RESUMO
BACKGROUND: The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden. METHODS: Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters. RESULTS: The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype. CONCLUSION: Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies.
Assuntos
Carcinoma/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , Individualidade , Mutação , Oncogenes/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/patologia , Carcinoma/cirurgia , Estudos de Coortes , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. OBJECTIVE: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. METHODS: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. RESULTS: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). CONCLUSIONS: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.
Assuntos
Fibrinólise/efeitos dos fármacos , Indóis/farmacologia , Inibidor 1 de Ativador de Plasminogênio , Tetrazóis/farmacologia , Adulto , Estudos Cross-Over , Método Duplo-Cego , Humanos , Modelos BiológicosRESUMO
BACKGROUND: Interaction of P-selectin with its glycoprotein ligand (P-selectin glycoprotein ligand type 1) mediates inflammatory processes that may also include vascular thrombosis. Platelet P-selectin expression is increased in patients with coronary heart disease, and its antagonism represents a potential future therapeutic target for the prevention and treatment of atherothrombosis. AIM: To investigate the effects of the novel small molecule P-selectin antagonist PSI-697 on thrombus formation in humans. METHODS AND RESULTS: In a double-blind randomized crossover study, thrombus formation was measured in 12 healthy volunteers, using the Badimon ex vivo perfusion chamber under conditions of low and high shear stress. Saline placebo, low-dose (2 m) and high-dose (20 m) PSI-697 and the glycoprotein IIb-IIIa receptor antagonist tirofiban (50 ng mL(-1)) were administered into the extracorporeal circuit prior to the perfusion chamber. As compared with saline placebo, blockade of platelet glycoprotein IIb-IIIa receptor with tirofiban produced 28% and 56% reductions in thrombus formation in the low-shear and high-shear chambers, respectively. PSI-697 caused a dose-dependent, but more modest, reduction in thrombus formation. Low-dose PSI-796 (2 m) reduced total thrombus area by 14% (P = 0.04) and 30% (P = 0.0002) in the low-shear and high-shear chambers, respectively. At the high dose (20 m), PSI-697 reduced total thrombus area by 18% (P = 0.0094) and 41% (P = 0.0008) in the low-shear and high-shear chambers, respectively. CONCLUSIONS: P-selectin antagonism with PSI-697 reduces ex vivo thrombus formation in humans. These findings provide further evidence that P-selectin antagonism may be a potential target for the prevention and treatment of cardiovascular disease.
Assuntos
Selectina-P/antagonistas & inibidores , Trombose/prevenção & controle , Adolescente , Adulto , Sangue/efeitos dos fármacos , Células Cultivadas , Estudos Cross-Over , Método Duplo-Cego , Humanos , Hidroxiquinolinas/farmacologia , Perfusão , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Trombose/tratamento farmacológico , Tirofibana , Tirosina/análogos & derivados , Tirosina/farmacologia , Adulto JovemRESUMO
The discipline of biochemical pharmacology emerged in the late 1940s as a result of an increasing emphasis on understanding drug mechanisms at the cellular level. This research approach has contributed significantly to the development of many new drug classes including antihypertensive, antifective, cholesterol lowering, anti-inflammatory, and anticancer agents, as well as antipsychotics, antidepressants and anxiolytics. Biochemical pharmacology remains a major tool in drug discovery, being employed in the search for novel therapeutics for the above and other conditions and clinical challenges, such as neurodegenerative disorders, for the treatment of pain, and for development of agents that do not induce, or can overcome, antibiotic/antiviral resistance. Together with chemical, molecular, genetic, physiological, and clinical sciences, biochemical pharmacology will in the coming decades continue to be a critical component of the drug discovery process.
Assuntos
Bioquímica , Farmacologia , Editoração/história , História do Século XX , História do Século XXIRESUMO
Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.
Assuntos
Artérias Carótidas/efeitos dos fármacos , Fator IX/fisiologia , Deficiência do Fator XI/patologia , Fator XI/fisiologia , Compostos Férricos/farmacologia , Hemofilia B/patologia , Animais , Artérias/efeitos dos fármacos , Artérias/lesões , Aspirina/farmacologia , Tempo de Sangramento , Velocidade do Fluxo Sanguíneo , Doenças das Artérias Carótidas/patologia , Cloretos , Relação Dose-Resposta a Droga , Genótipo , Heparina/química , Heparina/farmacologia , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária , Fluxo Sanguíneo Regional/efeitos dos fármacos , Trombose/patologia , Trombose/terapia , Fatores de TempoRESUMO
Brain ischemia elicits an intense inflammatory reaction as evidenced by endogenous activation of microglia and infiltration of leukocytes from the systemic circulation into the brain. A key issue regarding the well-described inflammation in brain injury is whether this reaction is of salutary or detrimental nature in the short and longterm post ischemia. In this brief review, evidence in support for the possible beneficial as well as detrimental role of inflammatory cells and mediators in ischemic brain injury is highlighted. We offer the opinion that both benefits and adverse effects of the inflammatory reaction at large depent on the levels of a specific mediator, the temporal relationships to the injury, the context in which the mediator operates and the spatial relationships to the injury.
