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BACKGROUND: Metastatic clear cell renal cell carcinoma (ccRCC) patients have <9% 5-year survival rate, do not respond well to targeted therapy and eventually develop resistance. A better understanding of molecular pathways of RCC metastasis is the basis for the discovery of novel prognostic markers and targeted therapies. METHODS: We investigated the biological impact of galectin-1 (Gal-1) in RCC cell lines by migration and invasion assays. Effect of Gal-1 expression on the mitogen-activated protein kinase pathway was assessed by proteome array. RESULTS: Increased expression of Gal-1 increased cell migration while knocking down Gal-1 expression by siRNA resulted in reduced cellular migration (P<0.001) and invasion (P<0.05). Gal-1 overexpression increased phosphorylation of Akt, mTOR and p70 kinase. Upon hypoxia and increased HIF-1α, Gal-1 increased in a dose-dependent manner. We also found miR-22 overexpression resulted in decreased Gal-1 and HIF-1α. Immunohistochemistry analysis showed that high Gal-1 protein expression was associated with larger size tumor (P=0.034), grades III/IV tumors (P<0.001) and shorter disease-free survival (P=0.0013). Using the Cancer Genome Atlas data set, we found that high Gal-1 mRNA expression was associated with shorter overall survival (41 vs 78 months; P<0.01). CONCLUSIONS: Our data suggest Gal-1 mediates migration and invasion through the HIF-1α-mTOR signaling axis and is a potential prognostic marker and therapeutic target.
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Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Galectina 1/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Serina-Treonina Quinasas TOR/metabolismo , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Galectina 1/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Renales/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Pronóstico , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
PURPOSE: Ruptured abdominal aortic aneurysm (RAAA) remains a lethal condition despite improvements in perioperative care. The consequences of RAAA are hypothesized to result from a combination of two ischemia/reperfusion events: hemorrhagic shock and lower torso ischemia. Ischemia/reperfusion results in tissue injury by diverse mechanisms, which include oxygen free radical-mediated injury produced from activated neutrophils, xanthine oxidase, and mitochondria. Oxygen-free radicals attack membrane lipids, resulting in membrane and subsequently cellular dysfunction that contributes to postoperative organ injury/failure. The purpose of this investigation was to quantify the oxidative injury that occurs as a result of the ischemia/reperfusion events in RAAAs and elective AAAs. METHODS: Blood samples were taken from 22 patients for elective AAA repair and from 14 patients for RAAA repair during the perioperative period. Plasma F(2)-isoprostanes were extracted, purified, and measured with an enzyme immunoassay. Aldehydes and acyloins were purified and quantified. Neutrophil oxidative burst was measured in response to a receptor independent stimulus (phorbol 12-myristate 13-acetate) with luminol-based chemiluminescence. RESULTS: Plasma from patients with RAAAs showed significantly elevated F(2)-isoprostane levels on arrival at hospital and were significantly elevated as compared with the levels of patients for elective repair throughout the perioperative period (two-way analysis of variance, P <.0001). Multiple regression showed a significant relationship between the phagocyte oxidative activity and F(2)-isoprostane levels (P <.013). Total acyloin levels were significantly higher in patients with RAAAs as compared with the levels in elective cases. CONCLUSION: The F(2)-isoprostane levels, specific markers of lipid peroxidation, showed that patients with RAAAs had two phases of oxidative injury: before arrival at hospital and after surgery. The significant relationship between the postoperative increases in F(2)-isoprostane levels and the neutrophil oxidant production implicates neutrophils in the oxidative injury that occurs after RAAA. New therapeutic interventions that attenuate neutrophil-mediated oxidant injury during reperfusion may decrease organ failure and ultimately mortality in patients with RAAAs.
