Mitochondrial DNA damage and repair during ischemia-reperfusion injury of the heart.

Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."

Nuclear factor kappa-B and the heart.

Nuclear factor kappa-B (NFkappaB), a redox-sensitive transcription factor regulating a battery of inflammatory genes, has been indicated to play a role in the development of numerous pathological states. Activation of NFkappaB induces gene programs leading to transcription of factors that promote inflammation, such as leukocyte adhesion molecules, cytokines, and chemokines, although some few substances with possible anti-inflammatory effects are also NFkappaB regulated. The present article reviews basic regulation of NFkappaB and its activation, cell biological effects of NFkappaB activation and the role of NFkappaB in apoptosis. Evidence involving NFkappaB as a key factor in the pathophysiology of ischemia-reperfusion injury and heart failure is discussed. Although activation of NFkappaB induces pro-inflammatory genes, it has lately been indicated that the transcription factor is involved in the signaling of endogenous myocardial protection evoked by ischemic preconditioning. A possible role of NFkappaB in the development of atherosclerosis and unstable coronary syndromes is discussed. Nuclear factor kappa-B may be a new therapeutic target for myocardial protection.

Unstable angina activates myocardial heat shock protein 72, endothelial nitric oxide synthase, and transcription factors NFkappaB and AP-1.

OBJECTIVE: Unstable angina may improve the clinical outcome of acute myocardial infarction, but increases the morbidity and mortality of open heart surgery. We hypothetized that unstable angina influences the myocardium, and investigated the expression of the inducible heat shock protein 72 (HSP72), constitutive HSP73, and endothelial nitric oxide synthase (eNOS), and activation of the transcription factors NFkappaB and AP-1 in cardiac tissue. METHODS: Biopsies were taken from the right atrium of 15 patients with unstable and 15 with stable angina undergoing coronary artery bypass grafting. Immunoblotting with monoclonal antibodies against HSP72, HSP73, and eNOS were performed on protein extracts, while nuclear proteins were assessed by electromobility shift assay. RESULTS: When calculating the optical density of the bands, patients with unstable angina had more than twice as much HSP72 and eNOS as stable patients (P<0.005), while HSP73 was similar in both groups. Nuclear translocation of NFkappaB and AP-1 was found in patients with anginal pain shortly before surgery, but not in stable patients or in patients without symptoms for 4 days or more prior to surgery. CONCLUSIONS: HSP72 and eNOS, which may be associated with cardioprotection in ischemic preconditioning, are increased in atrial tissue of patients with unstable angina. Activation of NFkappaB and AP-1, which regulate a battery of inflammatory genes, was found in hearts of unstable patients. NFkappaB activation may induce a myocardial proinflammatory state, possibly making the unstable myocardium more susceptible to the inflammation induced by open heart surgery.

Inhibition of lipoxygenase and cyclooxygenase augments cardiac injury by H2O2.

The role of arachidonic acid metabolites in the cardiac effects of toxic oxygen metabolites (TOM) was investigated in buffer-perfused rat hearts (Langendorff model). Hydrogen peroxide (H2O2, 200 microM) was given for 10 min to generate TOM, followed by 30 min recovery. H2O2 reduced left ventricular developed pressure (LVDP), increased left ventricular end-diastolic pressure (LVEDP), and increased coronary flow (CF). The hydroxyl radical scavenger thiourea inhibited the H2O2-induced effects. Perfusion with three lipoxygenase inhibitors, AA861, BWA4C, and diethylcarbamazine, in addition to H2O2, augmented the decrease of LVDP and the increase of LVEDP induced by H2O2. The cyclooxygenase inhibitor indomethacin had the same effects. The H2O2-induced increase in CF was not influenced by diethylcarbamazine, but inhibited by all other drugs. Control perfusion with drugs alone did not influence cardiac function. In conclusion, inhibition of lipoxygenase and cyclooxygenase augmented the depression of cardiac function induced by TOM. Leukotrienes and prostanoids appear to be protective against H2O2-induced cardiac injury.

Effects of a novel low-molecular weight antioxidant on cardiac injury induced by hydrogen peroxide.

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.

Hydrogen peroxide induces endothelial cell atypia and cytoskeleton depolymerization.

