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Methylprednisolone (MP), a synthetic glucocorticoid, is widely used clinically and experimentally as acute antiinflammatory treatment. The molecular actions of MP indicate that pretreatment with this drug may be cardioprotective. We investigated if giving rats MP prior to excising their hearts for Langendorff-perfusion protected cardiac function against oxidative stress, and if this was mediated by increasing antioxidant defence or influencing myocardial nitric oxide synthase (NOS). Rats (n=6-11 in each group) were injected with MP (40mg/kg i.m.) or vehicle 24 and 12 h before Langendorff-perfusion with 30 min global ischaemia and 60 min reperfusion, or 10 min perfusion with 180 micromol/L hydrogen peroxide. Other hearts were exposed to 30 min global ischaemia 5 days after MP-injection. Additional hearts were sampled before, during, and after ischaemia for analyzing tissue activity of antioxidant enzymes. Tissue endothelial and inducible NOS (eNOS and iNOS) were investigated by immunoblotting and semiquantitative RT-PCR in a time-course after MP injection. Pretreatment with MP improved left ventricular function and increased coronary flow during postischaemic reperfusion, and this effect was sustained 5 days afterwards. When exposing hearts to hydrogen peroxide, MP improved coronary flow. Catalase, glutathione peroxidase, and oxidized glutathione were increased during reperfusion of MP-treated hearts compared to vehicle only. MP did not influence eNOS at protein or mRNA level. iNOS could not be detected by immunoblotting, indicating low cardiac enzyme content. Its mRNA initially increased the first hour after injection, thereafter decreased. In conclusions, pretreating rats with MP protects the heart against ischaemia-reperfusion dysfunction. This effect could be due to increase of tissue antioxidant activity during reperfusion. MP did not influence cardiac eNOS. mRNA for iNOS was influenced by MP, but the corresponding protein could not be detected.

Reactive oxygen intermediates are important mediators of inflammation. We investigated if hydrogen peroxide (H2O2) induces tumour necrosis factor alpha (TNFalpha) expression in cultured human cells from umbilical vein endothelium (HUVEC), aortic smooth muscle cells (SMC), peripheral blood mononuclear cells (PBMC), or the cell line Mono Mac 6. Cultures were stimulated with 200 micromol/L H2O2 for 15 min. After 4 h cells were harvested, mRNA extracted, and amplified by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) with histone (H3) as reference gene. In HUVECs, mRNA for TNFalpha increased with a factor of 4 after stimulation (p < 0.001), in PBMC with a factor of 2 (p < 0.05), while mRNA from SMC and Mono Mac 6 did not increase significantly. Cellular TNFalpha protein in HUVECs was measured with flow cytometry (FACS) before and 6, 12, and 24 h after stimulation. TNFalpha protein was detectable in small, but reproducible amounts 12 h after stimulation, and increased further after 24 h. However, no secretion of TNFalpha was detected by ELISA. FACS analysis of the passaged HUVEC cultures did not reveal any contamination with non-endothelial cells. In conclusion, H2O2 induces TNFalpha mRNA in HUVECs and PBMC. In HUVECs an increase of intracellular TNFalpha protein was also detected, indicating that endothelial cells can produce small amounts of TNFalpha.

