Kirsti Berg

The Additive Contribution from Inflammatory Genetic Markers on the Severity of Cardiovascular Disease

Inflammation plays a key role in the development of atherosclerosis. Genetic differences in molecules related to inflammation have therefore been linked to the susceptibility for and severity of atherosclerosis. We hypothesized that the additive contribution from different genes of importance for inflammation would enhance the severity of cardiovascular disease. Blood samples were collected from 230 adults admitted for elective coronary angiography. A total of 130 patients had significant (>50%) stenosis in at least one main coronary artery branch and 100 had not. Six polymorphisms in five different genes were analysed: myeloperoxidase (MPO) −129G/A and −463G/A, toll‐like receptor 4 (TLR4) Asp299Gly, interleukin‐6 (IL6) −174G/C, surfactant protein D (SFTPD) Met11Thr and regulated upon normal T‐cell expressed and secreted (CCL5) −403G/A. The IL6 polymorphism was significantly associated (P = 0.017) to angiographic significant coronary artery disease, and this relation remained after adjustment for age, gender, smoking and hypercholesterolaemia (P = 0.007). The TLR4 (P = 0.050) and SFTPD (P = 0.058) polymorphisms were also associated with the presence of coronary stenosis in univariate but not in multivariate analyses. For MPO and CCL5 no associations were found. There was a significant linear association between the number of high‐risk gene variants (IL6−174CC, SFTPD 11CC and TLR4 299AA) and the proportion of patients with coronary artery disease (P < 0.0005). Inherited factors related to inflammation may increase susceptibility for severe coronary artery disease. Furthermore, the additive contribution from different inflammatory genetic markers strongly enhances the individual severity of cardiovascular disease.

Effects of manganese dipyridoxyl diphosphate, dipyridoxyl diphosphate--, and manganese chloride on cardiac function. An experimental study in the Langendorff perfused rat heart.

RATIONALE AND OBJECTIVES.Manganese dipyridoxyl diphosphate (MnDPDP) is a promising contrast agent for magnetic resonance imaging of the liver. The authors explored the possibility that high concentrations of MnDPDP may cause manganese ion (Mn++)-induced side effects on cardiac function. METHODS.Potential cardiodepression by MnDPDP, DPDP– –, and manganese chloride (MnCl2) (100-3,0000 µmol/ L) was investigated in the isolated rat heart, with left ventricular developed (systolic—end-diastolic) pressure and heart rate as the primary indices of cardiac function. RESULTS.During 5-minute exposures, 10% and 50% decreases in left ventricular developed pressure were observed for MnDPDP, 250 µmol/L and 1580 /µmoI/L; DPDP– –, less than 100 µmol/L and 1000 /µmoI/L; MnCl2, 30 /µmol/L and 250 /µmol/L. Heart rate changes were not observed with MnDPDP. Cardiodepression was reversed within 2 minutes during a 14- minute recovery period for all investigated concentrations of MnDPDP and was less rapid for the highest concentrations of MnCl2. CONCLUSIONS.Manganese dipyridoxyl diphosphate is well tolerated in the rat heart at concentrations as high as 200 to 250 limol/L and is approximately 10 times less cardiotoxic than MnCl2. Cardiodepressive effects of MnDPDP in the present rat heart model, perfused in the absence of blood and proteins, are related primarily to the release of free Mn++ions and in part to the simultaneous release of DPDP¯ ¯.

Myocardial manganese elevation and proton relaxivity enhancement with manganese dipyridoxyl diphosphate. Ex vivo assessments in normally perfused and ischemic guinea pig hearts.

