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MS463 TYPE 2 DIABETES-ASSOCIATED ATHEROTHROMBOSIS: INSIGHTS INTO PATHOGENESIS AND THERAPEUTIC TARGET
78th EAS Congress
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
Aim: The aim of this study is to examine the role of these protein-variants in calcification in vitro. Methods: Both variants of cDNA were cloned and inserted in a pSG5 expression vector (pSG5-Abcc6-C3H/He, pSG5-Abcc6-C57BL/6). We established a calcifying cell-culture-model using the mesenchymal stem-cellline C3H10T1/2 and adding inorganic phosphate to the media. Cells were transfected with empty pSG5 vector (pSG5), pSG5-Abcc6-C3H/He and pSG5Abcc6-C57BL/6 and analyzed 3, 7 and 21 days after induction of calcification by Alizarin-Red-S-staining and quantification using Randox Ca Kit. Results: The time-course-analysis revealed no calcification after 3, an initiation of calcification after 7 and a strong calcification after 21 days. The mean values of calcium-deposits were measured to be 9.42 mmol in pSG5, 13.76 mmol in pSG5-Abcc6-C3H/He and 9.13 mmol in pSG5-Abcc6-C57BL/6-transfected cells after 7 days (n = 3 × 3). Interestingly, a significant increase in calciumdeposits was found in pSG5-Abcc6-C3H/He- transfected cells compared to pSG5-transfected (13.75 vs. 9.42; p = 0.0063). A non significant decrease in calcium-deposits was observed in pSG5-Abcc6-C57BL/6-transfected cells compared to pSG5 (9.13 mmol vs. 9.42 mmol; p = 0.7321). Conclusion: Using cell-culture-model, we functionally demonstrate for the first time the effect of the amino-acid-substitutions found in the C3H/He-Abcc6 on calcification in vitro. MS461 VITAMIN E ISOMERS TOCOTRIENOLS CONFER RESISTANCE TO ISCHEMIC INJURY IN THE HYPERCHOLESTEROLEMIC RABBIT HEARTS D.K. Das. Cardiovascular Research Center, University of Connecticut Medical Center, Farmington, CT, USA Most clinical trials with a-tocopherol could not alter the levels of cholesterol and thus, have been deemed unsuccessful. Recently, tocotrienols, isomers of vitamin-E have been found to lower LDL levels. To explore the mechanisms of action, rabbits were kept on high cholesterol diet for 60 days and supplemented with tocotrienol a, tocotrienol d, or tocotrienol g for the last 30 days. The serum cholesterol levels were 24.4 mmol/L [tocotrienol a], 34.9 mmol/L [tocotrienol d], 19.8 mmol/L [tocotrienol g] vs.39.7 mmol/L [control]. Left ventricular function exhibited significantly improved recovery with tocotrienol g and tocotrienol a, but not with tocotrienol d. The myocardial infarct size showed a similar pattern: 33% [tocotrienol a], 23% tocotrienol g], and 47% [tocotrienol d]. To examine the molecular mechanisms, gene expression profile was determined using Atlas 1.2 and 1.2II followed by determination of gene profiles using PedQuest 8.3 software. Based on the genomics profiles, the following cholesterol-related proteins were examined: TGFb [cholesterol suppresses TGFb], ET-1 [increased by hypercholesterolemia] SPOT 14 [linked with hypercholesterolemia] and matrix metalloproteinase [MMP] 2 and MMP9 [cholesterol regulates MMP2 and MMP9 expression] in the heart. Consistent with the results of cardioprotective effects of tocotrienol a and g, these two isomers reduced ET-1, decreased MMP2 and MM9 expression, increased TGFb expression and reduced SPOT 14 expression, while tocotrienol d had no effects. The results demonstrate that tocotrienols a and g render the hypercholesterolemic hearts resistant to ischemia by lowering MMP2, MMP9, ET-1 and SPOT 14 and upregulating TGFb. MS462 INTERRELATIONSHIP OF CORONARY ARTERY CALCIFICATION, INFLAMMATION AND GLOBAL LEFT VENTRICULAR FUNCTION IN PATIENTS WITH CHRONIC CORONARY ARTERY DISEASE I. Burazor1 , M. Burazor2 , M. Krstic1 , Z. Radovanovic3 , D. Ilic3 , V. Atanaskovic1 , M. Lazovic1 , J. Djordjevic1 , V. Stojanovic1 . 