Ventricular fibrillation (VF) and ventricular tachycardia (VT) are common phenomena during reperfusion. In experimental research many hearts have to be excluded from haemodynamic evaluation because of severe arrhythmias. Theoretically, electroconversion or mechanical conversion (MC) might be used to convert VF or VT. MC induces a physical shock analogous to a chest thump. The aim of this study was to investigate the efficacy of MC in isolated, perfused rat hearts, and to see whether MC itself induced myocardial cell injury and functional impairment. Langendorff-perfused rat hearts (n = 89) from several experimental series subjected to 30 min of global ischaemia and 60 min of reperfusion were retrospectively analysed. Left ventricular systolic (LVSP), end-diastolic (LVEDP), and developed (LVDP) pressures, coronary flow (CF), and heart rate (HR) were measured. If VF or VT continued for 1 min during reperfusion, MC was attempted by a flick of the forefinger to the right ventricle. If VF or VT still occurred, MC was repeated. Hearts that did not have regular beating after 20 min of reperfusion were excluded. Release of cardiac troponin T (cTnT) was measured before ischaemia and after 20 min of reperfusion. Forty-four out of 89 hearts had VF or VT during reperfusion. Thirty-five hearts were converted, 18 of which were converted by one or two MCs only. The higher the total number of MCs employed, the more cTnT was released. After 20 min of reperfusion, LVEDP, LVDP and CF were better in hearts with a higher number of MCs and with increased release of cTnT. After 60 min of reperfusion, LVEDP was still improved in hearts with more cTnT release, whereas LVSP was lower, and LVDP and CF were independent of the number of MCs. There was no consistent correlation between release of cTnT and heart dysfunction. In conclusion, MC effectively converted VF or VT. MCs increased post-ischaemic myocardial cell damage, as judged from increased cTnT release. Post-ischaemic dysfunction was partly attenuated in hearts with multiple MCs, and did not correlate with release of cTnT. We feel that MCs should not be used in isolated, perfused hearts.

We investigated the effect of H290/51, a novel, low-molecular-weight inhibitor of lipid peroxidation, on cardiac ischemia–reperfusion injury. Lactate dehydrogenase (LD) release from cultured cardiomyocytes exposed to 1 h hypoxia and 4 h reoxygenation was measured after pretreatment with different concentrations of H290/51. In another series, Langendorff-perfused rat hearts were exposed to 30 min global ischemia and 60 min reperfusion (n = minimum 10 in each group): 1. Control ischemia–reperfusion. 2. Vehicle throughout the experiment. 3. Vehicle during stabilization, and H290/51 (10−6 mol/l) during reperfusion. 4. H290/51 throughout the experiments. During reoxygenation of isolated cardiomyocytes, H290/51 dose dependently inhibited LD release with an pIC50 value of 7.2 ± 0.4 (mean ± SEM), with 10−6 mol/l as the lowest efficient concentration. In isolated hearts ischemia–reperfusion induced severe reperfusion arrhythmias, reduced left ventricular developed pressure (LVDP) and coronary flow (CF), and increased LV end-diastolic pressure (LVEDP). LD activity in the effluent increased. H290/51 throughout perfusion (group 4) reduced the occurrence of severe reperfusion arrhythmias (p < .0001), attenuated the decrease of LVDP (p < .008), and CF (p < .006), the increase of LVEDP (p < .008), and the release of LD (p < .002). Tissue contents of thiobarbituric acid-reactive substances did not increase during reperfusion in controls, but was reduced in group 4 (p < .004). H290/51 given only during reperfusion (group 3) tended to improve cardiac function, but significantly so only for increase of CF (p < .01). The lipid peroxidation inhibitor H290/51 attenuated cardiac injury induced by ischemia–reperfusion.

Thioredoxin (Trx) and glutaredoxin (Grx) are both multifunctional redox-active proteins. In this study, Grx was identified in human plasma by immunoaffinity purification. The affinity-purified material from human plasma displayed a band of 12 kDa identical to recombinant human Grx by Western blotting and its glutathione-dependent reducing activity of β-hydroxyethyl disulfide. Competitive enzyme-linked immunosorbent assays (ELISA) showed that plasma levels (mean ± SD) of Grx and Trx in healthy volunteers (n = 41) were 456 ± 284 ng/ml and 28.5 ± 12.6 ng/ml, respectively. In cardiac surgical patients (n = 17), plasma Grx levels did not significantly change during cardiopulmonary bypass (CPB). In contrast, Trx levels in arterial plasma measured by sandwich ELISA and corrected for hemolysis were elevated during reperfusion of the postcardioplegic heart (p = .0001 at maximum), whereas by competitive ELISA Trx increased during surgical preparation for CPB, but decreased during CPB. When recombinant Trx was oxidized, immunoreactive Trx levels were decreased by competitive ELISA but not changed by sandwich ELISA. These results suggest that oxidized Trx is released into plasma during CPB. There was no significant difference in Trx and Grx levels between arterial and intracoronarial plasma samples, indicating no specific release by the postcardioplegic heart. Trx and Grx may be important components in the plasma defense against oxidative stress.

