Biomedical subjects

Michael Buerke

Publications and source records attributed to Michael Buerke.

27 records · Page 2Linked to original sources

PubMed2003

Gene expression profiling of human stenotic aorto-coronary bypass grafts by cDNA array analysis.

OBJECTIVE: Aorto-coronary bypass graft disease with its increasing clinical signification represents an unsolved problem in cardiological and heart surgery practice. Late occlusion of autologous saphenous vein grafts is due to medial and neointimal thickening secondary to migration and proliferation of smooth muscle cells (SMCs) and the subsequent formation of atherosclerotic plaques. This study is aimed at identifying differentially expressed genes in human stenotic bypass grafts to detect unknown pathomechanism and to identify novel targets for prophylactic treatment options. METHODS: Stenotic saphenous aorto-coronary bypass grafts (n=5) were retrieved during re-do aorto-coronary bypass surgery. Ungrafted saphenous vein segments (n=5) were taken from the same group of patients and served as internal controls. cDNA samples were prepared and hybridized to cDNA arrays. RESULTS: Some of the differentially expressed genes complied with expected gene expression including upregulation of c-jun and CDK10. In addition, previously unidentified gene expression patterns were detected such as upregulation of HSP70, fibronectin1, erbB3 proto-oncogene and c-myc. To confirm the latter finding, upregulation of c-myc in neointimal and medial SMCs of stenotic graft segments was confirmed by in situ hybridization studies and by immunohistochemistry. CONCLUSION: Gene expression patterns of human stenotic bypass grafts retrieved by re-do operations can be reliably analyzed by cDNA array technology. With this technique, new therapeutic targets in patients could be identified as shown by the findings regarding c-myc. c-myc is a proto-oncogene acting as a transcription factor and blocking c-myc has shown a reduction of neointima formation in animal models. Our study yields a rational for the use of antisense c-myc oligonucleotides to reduce neointima formation and to avoid stenosis in patients.

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PubMed2003

Leukotriene-mediated coronary vasoconstriction and loss of myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused rat hearts.

OBJECTIVE: hemolysin has been implicated as an important pathogenic factor in extraintestinal infections including sepsis. We investigated the effects of coronary administration of hemolysin on cardiac function in isolated rat hearts perfused at constant flow. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Isolated hearts were perfused with purified hemolysin for 60 min. MEASUREMENTS AND MAIN RESULTS: Low concentrations of the toxin in the perfusate (0.1-0.2 hemolytic units/mL) caused a dose-dependent coronary vasoconstriction with a marked increase in coronary perfusion pressure, which was paralleled by a decrease in left ventricular developed pressure (and the maximum rate of left ventricular pressure increase). Moreover, 0.2 hemolytic units/mL hemolysin evoked ventricular fibrillation within 10 mins of toxin application. These events were accompanied by the liberation of leukotrienes (LTC4, LTD4, LTE4, and LTB4), thromboxane A2, prostaglandin I2, and the cell necrosis markers lactate dehydrogenase and creatine kinase into the recirculating perfusate. The lipoxygenase inhibitor MK-886 fully blocked the toxin-induced coronary vasoconstrictor response and the loss of myocardial contractility and reduced the release of lactate dehydrogenase and creatine kinase. In contrast to this, the cyclooxygenase inhibitor indomethacin was entirely ineffective. In addition, hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886. CONCLUSIONS: Low doses of hemolysin cause strong coronary vasoconstriction, linked with loss of myocardial performance, release of cell injury enzymes, and electrical instability, with all events being largely attributable to toxin-elicited leukotriene generation in the coronary vasculature. Bacterial exotoxins such as hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock.

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PubMed2003

Fondaparinux and enoxaparin in comparison to unfractionated heparin in preventing thrombus formation on mechanical heart valves in an ex vivo rabbit model.

The aim of the present study was to investigate the efficacy of three different parenterally administered anticoagulants for the prevention of thrombus formation on artificial heart valves in an experimental rabbit model. Unfractionated heparin was administered intravenously in group I (n = 10), Enoxaparin subcutaneously in group II (n = 10), fondaparinux intravenously in group III (n = 10), and no medication was administered to group IV (n = 9). Leaflets from Sulzer Carbomedics bileaflet mechanical heart valves were placed in a flow chamber. The flow chamber was filled with blood in a continuous circulation between the carotid artery and the jugular vein. In group IV the flow chamber was clotted after a median of 15 minutes of circulation. Weight analysis before and after 1 h of perfusion showed that the median thrombus weight was 18.0 mg in group I, 17.7 mg in group II, 20.3 mg in group III, and 30.8 mg in group IV. Further analysis by electron microscopy showed similar results regarding deposition of fibrin, platelets, and erythrocytes on leaflet surfaces. Fondaparinux and subcutaneously administered enoxaparin were as effective as intravenously administered unfractionated heparin in preventing thrombus formation on artificial heart valve leaflets in our investigation. This rabbit model, in which the heart valve leaflets were exposed to rabbit blood for a short time under laminar flow, should be further evaluated with respect to whether it can provide information about anti-thrombotic regimens in patients after mechanical heart valve replacement.

