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ANTI-OXIDATIVE DEFENCE MARKERS FOR THE PREDICTION OF CARDIOVASCULAR RISK
78th EAS Congress
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
MS523 USING ImageJ FOR THE QUANTITATIVE ANALYSIS OF FLOW-BASED ADHESION ASSAYS IN REAL-TIME UNDER PHYSIOLOGIC FLOW CONDITIONS S. Meyer dos Santos1 , U. Klinkhardt1 , R. Schneppenheim2 , S. Harder1 . 1 Department of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, 2 Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany A simple and robust method is presented for analysing raw videomicroscopic data of flow-based adhesion assays using the freely available public domain software ImageJ. The depicted procedures were exemplified by analysing platelet interaction with immobilised von Wille-brand factor and fibrinogen in flowing blood under physiological wall shear rates. Platelets adhered to vWf in a shear-dependent manner (adherent platelets±SEM: 80±4 at 150 s−1 wall shear rate, 233±11 at 600 s−1 and 441±15 at 1800 s−1 ) and neutralising GPIba function abol-ished firm platelet adhesion. Furthermore, the described procedures enable the researcher to quantify the number of translocating platelets. The number of platelets translocating on vWf was likewise dependent on the applied wall shear rate (24±5 at 150 s−1 , 318±24 at 600 s−1 and 1142±89 at 1800 s−1 ). The suitability of the method was further demonstrated by the studying the adhesion to fibrinogen at 150 s−1 . Platelet pre-incubation with 100 nM Abcixi-mab, 10 mM Tirofiban or 100 mM Eptifibatide dropped GPIIb/IIIa-dependent stable platelet deposition on fibrinogen to baseline level. The presented method to analyse videomicroscopic recordings from flow-based adhesion assays offers the advantage to provide a simple and reli-able way to quantify flow-based adhesion assays and provides the researcher a tool to rapidly gain quantitative data for statistical analyses. It can easily be applied to study adhesion mechanisms of cells in non-fluorescent modes without the need to deviate from the presented protocol. MS524 PHENOTYPIC DIFFERENCES OF HUMAN NEUTROPHILS OF CARRIERS OF THE PSGL-1 A AND B-ALLELE IN BINDING TO P-SELECTIN UNDER FLOW CONDITIONS S. Meyer dos Santos, U. Klinkhardt, K. Lang, J. Parisius, K. Kuczka, S. Harder. Department of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, Germany P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins expressed on endothelial cells and supports the trafficking of leukocytes into inflamed tissues. PSGL-1 extracellular mucin domain contains decameric repeats that display genetic polymorphisms in the variable number of tandem repeats (VNTRs). The wildtype consists of 16 decameric repeats (designate A isoforms) and variants with 15 (B allele) and 14 (C allele) repeats that are possibly associated with reduced risk of vascular disease. We investigated the potential different adhesive capacity of these natural variants in native human neutrophils. Healthy volunteers were genotyped and the adhesion of neutrophils expressing the PSGL-1 isoforms A/A, A/B and B/B were studied under static and physiologic flow conditions. Homozygous B/B neutrophils attached significantly weaker to P-selectin at elevated shear rates than A/A and A/B neutrophils. No difference was found under static conditions and shear stress below 24 dyn/cm2 . B/B neutrophils rolled significantly faster than A/A neutrophils at 12 dyn/cm2 shear stress or higher. There was no difference in the adhesive capacity between A/A an A/B neutrophils. These data support the view that the role of the decamers is to extend the ligand binding domain far above the cell surface to support stable leukocyte adhesion and rolling. MS525 SMALL-SIZED HIGH-DENSITY LIPOPROTEIN PARTICLES ARE ASSOCIATED WITH REDUCED SURVIVAL RATE IN END-STAGE RENAL DISEASE PATIENTS A. Zeljkovic1 , J. Vekic1 , Z. Jelic-Ivanovic1 , V. Spasojevic-Kalimanovska1 , S. Spasic1 , S. Simic-Ogrizovic2 , V. Dopsaj1 . 