To evaluate the potential of tissue plasminogen activator (t-PA) as a marker of endothelial activation or injury, the dose-response relationship between reactive oxygen intermediates and t-PA release was investigated in isolated rat hearts. After stabilization the hearts were perfused for 10 minutes with different concentrations of hydrogen peroxide (H 2 O 2 ) (0 (control perfusion), 20, 40, 80, 120, 160, or 200 μM) (n=8 hearts/group), followed by 30 minutes recovery. Higher concentrations than 80 μM induced cardiac dysfunction and a dose-dependent release of lactate dehydrogenase, indicating myocyte injury. H 2 O 2 -concentrations of 80 μM and more caused a significant, but temporary t-PA release. Peak t-PA release occurred more rapidly with higher concentrations, but otherwise there was no difference dependent on the H 2 O 2 -dose. The effects of H 2 O 2 (120 or 200 μM) on t-PA release were also compared to the effects of bradykinin. Both were given for 10 minutes as above, and the procedure was repeated after 10 minutes recovery. Bradykinin (50 or 500 nM) released t-PA with the same magnitude, but with peak values occurring earlier than t-PA release induced by H 2 O 2 . Bradykinin, but not H 2 O 2 , induced t-PA release during the second exposure, suggesting different mechanisms of release. In conclusion: Perfusion with H 2 O 2 leads to a dose-dependent myocardial injury in isolated rat hearts. H 2 O 2 also causes an acute t-PA release without dose-dependency, suggesting an all or nothing response of the endothelium. t-PA may be used as an indicator of, but cannot quantify endothelial activation or injury. Copyright © 1997 Elsevier Science Ltd

Cold cardioplegic arrest causes reperfusion injury to both endothelium and myocardium. We investigated release of troponin-T (TnT), tissue plasminogen activator activity (t-PA) and histamine (HA) from the heart before and after 2h of cold crystalloid cardioplegia in eight Swedish landrace pigs. Coronary sinus blood flow was measured in an external shunt between the coronary sinus and the right atrium. TnT, t-PA and HA were measured concomitantly in arterial and coronary sinus plasma, and the cardiac release was calculated. Cardiac release of TnT increased from 18 (15-25) micrograms/min (median (central 90% percentile)) before cold cardioplegia to maximum 281 (132-510) micrograms/min 30 min after aortic declamping (p0.02 vs initial value). t-PA rose from -4 (-52-34) to maximum 249 (75-691) IU/min 2 min after declamping (p0.01) and thereafter returned to baseline levels. The net cardiac release of HA was 72 (-80-1321) nmol/min before cardioplegia, rising to 234 (-188-524) after 2 min of reperfusion (p0.02) and returning to baseline after 30 minutes. We conclude that the porcine heart releases t-PA, Tn-T and HA during postcardioplegic reperfusion. The differing kinetics of their release may indicate different affection of the myocardium and the endothelium. Tn-T, t-PA and HA are potential markers of myocardial and endothelial injury in the porcine heart.

