RESUMEN
BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) plays a key role in causing ischaemia/reperfusion (I/R) injury. I/R also causes activation of xanthine oxidase and dehydrogenase (XDH + XO) system that, via generated free radicals, causes organ damage. We investigated the effect of ischaemia, reperfusion and non-ischaemic prolonged perfusion (NIP) on TNF-alpha and XDH + XO production in an isolated perfused rat liver model. MATERIALS AND METHODS: Rat livers underwent 150 min NIP (control group) or two hours of ischaemia followed by reperfusion (I/R group). TNF-alpha (TNF-alpha mRNA and protein level), XDH + XO production and bile secretion were determined in tissue and effluent at baseline, at 120 min of ischaemia, after 30 min of reperfusion (I/R group) and after 120 and 150 min of prolonged perfusion (control). RESULTS: Unexpectedly, neither ischaemia nor reperfusion had any effect on TNF-alpha production. TNF-alpha in effluent was 11 +/- 4.8 pg mL(-1) at baseline, 7 +/- 3.2 pg mL(-1) at the end of ischaemia, and 13 +/- 5.3 pg mL(-1) after 30 min of reperfusion. NIP, however, caused a significant increase of TNF-alpha synthesis and release. TNF-alpha effluent level after 120 and 150 min of perfusion was 392 +/- 78.7 pg mL(-1) and 408 +/- 64.3 pg mL(-1), respectively. TNF-alpha mRNA in tissue was also significantly elevated compared to baseline levels (1.31 +/- 0.2 P < 0.001 and 1.38 P < 0.002, respectively). Decrease of liver function (expressed by bile secretion) during I/R and NIP was accompanied by significant XDH + XO elevation. CONCLUSION: This is the first evidence that NIP, and not I/R, is the decisive trigger for TNF-alpha production. This study leads to a better understanding of pathogenesis of liver I/R and perfusion damage.
Asunto(s)
Hígado/irrigación sanguínea , Daño por Reperfusión/etiología , Factor de Necrosis Tumoral alfa/fisiología , Xantina Deshidrogenasa/biosíntesis , Xantina Oxidasa/biosíntesis , Animales , Masculino , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To investigate alterations in endothelial nitric oxide synthase and inducible nitric oxide synthase mRNA expressions and nitric oxide release in the myocardium during ischaemia/reperfusion and determine whether these changes are ischaemic and/or reperfusion dependent. MATERIALS AND METHODS: Isolated rat hearts were perfused by a modified Langendorff system. Following 1 h of global cardioplegic ischaemia, left ventricle haemodynamic parameters were recorded at baseline and during 30 min of reperfusion. Levels of endothelial, inducible nitric oxide synthases mRNA expression and nitric oxide release were measured at baseline, after ischaemia and at 30 min of reperfusion. RESULTS: Global cardioplegic ischaemia caused a significant depression of left ventricular function and a decrease of coronary flow. Postischaemic intensities of the endothelial nitric oxide synthase mRNA bands were significantly lower than at baseline (P < 0.01). There were no significant differences in endothelial nitric oxide synthase mRNA band intensities immediately after ischaemia compared to the end of reperfusion, nor between the intensities of inducible nitric oxide synthase mRNA bands at baseline, at end of ischaemia and at end of reperfusion. Nitric oxide in the myocardial effluent was below detectable levels at all measured points. CONCLUSION: Ischaemic injury causes down-regulation of endothelial nitric oxide synthase mRNA expression, which is then associated with reduction of coronary flow during reperfusion, representing one possible mechanism of ischaemia/reperfusion injury. We did not find expected elevations of inducible nitric oxide synthase mRNA expression during ischaemia or reperfusion and we suggest that ischaemia/reperfusion injury is not associated with nitric oxide overproduction.