Manganese (Mn) dipyridoxyl diphosphate (MnDPDP) is the active component of a contrast medium for liver MRI. By being metabolized, MnDPDP releases Mn2+, which is taken up and retained in hepatocytes. The study examined whether MnDPDP elevates Mn content and enhances proton relaxivity in normal myocardium, but not in ischemic myocardium with reduced coronary flow and impaired metabolism. Isolated guinea pig hearts were perfused at normal flow or low flow, inducing global subtotal ischemia. Ventricular ATP and Mn contents, T1 and T2 were measured. At normal flow tissue Mn content increased from the control level of 4.1 to 70.4 µmol/100g dry wt with MnDPDP (3000 µM), while low‐flow perfusion with MnDPDP (3000 µM) resulted in a Mn content of 16.6 µmol/100 g dry wt. Prolonged ischemia (35 and 90 min) reduced tissue Mn down to the control level. T1 shortening closely paralleled myocardial Mn elevations during both normal and low‐flow perfusion. The use of a Mn2+‐releasing contrast agent like MnDPDP may be a promising principle in MRI assessments of myocardial function and viability in coronary heart disease by revealing a differential pattern of changes in T1 relative to coronary flow, cell Mn uptake and retention, ion channel function and metabolism. Copyright

Oxidative stress and inflammatory response during and following coronary interventions for acute myocardial infarction

Background: In acute myocardial infarction (AMI) treated with percutaneous coronary intervention (PCI), myocardial injury results from complex processes during both ischemia and reperfusion. Release of reactive oxygen species (ROS) may contribute to the accumulated myocardial damage. Aims: To examine by frequent sampling of peripheral blood oxidative stress and early inflammation in patients undergoing primary PCI for AMI. Secondly, to assess whether a correlation exists between these parameters and the extent of myocardial damage. Methods: Sixteen patients undergoing primary PCI within 6 h of AMI onset were included. Peripheral blood was sampled at start of procedure (t0) and repeatedly over 24 h following reperfusion. Main plasma analyses were: 8-iso-PGF2α (oxidative stress), 15-keto-dihydro-PGF2α (cyclooxygenase-mediated inflammation); and troponin-T (myocardial injury). Additional analyses included: total antioxidant status (TAS); vitamins; hsCRP and lipids. Results: 8-Iso-PGF2α increased following restoration of blood flow, returned to t0 values after 3 h and was reduced below t0 the following day. TAS decreased significantly from t0 to the next day. There was no significant correlation between 8-iso-PGF2α and troponin T values. 15-Keto-dihydro-PGF2α was elevated during the first hour. There was a major rise in hsCRP after 24 h. Conclusion: Following reperfusion by primary PCI in AMI, oxidative stress and an inflammatory response are induced immediately. A rise in 8-iso-PGF2α during ischemia indicate that ROS generation may also take place during severely reduced coronary blood flow and hypoxia. No direct relationship between 8-iso-PGF2α or 15-keto-dihydro-PGF2α and troponin T was evident. The present study adds to the increasingly complex pathophysiological roles of ROS acting both as signal molecules and as mediators of tissue injury.

Oxidative stress and myocardial damage during elective percutaneous coronary interventions and coronary angiography. A comparison of blood-borne isoprostane and troponin release.

The role of oxidative stress in clinical cardiology is still controversial. The aims of the present study were to examine if minor ischaemic episodes as may occur during elective percutaneous coronary intervention (PCI) induce oxidative stress and, eventually, if oxygen stress correlates with myocardial injury. Thirty eight and nine patients underwent PCI and diagnostic coronary angiography, respectively. Peripheral blood was sampled at different time points for plasma analyses of: 8-iso-PGF2α (free radical-mediated oxidative stress); 15-keto-dihydro-PGF2α (cyclooxygenase-mediated inflammation); troponin-T (myocardial injury); hsCRP, vitamin A and vitamin E; and, total antioxidants status (TAS). In both groups 8-iso-PGF2α increased transiently by approximately 80% (p<0.001) during the procedure. There was a minor troponin-T release (p<0.001) after PCI, but no correlation with 8-iso-PGF2α. Troponin-T did not increase after angiography. 15-keto-dihydro-PGF2α decreased by 50% after ended procedure, but increased by 100% after 24 h compared to baseline. hsCRP increased significantly (p<0.001) from baseline to the next day in the PCI-group, but not in the angiography group. Vitamins and TAS decreased slightly after the procedures. It is concluded that a moderate oxidative stress was induced by both elective PCI and coronary angiography but that no correlation was found between oxidative stress and myocardial injury in this setting. This indicates that other mechanisms than ischaemia–reperfusion episodes caused an elevation in plasma isoprostane such like the injury at a vascular site mutual for both procedures. A secondary finding from the study was elevated markers of early inflammatory response, not only after PCI, but also after angiography.