Reactive oxygen intermediates induce cell injury in a variety of pathophysiological conditions. Human umbilical cord vein endothelial cell (HUVEC) cultures were exposed to 1 or 200 microM H2O2 for 15 min, and observed after 15 min, or 1, 4, 24, or 120 h. Factor VIII and the cytoskeletal proteins vimentin and tubulin were visualized immunocytochemically. Release of lactate dehydrogenase (indices of cell membrane injury) did not increase after H2O2 exposure; nor was cellular expression of factor VIII affected. 200 microM H2O2 induced cell contraction after 15 min which disappeared after 1 and 4 h, but was evident again after 24 h. Immediately after exposure, the filamentous structure of vimentin and tubulin disappeared, but normalized after 1 h. After 120 h, the cytoskeleton filaments were coarsened and disorganized, and an abundance of multinucleated giant cells were observed. Catalase (150 U/ml) abolished all effects of H2O2. One microM H2O2 did not induce any changes in HUVEC. Thus, the present concentrations of H2O2 did not induce cell necrosis or altered expression of factor VIII. Early, reversible cell contraction and depolymerization of cytoskeletal proteins were observed, followed by a delayed contraction and cell atypia after 200 microM H2O2.

Measurements of plasma glutaredoxin and thioredoxin in healthy volunteers and during open-heart surgery.

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 beta-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 post-cardioplegic heart. Trx and Grx may be important components in the plasma defense against oxidative stress.

Effects of a novel, low-molecular weight inhibitor of lipid peroxidation on ischemia-reperfusion injury in isolated rat hearts and in cultured cardiomyocytes.

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.

Endothelial dysfunction in atherosclerotic mice: improved relaxation by combined supplementation with L-arginine-tetrahydrobiopterin and enhanced vasoconstriction by endothelin.

1. Mice lacking the apolipoprotein E and low density lipoprotein receptor genes (E degrees xLDLR degrees ) develop atherosclerosis and endothelial dysfunction. The aim of this study was to characterize the roles of L-arginine and tetrahydrobiopterin (BH(4)) for endothelium-dependent relaxation and the changes in the vasoconstrictor response to endothelin-1 (ET-1) in thoracic aortic rings of E degrees xLDLR degrees mice. 2. Histological examination revealed severe atherosclerosis of the thoracic aorta of E degrees xLDLR degrees mice. Relaxations induced by acetylcholine (Ach), but not that to sodium nitroprusside, were significantly impaired in E degrees xLDLR degrees mice compared to control mice indicating attenuated endothelium-dependent relaxations. 3. Preincubation with the nitric oxide (NO) substrate L-arginine did not affect, whereas the co-factor for NO synthase, BH(4), slightly improved the relaxations induced by Ach. Combined preincubation with L-arginine and BH(4) induced a pronounced enhancement of Ach-induced relaxations in E degrees xLDLR degrees mice. The relaxations induced by Ach in E degrees xLDLR degrees mice in the presence of L-arginine and BH(4) were not different from those observed in control mice. 4. Preincubation with superoxide dismutase did not affect Ach-induced relaxations in aorta from E degrees xLDLR degrees mice. 5. The contractile response to ET-1 was enhanced in E degrees xLDLR degrees mouse aorta. The contractions were abolished by the ET(A) receptor antagonist LU 135252. The ET(B) receptor agonist sarafotoxin 6c did not induce contractions or relaxations. 6. It is concluded that endothelial dysfunction of E degrees xLDLR degrees mouse aorta is reversed by combined administration of L-arginine and BH(4). In addition, the ET(A) receptor-mediated vasoconstriction by ET-1 is enhanced in E degrees xLDLR degrees mice.

Preconditioning improves cardiac function after global ischemia, but not after cold cardioplegia.

BACKGROUND: Ischemic preconditioning reduces infarct size and cardiac dysfunction during reperfusion. Preconditioning may offer myocardial protection in open heart operations. METHODS: 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). RESULTS: Preconditioning before warm ischemia attenuated the ischemia-induced increase of left ventricular end-diastolic pressure (3 +/- 1 versus 17 +/- 4 mm Hg; p < 0.01) (mean +/- standard error of the mean), the reduction of coronary flow (14 +/- 1 versus 9 +/- 0.5 mL/min; p < 0.001) and heart rate (252 +/- 19 versus 198 +/- 18 beats/min; p < 0.04), and the incidence of ventricular fibrillation (2 of 17 versus 10 of 17 hearts; p < 0.04) 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. CONCLUSIONS: 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.

Induction of inflammatory mediators during reperfusion of the human heart.