Asunto(s)
Miocardio/enzimología , Óxido Nítrico Sintasa/análisis , Óxido Nítrico/metabolismo , Animales , Endotelio Vascular/enzimología , Paro Cardíaco Inducido/métodos , Masculino , Isquemia Miocárdica/enzimología , Reperfusión Miocárdica/métodos , Óxido Nítrico Sintasa de Tipo II/análisis , ARN Mensajero/análisis , Ratas , Ratas Wistar , Disfunción Ventricular Izquierda/enzimología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
OBJECTIVE: The internal thoracic artery (ITA) is the most important graft in coronary artery bypass grafting. Its distal region is, however, prone to vasospasm. We studied the effects of nitroglycerin (NTG) and isosorbide-dinitrate (DSDN) on distal segments of left versus right ITA. METHODS: Rings of distal segments (6 to 9 mm proximal to bifurcation) of the human left and right ITA were studied. After baseline contraction of the rings, achieved using 60 mmol/L of KCl, they were exposed to increasing doses of ISDN and NTG (10 to 100 microg/ml), and dose-response curves were recorded. RESULTS: The contractile response of left ITA rings to KCl were significantly lower than those of right ITA rings (1.87 +/- 0.25 g versus 3.5 +/- 0.61 g, p < 0.005). Both nitrates inhibited the contractile response in a concentration-dependent manner, with relaxing effects of ISDN higher than those of NTG (p < 0.01) in both left and right ITA rings. CONCLUSIONS: The distal segment of the left ITA is less prone to vasospasm than that of the right. ISDN has a considerably higher relaxant effect on this segment than NTG. We therefore recommend favoring high doses of ISDN over NTG as an antispastic measure.
Asunto(s)
Dinitrato de Isosorbide/farmacología , Arterias Mamarias/efectos de los fármacos , Nitroglicerina/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Puente de Arteria Coronaria/métodos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Dinitrato de Isosorbide/administración & dosificación , Arterias Mamarias/fisiología , Nitroglicerina/administración & dosificación , Vasodilatadores/administración & dosificaciónRESUMEN
BACKGROUND: We investigated the role of tumor necrosis factor alpha (TNF-alpha) in protamine-induced cardiotoxicity and the possibility of preventing or decreasing this effect by anti TNF-alpha antibodies and heparin. METHODS: Isolated rat hearts were perfused for 60 min with Krebs-Henseleit solution (KH). The control group was perfused with KH alone, the KH > protamine > KH group was treated from the 20th to the 40th minute with protamine, and the KH + anti-TNF > protamine + anti-TNF > KH + anti-TNF group was treated the same as the KH > protamine > KH group but with anti-TNF-alpha antibodies added throughout perfusion. The KH + heparin > protamine + heparin > KH + heparin group was treated the same as the KH > protamine > KH group but with heparin added to KH throughout perfusion. The KH > protamine > KH + heparin was perfused the same as the KH> protamine > KH group but with heparin added to KH for the last 20 min. Left ventricular (LV) function and coronary flow were measured every 10 min. TNF-alpha was measured in the coronary sinus effluent. Left ventricular TNF messenger RNA was determined in the control and KH > protamine > KH groups at baseline and after the 40-min perfusion. RESULTS: Protamine caused a significant decrease of peak systolic pressure and dP/dt (to 25% of baseline). Significant amounts of TNF-alpha in the effluent in the KH > protamine > KH group (102.3 +/- 15.5 pg/min) and TNF messenger RNA expression in left ventricular samples were detected. TNF-alpha was below detectable concentrations in the control, KH + anti-TNF > protamine + anti-TNF > KH + anti-TNF, and KH + heparin > protamine + heparin > KH + heparin groups. TNF-alpha concentrations correlated with depression of LV peak systolic pressure (r = 0.984; P = 0.01) and first derivate of the increase of LV pressure (r = 0.976; P = 0.001). Heparin improved LV recovery and decreased protamine-induced TNF-alpha release (KH > protamine > KH + heparin group). CONCLUSIONS: Anti-TNF-alpha antibodies and heparin prevent protamine-induced TNF-alpha release and depression of LV function. Heparin improves protamine-induced depression of cardiac function.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Anticoagulantes/uso terapéutico , Cardiopatías/prevención & control , Antagonistas de Heparina/toxicidad , Heparina/uso terapéutico , Protaminas/toxicidad , Factor de Necrosis Tumoral alfa/inmunología , Animales , Cardiopatías/inducido químicamente , Hemodinámica/efectos de los fármacos , Técnicas In Vitro , Masculino , Protaminas/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Impaired handling of apoptotic cells has been suggested as an important factor in the development of systemic lupus erythematosus (SLE), and a role for complement in the removal of apoptotic cells was shown recently. We studied the in vitro function of macrophages from 40 patients with SLE and their matched controls in the removal of heterologous apoptotic cells opsonized by iC3b. Interaction index of apoptotic cells opsonized by iC3b was significantly lower in patients with SLE and averaged 71% +/- 37 of that of healthy individuals (p < 0.002) and 69% +/- 35 of patients with rheumatoid arthritis (p < 0.007). SLE patients had increased apoptosis of both freshly isolated monocytes (p < 0.001) and maturing macrophages (p < 0.04) that led to decreased density of monocyte-derived macrophages. Apoptosis was inhibited by adding soluble Fas receptor indicating Fas-mediated apoptosis. As demonstrated in both healthy controls and patients with SLE, decreased macrophage density by itself caused significant decreased uptake of apoptotic cells by the remaining macrophages. Maintaining normal density in SLE patients either by an increased initial density or by using soluble Fas restored the interaction capacity of the individual macrophages in the majority of patients. We concluded that impaired in vitro interaction of iC3b-opsonized apoptotic cells with macrophages from patients with SLE was mainly associated with Fas-dependent accelerated apoptosis of the monocytes/macrophages. Accelerated apoptosis of phagocytes may represent a novel in vitro mechanism of impairment of interaction with apoptotic cells that, apart from reducing the number of professional phagocytes, alters the function of the remaining macrophages.