Acetylsalicylic acid treatment until surgery reduces oxidative stress and inflammation in patients undergoing coronary artery bypass grafting

OBJECTIVES Acetylsalicylic acid (ASA) is a cornerstone in the treatment of coronary artery disease (CAD) due to its antiplatelet effect. Cessation of aspirin before coronary artery bypass grafting (CABG) is often recommended to avoid bleeding, but the practice is controversial because it is suggested to worsen the underlying CAD. The aims of the present prospective, randomized study were to assess if ASA administration until the day before CABG decreases the oxidative load through a reduction of inflammation and myocardial damage, compared with patients with preoperative discontinuation of ASA. METHODS Twenty patients scheduled for CABG were randomly assigned to either routine ASA-treatment (160 mg daily) until the time of surgery (ASA), or to ASA-withdrawal 7 days before surgery (No-ASA). Blood-samples were taken from a radial artery and coronary sinus, during and after surgery and analysed for 8-iso-prostaglandin (PG) F2α; a major F2-isoprostane, high-sensitivity C-reactive protein (hs-CRP), cytokines and troponin T. Left ventricle Tru-Cut biopsies were taken from viable myocardium close to the left anterior descending artery just after connection to cardiopulmonary bypass, and before cardioplegia were established for gene analysis (Illumina HT-12) and immunohistochemistry (CD45). RESULTS 8-Iso-PGF2α at baseline (t1) were 111 (277) pmol/l and 221 (490) pmol/l for ASA and No-ASA, respectively (P = 0.065). Area under the curve showed a significantly lower level in plasma concentration of 8-iso-PGF2α and hsCRP in the ASA group compared with the No-ASA group with (158 pM vs 297 pM, P = 0.035) and hsCRP (8.4 mg/l vs 10.1 mg/l, P = 0.013). All cytokines increased during surgery, but no significant differences between the two groups were observed. Nine genes (10 transcripts) were found with a false discovery rate (FDR) <0.1 between the ASA and No-ASA groups. CONCLUSIONS Continued ASA treatment until the time of CABG reduced oxidative and inflammatory responses. Also, a likely beneficial effect upon myocardial injury was noticed. Although none of the genes known to be involved in oxidative stress or inflammation took a different expression in myocardial tissue, the genetic analysis showed interesting differences in the mRNA level. Further research in this field is necessary to understand the role of the genes.

Cardiac Metal Contents After Infusions of Manganese: An Experimental Evaluation in the Isolated Rat Heart

RATIONALE AND OBJECTIVES Manganese dipyridoxyl diphosphate (MnDPDP), a contrast agent for liver MRI, releases free Mn2+ in a graded manner. The aim of the study was to compare the effects of brief versus prolonged infusions of MnDPDP and manganese chloride (MnCl2) on cardiac function, metabolism, Mn accumulation, and tissue metal content. METHODS Isolated perfused rat hearts received 1-minute or 10-minute infusions of MnDPDP (100 microM, 1000 microM) or of MnCl2 (10 microM, 100 microM). Physiologic indices were measured intermittently, and tissue high-energy phosphate compounds and Ca/Fe/Mg/Mn/Zn contents were measured after a standardized Mn washout. RESULTS One-minute and 10-minute infusions induced, respectively, minor and marked depressions of contractile function and corresponding elevations in myocardial Mn content. MnCl2 was markedly more potent than MnDPDP. Ten-minute infusions of the highest concentration of MnDPDP and MnCl2 lowered tissue Mg and elevated tissue Ca (MnCl2), whereas high-energy phosphates were unaffected. CONCLUSIONS Mn uptake after Mn infusion is strongly related to the duration, concentration, and dose of free Mn ions. Differences in Mn accumulation between MnDPDP and MnCl2 were more pronounced after the 10-minute infusion.

Effects of MnDPDP, DPDP--, and MnCl2 on cardiac energy metabolism and manganese accumulation. An experimental study in the isolated perfused rat heart.