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PubMed2002

Simvastatin inhibits inflammatory properties of Staphylococcus aureus alpha-toxin.

BACKGROUND: Simvastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, has been shown to lower serum cholesterol levels in clinical use. Moreover, statins exert beneficial effects in vascular diseases by inhibition of leukocyte rolling, adherence, and transmigration. The aim of this study was to determine if pretreatment with simvastatin attenuates Staphylococcus aureus alpha-toxin-induced increase in leukocyte-endothelial interactions during exotoxemia. METHODS AND RESULTS: The effects of simvastatin on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation. Simvastatin (50 or 100 microg/kg) was administered 18 hours before the study. Activation of microcirculation was induced by bolus administration of 40 microg/kg S aureus alpha-toxin. Exotoxemia resulted in a significant and time-dependent increase in leukocyte rolling, adherence, and transmigration of leukocytes as well as P-selectin expression on the intestinal vascular endothelium. Pretreatment with simvastatin significantly inhibited exotoxin-induced leukocyte rolling from 71+/-10 to 14+/-4.7 cells/min (P<0.01) and adherence from 14+/-3.5 to 0.4+/-0.2 cells (P<0.01). In addition, simvastatin pretreatment significantly inhibited transmigration of leukocytes from 10.5+/-1.2 to 4.2+/-0.9 (P<0.05) cells. Immunohistochemical detection of endothelial cell adhesion molecule P-selectin showed a 50% decrease in endothelial cell surface expression after simvastatin treatment. Furthermore, simvastatin treatment resulted in enhanced expression of endothelial cell NO synthase III in the intestinal microcirculation. CONCLUSIONS: These results demonstrate that simvastatin interferes with exotoxin-induced leukocyte-endothelial cell interactions, which may be relevant in various infectious diseases. Statin treatment may offer a new therapeutic strategy for these clinical conditions.

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PubMed2002

Two-dimensional analysis of myocardial protein expression following myocardial ischemia and reperfusion in rabbits.

Myocardial ischemia and reperfusion injury (MI/R) can be related to leukocyte activation with subsequent release of cytokines and oxygen derived free radicals. Activation of the complement system has been implicated in the pathogenesis of myocardial ischemia and reperfusion injury. Inflammatory injury will subsequently result in cellular activation and protein synthesis. In the present study we analyzed the myocardial protein expression and its pattern following myocardial ischemia and reperfusion, with and without complement inhibition with the synthetic serine protease inhibitor Futhan/nafamstat mesilate (FUT-175) known to inhibit classical and alternative complement pathway in a rabbit model of myocardial ischemia and reperfusion (60 min I+180 min R). FUT-175 significantly reduced myocardial necrosis, i.e. creatine kinase release which were analyzed for the three groups (p<0.05). Similarly, histological analysis demonstrated preservation of myocardial tissue injury and reduced leukocyte accumulation following FUT-175 treatment. Further, the myocardial protein expression was analyzed by two-dimensional gel electrophoresis following MI/R in the different groups. The protein patterns were evaluated by means of MELANIE III, a computer assisted gel analysis system. The biochemical identification of the proteins of interest was, achieved using nanohigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry. On average, 509 +/- 25 protein spots were found on the gels. A pattern of 480 spots with identical positions was found on every gel of five animals of each group. We analyzed ten spots which were significantly altered (i.e., in eight spots we observed decreased protein expression and in two spots we observed increased expression, vehicle vs. sham), by using mass spectrometry. Superoxide dismutase precursor and alphaB-crystallin were identified. We compared sham group vs. vehicle group and vehicle group vs. FUT-175 treated animals. Expression of the two identified proteins decreased by half the amount in the vehicle group when compared to sham treated animals. Treatment with FUT-175 preserved significantly superoxide dismutase precursor and alphaB-crystallin protein expression when compared to vehicle animals. The results present marked differences in myocardial protein expression after ischemia and reperfusion and following treatment with the complement inhibitor FUT-175. Our results illustrate the application of proteomics to discover possible new therapeutic targets or to detect unexpected effects of pharmacological inhibitors.

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PubMed2002

Cardioprotective effects of the serine protease inhibitor aprotinin after regional ischemia and reperfusion on the beating heart.