Asunto(s)
Apoptosis , Complemento C3b/inmunología , Lupus Eritematoso Sistémico/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Receptor fas/fisiología , Supervivencia Celular , Células Cultivadas , Humanos , Cinética , Lupus Eritematoso Sistémico/patología , Proteínas Opsoninas/inmunología , Receptores de Complemento/fisiología , Timo/inmunologíaRESUMEN
OBJECTIVES: The purpose of this study is to assess the role of the nitric oxide (NO) pathway in protamine-induced cardiotoxicity and to formulate a possible explanation for this adverse effect. METHODS: Isolated rat hearts were perfused by Krebs--Henseleit (KH) solution using a modified Langendorff model. They were randomized into three groups: A, 40 min perfusion with KH solution; B, 20 min perfusion with KH solution and 20 min with protamine; C, as B but Ng-monomethyl-L-arginine (L-NMMA), a non-selective inhibitor of the NO pathway, was added during 40 min of the perfusion period. Left ventricular (LV) function was measured every 10 min. NO and tumor necrosis factor-alpha (TNF) were detected in the effluent from the coronary sinus (CS) and in the supernatant of the cardiac myocytes culture. Nitric oxide synthases (NOS) mRNA levels were determined in groups A and B from LV samples at baseline and after 40 min of perfusion. RESULTS: We found that protamine at a dose of 12 microg/ml causes significant depression of LV function (decreased peak systolic pressure to 22.5+/-3.2% and dP/dt max to 22.9+/-3.1%). L-NMMA did not prevent protamine cardiotoxicity. NOS mRNA was not detected from LV samples in any group. The NO in the effluent from the CS and from the supernatant of the cardiomyocytes culture was below detectable levels. However, a significant amount of TNF was measured in the effluent from the CS (108+/-17 pg/min for group B and 117+/-13 pg/min for group C) and in the supernatant of the cardiomyocytes culture (65+/-21 pg/ml). CONCLUSIONS: This study suggests that direct protamine-induced cardiotoxicity does not depend on the NO pathway. Our finding that protamine induced TNF release by cardiomyocytes can shed new light on the understanding of protamine cardiotoxicity.
Asunto(s)
Corazón/efectos de los fármacos , Antagonistas de Heparina/toxicidad , Óxido Nítrico/metabolismo , Protaminas/toxicidad , Animales , Masculino , Miocardio/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Perfusión , ARN Mensajero/genética , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
OBJECTIVE: The left internal thoracic artery (LITA) is the most important graft for coronary artery bypass grafting (CABG). Its distal region is, however, prone to vasospasm. The effect of nitroglycerin (NTG) and isosorbide-dinitrate (ISDN) on different segments of this region was studied. METHODS: Rings of three segments of the LITA were studied: 6-9 mm proximal to the bifurcation (part A); 1-3 mm proximal to the bifurcation (part B); and 3-6 mm distal to the bifurcation (part C). After baseline, maximal contraction of the rings was achieved using 60 mmol/l of KCl, they were exposed to increasing doses of ISDN and NTG (10-100 microg/ml), and dose-response curves were recorded. RESULTS: The contractile response of part A to KCl was significantly lower than that of parts B and C (1.87+/-0.25 versus 4.05+/-0.39 and 7.64+/-0.54 g, respectively; P<0.001). Both nitrates inhibited the contractile response in a concentration-dependent manner. The relaxing effects of both nitrates on part A was most pronounced (P<0.01), with the effect of ISDN being higher than that of NTG (P<0.01). CONCLUSIONS: The region 6-9 mm proximal to the LITA bifurcation is less prone to vasospasm, and has greater relaxation responses to ISDN and NTG than the more vasospastic distal parts of the LITA. We recommend avoiding the use of the very distal part of this artery during CABG, and to use high doses of ISDN rather than NTG as an anti-spastic measure.