Objectives Bone morphogenetic proteins (BMP) may have multiple actions on cardiac cells. The aim of our study is to investigate the effect of BMP4 on oxidative stress-induced cardiomyocyte cell death and the possible signalling pathway. Methods All experiments were conducted using the immortalised cardiomyocyte-like HL-1 cells. 1, Oxidative stress was induced by hydrogen peroxide (H 2 O 2 ). HL-1 cells were stimulated with incremental concentrations of H 2 O 2 (10, 50, 100, 200, 300, 400, 500 µM) for 4 hours, and cell viability was evaluated to establish the optimal model of H 2 O 2 -induced injury. 2, To study the effect of BMP4 on cell viability, HL-1 cells were seeded on 96-well plates and pretreated for 24 hours with different concentrations of human recombinant BMP4 (0,10, 50, 100ng/ml) prior to the application of optimal concentrated H 2 O 2 For 4 hours. 3, To define the signalling mechanism downstream to BMP4 effects, Proteins were isolated from cells 30min and 24h after treatment of BMP4, and subjected to western blotting with antibodies against phosphorylated Smad1/5/8 and the downstream protein-inhibitor of differentiation-1 (ID-1). The western blot analysis expression value was calculated relative to that of actin. Cell viability was detected by using Lactate dehydrogenase (LDH) cytotoxicity detection kit. Results 300uM H 2 O 2 induces 57.8 ± 5.1% death of HL-1 cells, and was selected for further experiments. HL-1 cells pretreated with 100ng/ml recombinant BMP4 had a significiant reduction of H 2 O 2 -mediated cell death (38.4 ± 2.1%, P Conclusions Recombinant BMP4 protect HL-1 cells from oxidative stress. Smad1/5/8 and ID-1 may be involved in the protective BMP4 signalling pathway.

Ischemic preconditioning reduces infarct size and cardiac dysfunction during reperfusion. Preconditioning may offer myocardial protection in open heart operations.The effect of preconditioning before ischemia and cardioplegia was investigated in Langendorff-perfused rat hearts in the following groups. First, group 1 received two episodes of 3-minute ischemia and 5-minute reperfusion before 25 minutes of global (37 degrees C) ischemia and 60 minutes of reperfusion. Group 2 served as ischemic controls to group 1. Groups 3, 5, and 7 were preconditioned as described, before 3.5, 4, or 5 hours of cold (6 degrees to 8 degrees C) St. Thomas' II cardioplegia and 1 hour of reperfusion (37 degrees C). Groups 4, 6, and 8 were cardioplegic controls to groups 3, 5, and 7 (n = 17 in groups 1 and 2, and n = 10 in groups 3 to 8).Preconditioning before warm ischemia attenuated the ischemia-induced increase of left ventricular end-diastolic pressure (3 +/- 1 versus 17 +/- 4 mm Hg; p0.01) (mean +/- standard error of the mean), the reduction of coronary flow (14 +/- 1 versus 9 +/- 0.5 mL/min; p0.001) and heart rate (252 +/- 19 versus 198 +/- 18 beats/min; p0.04), and the incidence of ventricular fibrillation (2 of 17 versus 10 of 17 hearts; p0.04) at the start of reperfusion. However, preconditioning did not influence postischemic cardiac function or the release of lactate dehydrogenase in any of the cardioplegia groups.Ischemic preconditioning improved post-ischemic cardiac function after warm global ischemia, but did not protect cold cardioplegic hearts, perhaps because of the time span used.

Thrombomodulin is a potential marker of endothelial injury. Plasma thrombomodulin was measured in concomitant arterial and coronary sinus samples in 9 patients undergoing elective coronary artery bypass surgery with cardiopulmonary bypass (CPB, 88 +/- 14 min) (mean +/- SD) and cold, crystalloid, antegrade cardioplegia (44 +/- 14 min). Arterial thrombomodulin was 17 +/- 6 ng/ml before surgery, and decreased to 10 +/- 5 ng/ml after heparinization (p0.008 compared to initial value). During CPB thrombomodulin increased, with a maximal level of 23 +/- 7 ng/ml (p0.008 vs initial value) 40 min after aortic declamping. No difference between arterial and coronary sinus concentrations was detected during reperfusion of the heart. In conclusion, plasma thrombomodulin is decreased by heparin, and increased during CPB. Consequently, thrombomodulin may be used to evaluate endothelial injury during CPB. However, as there is no specific intracoronary release of thrombomodulin during reperfusion, thrombomodulin is not a suitable marker of coronary endothelial injury after cardioplegia.