BACKGROUND: Cardioplegia and reperfusion may induce an inflammatory reaction, which may contribute to postoperative morbidity and mortality. METHODS: Gene expression of cytokines, adhesion molecules, and vasoactive substances was evaluated in left ventricular biopsies taken before cardioplegia (lasting approximately 70 minutes) and after reperfusion (approximately 40 minutes) from 19 patients (5 with valvular or combined disease, 7 with stable angina pectoris, 7 with unstable angina). mRNA was extracted and amplified with a semiquantitative reverse transcription polymerase chain reaction. RESULTS: Cardioplegia-reperfusion increased mRNA for E-selectin by a factor of 17 +/- 5 (p < 0.002) (mean +/- SEM), interleukin-1beta, with 9 +/- 3 (p < 0.007), tumor necrosis factor-alpha with 6 +/- 3 (p < 0.05), interleukin-2 receptor alpha chain CD25 with 2 +/- 0.6 (p < 0.04), and intercellular adhesion molecule-1 with 2 +/- 0.4 (p < 0.005). Before cardioplegia, mRNA for endothelial nitric oxide synthase was predominantly detected in unstable angina patients, and increased by a factor of 11 +/- 6 (p < 0.02) during reperfusion. mRNA for endothelin-1 decreased by a factor of 0.5 +/- 0.1 (p < 0.0005). The changes were more pronounced in unstable patients. The transcription factor nuclear factor kappa B (NFkappaB), which regulates expression of inflammatory mediators, was activated during reperfusion (n = 10, p < 0.0001). CONCLUSIONS: Open heart surgery induces an inflammatory response in the human heart, which is more pronounced in patients with unstable angina. It involves NFkappaB activation and expression of several NFkappaB-regulated genes.

Gene expression of inflammatory mediators in different chambers of the human heart.

BACKGROUND: Inflammatory genes may be unevenly expressed in different heart chambers. METHODS: Biopsies were taken simultaneously from the right atrium (RA), left atrium (LA), and left ventricle (LV) of 19 patients before cardioplegic arrest during open heart surgery. The mRNA expression of tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), inducible and endothelial nitric oxide synthase (iNOS and eNOS), endothelin-1 (ET-1), E-selectin (CD62E), intercellular adhesion molecule-1 (ICAM-1) and its ligand CD18, and CD25 was evaluated with semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Expression of TNFalpha mRNA was higher in RA than LA and LV (p<0.05), whereas IL-1beta was more expressed in LA than RA (p<0.05), which was higher than LV (p<0.0001). There were no significant regional differences in the expression of ICAM-1, CD62E, CD25, iNOS, and eNOS. CD18 was higher in RA than LA (p<0.05); ET-1 was more expressed in RA than LV (p<0.04). Patients with stable angina had no expression of eNOS. CONCLUSIONS: Gene expression of inflammatory mediators was detected in the hearts of patients with different cardiovascular disorders, and was unevenly distributed in different heart chambers. Cardiac biopsies should be taken from the same site.

Preconditioning protects the severely atherosclerotic mouse heart.

BACKGROUND: Coronary atherosclerosis has profound effects on vascular and myocardial biology, and it has been speculated that the atherosclerotic heart does not benefit from ischemic preconditioning. METHODS: To investigate if atherosclerosis would influence the preconditioning response, Apolipoprotein E/low density lipoprotein (LDL) receptor double knockout mice (ApoE/LDLr-/-) were fed an atherogenic diet (21% fat, 0.15% cholesterol) for 6 to 8 months. At that time, extensive atherosclerotic lesions throughout the coronary tree were seen in transverse sections stained with Oil Red-O. Hearts of ApoE/LDLr-/- mice were Langendorff-perfused with 40 minutes of global ischemia and 60 minutes reperfusion, and compared with C57BL/6 controls. Preconditioning with two episodes of 2 minutes of ischemia and 5 minutes reperfusion, or exposing the mice to a hyperoxic environment (O2 > 98%) for 60 minutes before heart perfusion, was performed. RESULTS: Hearts of mice with coronary atherosclerosis had worse postischemic function, and increased infarct size and troponin T release compared to hearts of C57BL/6 mice. Ischemic preconditioning improved postischemic ventricular function, and reduced myocardial infarct size and troponin T release in both normal and ApoE/LDLr-/- mice. The effects were most pronounced in ApoE/LDLr-/- hearts. Exposure to hyperoxia exerted a similar protection of function and cell viability of ApoE/LDLr-/- mice hearts. CONCLUSIONS: These findings suggest that the severely atherosclerotic heart may be protected by preconditioning induced by ischemia or hyperoxia.

The effect of exogenous adenosine on functional injury caused by hydrogen peroxide in the isolated rat heart.