RATIONALE AND OBJECTIVES Recent studies indicate that manganese dipyridoxyl diphosphate (MnDPDP) may function as a slow release agent for manganese ions (Mn++) and that MnDPDP is approximately 10 times less potent than manganese chloride (MnCl2) in depressing cardiac function. The authors examined the possibility that MnDPDP and MnCl2 may influence cardiac metabolism and enzyme release and lead to a tissue accumulation of Mn. METHODS Manganese DPDP, DPDP--, or MnCl2 (1000 microM) was infused in isolated rat hearts, which were freeze-clamped at various time intervals during infusion (5 minutes) and recovery (14-minute washout). Enzyme (lactate dehydrogenase) release, tissue high energy phosphates, Mn contents, and physiologic indices were measured at various time intervals. RESULTS No significant differences were noted for: lactate dehydrogenase in the treated groups; tissue creatine phosphate (CrP) and adenosine triphosphate in MnDPDP, DPDP--, and control groups; and tissue Mn in DPDP-- and control groups. Manganese-chloride and MnDPDP-treated hearts accumulated and retained Mn in an 8:1 ratio. Manganese chloride depressed cardiac function more effectively than MnDPDP. CONCLUSIONS The study has shown that: heart tissue uptake and retention of Mn++ is rapid and effective; MnCl2 is approximately eight times more potent than MnDPDP in promoting these effects; and a rise in tissue Mn content to eight to nine times (MnDPDP) or 60 to 70 times (MnCl2) the normal level does not lead to acute side effects on cardiac energy metabolism, function, and enzyme release. The study indicates that MnDPDP may act like a slow release compound for Mn++ ions.

Oxidative stress during coronary artery bypass operations: importance of surgical trauma and drug treatment.

Objective. To investigate oxidative stress and myocardial injury at different stages of coronary artery bypass grafting (CABG). Design. Twenty patients underwent CABG with use of cardiopulmonary bypass (CPB) and with intermittent sampling of plasma and urine. Main markers were: 8-iso-PGF2α (oxidative stress); troponin T (myocardial injury); and 15-keto-dihydro-PGF2α and hsCRP (inflammation). Results. Plasma 8-iso-PGF2α increased after start of surgery, but there was no further rise during CPB or after aortic cross-clamp release and no significant myocardial arterio-venous differences. An increase in troponin T was seen early after the operation, but no relationship was established between 8-iso-PGF2α and troponin T. 8-iso-PGF2α levels were elevated by preoperative withdrawal of acetylsalicylic acid (ASA) but reduced by intraoperative use of heparin. 15-keto-dihydro-PGF2α was elevated during operation and hsCRP following operation. Conclusions. In the present study oxidative stress was multifactorial in origin with main impacts from surgical trauma, less from CPB and little if any from myocardial ischemia-reperfusion events. In addition, cardiovascular drugs in common use like ASA and heparin seemed to influence the pro- and antioxidant balance, a finding that has to be confirmed in future studies.

A High Precision Method for Quantitative Measurements of Reactive Oxygen Species in Frozen Biopsies

Objective An electron paramagnetic resonance (EPR) technique using the spin probe cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) was introduced as a versatile method for high precision quantification of reactive oxygen species, including the superoxide radical in frozen biological samples such as cell suspensions, blood or biopsies. Materials and Methods Loss of measurement precision and accuracy due to variations in sample size and shape were minimized by assembling the sample in a well-defined volume. Measurement was carried out at low temperature (150 K) using a nitrogen flow Dewar. The signal intensity was measured from the EPR 1st derivative amplitude, and related to a sample, 3-carboxy-proxyl (CP•) with known spin concentration. Results The absolute spin concentration could be quantified with a precision and accuracy better than ±10 µM (k = 1). The spin concentration of samples stored at −80°C could be reproduced after 6 months of storage well within the same error estimate. Conclusion The absolute spin concentration in wet biological samples such as biopsies, water solutions and cell cultures could be quantified with higher precision and accuracy than normally achievable using common techniques such as flat cells, tissue cells and various capillary tubes. In addition; biological samples could be collected and stored for future incubation with spin probe, and also further stored up to at least six months before EPR analysis, without loss of signal intensity. This opens for the possibility to store and transport incubated biological samples with known accuracy of the spin concentration over time.