Myocardial ischemia-reperfusion injury increases both tissue levels and release of histamine. To study the possible effects of ischemia-reperfusion on histamine metabolism tissue activities of histidine decarboxylase (HDC), histamine N-methyl transferase (HNMT) and diamine oxidase (DAO) were investigated in isolated rat hearts subjected to either 20 min global ischemia and 40 min reperfusion (n = 10) or control perfusion (n = 8). Histamine in the coronary effluent increased from 21 +/- 4 nmol/min (mean +/- SEM) before ischemia to 55 +/- 5 and 50 +/- 7 nmol/min after 4 and 10 min reperfusion (p0.004 and p0.004). Tissue HDC activity did not change during observation in any group. HNMT activity was unchanged in controls, but increased from 0.37 +/- 0.04 to 0.84 +/- 0.18 and 0.96 +/- 0.22 pmol methylhistamine/mg protein hour after 4 and 10 min reperfusion (p0.008 and p0.01). DAO decreased similarly in controls and ischemic-reperfused hearts during observation. In conclusion, the previously observed increase of tissue histamine during reperfusion cannot be explained by increased histamine synthesis or decreased histamine catabolism.

H290/51, an indenoindole derivative, is a novel low-molecular weight (287.8) inhibitor of lipid peroxidation. Its effect on cardiac injury induced by exogenous reactive oxygen intermediates (ROI) was investigated. ROI were generated by adding H2O2 (180 mu M) to the perfusate of isolated rat hearts (Langendorff model, n = 9) for 10 min. H2O2 reduced left ventricular developed pressure (LVDP = left ventricular systolic pressure -- left ventricular end-diastolic pressure) from 90 +/- 6 to a minimum of 25 +/- 2 mmHg (mean +/- SEM) after 10 min (p < 0.001), elevated left ventricular end-diastolic pressure (LVEDP) from 0 to 32 +/- 7 mmHg after 20 min (p < 0.0001), and increased coronary flow (CF). Lactate dehydrogenase (LDH) release in the coronary effluent and thiobarbituric acid-reactive substances (TBARS) in cardiac tissue increased (TBARS from 0.6 +/- 0.04 to 3.1 +/- 0.4 nmol/g tissue after 10 min of H2O2 administration, p < 0.001). Addition of H290/51 (1 mu M, n = 12) from the start of H2O2 exposure, attenuated the H2O2-induced increase of LVEDP (9 +/- 3 mmHg at 20 min, p < 0.006) and reduced the release of LDH (p < 0.02 at 30 min). LVDP was not significantly influenced. The increase of TBARS was abolished by H290/51 (p < 0.001). In conclusion, H290/51 inhibited lipid peroxidation, and attenuated functional and biochemical injury induced by H2O2 exposure.

Histamine has inotropic, chronotropic, arrhythmogenic, and vasoactive effects, and is released from the heart in ischaemia-reperfusion injury. The effect of ventricular fibrillation (VF) and defibrillation (DEF) on histamine release was investigated in 9 anaesthetized patients undergoing transvenous implantation of ICD. Concomitant arterial and coronary sinus (CS) blood samples were drawn before induction of VF (duration 20 seconds), immediately after, and 2 and 5 min after DEF (18-24 Joules). Basal arterial histamine was 2.5 +/- 6 nmol/l, and did not increase after VF. The histamine level in CS was 1.1 +/- 0.2 nmol/l before VF (p0.008 compared to arterial), and increased to 2.5 +/- 0.6 nmol/l immediately after (p0.045 compared to basal), to 3 +/- 1.1 nmol/l 2 min after (p0.45), and to 2.4 +/- 0.8 nmol/l 5 min after VF. In the basal state there was an uptake of histamine across the coronary circulation. After VF/DEF the level of histamine increased in coronary venous blood, suggesting cardiac release of histamine.