Adenosine is an endogenous cardioprotective substance. The present study examines whether exogenous adenosine attenuates cardiac injury induced by oxidative stress. Rat hearts (Langendorff model) were perfused with H2O2 (180 microM) for 10 min, then recovered for 60 min (n = 10). In other groups adenosine 55 microM, 11 0 microM, or 220 microM (n = 10 in each) was given in addition to H2O2 throughout perfusion. Control perfusion with Krebs Henseleit only (n = 7), adenosine 110 microM throughout perfusion (n = 7), and adenosine 110 microM as an intervention (n = 7) was performed. The hearts were paced at 320 beats/min. Left ventricular systolic (LVSP) and end-diastolic (LVEDP) pressures were measured together with coronary flow (CF), and left ventricular developed pressure (LVDP = LVSP - LVEDP) was calculated. H2O2 decreased LVSP from 105 +/- 8 to 60 +/- 5 mmHg (mean +/- SEM) after 10 min infusion (p < 0.008). Adenosine did not attenuate the decrease of LVSP. LVEDP increased from 0 to 59 +/- 10 mmHg (p < 0.004) and 62 +/- 11 mmHg 5 and 15 min after end of infusion of H2O2, respectively. Neither 55 microM nor 220 microM adenosine inhibited the H2O2-induced increase of LVEDP. Adenosine 110 microM attenuated the increase after 15 (15 +/- 4 mmHg, p < 0.004) and 25 min observation (26 +/- 7 mmHg, p < 0.012). Adenosine did not attenuate the reduction of LVDP. CF initially increased during infusion of H2O2, thereafter decreased. Hearts given adenosine had higher basal CF, and CF did not increase after H2O2. Control perfusion with adenosine, given throughout perfusion or as an intervention, increased CF and tended to increase LVSP. In summary, adenosine did not inhibit H2O2-induced depression of contractility or reduction of CF. One concentration of adenosine (110 microM) attenuated H2O2-induced impairment of relaxation. Exogenous adenosine does not have an important influence on functional injury caused by exogenous oxidants.

Systemic release of thrombomodulin, but not from the cardioplegic, reperfused heart during open heart surgery.

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 (p < 0.008 compared to initial value). During CPB thrombomodulin increased, with a maximal level of 23 +/- 7 ng/ml (p < 0.008 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.

Oxidative stress and release of tissue plasminogen activator in isolated rat hearts.

UNLABELLED: 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 (H2O2) (0 (control perfusion), 20, 40, 80, 120, 160, or 200 microM) (n = 8 hearts/group), followed by 30 minutes recovery. Higher concentrations than 80 microM induced cardiac dysfunction and a dose-dependent release of lactate dehydrogenase, indicating myocyte injury. H2O2-concentrations of 80 microM 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 H2O2-dose. The effects of H2O2 (120 or 200 microM) 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 H2O2. Bradykinin, but not H2O2, induced t-PA release during the second exposure, suggesting different mechanisms of release. IN CONCLUSION: Perfusion with H2O2 leads to a dose-dependent myocardial injury in isolated rat hearts. H2O2 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.

Release of tissue plasminogen activator during reperfusion after different times of ischaemia in isolated, perfused rat hearts.

Tissue plasminogen activator (t-PA) is a potential marker of endothelial cell activation or injury. The relationship between duration of ischaemia and release of t-PA during reperfusion was investigated in isolated rat hearts exposed to either 5, 10, 20, 30, 40, or 60 min of global, normothermic ischaemia followed by 30 min of reperfusion (n = 8 in each group). t-PA activity was measured (chromogenic peptide substrate assay) in the effluent before ischaemia, and after 2.5, 5, 7.5, 10, 20, and 30 min of reperfusion. Release of lactate dehydrogenase (LD), a marker of myocyte injury, was measured before ischaemia and after 5 min reperfusion. Left ventricular pressures were measured by a balloon in the left ventricle. Ischaemia for 20 min or less had only minor effects on cardiac function. Thirty min or more of ischaemia induced ventricular fibrillation during reperfusion in most hearts. After ischaemia t-PA outflow increased, but without any significant difference between groups. Peak release occurred after either 2.5 or 5 min of reperfusion. After 10 min reperfusion the release was not different from the basal value. In contrast, postischaemic release of LD correlated to the length of ischaemia. To conclude, t-PA release from the ischaemic-reperfused rat heart is independent of the length of ischaemia. Thus the potential of t-PA to quantify endothelial injury appears to be limited.

Reactive oxygen intermediates and ischemia-reperfusion injury release tissue plasminogen activator from isolated rat hearts.

Tissue plasminogen activator (t-PA) is a marker of endothelial cell injury or activation. The release of t-PA from isolated rat hearts (Langendorff model) subjected to ischemia-reperfusion or reactive oxygen intermediates (ROI) generated by H2O2 was investigated. H2O2 (200 microM) increased t-PA activity in the coronary effluent to 305 +/- 84% of initial value (mean +/- SEM, p < 0.04 vs controls) at the end of a 10 min intervention. The hydroxyl radical scavenger thiourea (10 mM) only partially inhibited the increase (175 +/- 27%, p < 0.01 compared to controls). 20 min normothermic ischemia increased t-PA activity to 416 +/- 108% (p < 0.005 compared to controls) at the start of reperfusion. In conclusion, cardiac injury by ischemia-reperfusion or ROI increases release of t-PA.

Release of von Willebrand factor by cardiopulmonary bypass, but not by cardioplegia in open heart surgery.

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, p < 0.0026). 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, p < 0.0026). 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.

Hydrogen peroxide induces mRNA for tumour necrosis factor alpha in human endothelial cells.

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.