1 Cardiology, Clinical Center in Nis, 2 Cardiology, University Clinical Center in Nis, 3 Institute for Radiology, Clinical Center, Nis, Serbia Coronary calcium assessment is being increasingly used in clinical practice. We aimed to evaluate the possible relationship between coronary calcification, inflammation and global LV function obtained by using 3D imaging modality. Methods: Out of 598 persons who were referred to 64 slices MSCT, the total of 121 patients (age 50 to 69 years, 65% males) with chronic coronary artery disease entered the study. Total Agatston (CAC) score was calculated. Non invasive coronary angiography was performed to detect significant lesions (> 50% diameter reduction). Global LV function was assessed (ejection fraction, stroke volume, cardiac output, LV volume, LV mass, end-systolic and enddiastolic volume). C-reactive protein and leucocytes count were determined. Results: All patients had coronary artery calcifications. CAC score between 1 and 100 was detected in 74% of patients, between 101 and 399 in 21%, between 400 and 999 in 25%. The total of 25% had CAC score above 1000. Significant lesions were detected in 16% of patients. There was a correlation between CAC and global left ventricular function. Higher CAC correlated with lower ejection fraction (p = 0.01), stroke volume (p = 0.05), end -systolic volume (p = 0.01) and end-diastolic volume (p = 0.05). Also, CAC correlated with years of age, male gender and leucocytes count (p = 0.05). No correlation was found between CAC and C-reactive protein. Conclusions: The results of our study suggest that coronary artery calcification is related to global left ventricular function and leucocytes count in patients with chronic coronary artery disease.
203
MS463 TYPE 2 DIABETES-ASSOCIATED ATHEROTHROMBOSIS: INSIGHTS INTO PATHOGENESIS AND THERAPEUTIC TARGET K. Ahmed1,2 , S. Muniandy2 , I.S. Ismail3 . 1 Medical Sciences, Faculty of Dentistry, Ibb University, Ibb, Yemen, 2 Molecular Medicine, 3 Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Introduction: Increased atherothrombotic tendency was found in patients with diabetes. Ne -(carboxymethyl)lysine (CML) is a product of proteins glycoxidation and its effect on gene expression of the fibrinolysis regulator; plasminogen activator inhibitor-1 (PAI-1) have not been elucidated. Objectives: This study was aimed to establish a mechanism by which CML could lead to coronary artery atherosclerosis in patients with type 2 diabetes. In addition, the hypothesis that atherothrombogenic abnormalities are enhanced by CML was elucidated. Patients and Methods: Levels of CML, PAI-1, and tPA in patients with T2DM with and without CAD as well as healthy control subjects were examined. Enhanced oxidative stress and impaired LDL metabolism due to CML were also investigated. Multiplex Real-Time PCR was used to evaluate the gene expression of PAI-1 due to higher levels of CML. Results: Serum levels of CML and PAI-1 were significantly higher while tissue plasminogen activator (tPA) was lower in T2DM with CAD patients than diabetic patients without CAD or healthy control subjects. Metabolism of LDL from T2DM patients with CAD was completely impaired. Furthermore, CML levels were correlated to the overexpression of PAI-1 genes in T2DM patients compared to healthy subjects. Conclusions: The present study showed that CML directly enhanced PAI-1 secretion and gene expression in the vascular cells. The hypersecretion of CML might be one of the mechanisms underlying the impaired fibrinolysis and increased tendency for thrombosis observed in patients with T2DM and CAD. Thus, CML may be considered as a therapeutic target in patients with type 2 diabetes-induced atherothrombosis. MS464 THE ROLE OF FHL2 GENE IN FORMATION OF ATHEROSCLEROTIC LESION P.-H. Chu1 , H.-I. Yeh2 . 