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Aneurisma Roto/fisiopatología , Aneurisma de la Aorta Abdominal/fisiopatología , Biomarcadores/sangre , Dinoprost/sangre , Neutrófilos/fisiología , Estrés Oxidativo , Daño por Reperfusión , Aldehídos/sangre , Aneurisma Roto/sangre , Aneurisma Roto/cirugía , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/cirugía , Dinoprost/análogos & derivados , Alcoholes Grasos/sangre , Humanos , Técnicas In Vitro , Isquemia/sangre , Mediciones Luminiscentes , Modelos Cardiovasculares , Neutrófilos/efectos de los fármacos , Estallido Respiratorio , Choque Hemorrágico/sangre , Acetato de TetradecanoilforbolRESUMEN
Revascularization of an ischemic lower extremity is associated with high morbidity (20-30%) and perioperative mortality (10-20%) regardless of the mode of intervention, surgical or thrombolytic, considered to be due to polymorphonuclear (PMN) activation and mediator release. In this study, the safety and feasibility of cell-free extracorporeal perfusion of the limb with a solution designed to minimize both local and systemic injury was tested. Methods. Patients with severe limb ischemia (sensory/motor loss, rest pain/gangrene) were studied prospectively by random assignment into the treatment arm (n = 14) or control arm (n = 21). Surgical management consisted of restorative procedure, thrombectomy or embolectomy (n = 21), or reconstruction (n = 14). Reperfusion of the ischemic limb was achieved with hypertonic, hyperoncotic perfusate containing anti-oxidants delivered via the arterial tree (mean volume 1835 +/- 824 ml) with initial venous drainage (mean volume 775 +/- 263 ml) in the restorative group. Means were compared by paired t test. Results. No adverse systemic effects were detected after limb perfusion (electrolytes, coagulation, platelet function, CBC). Rapid lactate wash-out was observed within 30 min of perfusion (preperfusion 3.2 +/- 4.1 mM, 30 min postperfusion 0.7 +/- 0.71 mM, P < 0.01). Blunting of PMN activation was shown by chemiluminescence (CL) analysis (preischemic CL: 0.68 +/- 0.2; 30 min CL: 0.47 +/- 0. 2; P < 0.013). F2-isoprostanes, a marker of free radical-mediated systemic lipid peroxidation, were significantly reduced in patients treated with study perfusion method (70.55 +/- 39.54 versus control 194.38 +/- 25.24, P < 0.005). Mortality with treatment was 0/14 versus 5/21 in the control. Complication frequency: MI 0/14 vs 3/21; renal 0/14 vs 1/21; leg edema 1/14 vs 5/21; amputations 2/14 vs 1/21. Conclusion. Modification of limb perfusion in patients with severe limb ischemia, using our simple and rapid (15-20 min) method provides beneficial systemic effects.
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Isquemia/terapia , Pierna/irrigación sanguínea , Perfusión , Terapia Recuperativa , Estudios de Cohortes , Humanos , Isquemia/cirugía , Perfusión/efectos adversos , Estudios Prospectivos , Terapia Recuperativa/efectos adversos , Resultado del TratamientoRESUMEN
INTRODUCTION: In the Olympus uric acid procedure, uric acid is converted by uricase to allantoin and hydrogen peroxide, which is reacted in a Trinder reaction to produce a chromophore read bichromatically at 520 and 660 nm. Repeated difficulty was encountered in obtaining uric acid results on samples from myeloma patients with known IgM paraproteins. Large absorbances in sample blanks were due to a visible precipitation observed in the reaction cuvettes. OBJECTIVE: To alter the Olympus method (OM) to eliminate the interference by IgM, and to verify the modified method (MM). METHODS: Dilution of the sample blank by saline was substituted for water in the MM, with small alterations in the reaction timing sequence necessary to accommodate the instrument requirements. RESULTS: A comparison of uric acid results obtained from nonmyeloma patient samples using the OM and the MM showed a good correlation (r = 0.970), and no statistical difference between the two means using a paired t-test. A similar comparison performed using the samples containing IgA and IgG paraproteins also revealed a good correlation (r = 0.981), and no statistical difference between the two means. Results on IgM containing specimens were assessed indirectly because the samples could not be assayed with the OM. First, removal of detectable levels of proteins using a 20% TCA solution did not affect the measurement of uric acid. Second, protein-free supernatants from IgM containing samples were measured by the OM and compared with the corresponding serum samples measured by the MM. There was good correlation between the two methods (r = 0.945), and no statistical difference between the means using a paired t-test. CONCLUSION: The modified method is satisfactory for routine analysis of samples, including those with IgM paraproteins.