Histamine is released from the heart during ischaemia-reperfusion injury. As histamine has cardiac effects, we investigated the role of histamine in ischaemia-reperfusion injury of isolated rat hearts. A Langendorff-model with 30 min global (37 degrees C) ischaemia followed by 60 min reperfusion was employed. The effects of ischaemia alone (n = 10, group 1.1 + n = 10, group 2.1, 2 different series), and ischaemia with H1- and H2-receptor blockade with cimetidine (10 microM, n = 10), chlorpheniramine (10 microM, n = 8), terfenadine (10 microM, n = 8), and promethazin (10 microM, n = 9), or both cimetidine and chlorpheniramine (n = 8), were studied. Histamine was measured in the coronary effluent and cardiac tissue of group 1.1. Release of histamine increased from 6.5 +/- 1 pmol min-1 before ischaemia to 19 +/- 3 pmol min-1 at the start of reperfusion. Ischaemia decreased left ventricular developed pressure to 18 +/- 11% (1.1) and 50 +/- 11% (2.1) of initial value (mean +/- SEM) at the start of reperfusion. Left ventricular end-diastolic pressure increased from 0 to 79 +/- 8 mmHg (1.1) and 39 +/- 9 (2.1) mmHg, while left ventricular systolic pressure was unchanged (101 +/- 12% in 1.1 and 101 +/- 10% in 2.1). Severe arrhythmias were induced in 90 (1.1) and 30 (2.1)% of the hearts, while coronary flow decreased during reperfusion. H2-blockade did not modify the changes in left ventricular pressures, coronary flow, or heart rate induced by ischaemia. Three different H1-blockers increased left ventricular systolic pressure, inhibited the decrease of developed pressure, attenuated the increase of end-diastolic pressure, and totally inhibited reperfusion arrhythmias. The effect of both blockers together was similar to that of H1-blockers alone. Coronary flow was increased during reperfusion in two of the groups with H1-blocker compared with ischaemic controls. Increased release of histamine from ischaemic-reperfused rat hearts concurred with depression of left ventricular function and arrhythmias during early reperfusion. Cardiac dysfunction during reperfusion was attenuated by three different H1-receptor blockers.

von Willebrand Factor (vWF) is released from endothelial cells. Increased vWF in the coronary circulation during cardiac surgery could be a potential indicator of coronary endothelial injury or stimulation, and thus a possible tool to evaluate regimens of myocardial protection. Release of vWF was investigated in 12 patients undergoing coronary artery bypass surgery with cardiopulmonary bypass (CPB). Concomitant samples of arterial and coronary sinus blood for measurement of vWF (antigen method) were drawn before start of CPB and 1, 4, 10 and 30 min after release of the aortic cross clamp. Additional arterial samples were drawn pre-, per-, and postoperatively. Preoperative arterial vWF was 1.58 +/- 0.59 IU/ml (mean +/- SD), and increased during CPB (highest level 2.37 +/- 0.76 IU/ml, p0.0026). No difference between arterial and coronary sinus vWF levels was found. Arterial vWF increased further the first postoperative day (3.96 +/- 0.92 IU/ml, p0.0026). In conclusion, systemic vWF is increased during CPB, and may be a possible marker of endothelial injury/activation to evaluate deleterious effects of different equipment for CPB. Reperfusion of the ischaemic, cardioplegic heart did not release vWF in the coronary circulation.

Production of oxygen free radicals and subsequent lipid peroxidation are thought to occur during cardiopulmonary bypass (CPB) and myocardial ischaemia- reperfusion injury. Malondialdehyde (MDA), a lipid peroxidation product, was measured simultaneously in arterial and coronary sinus blood before CPB and after release of the aortic crossclamp. Additional arterial samples were drawn pre-, per-, and postoperatively. Thirteen patients scheduled for coronary artery and/or valvular surgery were studied. Cold, crystalloid, cardioplegic arrest (54 [35-120] minutes, median [range]) was induced retrogradely. Preoperatively, arterial MDA was 0.78 ± 0.4 (mean ± SD) μmol/l, and increased during CPB (highest level 3.66 ± 1.08 μmol/l, p < 0.002, 30 minutes after the start of reperfusion). Arterial MDA was still increased four hours after the end of CPB (3.17 ± 0.88 μmol/l, p < 0.003), but had returned to normal the first postoperative day. No difference was found between arterial and coronary sinus samples at any time. In conclusion, MDA increased in arterial blood during CPB, indicating that lipid peroxidation occurred. There was no intracoronary release of MDA during reperfusion of the ischaemic heart.