1 Cardiolgoy, Chang Gung University, Chang Gung Memorial Hospital, 2 Mackay Memorial Hospital, Taipei, Taiwan R.O.C. Aims: FHL2, a member of the four and a half LIM domain (FHL), may play an important role in the circulatory system and in particular atherosclerosis. Main methods: To investigate the role of FHL2 in atherogenesis, FHL2-null and wild-type control male mice were fed either a normal chow (NC) or a cholesterol enriched diet (CED). Key findings: At 3 months post CED, aortic atherosclerotic plaques were observed in both control and FHL2-null mice. Lesions in control mice increased dramatically by 6 months of CED. In contrast, lesion size did not increase during this time in CED fed FHL2-null mice. Relative to control mice on a NCD, control mice on a CED exhibited lower circulating nitric oxide (NO) levels, and decreased expression of connexin37 (Cx37) and Cx40 in aortic endothelium. In contrast, FHL2-null mice on a CED maintained similar levels of circulating NO as FHL2-null mice fed a NCD. Cxs levels in aortic endothelium of FHL2-null mutants on a NCD were lower relative to control mice on a NCD, and did not decrease with CED. Significance: Our data demonstrate a role for FHL2 in atherogenesis, the regulation of circular NO release, and expression of gap junctions within aortic endothelium. MS465 PMN MEMBRANE FLUIDITY, CITOSOLIC Ca2+ CONTENT AND BETA2-INTEGRIN PATTERN IN VASCULAR ATHEROSCLEROTIC DISEASE G. Caimi, E. Hopps, B. Canino, M. Montana, R. Lo Presti. Dipartimento di Medicina Interna. Malattie Cardiovascolari e Nefrourologiche, Universita` di Palermo, Palermo, Italy In atherosclerosis and in diabetes mellitus (DM) polymorphonuclear cells (PMN) play a role in the organ injury. The PMN-mediated tissue damage can be exacerbated by a PMN functional abnormality and among the parameters that reflect this PMN dysfunction we can include membrane fluidity and cytosolic Ca2+ content. Both leukocyte-endothelium and leukocyte-platelet interactions, mediated especially by b2 -integrins, are involved in atherogenesis. We evaluated PMN membrane fluidity and cytosolic Ca2+ content in subjects with vascular atherosclerotic disease (VAD) with (n = 52) and without DM2 (n = 92), subdivided according to the extent of vascular disease and the examination of the PMN integrin pattern (CD11a, CD11b, CD11c and CD18) at baseline and after activation in a subgroup of VAD subjects with (n = 21) and without DM (n = 27). There was no difference in membrane fluidity between controls and VAD subjects, mono or polyvascular, while the cytosolic Ca2+ content was increased in VAD subjects (mono and polyvascular). The baseline evaluation of the integrin pattern showed that CD11a and CD18 were increased, while CD11b and CD11c were decreased. After activation we observed, in VAD subjects with and without DM, a decrease of CD11a and an increase of CD11b; we noted also a decrease of CD18 in VAD subjects without DM
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
Aim: The aim of this study is to examine the role of these protein-variants in calcification in vitro. Methods: Both variants of cDNA were cloned and inserted in a pSG5 expression vector (pSG5-Abcc6-C3H/He, pSG5-Abcc6-C57BL/6). We established a calcifying cell-culture-model using the mesenchymal stem-cellline C3H10T1/2 and adding inorganic phosphate to the media. Cells were transfected with empty pSG5 vector (pSG5), pSG5-Abcc6-C3H/He and pSG5Abcc6-C57BL/6 and analyzed 3, 7 and 21 days after induction of calcification by Alizarin-Red-S-staining and quantification using Randox Ca Kit. Results: The time-course-analysis revealed no calcification after 3, an initiation of calcification after 7 and a strong calcification after 21 days. The mean values of calcium-deposits were measured to be 9.42 mmol in pSG5, 13.76 mmol in pSG5-Abcc6-C3H/He and 9.13 mmol in pSG5-Abcc6-C57BL/6-transfected cells after 7 days (n = 3 × 3). Interestingly, a significant increase in calciumdeposits was found in pSG5-Abcc6-C3H/He- transfected cells compared to pSG5-transfected (13.75 vs. 9.42; p = 0.0063). A non significant decrease in calcium-deposits was observed in pSG5-Abcc6-C57BL/6-transfected cells compared to pSG5 (9.13 mmol vs. 9.42 mmol; p = 0.7321). Conclusion: Using cell-culture-model, we functionally demonstrate for the first time the effect of the amino-acid-substitutions