1 Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, 2 Nephrology Clinic, Clinical Center of Serbia, Belgrade, Serbia Introduction: Dyslipidemia is commonly seen in patients with end-stage renal disease (ESRD). Small-sized high-density lipoprotein (HDL) particles are feature of atherogenic dyslipidemia, but their predictive role for ESRD progression is uncertain. Objective: This prospective study investigates the effect of small-sized HDL particles alone and in combination with increased high sensitivity C-reactive protein (hsCRP) levels on survival of ESRD patients treated by haemodialysis (HD). Methods: We performed 36 months follow-up study in 122 HD patients. HDL size and subclass distribution were determined by gradient gel electrophoresis, while serum hsCRP and lipid parameters were measured by standard laboratory methods. Baseline characteristics of the patients were evaluated for the prediction of mortality. Results: The carriers of small-sized HDL particles were more prevalent among patients who died (n = 24) compared with those who survived (n = 98, P < 0.05). Serum hsCRP levels were significantly higher in deceased patients (P < 0.05). Univariate Cox regressions analysis showed that patients with small-sized HDL particles had 2.8-fold higher risk of adverse disease outcome (P < 0.05). In
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addition, concomitant presence of small-sized HDL particles and increased hsCRP concentration was significantly associated with reduced survival rate (HR = 3.907; P < 0.05). Observed relationships persisted after adjustment for serum lipid and lipoprotein concentrations. Conclusions: Both dyslipidemia and inflammation play significant role in the progression of ESRD. Our results indicate that small-sized HDL particles alone and combined with elevated hsCRP concentrations are independent predictors of reduced survival in HD patients. MS526 UK INPRACTICE STUDY: CONCORDANCE BETWEEN APOLIPOPROTEIN (Apo) B LEVELS AND LDL-CHOLESTEROL AMONG PATIENTS ON STATINS & NON-STATIN TREATMENT I. Idris1 , H. Tate2 , A. Ahmad3 , T. Mccormack4 . 1 Sherwood Forest Hospitals Foundation Trust, Mansfield, 2 MSD, Hoddesdon, 3 Medical, MSD, Welwyn Garden City, 4 Whitby Group Practice, Whitby, UK Background: ApoB is known to be a superior predictor of LDL-C particle number and CVD risk compared with LDL-C. In patients not taking lipid lowering therapy, current evidence has questioned the concordance between LDL-C with ApoB levels. However the concordance in patients receiving lipid lowering therapies is unclear. This analysis examines the LDL-C and ApoB levels among patients receiving statins and non-statin treatment. Methods: We performed post hoc analysis of the INPRACTICE* data looking at the concordance between LDL-C with ApoB values. This analysis involved 760 high-risk CVD patients from 34 primary care centres initially treated with simvastatin (S) 40 mg at baseline subsequently randomized to adding ezetimibe (E) to S 40 mg or changed to atorvastatin (A) 40 mg or to rosuvastatin (R) 5−10 mg for 6 weeks. Graphs and cross-tabulated quintile tables were assessed. Results: Based on the post-6-weeks treatment values showed that for all treatment groups there was a similar relationship between LDL-C and ApoB; Pearson correlation coefficients were 0.84 (E+S), 0.82 (A) and 0.83 (R). Proportions of patients who fell into the same quintile were 49% (E+S), 46% (A) and 44% (R). Kappa analysis confirmed fair agreement for all treatment groups; 60 (E+S), 0.58 (A) and 0.55 (R). Proportions of patients achieving ApoB< 90 mg/dl were 85% (E+S), 72% (A) and 58% (R); P < 0.0001. Conclusion: There was a similar concordance pattern between LDL-C and ApoB in patients treated with E+S compared to A and R. A higher percentage of patients achieved an ApoB level of < 90 mg/dl whilst treated with E+S compared to A and R. *Presented at ESC 2009 MS527 EVALUATION OF