Asunto(s)
Dinitrato de Isosorbide/farmacología , Arterias Mamarias/efectos de los fármacos , Nitroglicerina/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Análisis de Varianza , Puente de Arteria Coronaria/métodos , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Humanos , Arterias Mamarias/trasplante , Probabilidad , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: The purpose of this study was to explore interactions between paracrine angiotensin II (Ang-II) and tumor necrosis factor-alpha (TNF-alpha) during myocardial ischemia. BACKGROUND: Ischemic myocardium releases significant amounts of TNF-alpha. This paracrine release correlated with postischemic myocardial injury. Other studies showed myocardial protection obtained by the use of angiotensin-converting enzyme inhibitors (i.e., captopril) and the Ang-II type 1 receptor antagonist losartan after ischemia. The possibility that these agents decrease TNF-alpha synthesis has not yet been investigated. METHODS: Using the modified Langendorff model, isolated rat hearts underwent either 90 min of nonischemic perfusion (control group) or 1 h of global cardioplegic ischemia. In both groups, either captopril (360 micromol/liter) or losartan (182.2 micromol/liter) was added before ischemia. The hearts were assayed for messenger ribonucleic acid (mRNA) expression and effluent TNF-alpha levels. In addition, cardiac myocytes were incubated in cell culture with Ang-II. RESULTS: After ischemia, TNF-alpha mRNA expression intensified from 0.63 +/- 0.06 (control group) to 0.92 +/- 0.12 (p < 0.03), and effluent TNF-alpha levels were 711 +/- 154 pg/ml. The TNF-alpha mRNA expression declined to 0.46 +/- 0.07 (p < 0.01) and 0.65 +/- 0.08 (p < 0.02) in captopril- and losartan-treated hearts, respectively. Effluent TNF-alpha was below detectable levels. Concentrations of TNF-alpha in supernatants of incubated cardiac myocytes treated with 10 and 50 nmol/liter of Ang-II were 206.0 +/- 47.0 pg/ml and 810 +/- 130 pg/ml, respectively (p < 0.004). When pretreated with 700 micromol/liter of losartan, TNF-alpha was below detectable levels. CONCLUSIONS: This study presents an original explanation for previously reported myocardial protection after ischemia, obtained by the use of captopril and losartan. These drugs reduce TNF-alpha synthesis, providing strong evidence of active interactions between paracrine TNF-alpha and Ang-II in the evolution of the ischemic cascade.
Asunto(s)
Angiotensina II/fisiología , Daño por Reperfusión Miocárdica/fisiopatología , Comunicación Paracrina/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Animales Recién Nacidos , Captopril/farmacología , Células Cultivadas , Losartán/farmacología , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: Protamine is commonly used in cardiac surgery to reverse the anticoagulant effects of heparin. We investigated the role of different nitric oxide synthase pathways in the response of the human internal thoracic artery to protamine and evaluated whether heparin could prevent this effect. METHODS: A tension-recording method was used to obtain baseline measurements of contractions of human internal thoracic artery rings achieved with norepinephrine. Isolated internal thoracic artery rings were suspended in two organ chambers. One contained Krebs-Henseleit solution and served as control. The other contained a heparin or Nomega-Nitro-L-arginine (L-NAM, an inhibitor of both endothelial and inducible nitric oxide synthase) or a specific inhibitor of inducible nitric oxide synthase, aminoguanidine. Increasing doses of protamine were added to both chambers and dose-response curves were obtained. RESULTS: Protamine was found to relax contracted internal thoracic arteries 56% +/- 4.7% of baseline measurements in a concentration-dependent manner. When L-NAM was added, protamine caused only a slight decrease of tension. There were no differences in the relaxing effect of protamine in the presence of aminoguanidine or heparin. CONCLUSIONS: Protamine induces nitric oxide-dependent relaxation of the internal thoracic artery by activation of endothelial nitric oxide synthase pathway. Heparin could not prevent this relaxing effect of protamine.