found in the C3H/He-Abcc6 on calcification in vitro. MS461 VITAMIN E ISOMERS TOCOTRIENOLS CONFER RESISTANCE TO ISCHEMIC INJURY IN THE HYPERCHOLESTEROLEMIC RABBIT HEARTS D.K. Das. Cardiovascular Research Center, University of Connecticut Medical Center, Farmington, CT, USA Most clinical trials with a-tocopherol could not alter the levels of cholesterol and thus, have been deemed unsuccessful. Recently, tocotrienols, isomers of vitamin-E have been found to lower LDL levels. To explore the mechanisms of action, rabbits were kept on high cholesterol diet for 60 days and supplemented with tocotrienol a, tocotrienol d, or tocotrienol g for the last 30 days. The serum cholesterol levels were 24.4 mmol/L [tocotrienol a], 34.9 mmol/L [tocotrienol d], 19.8 mmol/L [tocotrienol g] vs.39.7 mmol/L [control]. Left ventricular function exhibited significantly improved recovery with tocotrienol g and tocotrienol a, but not with tocotrienol d. The myocardial infarct size showed a similar pattern: 33% [tocotrienol a], 23% tocotrienol g], and 47% [tocotrienol d]. To examine the molecular mechanisms, gene expression profile was determined using Atlas 1.2 and 1.2II followed by determination of gene profiles using PedQuest 8.3 software. Based on the genomics profiles, the following cholesterol-related proteins were examined: TGFb [cholesterol suppresses TGFb], ET-1 [increased by hypercholesterolemia] SPOT 14 [linked with hypercholesterolemia] and matrix metalloproteinase [MMP] 2 and MMP9 [cholesterol regulates MMP2 and MMP9 expression] in the heart. Consistent with the results of cardioprotective effects of tocotrienol a and g, these two isomers reduced ET-1, decreased MMP2 and MM9 expression, increased TGFb expression and reduced SPOT 14 expression, while tocotrienol d had no effects. The results demonstrate that tocotrienols a and g render the hypercholesterolemic hearts resistant to ischemia by lowering MMP2, MMP9, ET-1 and SPOT 14 and upregulating TGFb. MS462 INTERRELATIONSHIP OF CORONARY ARTERY CALCIFICATION, INFLAMMATION AND GLOBAL LEFT VENTRICULAR FUNCTION IN PATIENTS WITH CHRONIC CORONARY ARTERY DISEASE I. Burazor1 , M. Burazor2 , M. Krstic1 , Z. Radovanovic3 , D. Ilic3 , V. Atanaskovic1 , M. Lazovic1 , J. Djordjevic1 , V. Stojanovic1 . 1 Cardiology, Clinical Center in Nis, 2 Cardiology, University Clinical Center in Nis, 3 Institute for Radiology, Clinical Center, Nis, Serbia Coronary calcium assessment is being increasingly used in clinical practice. We aimed to evaluate the possible relationship between coronary calcification, inflammation and global LV function obtained by using 3D imaging modality. Methods: Out of 598 persons who were referred to 64 slices MSCT, the total of 121 patients (age 50 to 69 years, 65% males) with chronic coronary artery disease entered the study. Total Agatston (CAC) score was calculated. Non invasive coronary angiography was performed to detect significant lesions (> 50% diameter reduction). Global LV function was assessed (ejection fraction, stroke volume, cardiac output, LV volume, LV mass, end-systolic and enddiastolic volume). C-reactive protein and leucocytes count were determined. Results: All patients had coronary artery calcifications. CAC score between 1 and 100 was detected in 74% of patients, between 101 and 399 in 21%, between 400 and 999 in 25%. The total of 25% had CAC score above 1000. Significant lesions were detected in 16% of patients. There was a correlation between CAC and global left ventricular function. Higher CAC correlated with lower ejection fraction (p = 0.01), stroke volume (p = 0.05), end -systolic volume (p = 0.01) and end-diastolic volume (p = 0.05). Also, CAC correlated with years of age, male gender and leucocytes count (p = 0.05). No correlation was found between CAC and C-reactive protein. Conclusions: The results of our study suggest that coronary artery calcification is related to global left ventricular function and leucocytes count in patients with chronic coronary artery disease.