LIPID, INFLAMMATORY AND OXIDATIVE STRESS/ANTI-OXIDATIVE DEFENCE MARKERS FOR THE PREDICTION OF CARDIOVASCULAR RISK N. Bogavac-Stanojevic, Z. Jelic-Ivanovic, S. Spasic, V. SpasojevicKalimanovska, J. Kotur-Stevuljevic. Institute of Medical Biochemistry, Faculty of Pharmacy, University in Belgrade, Belgrade, Serbia Objectives: Atherosclerosis is a complex disease caused by a variety of risk factors such as dyslipidemia, inflammation and oxidative stress. However, none of the aforementioned risk factors used as a single biomarker is able to predict coronary artery disease (CAD) with sufficient accuracy. Measurement of multiple biomarkers may be an effective strategy to improve prediction of CAD. Methods: The effectiveness of apolipoproteins, lipoproteins, inflammatory and oxidative stress/anti-oxidative defence risk parameters supplementary to Framingham scoring data (FRS) was established in 188 CAD patients and 197 controls. The discriminative ability of the models was evaluated using receiver-operating characteristic (ROC) curves with the predictive probabilities from logistic regression models. The jackknife technique was used to detect the outliers. Results: The model, consisting of FRS, superoxide dismutase (SOD) and superoxide anion had outstanding discriminative ability with the areas under the curves (AUC) value of 0.924. One more model (FRS plus SOD) had an outstanding AUC value (0.906). All other models had excellent discriminative abilities (AUC values between 0.854 and 0.887). Results obtained using the jackknife technique indicate that our original model estimates were stable and were not affected by influential outlier cases within the study sample. Conclusions: Our results support the feasibility of using oxidative stress/antioxidative defence markers in the clinical laboratory. Prospective studies validating the utility of novel markers in combination with FRS are needed to fully determine the most effective model for coronary risk assessment. Funding: This work was supported by a grant from the Ministry of Science, Republic of Serbia.
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
MS523 USING ImageJ FOR THE QUANTITATIVE ANALYSIS OF FLOW-BASED ADHESION ASSAYS IN REAL-TIME UNDER PHYSIOLOGIC FLOW CONDITIONS S. Meyer dos Santos1 , U. Klinkhardt1 , R. Schneppenheim2 , S. Harder1 . 1 Department of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, 2 Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany A simple and robust method is presented for analysing raw videomicroscopic data of flow-based adhesion assays using the freely available public domain software ImageJ. The depicted procedures were exemplified by analysing platelet interaction with immobilised von Wille-brand factor and fibrinogen in flowing blood under physiological wall shear rates. Platelets adhered to vWf in a shear-dependent manner (adherent platelets±SEM: 80±4 at 150 s−1 wall shear rate, 233±11 at 600 s−1 and 441±15 at 1800 s−1 ) and neutralising GPIba function abol-ished firm platelet adhesion. Furthermore, the described procedures enable the researcher to quantify the number of translocating platelets. The number of platelets translocating on vWf was likewise dependent on the applied wall shear rate (24±5 at 150 s−1 , 318±24 at 600 s−1 and 1142±89 at 1800 s−1 ). The suitability of the method was further demonstrated by the studying the adhesion to fibrinogen at 150 s−1 . Platelet pre-incubation with 100 nM Abcixi-mab, 10 mM Tirofiban or 100 mM Eptifibatide dropped GPIIb/IIIa-dependent stable platelet deposition on fibrinogen to baseline level. The presented method to analyse videomicroscopic recordings from flow-based adhesion assays offers the advantage to provide a simple and reli-able way to quantify flow-based adhesion assays and provides the researcher a tool to rapidly gain quantitative data for statistical analyses. It can easily be applied to study adhesion mechanisms of cells in non-fluorescent