OBJECTIVE: Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal to preserve myocardium. However, reperfusion itself contributes to an additional myocardial injury (ie, reperfusion injury), which has been attributed to neutrophil infiltration with subsequent release of proteases and oxygen-derived radicals. We studied the effects of the serine protease inhibitor aprotinin (Trasylol) on myocardial ischemia and reperfusion in a rat model. METHODS: The effects of aprotinin (5000 and 20,000 U/kg) were examined in vivo in a rat model of regional myocardial ischemia (20 minutes) and long-term reperfusion (24 hours). Cardioprotecive effects were determined by means of measurement of creatine kinase and myeloperoxidase activity within the myocardium, as well as histochemical analysis. RESULTS: Aprotinin (20,000 U/kg) administrated 2 minutes before reperfusion significantly attenuated myocardial injury expressed as creatine kinase washout compared with that seen in vehicle-treated rats (65 +/- 25 vs 585 +/- 98 creatine kinase difference in units per 100 mg, P <.01). Administration of 5000 U/kg of the protease inhibitor resulted in partial inhibition of myocardial reperfusion injury. Moreover, cardiac myeloperoxidase activity in the ischemic myocardium, a marker of neutrophil accumulation, was significantly reduced after aprotinin treatment. Histologic analysis of the reperfused myocardium demonstrated reduced polymorphonuclear leukocyte infiltration and reduced tissue injury. Furthermore, aprotinin treatment resulted in decreased induction of cardiac myocyte apoptosis compared with that seen in vehicle-treated rats. CONCLUSIONS: Inhibition of serine proteases with aprotinin appears to be an effective means of preserving ischemic myocardium from reperfusion injury, even after 24 hours of reperfusion. Aprotinin might exert cardioprotection through inhibition of polymorphonuclear leukocyte-induced myocardial injury and inhibition of reperfusion-induced apoptosis of cardiac myocytes.

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PubMed2002

Staphylococcus aureus alpha toxin mediates polymorphonuclear leukocyte-induced vasocontraction and endothelial dysfunction.

The effect of Staphylococcus aureus alpha toxin (alpha-toxin) on selectin-mediated neutrophil adhesion was investigated in polymorphonuclear leukocyte- (PMN) induced vasocontraction and endothelial dysfunction. Adherence of human PMNs to rat aortic endothelium increased significantly following stimulation of the endothelium with alpha-toxin (0.1, 0.5, and 1 microg/mL). This effect could be significantly attenuated by monoclonal antibodies directed against P-selectin or fucoidin, a carbohydrate known to block selectins. Unstimulated human PMNs (10(6)cells/mL) were added to organ chambers containing rat aortic rings stimulated with alpha-toxin (0.5 microg/mL). PMNs elicited a significant vasocontraction in alpha-toxin-stimulated, but not in control aortic, rings (142+/-12 mg versus 12+/-4 mg, P < 0.05). This PMN-induced vasocontraction was virtually blunted by pretreatment with MAb directed against P-selectin or fucoidin (P < 0.05). Endothelial function as assessed by endothelium-dependent vasorelaxation to acetylcholine was substantially inhibited after induction of PMN-induced vasocontraction in alpha-toxin-stimulated aortic rings. This endothelial dysfunction was reduced by P-selectin MAb or fucoidin. In contrast, endothelium-independent relaxation to sodium nitrite was not altered by PMN incubation, indicating that vascular smooth muscle function was unaffected. Thus, PMN-endothelial interaction following S. aureus a-toxin activation of the vascular endothelium is at least, in part, mediated by selectins. As a consequence, PMN-induced vasocontraction and endothelial dysfunction occur. Such mechanisms may be involved in microcirculation abnormalities encountered in sepsis or septic shock due to S. aureus infection.

Animals↗

PubMed2002

Staphylococcal alpha-toxin provokes neutrophil-dependent cardiac dysfunction: role of ICAM-1 and cys-leukotrienes.

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.

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PubMedDate not supplied

Proliferative activity in stenotic human aortocoronary bypass grafts.

BACKGROUND: Aortocoronary bypass graft disease is responsible for long-term failure of autologous vein grafts. The analyses of proliferation and cell type characterisation in human bypass grafts harvested during re-do surgery make it possible to investigate the cellular processes leading to bypass graft failure. METHODS: 30 stenotic vein grafts and 25 control veins were explantated during re-do heart surgery procedures. The total area and cell count of the neointima, media and adventitia were calculated computer-assisted. Actively proliferating cells were identified using antibody to Ki-67 and positive cells were determined by double-label immunocytochemistry with SMC alpha-actin, CD 31 (endothelial cells), CD 68 (macrophages) and CD 45 (T-lymphocytes). RESULTS: Active proliferation was detected in different cell types with an average proliferation index of 0.15%, 0.18% and 0.086% for neointima, media and adventitia. Only 9% of proliferating cells in the neointima were SMC (not identified cells 40%); correspondingly, 14% SMC (not identified cells 33%) were detected in the media. Endothelial cells turned out to be the predominant proliferating cell type in all sections of the vessel wall. CONCLUSION: Proliferation in our series of stenotic vein grafts occurred at a low level, but was significantly higher compared to native control veins. While proliferation may play an important role in early lesions, our data clearly show low proliferation activity in advanced graft lesions. The identification of proliferating macrophages and T-lymphocytes implicate an additional inflammatory component in the development of human bypass graft disease. SUMMARY: To clarify the role of cellular proliferation in human aortocoronary bypass grafts, we characterized the cellular composition and proliferation index in 30 stenotic saphenous vein grafts in comparison to 25 native veins. Proliferation in our series of stenotic vein grafts occurred at a low level, but was significantly higher compared to native control veins.

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