203
MS463 TYPE 2 DIABETES-ASSOCIATED ATHEROTHROMBOSIS: INSIGHTS INTO PATHOGENESIS AND THERAPEUTIC TARGET K. Ahmed1,2 , S. Muniandy2 , I.S. Ismail3 . 1 Medical Sciences, Faculty of Dentistry, Ibb University, Ibb, Yemen, 2 Molecular Medicine, 3 Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Introduction: Increased atherothrombotic tendency was found in patients with diabetes. Ne -(carboxymethyl)lysine (CML) is a product of proteins glycoxidation and its effect on gene expression of the fibrinolysis regulator; plasminogen activator inhibitor-1 (PAI-1) have not been elucidated. Objectives: This study was aimed to establish a mechanism by which CML could lead to coronary artery atherosclerosis in patients with type 2 diabetes. In addition, the hypothesis that atherothrombogenic abnormalities are enhanced by CML was elucidated. Patients and Methods: Levels of CML, PAI-1, and tPA in patients with T2DM with and without CAD as well as healthy control subjects were examined. Enhanced oxidative stress and impaired LDL metabolism due to CML were also investigated. Multiplex Real-Time PCR was used to evaluate the gene expression of PAI-1 due to higher levels of CML. Results: Serum levels of CML and PAI-1 were significantly higher while tissue plasminogen activator (tPA) was lower in T2DM with CAD patients than diabetic patients without CAD or healthy control subjects. Metabolism of LDL from T2DM patients with CAD was completely impaired. Furthermore, CML levels were correlated to the overexpression of PAI-1 genes in T2DM patients compared to healthy subjects. Conclusions: The present study showed that CML directly enhanced PAI-1 secretion and gene expression in the vascular cells. The hypersecretion of CML might be one of the mechanisms underlying the impaired fibrinolysis and increased tendency for thrombosis observed in patients with T2DM and CAD. Thus, CML may be considered as a therapeutic target in patients with type 2 diabetes-induced atherothrombosis. MS464 THE ROLE OF FHL2 GENE IN FORMATION OF ATHEROSCLEROTIC LESION P.-H. Chu1 , H.-I. Yeh2 . 1 Cardiolgoy, Chang Gung University, Chang Gung Memorial Hospital, 2 Mackay Memorial Hospital, Taipei, Taiwan R.O.C. Aims: FHL2, a member of the four and a half LIM domain (FHL), may play an important role in the circulatory system and in particular atherosclerosis. Main methods: To investigate the role of FHL2 in atherogenesis, FHL2-null and wild-type control male mice were fed either a normal chow (NC) or a cholesterol enriched diet (CED). Key findings: At 3 months post CED, aortic atherosclerotic plaques were observed in both control and FHL2-null mice. Lesions in control mice increased dramatically by 6 months of CED. In contrast, lesion size did not increase during this time in CED fed FHL2-null mice. Relative to control mice on a NCD, control mice on a CED exhibited lower circulating nitric oxide (NO) levels, and decreased expression of connexin37 (Cx37) and Cx40 in aortic endothelium. In contrast, FHL2-null mice on a CED maintained similar levels of circulating NO as FHL2-null mice fed a NCD. Cxs levels in aortic endothelium of FHL2-null mutants on a NCD were lower relative to control mice on a NCD, and did not decrease with CED. Significance: Our data demonstrate a role for FHL2 in atherogenesis, the regulation of circular NO release, and expression of gap junctions within aortic endothelium. MS465 PMN MEMBRANE FLUIDITY, CITOSOLIC Ca2+ CONTENT AND BETA2-INTEGRIN PATTERN IN VASCULAR ATHEROSCLEROTIC DISEASE G. Caimi, E. Hopps, B. Canino, M. Montana, R. Lo Presti. Dipartimento di Medicina Interna. Malattie Cardiovascolari e Nefrourologiche, Universita` di Palermo, Palermo, Italy In atherosclerosis and in diabetes mellitus (DM) polymorphonuclear cells (PMN) play a role in the organ injury. The PMN-mediated tissue damage can be exacerbated by a PMN functional abnormality and among the parameters that reflect this PMN dysfunction we can include membrane fluidity and cytosolic Ca2+ content. Both leukocyte-endothelium and leukocyte-platelet interactions, mediated especially by b2 -integrins, are involved in atherogenesis. We evaluated PMN membrane fluidity and cytosolic Ca2+ content in subjects with vascular atherosclerotic disease (VAD) with (n = 52) and without DM2 (n = 92), subdivided according to the extent of vascular disease and the examination of the PMN integrin pattern (CD11a, CD11b, CD11c and CD18) at baseline and after activation in a subgroup of VAD subjects with (n = 21) and without DM (n = 27). There was no difference in membrane fluidity between controls and VAD subjects, mono or polyvascular, while the cytosolic Ca2+ content was increased in VAD subjects (mono and polyvascular). The baseline evaluation of the integrin pattern showed that CD11a and CD18 were increased, while CD11b and CD11c were decreased. After activation we observed, in VAD subjects with and without DM, a decrease of CD11a and an increase of CD11b; we noted also a decrease of CD18 in VAD subjects without DM