modes without the need to deviate from the presented protocol. MS524 PHENOTYPIC DIFFERENCES OF HUMAN NEUTROPHILS OF CARRIERS OF THE PSGL-1 A AND B-ALLELE IN BINDING TO P-SELECTIN UNDER FLOW CONDITIONS S. Meyer dos Santos, U. Klinkhardt, K. Lang, J. Parisius, K. Kuczka, S. Harder. Department of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, Germany P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins expressed on endothelial cells and supports the trafficking of leukocytes into inflamed tissues. PSGL-1 extracellular mucin domain contains decameric repeats that display genetic polymorphisms in the variable number of tandem repeats (VNTRs). The wildtype consists of 16 decameric repeats (designate A isoforms) and variants with 15 (B allele) and 14 (C allele) repeats that are possibly associated with reduced risk of vascular disease. We investigated the potential different adhesive capacity of these natural variants in native human neutrophils. Healthy volunteers were genotyped and the adhesion of neutrophils expressing the PSGL-1 isoforms A/A, A/B and B/B were studied under static and physiologic flow conditions. Homozygous B/B neutrophils attached significantly weaker to P-selectin at elevated shear rates than A/A and A/B neutrophils. No difference was found under static conditions and shear stress below 24 dyn/cm2 . B/B neutrophils rolled significantly faster than A/A neutrophils at 12 dyn/cm2 shear stress or higher. There was no difference in the adhesive capacity between A/A an A/B neutrophils. These data support the view that the role of the decamers is to extend the ligand binding domain far above the cell surface to support stable leukocyte adhesion and rolling. MS525 SMALL-SIZED HIGH-DENSITY LIPOPROTEIN PARTICLES ARE ASSOCIATED WITH REDUCED SURVIVAL RATE IN END-STAGE RENAL DISEASE PATIENTS A. Zeljkovic1 , J. Vekic1 , Z. Jelic-Ivanovic1 , V. Spasojevic-Kalimanovska1 , S. Spasic1 , S. Simic-Ogrizovic2 , V. Dopsaj1 . 1 Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, 2 Nephrology Clinic, Clinical Center of Serbia, Belgrade, Serbia Introduction: Dyslipidemia is commonly seen in patients with end-stage renal disease (ESRD). Small-sized high-density lipoprotein (HDL) particles are feature of atherogenic dyslipidemia, but their predictive role for ESRD progression is uncertain. Objective: This prospective study investigates the effect of small-sized HDL particles alone and in combination with increased high sensitivity C-reactive protein (hsCRP) levels on survival of ESRD patients treated by haemodialysis (HD). Methods: We performed 36 months follow-up study in 122 HD patients. HDL size and subclass distribution were determined by gradient gel electrophoresis, while serum hsCRP and lipid parameters were measured by standard laboratory methods. Baseline characteristics of the patients were evaluated for the prediction of mortality. Results: The carriers of small-sized HDL particles were more prevalent among patients who died (n = 24) compared with those who survived (n = 98, P < 0.05). Serum hsCRP levels were significantly higher in deceased patients (P < 0.05). Univariate Cox regressions analysis showed that patients with small-sized HDL particles had 2.8-fold higher risk of adverse disease outcome (P < 0.05). In
215
addition, concomitant presence of small-sized HDL particles and increased hsCRP concentration was significantly associated with reduced survival rate (HR = 3.907; P < 0.05). Observed relationships persisted after adjustment for serum lipid and lipoprotein concentrations. Conclusions: Both dyslipidemia and inflammation play significant role in the progression of ESRD. Our results indicate that small-sized HDL particles alone and combined with elevated hsCRP concentrations are independent predictors of reduced survival in HD patients. MS526 UK INPRACTICE STUDY: CONCORDANCE BETWEEN APOLIPOPROTEIN (Apo) B LEVELS AND LDL-CHOLESTEROL AMONG PATIENTS ON STATINS & NON-STATIN TREATMENT I. Idris1 , H. Tate2 , A. Ahmad3 , T. Mccormack4 . 