. However, the potential roles and pathogenic mechanisms of AGE in vascular smooth muscle cell(VSMC) proliferation and ISR remain unclear. We sought to determine the AGEs related pathobiological mechanism of diabetic vasculopathy. Serum levels of AGEs were analyzed by fluorescent intensity method in the diabetic PCI patients. High AGEs level was independent risk factors for ISR by multivariate analysis. The RAGE expression in human atheroma was assessed by immunohistochemistry. Rat aortic smooth muscle cell(RASMC) culture was done with AGEs stimulation. Western blotting was performed to assess the activation of mitogen-associated protein kinase(MAPK) system in the cultured VSMC. AGEs stimulated VSMC proliferation and were associated with increased phosphorylation of ERK, JUN, and p-38 by time and dose dependent manner. The ERKs activation by AGEs was decreased by RNA interference for RAGE, indicating that AGEs activated ERKs via RAGE. AGEs may play a key role in VSMC proliferation and increase ISR in patients with diabetes melitus. Activation of VSMC proliferation by MAPK system and increased formation of ROS may be the possible mechanism of AGEs induced diabetic vasculopathy PO9-236
CARDIOVASCULAR PATHOLOGY IN PATIENTS WITH CHRONIC KIDNEY DISEASE IS ASSOCIATED WITH ENHANCED CYTOKINE SECRETION BY MONOCYTES
V.J.U. Galchinskaya, P.S. Semenovykh. Department of Nephrology, Institute of Therapy of AMS of Ukraine, Kharkov, Ukraine The chronic kidney disease (CKD) is strongly associated with increased risk of cardiovascular pathology. Chronic inflammation and activation of immune cells, in particular monocytes may promote the development of atherogenesis in the coronary vessel as well as progression of renal pathology. This research was conducted to investigate the production of proinflammatory cytokines - interleukin 1β (IL-1β) and interleukin 6 (IL-6) by monocytes in patients with CKD and coronary artery disease. The investigation was done on freshly isolated peripheral blood-derived monocytes from patients with CKD and coronary artery disease combination (basic group) and patients with CKD without cardiovascular pathology
(control group). IL-1β and IL-6 levels in incubation medium of monocytes was determined by ELISA. To stimulate cellular response angiotensin II (10E-7 M) was used. Our results showed the enhanced cytokine production by monocytes in patients with combination of CKD and coronary artery disease. In control group levels of IL-1β and IL-6 in the incubation media of monocytes were (375,4±38,9) pg/ml and (120±16,9) pg/ml, P<0,05, respectively. Monocytes from patients with CKD associated with coronary artery disease showed signifi-cantly more intensive production of IL-1β and IL-6 – (607,3±69,4) pg/ml and (309±29,8) pg/ml, P<0,05, respectively. These findings suggest that high levels of IL-1β and IL-6 in the isolated monocytes from patients with coronary artery disease testify to the pathogenic link between cardiovascular pathology and increased production of cytokines in CKD. Such effects of cytokines as heart remodelling, disturbance of an endothelium-dependent arterioles relaxation and intensification of cardiomyocytes apop-tosis may underlie this phenomenon. PO9-237
IDENTIFICATION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 GENE EXPRESSION IN HUMAN CAROTID PLAQUES
M. Zivkovic 1 , D.J. Radak 2 , A. Stankovic 1 , T. Djuric 1 , A. Rakovic 1 , S. Radak 2 , D. Alavantic 1 . 1 Laboratory for Radiobiology and Molecular Genetics, VINCA Institute of Nuclear Sciences, Belgrade, Serbia; 2 Vascular Surgery Clinic, Dedinje Cardiovascular Institute, Belgrade, Serbia Background and Aims: Atherosclerosis is now recognized as a disease of arterial inflammation. Monocyte chemoattractant protein-1 (MCP-1) is the key chemokine that regulates migration and infiltration of monocytes/macrophages in vascular wall. MCP-1 expression is increased in atherosclerotic plaques, although most of the results come from experimental models and cell cultures. The aim of our study was to investigate gene expression of MCP-1 in human carotid plaques and controls (normal artery and artery with kinking) and to compare it between two groups of clinically defined plaque phenotypes: stable (fibrolipid, moderately or highly echogen) and unstable (complicated with low echogenicity and ulcerations). Methods: Atherosclerotic plaque from 26 patients (12 stable and 14 unstable) undergoing carotid endaterectomy was investigated. Real-time PCR was performed using ABI Prism 7500 for relative quantification of MCP-1 gene expression. The 2-Ct method was used to analyze the results. Results: The MCP-1 expression was 3.5-fold significantly higher in plaques compared to control artery, as well as compared to kinking. We did not detect significantly different expression of MCP-1 between stable and unstable plaques. Conclusion: Our results confirm that MCP-1 overexpression is associated with human carotid atherosclerosis. According to our results there is no evidence that MCP-1 expression is significantly associated with specific plaque phenotype and vulnerability. These results should be confirmed on larger sample. PO9-238
FOLIC ACID REDUCES CHEMOKINE MCP-1 RELEASE AND EXPRESSION IN RATS WITH HYPERHOMOCYSTEINEMIA
M. Li, J. Chen, Y. Li, Y. Feng, Q. Zeng, Y. Liao. Institute of Cardiovasoloy, Union Hospital, Tongji Medical College, Huazhong Science and Technology University. No.1277, Wuhan, China Objective: To investigate effects of folate on the monocyte MCP-1 expression and release in rats with hyperhomoysteinemia. Methods and Results: Thirty male SD rats were randomly divided into 3 groups: Control, high homocysteinemia (Hhcy) and folate treatment (FA) group. They were fed with nomal diet, normal diet enriched by 1.7% Met and normal diet plus 1.7% Met and 0.006% folate for 45 days.Our study was showing: (1) A high methionine diet for 45 days is sufficient to induce hyperhomocysteinemia; folate supplementation to the rats fed the high-methionine diet prevented an elevation of Hcy levels in the blood (P<0.01). (2) Compared with control group, Hhcy group had elevated plasma levels of MCP-1, and homocysteine was sigificantly correlated with MCP-1 (P<0.05). (3) The protein and mRNA expression of MCP-1 in aorta was higher in rats of Hhcy group than in rats of Control group. (4) Most importantly, after folic acid supplementation, the lowering of homocysteine levels was accompanied by a marked reduction of MCP-1 expressed in
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
PO9-235
75