Asunto(s)
Puente de Arteria Coronaria , Circulación Coronaria/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Protaminas/farmacología , Vasodilatación/efectos de los fármacos , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Humanos , Arterias Torácicas/efectos de los fármacos , Arterias Torácicas/trasplanteRESUMEN
BACKGROUND: Increasing evidence suggests that a locally integrated or intramyocardial renin-angiotensin system plays a significant role in ischemia-reperfusion injury. We evaluated the effects of losartan, an angiotensin II type 1 receptor blocking agent, on ischemic and nonischemic isolated rat hearts. METHODS: Using the modified Langendorff model, hearts were perfused with either low or high doses of losartan (18.2 mmol/L or 182.2 mmol/L, respectively) or with saline added to Krebs-Henseleit solution during phase I of the study. During phase II, hearts were exposed to a 60-minute period of global ischemia. Ischemic arrest was induced with warm cardioplegic solution (KCl, 16 mEq/L) containing either high-dose losartan (182.2 mmol/L) or Krebs-Henseleit solution only. RESULTS: During phase I of the study, no statistically significant differences were observed between the low-dose losartan group and the control group. However, hearts treated with high-dose losartan demonstrated an increase in peak systolic pressure, maximum first derivative of pressure, pressure-time integral, coronary flow, and oxygen consumption (p < 0.0001). During phase II, hearts treated with losartan showed a significantly better recovery on reperfusion, as reflected by better contractility (p < 0.001), higher oxygen consumption (p < 0.001), higher coronary flow (p < 0.0001), and lower creatine phosphokinase levels (41.1 +/- 1.7 versus 73.3 +/- 5.6 U/L; p < 0.001). CONCLUSIONS: High doses of losartan have a positive inotropic effect on normally perfused hearts. Given in cardioplegic solution, the drug has a significant protective effect on ischemic isolated rat hearts.
Asunto(s)
Angiotensina II/antagonistas & inhibidores , Corazón/fisiopatología , Losartán/farmacología , Isquemia Miocárdica/fisiopatología , Animales , Circulación Coronaria/efectos de los fármacos , Paro Cardíaco Inducido , Hemodinámica , Masculino , Isquemia Miocárdica/metabolismo , Reperfusión Miocárdica , Miocardio/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
OBJECTIVES: This study sought to assess the importance of locally released or paracrine myocardial tumor necrosis factor-alpha (TNF-alpha) in the evolution of postischemic myocardial dysfunction and to use immunohistochemical studies to localize TNF-alpha within the myocardium. BACKGROUND: TNF-alpha is implicated as a systemic mediator in the development of myocardial ischemia-reperfusion injury by promoting leukocyte myocardial infiltration, and it has been shown to originate from noncardiac peripheral mononuclear cells. We have recently documented in a blood-free environment the release of TNF-alpha from the ischemic-reperfused myocardium. METHODS: Isolated rat hearts undergoing 1 h of global cardioplegia-induced ischemia and 30 min of reperfusion were investigated with use of the modified Langendorff model. Hearts were randomly divided into three subgroups: group A, control group; and groups B and C, isolated hearts receiving cardioplegic solution containing monoclonal hamster antimurine TNF-alpha antibodies (group B) or hamster IgG (group C). RESULTS: Significant amounts of TNF-alpha were detected in group A and group C effluent on 1 min of reperfusion (752 +/- 212 and 958 +/- 409 pmol/ml, respectively). However, in group B, TNF-alpha was below detectable levels. In this group, postischemic left ventricular peak systolic pressures, first derivative of the rise in left ventricular pressure (dP/dtmax), pressure-time integral, coronary flow and O2 consumption improved (analysis of variance [ANOVA] p < 0.0001 for all variables) compared with values in groups A and C; creatine kinase levels decreased (p < 0.005); and myocardial structure was preserved. Immunohistochemical staining localized TNF-alpha to cardiac myocytes and to endothelial cells. CONCLUSIONS: Anti-TNF-alpha neutralizes local TNF-alpha release from cardiac myocytes after ischemia and improves myocardial recovery during reperfusion, indicating that postischemic paracrine TNF-alpha release plays an active role in myocardial dysfunction.