1 Sherwood Forest Hospitals Foundation Trust, Mansfield, 2 MSD, Hoddesdon, 3 Medical, MSD, Welwyn Garden City, 4 Whitby Group Practice, Whitby, UK Background: ApoB is known to be a superior predictor of LDL-C particle number and CVD risk compared with LDL-C. In patients not taking lipid lowering therapy, current evidence has questioned the concordance between LDL-C with ApoB levels. However the concordance in patients receiving lipid lowering therapies is unclear. This analysis examines the LDL-C and ApoB levels among patients receiving statins and non-statin treatment. Methods: We performed post hoc analysis of the INPRACTICE* data looking at the concordance between LDL-C with ApoB values. This analysis involved 760 high-risk CVD patients from 34 primary care centres initially treated with simvastatin (S) 40 mg at baseline subsequently randomized to adding ezetimibe (E) to S 40 mg or changed to atorvastatin (A) 40 mg or to rosuvastatin (R) 5−10 mg for 6 weeks. Graphs and cross-tabulated quintile tables were assessed. Results: Based on the post-6-weeks treatment values showed that for all treatment groups there was a similar relationship between LDL-C and ApoB; Pearson correlation coefficients were 0.84 (E+S), 0.82 (A) and 0.83 (R). Proportions of patients who fell into the same quintile were 49% (E+S), 46% (A) and 44% (R). Kappa analysis confirmed fair agreement for all treatment groups; 60 (E+S), 0.58 (A) and 0.55 (R). Proportions of patients achieving ApoB< 90 mg/dl were 85% (E+S), 72% (A) and 58% (R); P < 0.0001. Conclusion: There was a similar concordance pattern between LDL-C and ApoB in patients treated with E+S compared to A and R. A higher percentage of patients achieved an ApoB level of < 90 mg/dl whilst treated with E+S compared to A and R. *Presented at ESC 2009 MS527 EVALUATION OF LIPID, INFLAMMATORY AND OXIDATIVE STRESS/ANTI-OXIDATIVE DEFENCE MARKERS FOR THE PREDICTION OF CARDIOVASCULAR RISK N. Bogavac-Stanojevic, Z. Jelic-Ivanovic, S. Spasic, V. SpasojevicKalimanovska, J. Kotur-Stevuljevic. Institute of Medical Biochemistry, Faculty of Pharmacy, University in Belgrade, Belgrade, Serbia Objectives: Atherosclerosis is a complex disease caused by a variety of risk factors such as dyslipidemia, inflammation and oxidative stress. However, none of the aforementioned risk factors used as a single biomarker is able to predict coronary artery disease (CAD) with sufficient accuracy. Measurement of multiple biomarkers may be an effective strategy to improve prediction of CAD. Methods: The effectiveness of apolipoproteins, lipoproteins, inflammatory and oxidative stress/anti-oxidative defence risk parameters supplementary to Framingham scoring data (FRS) was established in 188 CAD patients and 197 controls. The discriminative ability of the models was evaluated using receiver-operating characteristic (ROC) curves with the predictive probabilities from logistic regression models. The jackknife technique was used to detect the outliers. Results: The model, consisting of FRS, superoxide dismutase (SOD) and superoxide anion had outstanding discriminative ability with the areas under the curves (AUC) value of 0.924. One more model (FRS plus SOD) had an outstanding AUC value (0.906). All other models had excellent discriminative abilities (AUC values between 0.854 and 0.887). Results obtained using the jackknife technique indicate that our original model estimates were stable and were not affected by influential outlier cases within the study sample. Conclusions: Our results support the feasibility of using oxidative stress/antioxidative defence markers in the clinical laboratory. Prospective studies validating the utility of novel markers in combination with FRS are needed to fully determine the most effective model for coronary risk assessment. Funding: This work was supported by a grant from the Ministry of Science, Republic of Serbia.