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Análisis Químico de la Sangre/métodos , Inmunoglobulina M/química , Mieloma Múltiple/sangre , Ácido Úrico/sangre , HumanosRESUMEN
A rapid (30 min) whole blood assay for the detection of lipopolysaccharide (LPS) is described. This chemiluminescent (CL) assay utilizes the CR1 and CR3 receptor-induced oxidant production of polymorphonuclear leucocytes as a detection platform. The differential priming of neutrophils in whole blood by LPS-antibody complexes allows the specificity of the assay to be achieved. Oxidant released in response to complement opsonized zymosan results in luminol oxidation and subsequent light emission. This is dependent on heat labile putative complement proteins in the plasma. The assay consists of a control which measures baseline whole blood neutrophil oxidant production. The test assay contains murine monoclonal IgM antibody against the Lipid A epitope of LPS and measures the enhanced chemiluminescent response of the neutrophils in the presence of LPS-antibody complexes. Maximal sensitivity of the CL assay is dependent upon optimal antigen-antibody equivalence and duration of pre-incubation with the whole blood sample. The quantification of LPS is possible by inclusion of a positive control containing a maximally reactive LPS dose (800 pg/ml Escherichia coli 055:B5 LPS at an antibody concentration of 0.8 microg/assay). The CL assay is insensitive to variations in patient neutrophil concentration over a minimum range of 0.5 to 20 x 10(9) cells/l. The CL assay is widely reactive with the LPS of many strains of gram negative bacteria but not with the cell wall products of gram positive bacteria or Candida and Aspergillus. In comparison to acid extraction chromogenic LAL, the CL assay demonstrates superior recovery precision and accuracy in in vitro studies. This was reproducible over a wide range of LPS concentrations (0.017-1.6 EU/ml or 20-2000 pg/ml). This assay may be a clinically useful tool for the diagnosis of infection or endotoxin in patients.
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Anticuerpos Antibacterianos , Bioensayo/métodos , Infecciones por Bacterias Gramnegativas/diagnóstico , Lipopolisacáridos/sangre , Activación Neutrófila , Sepsis/diagnóstico , Anticuerpos Monoclonales , Células HL-60 , Humanos , Lipopolisacáridos/inmunología , Mediciones Luminiscentes , Luminol , Antígeno de Macrófago-1 , Oxidación-Reducción , Receptores de Complemento 3b , Sensibilidad y Especificidad , Manejo de Especímenes , Factores de TiempoRESUMEN
Reperfusion of acutely ischemic skeletal muscle is associated with neutrophil activation, which may augment local injury or cause damage to distant organs. Polymorphonuclear neutrophil glycoprotein CD18 plays a role in this injury, since its blockade substantially reduces damage; however, its mechanisms of control during reperfusion are poorly understood. The purpose of this study was to investigate the importance of circulating plasma factors to CD18-dependent neutrophil function during reperfusion and to relate these to quantitative expression of CD18. Eight rabbits were subjected to hindlimb ischemia for 5 h, followed by 48 h of reperfusion. Plasma collected at seven intervals was incubated with unstimulated neutrophils from uninjured rabbits. CD18-specific neutrophil activation was evaluated by quantifying adherence to protein-coated polystyrene and by measuring oxidant production, detected by chemiluminescence after exposure to complement-opsonized zymosan. CD18 was quantified cytofluorometrically. Plasma collected at end ischemia and during early reperfusion affected no significant alterations of adhesion, oxidant production, or CD18. Late reperfusion plasma (between 8 and 48 h) significantly increased adherence and oxidant production (to 4.11 +/- 0.61 and 2.60 +/- 0.32 times the values of preischemic plasma, P < 0.006). Peak adherence, oxidant production, and CD18 expression were evoked synchronously by 24 h plasma. CD18 expression increased only at 24 h and did not increase proportional to increases in adherence and oxidant production. Control plasma (nonischemic, n = 5) elicited no significant differences of any inflammatory measure during sham ischemia or reperfusion. These results indicate that endogenous mediators may evoke a progressive systemic inflammatory response after ischemia by stimulating CD18-dependent neutrophil function in a delayed but prolonged manner.