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Masculino , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/inmunología , Miocardio/metabolismo , Miocardio/patología , Técnicas de Cultivo de Órganos , Consumo de Oxígeno , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Previous studies have shown that long-term treatment with the angiotensin-converting enzyme inhibitor captopril attenuates left ventricular dilatation and improves survival after extensive myocardial infarction. However, there is only sparse evidence of the immediate effects of the drug on hearts undergoing global ischemia and reperfusion. The purpose of this study was to investigate the direct effect of captopril, given in cardioplegia or after ischemia, on the functional recovery of the reperfused myocardium. METHODS: Isolated rat hearts undergoing warm cardioplegic arrest followed by 1 hour of global ischemia and 30 minutes of reperfusion were studied using the modified Langendorff model. RESULTS: After ischemia, hearts receiving captopril (360 mumol/L) either in the cardioplegic solution (n = 9) or during reperfusion (n = 9) developed higher pressure (p < 0.001), greater first derivative of the rise in left ventricular pressure (p < 0.01 and p < 0.001, respectively), greater first derivative of the fall in left ventricular pressure (p < 0.001 and p < 0.002), higher pressure-time integral (p < 0.001), greater coronary flow (p < 0.001), and higher oxygen consumption values (p < 0.001 and p < 0.003) compared with the control group (n = 9). Hearts receiving captopril both in the cardioplegia and during reperfusion (n = 9) had the best recovery of all three groups and lower levels of creatine kinase (47.8 +/- 5.9 U/L versus 73.3 +/- 5.6 U/L; p < 0.01) compared with the control group. CONCLUSIONS: Captopril given in cardioplegia and in reperfusion has a favorable, protective, and additive effect on the recovery of isolated rat hearts undergoing global ischemia and reperfusion; hemodynamic performance improves, coronary flow and oxygen consumption increase, and myocardial damage decreases.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Captopril/uso terapéutico , Daño por Reperfusión Miocárdica/prevención & control , Animales , Circulación Coronaria/efectos de los fármacos , Masculino , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Wistar , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacos , Presión Ventricular/efectos de los fármacosRESUMEN
Complement in the respiratory tract protects the host from invading micoorganisms and other inhaled insults, but may damage normal tissue. Recently we reported that human respiratory epithelium from the nose to the alveoli expresses three cell-membrane regulators of complement activation: membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF; CD55), and CD59. In this study we investigated whether two of these complement-regulatory proteins, DAF and CD59, protect human nasal epithelial cells from complement-mediated lysis. Treatment of nasal epithelial cells in suspension with 50% or 100% normal human serum (NHS) lysed small percentages of cells (8% and 16%, respectively). Addition of complement activators, rabbit serum antinasal epithelial cells (anti-NEC), or lipopolysaccharide (LPS) increased cell lysis in the presence of 50% NHS in a dose-dependent manner up to 50% and 35% lysis, respectively. Human serum deficient in C3 or C7 did not lyse nasal epithelial cells even in the presence of anti-NEC. To assay the contribution of DAF and CD59 to cell protection against lysis, nasal epithelial cells in suspension were treated with appropriate blocking antibodies. Both anti-DAF and anti-CD59 markedly increased the susceptibility of human nasal epithelial cells to lysis by complement. At 50% NHS, anti-DAF and anti-CD59 antibodies increased epithelial cell lysis from 8% to 24% and 67%, respectively. A similar pattern of response to complement was demonstrated by monolayers of substrate-anchored cultured cells. These results indicate that DAF and CD59 protect human nasal epithelial cells from complement-mediated lysis; however, intense activation of complement may overcome this protection, leading to cell death and tissue injury. We speculate that imbalance between complement regulation and complement activation in the human respiratory tract in disease may result in tissue injury and impaired tissue function.