Release of histamine (H) by ischemia-reperfusion injury was investigated in isolated rat hearts (Langendorff model). The effect of 10, 15, 20, 25, 30, 40 and 60 min ischemia (n = 10 each) on H in the coronary effluent and in cardiac tissue was studied after 4 min reperfusion. Release of creatine kinase and lactate dehydrogenase in the coronary effluent increased with time of ischemia. Tissue H increased from 95 +/- 10 ng/g rat heart (mean +/- SEM) before ischemia to max 148 +/- 10 ng/g after 20 min ischemia (p0.002), and increased also after 15 (p0.01), 25 (p0.01), and 30 min (p0.045). H in the coronary effluent increased after 15 (from 16 +/- 3 to 26 +/- 2 pmol/min, p0.044), 30 (26 +/- 6 pmol/min, p0.027), and 60 min ischemia (47 +/- 6 pmol/min, p0.0044). Release of H during ischemia-reperfusion is neither dependent on the severity of the ischemic insult, nor on the level of tissue H.

Although necessary for the ultimate tissue survival, reperfusion may paradoxically exacerbate the ischemic injury. Ischemia and reperfusion injury is intimately woven together. The relative role of reperfusion injury is not clarified and probably varies with the ischemic insult: Reperfusion is always preceded by ischemia, and some of the reperfusion-related events may represent a process continuing from the ischemic period; thus the proper designation should be ischemia-reperfusion injury. The reperfusion-related events are: arrhythmias, myocardial stunning with both systolic and diastolic dysfunction, and low reflow and microvascular stunning. Of pathogenetic importance are the mode and speed of reperfusion as well as the initiation of an intracoronary inflammatory reaction during reperfusion, including endothelium-leukocyte interaction, platelets, generation of oxygen free radical, generation and release of arachidonic acid metabolites, platelet activating factor, endothelium derived relaxing factor, endothelins, kinins, and histamine, complement activation, disturbances in calcium homeostasis, and disturbances in lipid and fatty acid metabolism.

Histamine is synthetized in the heart, and released by ischemia-reperfusion injury in several species. Histamine has arrhythmogenic, chronotropic, inotropic and vasoactive effects. Cardiac histamine release during ischemia-reperfusion may be mediated by toxic oxygen metabolites. We studied the effect of ischemia-reperfusion and toxic oxygen metabolites on release and synthesis of histamine in the isolated rat heart (Langendorff model). The following groups were included: I, (n = 10) control perfusion for 60 min; II, (n = 7) H2O2 (200 microM) was given for 10 min followed by 50 min recovery; III, (n = 7) thiourea (15 mM) was given in addition to H2O2; IV, (n = 7) thiourea given alone; V, (n = 7) catalase (150 U/ml) plus H2O2; VI, (n = 7) 20 min ischemia followed by 40 min reperfusion. The contents of histamine in the coronary effluent and in cardiac tissue were measured repeatedly (radioenzymatic method). Ischemia-reperfusion and toxic oxygen metabolites increased release of histamine in the coronary effluent. Concomitantly the histamine contents in cardiac tissue increased, indicating increased synthesis of histamine.