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Antígenos CD18/inmunología , Isquemia/inmunología , Músculo Esquelético/irrigación sanguínea , Activación Neutrófila/fisiología , Neutrófilos/inmunología , Plasma/fisiología , Animales , Adhesión Celular , Femenino , Isquemia/sangre , Neutrófilos/fisiología , Conejos , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/inmunología , Daño por Reperfusión/fisiopatologíaRESUMEN
Sequential ischemia/reperfusion in a paired canine gracilis muscle model resulted in significant muscle salvage. In this model, one randomly chosen gracilis muscle was subjected to 5 h of ischemia followed by 48 h of in vivo reperfusion. The contralateral (second) muscle was then made ischemic and reperfused using the same protocol. Muscle necrosis was determined at the end of 48 h of reperfusion. A mean 60% reduction in muscle necrosis was observed in the second group of muscles. Analysis of tissue adenine nucleotides indicated that significant sparing of ATP utilization occurred in the second muscle group during ischemia. Preliminary analysis of tissue heat shock proteins (HSP) showed that the second group of muscles had a different pattern of HSP expression before the onset of ischemia. The results suggest that reduced ATP utilization and altered HSP expression in the second muscle play a role in the tissue salvage observed in this sequential muscle ischemia model.
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Isquemia/fisiopatología , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/fisiopatología , Reperfusión , Adenosina Trifosfato/metabolismo , Animales , Perros , Femenino , Proteínas de Choque Térmico/metabolismo , Isquemia/patología , Masculino , Músculo Esquelético/patología , NecrosisRESUMEN
Reperfusion of ischemic skeletal muscle is associated with neutrophil (PMN) adherence to damaged endothelium and PMN-mediated tissue destruction. Neutrophils may attach to endothelium through surface adhesive molecules, such as CD18. The purpose of this study was to determine whether monoclonal antibody blockade of CD18 would reduce skeletal muscle necrosis associated with ischemia and reperfusion. In rabbits, an entire hindlimb was rendered ischemic for 4 hr, followed by 48 hr of in vivo reperfusion. Animals were allocated to one of five treatment groups: ischemia/reperfusion without treatment (I/R controls), I/R plus treatment with the anti-CD18 antibody IB4 (end-ischemic 2 mg/kg dose), I/R plus treatment with an identical dose of isotype-matched control Ig, I/R plus anterior compartment fasciotomy, or I/R plus both IB4 and fasciotomy. After 48 hr of reperfusion anterior tibial muscle necrosis was assessed (by tetrazolium staining and computerized planimetry), wet:dry muscle weights (W:D) were determined, and muscle PMN sequestration was measured by myeloperoxidase (MPO) activity. IB4-treated animals exhibited markedly reduced muscle MPO activity, compared to untreated animals. Although all interventions reduced edema formation (W:D ratios), none did so significantly. IB4 treatment reduced muscle necrosis when used alone (to 28 +/- 7%, vs. 48% +/- 6% in untreated controls), however this was not statistically significant (P = 0.06).2+ Fasciotomy significantly reduced necrosis (to 22 +/- 2%, P < 0.05); however, the addition of IB4 to fasciotomy resulted in necrosis that was significantly lower than that after fasciotomy alone (12 +/- 4%, P < 0.05 vs fasciotomy group) and the least necrosis of any group.(ABSTRACT TRUNCATED AT 250 WORDS)
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Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/fisiología , Isquemia , Músculos/irrigación sanguínea , Daño por Reperfusión/prevención & control , Animales , Antígenos CD/inmunología , Antígenos CD18 , Femenino , Músculos/patología , Necrosis , Neutrófilos/fisiología , Tamaño de los Órganos , Peroxidasa/metabolismo , ConejosRESUMEN