Asunto(s)
Proteínas Inactivadoras de Complemento/fisiología , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica , Mucosa Nasal/inmunología , Adolescente , Adulto , Antígenos CD/fisiología , Antígenos CD55/fisiología , Antígenos CD59/fisiología , Células Cultivadas , Niño , Epitelio/inmunología , Humanos , Proteína Cofactora de Membrana , Glicoproteínas de Membrana/fisiología , Persona de Mediana EdadRESUMEN
OBJECTIVES: The purpose of this study was to examine whether tumor necrosis factor-alpha (TNF-alpha) is released directly from the ischemic myocardium undergoing reperfusion. BACKGROUND: Tumor necrosis factor-alpha is a protein hormone produced by systemic leukocytes (primarily by activated macrophages). It has been implicated as a systemic mediator in the development of septic shock and other pathologic conditions. Serum TNF-alpha has also been detected in a variety of cardiac disease states and after myocardial ischemia-reperfusion injury. METHODS: Nine isolated rat hearts undergoing 30 min of perfusion, followed by warm cardioplegic arrest, 1 h of global ischemia and 30 min of reperfusion, were investigated using the modified Langendorff model. RESULTS: Significant amounts of TNF-alpha (752 +/- 212 pmol/ml) were detected in the effluent during the first minute of reperfusion. Tumor necrosis factor-alpha levels correlated with postischemic deterioration in peak systolic pressures (r = 0.7882, p = 0.012), dP/dt max (r = 0.6795, p = 0.044), time-pressure integral (r = 0.7661, p = 0.0016) and postischemic creatine kinase levels (r = 0.8367, p = 0.005). The deterioration in coronary flow, however, was inversely correlated with TNF-alpha levels (r = -0.7581, p = 0.018). CONCLUSIONS: To our knowledge, this study is the first to suggest that the isolated rat myocardium synthesizes and releases TNF-alpha in response to ischemia and reperfusion, which directly correlates with the postischemic deterioration in myocardial mechanical performance and the amount of cellular necrosis.
Asunto(s)
Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Masculino , Reperfusión Miocárdica , Daño por Reperfusión Miocárdica/fisiopatología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Recent evidence suggests that complement is activated in human nasal airways in inflammatory states. Activated complement protects the nasal mucosa against microorganisms, but also has the potential to lyse the host's normal cells. Complement-mediated cell lysis depends on adsorption of complement to the cell membrane and on uninterrupted activation of the complement cascade upon the same cell membrane. In the present study, the authors investigated first whether key complement components, C3-related fragments, are adsorbed to nasal epithelial cell membrane. Second, we investigated whether nasal epithelium expresses cell membrane complement regulatory proteins that are known as interruptors of complement activation. Studies were done using fresh nasal mucosa obtained at turbinectomies from allergic rhinitis and vasomotor rhinitis patients. In addition, in order to establish an in vitro model, studies were also done using primary cell cultures of nasal epithelium. We have found that complement C3-related fragments are present on cell membranes of fresh nasal epithelium and that C3-related fragments are adsorbed to the epithelial cell membrane in nasal mucosa tissue segments and in cell cultures that were incubated with autologous serum. Adsorption of C3-related fragments to the cell membrane of cultured nasal epithelial cells was found by flow cytometry analysis to be concentration-dependent. In addition, we found that nasal epithelium in fresh tissue and in cell culture express three cell membrane complement regulatory proteins: membrane cofactor protein (MCP, CD46), decay-accelerating factor (DAF, CD55), and CD59. Our findings in fresh nasal epithelium suggest that complement activation may occur upon the nasal epithelial cell membrane during inflammation in vivo and that nasal epithelium might regulate this complement activation. Our in vitro cell culture model will allow further investigations of complement activation and regulation upon the human nasal epithelial cell membrane.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD55/biosíntesis , Antígenos CD59/biosíntesis , Activación de Complemento/fisiología , Complemento C3c/inmunología , Proteínas Inactivadoras de Complemento/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Mucosa Nasal/inmunología , Adolescente , Adsorción , Adulto , Anciano , Células Cultivadas , Epitelio/inmunología , Femenino , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Masculino , Proteína Cofactora de Membrana , Persona de Mediana Edad , Mucosa Nasal/metabolismoRESUMEN