Large burns cause systemic inflammation and myocardial depression. We hypothesized that small burns affect cardiac tolerance to ischemia, and that tumor necrosis factor alpha (TNFalpha) signaling through endothelin-1 (ET) and nuclear factor kappa B (NF kappaB) are associated.Mice were randomly assigned to four groups: burn (caused by boiling water on2% of the body surface area), sham, burn+etanercept (TNFalpha blocker) treatment and sham+etanercept treatment. Twenty-four hours later, hearts were isolated and subjected to global ischemia followed by reperfusion. Additional hearts and burned skin lesions were sampled to evaluate expression of TNFalpha (immunoblotting) and endothelin-1 (radioimmunoassay). A NF kappaB-luciferase reporter mouse was used to evaluate NF kappaB activation.Baseline cardiac function before ischemia (BI) was only negligibly influenced by burn or etanercept, but was reduced by burn+etanercept. Burn markedly impaired post-ischemic left ventricular function and increased infarct size in comparison with sham-treated mice. Cardiac, but nut cutaneous, expression of TNFalpha was increased in burned mice, while cardiac NF kappaB and endothelin-1 were not influenced. TNFalpha blockade reduced the detrimental effects of burn on cardiac tolerance to ischemia.Small cutaneous burns, that did not influence baseline heart function, impaired the tolerance to ischemia. This effect may be mediated through TNFalpha, but does not involve signaling through NF kappaB or endothelin-1.

Apoptosis and angiogenesis may be involved in the pathogenesis of atherosclerosis and plaque destabilization. In this study, we investigated if apoptosis and angiogenesis were induced in the unstable human coronary atherosclerotic plaque compared to stable atherosclerotic plaque.Atherosclerotic plaques from patients with stable (n = 9) and unstable angina (n = 13) were obtained by directional coronary atherectomy performed during percutaneous transluminal coronary angioplasty. Apoptosis was detected by terminal deoxynucleotidyl transferase end labelling (TUNEL), as well as by immunostaining for caspase 3, Bax and Bcl-2. Neovascularization was determined by immunostaining for the endothelial cell-specific CD31, vascular endothelial growth factor (VEGF-A), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hypoxia inducible factor-1alpha (HIF-alpha), and the sections were quantified blindly.The apoptotic nuclei were more frequently found in the unstable coronary atherosclerotic plaques. When the number of apoptotic cells was quantified, an increased apoptotic index was found in the unstable plaques (P = 0.04). The positive staining for caspase-3 was increased in the unstable plaques (P = 0.0008), while no difference in either Bax or Bcl-2 was found between groups. Neovascularization, as evidenced by lumens surrounded by a CD31 positive endothelial layer, was more frequently present in the plaques from patients with unstable angina (P = 0.04). The number of cells with positive staining for VEGF-A was increased in unstable plaques (P = 0.005). No difference of Ang I, Ang II, HIF1-alpha was found between groups.In unstable human coronary plaques, apoptosis probably involving caspase 3 was found. The plaques had an increased neovascularization, probably induced by VEGF-A. These factors may contribute to explaining plaque destabilization and intraplaque haemorrhage.

Tissue plasminogen activator (t-PA) as a possible marker of endothelial injury during elective coronary artery bypass surgery was studied. T-PA antigen and activity were measured in arterial and coronary venous plasma in 14 patients, and compared to the markers of myocyte injury creatine kinase (CK-MB) and troponin-T (TnT). Cardiopulmonary bypass (CPB) lasted 86 (55-107) min, and aortic cross-clamping (cold, crystalloid cardioplegia) lasted 41 (25-62) min (median (central 90% percentile)). Blood flow in the great cardiac vein was measured by retrograde thermodilution, and increased from 49 (27-90) ml/min before CPB to a maximum of 92 (55-125) ml/min 40 min after declamping (not significant). CK-MB, TnT, and t-PA antigen and activity all increased during CPB, and were significantly higher in coronary sinus than arterial plasma after declamping the aorta. Net cardiac release ([coronary sinus-arterial concentration] x coronary flow) of TnT increased after the aorta was declamped, and was higher in the seven patients with the longest cross-clamping time than in the seven with the shortest time (p0.01). Cardiac release of CK-MB and t-PA antigen also increased after declamping, but with no significant difference between long and short cross-clamp times. t-PA activity, however, increased more in the patients with the longest cross-clamp times (p0.008). In conclusion, CK-MB, TnT and t-PA were released from the postcardioplegic heart. Release of t-PA indicates that postcardioplegic coronary endothelial activation or injury occurred t-PA activity as well as TnT increased more in patients with long times of cross-clamping, indicating that t-PA activity may be a possible marker of postcardioplegic endothelial injury or activation.