X-ray photoelectron spectroscopy was used to characterize modified surfaces of a biomedically important polymer, poly(ethylene terephthalate). Several modification schemes were investigated and direct silanization with 3-aminopropyltriethoxysilane was found to be the optimum procedure, resulting in an aminated surface. Surface coverage of up to 100% was achieved with retention of the polymeric structural integrity. Further activation of the silanized surface was accomplished with two cross-linkers, glutaraldehyde and sebacoyl chloride. A simple biomolecule, L-cysteine, was successfully immobilized onto a surface pre-treated with 3-aminopropyltriethoxysilane and glutaraldehyde, with a coverage of 42%.
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Tereftalatos Polietilenos/química , Materiales Biocompatibles , Indicadores y Reactivos , Microscopía Electrónica de Rastreo , Propiedades de SuperficieRESUMEN
PURPOSE: The mechanism by which some graft materials are more thrombogenic than others is poorly understood. We hypothesized that differential induction of macrophage procoagulant activity (PCA) by various materials may contribute to variable thrombogenicity. METHODS: Thioglycollate-elicited murine peritoneal macrophages were added to disks of Dacron and expanded polytetrafluoroethylene (ePTFE). After adherence, macrophages were incubated with and without endotoxin (lipopolysaccharide) and then recovered by sonication for determination of PCA with a one-step clotting bioassay. RESULTS: PCA was significantly higher in cells after incubation on Dacron compared with ePTFE both in the absence of lipopolysaccharide (243 +/- 76 vs 68 +/- 39 mU, n = 4) and after stimulation with lipopolysaccharide (491 +/- 137 vs 139 +/- 41 mU, n = 4) (p < 0.01, analysis of variance). Using factor-deficient plasmas, we found that this PCA was consistent with tissue factor. This differential induction of PCA was related to increased macrophage adherence to Dacron compared to that to ePTFE (9374 +/- 1158 vs 2111 +/- 330 cells/mm2; n = 4; p < 0.01, analysis of variance). CONCLUSIONS: The thrombogenic nature of Dacron correlates with its ability to adhere macrophages and induce PCA. Strategies aimed at modulating these effects may reduce the thrombogenicity of vascular grafts and therefore potentially the incidence of graft thrombosis.
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Factores de Coagulación Sanguínea/fisiología , Coagulación Sanguínea/fisiología , Prótesis Vascular/efectos adversos , Macrófagos/fisiología , Tereftalatos Polietilenos/efectos adversos , Politetrafluoroetileno/efectos adversos , Análisis de Varianza , Animales , Bioensayo , Femenino , Radioisótopos de Indio , RatonesRESUMEN
Skeletal muscle ischemia results in energy depletion and intracellular acidosis. Reperfusion is associated with impaired adenine nucleotide resynthesis, edema formation, and myocyte necrosis. The purpose of these studies was to define the time course of cellular injury and adenine nucleotide depletion and resynthesis in postischemic skeletal muscle during prolonged reperfusion in vivo. The isolated canine gracilis muscle model was used. After 5 h of ischemia, muscles were reperfused for either 1 or 48 h. Lactate and creatine phosphokinase (CPK) release during reperfusion was calculated from arteriovenous differences and blood flow. Adenine nucleotides, nucleosides, bases, and creatine phosphate were quantified by high-performance liquid chromatography, and muscle necrosis was assessed by nitroblue tetrazolium staining. Reperfusion resulted in a rapid release of lactate, which paralleled the increase in blood flow, and a delayed but prolonged release of CPK. Edema formation and muscle necrosis increased between 1 and 48 h of reperfusion (P less than 0.05). Recovery of energy stores during reperfusion was related to the extent of postischemic necrosis, which correlated with the extent of nucleotide dephosphorylation during ischemia (r = 0.88, P less than 0.001). These results suggest that both adenine nucleotide resynthesis and myocyte necrosis, which are protracted processes in reperfusing skeletal muscle, are related to the extent of nucleotide dephosphorylation during ischemia.