Complement in the human respiratory tract protects the host from invading microorganisms and from other inhaled insults. However, complement may also lyse the host's respiratory tract cells, leading to tissue injury. In many extrapulmonic tissues, cells express cell-membrane complement regulatory glycoproteins that protect the cells from complement-induced lysis. To determine whether these glycoproteins are expressed in human respiratory tract tissue, we studied tissue biopsies of healthy and diseased human respiratory tract from nose to alveoli for the presence of four cell-membrane complement regulatory glycoproteins (membrane cofactor protein [MCP], decay-accelerating factor [DAF], CD59, and complement receptor type 1 [CR1]) using an immunoperoxidase technique. In addition, to establish a model for in vitro studies of these glycoproteins in respiratory cells, we studied whether they are expressed in cultured nasal epithelial cells, using the same technique. Altogether, 26 tissue specimens from 22 patients were studied. We found that normal human respiratory tract from nose to alveoli express MCP, DAF, and CD59, but not CR1, and that this expression increases in inflammation and in lung cancer. In addition, expression in nasal epithelial cells is retained under cell culture conditions. These findings suggest that human respiratory tract tissue may regulate complement activation on its surface in order to avoid self-injury. We propose that imbalances in the mechanism that regulates cell-membrane complement may predispose the respiratory tract to tissue injury and disease, and that iatrogenic modulation of such imbalances may help to prevent these adverse consequences.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Glicoproteínas de Membrana/metabolismo , Sistema Respiratorio/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Proteínas Sanguíneas/metabolismo , Antígenos CD55 , Antígenos CD59 , Células Cultivadas , Proteínas Inactivadoras de Complemento/metabolismo , Femenino , Humanos , Inflamación , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Proteína Cofactora de Membrana , Persona de Mediana Edad , Receptores de Complemento/metabolismo , Sistema Respiratorio/patología , Enfermedades Respiratorias/metabolismo , Enfermedades Respiratorias/patologíaRESUMEN
Airway inflammation is characterized by intraluminal influx of inflammatory cells, exudation of plasma, and increased procoagulant activity. We speculated that inflammatory cells might adhere to the airway surface epithelium in order to better localize and regulate airway inflammatory responses. Therefore, in this study, we asked whether neutrophils adhere to airway epithelial cells, whether serum or plasma factors increase adhesion, and, if so, what the characteristics of the involved adhesion molecules are. To answer these questions, we incubated human 51Cr-labeled neutrophils from peripheral blood with dog tracheal epithelial cells in culture in the presence or absence of normal human serum or plasma. After 30 min, nonadhering neutrophils were centrifuged away and neutrophil adhesion was assessed by radioassay. We found that unstimulated adhesion of neutrophils to cultured epithelial cells was quite low (< 6%). However, incubation with 10% serum or plasma increased adhesion of neutrophils to epithelial cells dramatically (up to a mean of 71%). The serum-induced increase in adhesion was concentration dependent; even 1% serum was effective (19% adhesion). Serum adhesion factor acted selectively on epithelial surfaces, was heat sensitive, had a molecular weight > 12,000, and depended on the presence of divalent cations. mAb 60.3 (anti-CD18) and mAb anti-Mol (anti-CD11b, anti-CR3) inhibited serum-induced adhesion by > 50% each. We conclude that normal serum and plasma contain a potent adhesion factor that induces adhesion of neutrophils to tracheal epithelium in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antígenos CD/inmunología , Proteínas Sanguíneas/fisiología , Complemento C3b/fisiología , Neutrófilos/fisiología , Tráquea/citología , Animales , Anticuerpos Monoclonales , Antígenos CD11 , Antígenos CD18 , Cationes Bivalentes/farmacología , Adhesión Celular , Células Cultivadas , Perros , Femenino , Calor , Humanos , Inmunohistoquímica , Masculino , Peso Molecular , Neutrófilos/citología , Neutrófilos/inmunología , RadioinmunoensayoRESUMEN
Glycerol-induced acute renal failure (ARF) in rats is a model of acute trauma in which intra-muscular injection of 50% glycerol causes rapid myoglobinuria, oliguria, and a rapid reduction in glomerular filtration rate. We found that plasma tumor necrosis factor-alpha (TNF-alpha) is rapidly induced in glycerol injected rats. It can be detected in some animals as early as 30 minutes post-injection, peaks at one hour (range: 4 to 32 U/ml) with no significant difference between blood from renal vein and vena cava, and decreases by three hours. None was detected in control saline injected rats (P < 0.001). Four out of five rats infused with neutralizing anti-TNF-alpha antiserum (200 microliters/300 g body wt) immediately prior to glycerol injection had significantly protected kidney function (P = 0.001). In these rats, plasma urea (104.8 +/- 58.9 mg%) and creatinine (1.16 +/- 0.38 mg%) were lower and creatinine clearance higher (0.34 +/- 011 ml/min) than in glycerol injected animals pretreated with normal serum (291.8 +/- 41.8 mg%, 3.15 +/- 0.74 mg%, and 0.03 +/- 0.03 ml/min, respectively) or animals injected with glycerol alone (302.6 +/- 76.8 mg%, 3.45 +/- 0.97 mg%, and 0.03 +/- 0.03 ml/min, respectively). These results imply a direct role for TNF-alpha in pathogenesis of glycerol induced ARF in rats.