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Nucleótidos de Adenina/biosíntesis , Isquemia/metabolismo , Músculos/irrigación sanguínea , Daño por Reperfusión/metabolismo , Animales , Creatina Quinasa/metabolismo , Perros , Isquemia/patología , Lactatos/metabolismo , Ácido Láctico , Músculos/metabolismo , Músculos/patología , Necrosis , Nitroazul de Tetrazolio , Tamaño de los Órganos , Fosfocreatina/metabolismo , Flujo Sanguíneo Regional , Daño por Reperfusión/patología , Factores de TiempoRESUMEN
In this clinical study we have prospectively measured plasma phospholipase A2 (PLA2) activity and tumor necrosis factor (TNF) levels in ventilated intensive care unit (ICU) patients with (n = 9) and without (n = 12) evidence of respiratory distress syndrome (ARDS) and multiple-organ failure (MOF). The median peak TNF concentration in control patients was 40 ng/L (range less than 40-100 ng/L) and in ARDS patients 231 ng/L (range 100-2550 ng/L; p less than 0.001). All of the control patients were discharged alive from the ICU, whereas 6 of 9 ARDS patients died in the ICU. In 6 ARDS patients, it was possible to measure more than 4 consecutive plasma TNF levels. Of these 6 patients, the 3 with persistent elevations in systemic TNF above 230 ng/L succumbed (p less than 0.05, one-tailed). Patients with ARDS also had parallel elevations in plasma PLA2 activity above controls. These elevations were significant for arterial PLA2 activity but not for venous PLA2 activity. Our study suggests that serial measurement of plasma (arterial or venous) TNF levels may have (1) prognostic and (2) etiologic significance in ICU patients with ARDS and MOF.
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Insuficiencia Multiorgánica/sangre , Fosfolipasas A/sangre , Síndrome de Dificultad Respiratoria/sangre , Factor de Necrosis Tumoral alfa/análisis , Cuidados Críticos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Fosfolipasas A2 , Pronóstico , Estudios ProspectivosRESUMEN
The authors have modified ePTFE by hydroxylating the surface using aluminum deposition and removal with sodium hydroxide. This process has no effect on the microfibrillar structure, but reduces the hydrophobicity of ePTFE. There were significantly greater numbers of rat aortic endothelial cells on the surface of modified as compared to control ePTFE after 14 days (469 +/- 44 vs. 4 +/- 3 cells/mm2, p less than 0.01, paired t-test). This simple chemical modification facilitates endothelialization, without using thrombogenic cell adhesives.
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Prótesis Vascular , Adhesión Celular/fisiología , División Celular/fisiología , Endotelio Vascular/citología , Politetrafluoroetileno , Animales , Hidroxilación , Masculino , Diseño de Prótesis , Ratas , Ratas Endogámicas , Propiedades de SuperficieRESUMEN
Biomaterials activate the complement system which is important since C3a promotes platelet aggregation and release, and C5a activates neutrophils that may augment coagulation. Tiny air nuclei (microbubbles) are found in the surface roughness of biomaterials on exposure to a liquid, therefore two interfaces exist: (a) a blood/biomaterial, and (b) a blood/air interface. Experiments were carried out that documented that air bubbles activate complement and augment in vitro platelet aggregation in human plasma. The air nuclei were removed from the surface of silicone rubber by a technique termed denucleation to determine if complement activation and platelet aggregation could be reduced. We observed a significant reduction in C3a and C5a in the plasma samples incubated with denucleated silicone rubber as compared to the control samples (p less than 0.001, ANOVA). The plasma incubated with the denucleated silicone caused reduced platelet aggregation as compared to the plasma incubated with the control silicone when added to a platelet suspension (p less than 0.001, ANOVA). Surface chemical analysis by x-ray photo-electron spectroscopy (XPS) showed no change in the silicone rubber surface after the denucleation procedure.
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Materiales Biocompatibles , Recolección de Muestras de Sangre , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Agregación Plaquetaria , Elastómeros de Silicona , Aire , Análisis de Varianza , Femenino , HumanosRESUMEN
After skeletal muscle ischemia, tissue damage is augmented during reperfusion. White blood cells (WBCs) and complement proteins may participate in the reperfusion injury. The purpose of this study was to define the kinetics of classical and alternative pathway complement activation and WBC sequestration by postischemic skeletal muscle during the first 48 h of reperfusion in vivo. The isolated canine gracilis muscle model was used. Systemic levels of the complement proteins factor B (alternative pathway) and C4 (classical pathway) were quantitated by hemolytic assay. WBC sequestration was measured by gracilis arterial-venous WBC differences and tissue myeloperoxidase activity. Reperfusion was associated with an 18% decrease in systemic factor B levels but no consistent change in systemic C4 levels. WBCs were sequestered during the first 4 h of reperfusion, and tissue myeloperoxidase activity was elevated 97-fold after 48 h of reperfusion. These results suggest that skeletal muscle ischemia-reperfusion stimulates 1) activation of the alternative but not the classical complement pathway and 2) an immediate and prolonged sequestration of WBCs.
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Activación de Complemento , Isquemia/sangre , Leucocitos/fisiología , Músculos/irrigación sanguínea , Reperfusión , Animales , Complemento C4/análisis , Factor B del Complemento/análisis , Perros , Músculos/enzimología , Peroxidasa/metabolismoRESUMEN
Previous studies in our laboratory have demonstrated the peroxidation of myocardial phospholipid in a canine model of reversible global normothermic ischemia and reperfusion while on cardiopulmonary bypass. The present study examines the distribution of phospholipid peroxidation products in three major cellular organelle fractions of myocardium prepared by established centrifugal fractionation procedures (sarcolemma, sarcoplasmic reticulum, and mitochondria). These organelles were isolated from control (nonischemic) and ischemic-reperfused myocardium harvested during early reperfusion (5 min), when previous studies indicated maximal peroxidative injury in whole myocardial biopsies. Utilizing a more rapid analytic procedure for measuring phospholipid containing the conjugated diene chromophore in the polyunsaturated fatty acyl substituents, we were able to establish the fidelity of this procedure by comparing the results obtained with it to the previous more laborious analytic procedure (involving phospholipid hydrolysis with phospholipase A2 and subsequent derivatization for high-pressure liquid chromatography followed by gas chromatographic-mass spectrometric analysis). Analysis of phospholipid extracts from organelle fractions for evidence of peroxidative conjugated diene formation revealed that sarcolemmal membranes had the highest content of oxidized phospholipid containing the conjugated diene chromophore (mean 2.2 +/- 1.2 nmol phospholipid-conjugated diene/mumol phospholipid phosphorus, P less than 0.02 compared with control). Both sarcoplasmic reticulum and mitochondrial membranes were also peroxidized but to a much smaller extent (mean 0.4 +/- 0.2 and 0.3 +/- 0.25 nmol phospholipid conjugated diene/mumol phospholipid phosphorus).
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Peróxidos Lipídicos/análisis , Daño por Reperfusión Miocárdica/patología , Miocardio/análisis , Animales , Calcio/farmacocinética , Canales de Calcio/fisiología , Canales de Calcio/ultraestructura , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular/fisiología , Perros , Miocardio/patología , Miocardio/ultraestructura , Orgánulos/análisis , Orgánulos/ultraestructuraRESUMEN
Prolonged ischemia to skeletal muscle as occurs after an acute arterial occlusion results in alterations in adenine nucleotide metabolism. Adenosine triphosphate continues to be used for cellular functions, and an ischemia-induced degradation of phosphorylated adenine nucleotides is initiated. In this experiment we demonstrated the time-dependent aspect of adenine nucleotide depletion during ischemia and the production of large quantities of soluble precursors. In addition, we studied the rate of conversion of xanthine dehydrogenase to xanthine oxidase, a potential source of oxygen-free radicals, after controlled periods of total normothermic ischemia (4 hours and 5 hours) and during the reperfusion phase. During ischemia complete depletion of creatine phosphate occurred in both groups, and adenosine triphosphate fell from 22.1 +/- 1.3 to 10.3 +/- 1.4 mumol/gm dry weight after 4 hours and from 21.6 +/- 0.7 to 3.9 +/- 0.8 mumol/gm dry weight after 5 hours (p less than 0.05). During reperfusion, creatine phosphokinase resynthesis occurred in both groups, but adenosine triphosphate levels were not significantly increased (p greater than 0.05). A washout of lipid soluble products of adenine nucleotide metabolism occurred equally in both groups. The relationship between phosphorylated adenine nucleotides as measured by the energy charge potential fell significantly in both groups (p less than 0.05), but after the shorter period of ischemia (4 hours it returned to normal during early reperfusion but did not after 5 hours of ischemia. There was 21% +/- 4% necrosis after 4 hours and 51% +/- 8% after 5 hours of ischemic stress when assessed at 48 hours. In conclusion, the degree of adenine nucleotide degeneration as determined primarily by the length of the ischemic period, may be the most important determinant of the ultimate extent of skeletal muscle ischemic necrosis that results from an acute interruption of circulation.
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Nucleótidos de Adenina/metabolismo , Isquemia/metabolismo , Músculos/metabolismo , Xantina Oxidasa/biosíntesis , Nucleótidos de Adenina/análisis , Animales , Perros , Técnicas In Vitro , Músculos/irrigación sanguínea , Fosfocreatina/análisis , Purinas/análisis , Xantina Deshidrogenasa/metabolismoRESUMEN
The production and prevention of calcium paradox injury in myocardium was studied in a canine model of cardiopulmonary bypass with multidose, moderately hypothermic, crystalloid cardioplegic solution. During 4 1/2 hours of global ischemia, three groups of six dogs each received one of three histidine-buffered cardioplegic solutions (500 ml initially and 250 ml every 30 minutes) at 27 degrees C. Group 1 cardioplegic solution was calcium free, group 2 solution contained a trace amount of calcium chloride (70 mumols /L), and group 3 cardioplegic solution was calcium free but contained diltiazem (150 micrograms/kg body weight). Left ventricular function measured as percent control of developed pressure revealed significantly greater (p less than 0.05) recovery in groups 2 and 3. Triphenyltetrazolium chloride staining showed 35% +/- 9% (mean +/- standard error) of heart mass necrosis in group 1 versus 0% and 0.5% +/- 0.4% in groups 2 and 3, respectively (p less than 0.001). Electron microscopy revealed ultrastructural changes characteristic of calcium paradox injury in group 1 myocardium. Calcium paradox injury was produced in an in vivo model of global myocardial ischemia and multidose cardioplegia despite moderate hypothermia and non-coronary collateral flow. The addition of either trace levels of calcium or diltiazem to the cardioplegic solution was effective in preventing this injury.