Assuntos
Isquemia Encefálica/complicações , Isquemia Encefálica/imunologia , Encéfalo/imunologia , Encefalite/etiologia , Encefalite/imunologia , Regulação da Expressão Gênica/imunologia , Mediadores da Inflamação/imunologia , Animais , HumanosRESUMO
Activation of the extracellular-signal-responsive kinase (ERK 1/2) by MAP kinase/ERK kinase (MEK1/2) following ischemia/reperfusion in the brain has been associated with cell death since inhibition of MEK1/2 provides neuroprotection in cerebral ischemia injury. Since inflammation has been implicated in ischemic brain injury, the present study investigated whether MEK1/2 modifies expression of two key inflammatory cytokines, IL-1beta and TNFalpha, that have been shown to exacerbate ischemic brain injury. A mouse model of transient cerebral ischemia was deployed to test the effect of selective MEK1/2 inhibitor (SL327) on infarct size and cytokine expression. SL327 (100 mg/kg, i.p.) administered 15 min prior to ischemia resulted in 64% reduction in infarct size over controls (n = 8, P < 0.01). Under the same condition, SL327 significantly reduced peak expression of IL-1beta mRNA (59% reduction compared to vehicle, P < 0.01, n = 4) but not TNF-alpha mRNA. A parallel reduction in IL-1beta protein (67%, P < 0.05, n = 6) was also observed using ELISA analysis. These data suggest that the neuroprotective effect of MEK1/2 inhibition may be mediated by suppression of IL-1beta. The study also demonstrates for the first time that these two cytokines are differentially regulated by kinase mediated signaling pathways.
Assuntos
Isquemia Encefálica/metabolismo , Interleucina-1/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Aminoacetonitrila/análogos & derivados , Animais , Encéfalo/metabolismo , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteases/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
The availability of potent and orally active nonpeptide endothelin (ET) receptor antagonists has generated a host of information on the pathophysiological role of ET-1 in a number of preclinical models including hypertension, renal failure, heart failure and pulmonary hypertension. Convincing data are available to show that ET-1 receptor antagonists are beneficial in humans as far as reversal of deranged systemic and regional hemodynamics associated with CHF and pulmonary hypertension. As in other disease areas, the issue of whether ET(A)-selective or ET(A/B) antagonists are more suited for CHF treatment remains unresolved. ET(B) receptors may mediate some critical processes in the kidney such as sodium and water excretion in addition to releasing vasodilator substances such as NO and prostacyclin from endothelial cells. In heart failure and chronic renal diseases, preservation of ET(B)-mediated responses in the kidney and pulmonary endothelium might be beneficial. On the other hand, blockade of ET(B)-mediated vasoconstriction, smooth muscle cell proliferation and fibrosis by ET(B) antagonists might be beneficial. In clinical trials so far, the hemodynamic effects of mixed antagonists of ET receptors and ET(A) selective antagonists seem equivalent.
Assuntos
Endotelinas/fisiologia , Insuficiência Cardíaca/fisiopatologia , Receptores de Endotelina/fisiologia , Bosentana , Ensaios Clínicos como Assunto , Antagonistas dos Receptores de Endotelina , Insuficiência Cardíaca/tratamento farmacológico , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Humanos , Sulfonamidas/efeitos adversos , Sulfonamidas/uso terapêuticoRESUMO
BACKGROUND AND PURPOSE: CD44 is a transmembrane glycoprotein involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. The present report describes the discovery of upregulated CD44 gene expression and its spatial and temporal distribution in the brain after focal stroke. METHODS: Rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). Suppression subtractive hybridization (SSH) strategy was used to identify differentially expressed genes. Northern blotting and real-time polymerase chain reaction were used to evaluate the expression of CD44 and hyaluronan synthase 2 (HAS-2) mRNA. Western blotting and immunohistochemistry were used to examine CD44 expression and cellular distribution. RESULTS: CD44 upregulation after focal stroke was discovered by the SSH approach and confirmed by DNA sequencing. Northern blot using a pooled poly(A)+ RNA revealed 3 splice variants of CD44 mRNA, and their inducible expression started at 6 hours (5.3-fold increase over sham operation), peaked at 24 hours (28.6-fold increase), and persisted up to 72 hours (17.8-fold increase) after MCAO. A parallel induction profile of HAS-2 mRNA was observed in the ischemic brain tissue. The levels of CD44 were markedly elevated at 6 hours (1.8-fold increase over sham; n=3), 24 hours (2.9-fold, peak induction; P<0.01), and 72 hours (2.4-fold increase; P<0.05) after MCAO by means of Western analysis. Immunohistochemical and confocal microscopy confirmed that constitutive expression of CD44 is limited to microvessels in normal brain but is strongly induced after ischemia, where the immunoreactive signal mainly resided in endothelial cells and monocytes. Double-labeling immunohistochemistry demonstrated that a marked induction of CD44 in the ischemic lesion is dominantly located in microglia and a subset of macrophages. CONCLUSIONS: The discovery of concomitant induction of CD44 and HAS-2 mRNA expression and the localization of CD44 in the microglia, macrophages, and microvessels of the ischemic brain tissue suggest that an active interaction between CD44 and hyaluronan may occur and play a role in the known inflammatory response and tissue remodeling after stroke.
Assuntos
Perfilação da Expressão Gênica , Receptores de Hialuronatos/biossíntese , Hibridização de Ácido Nucleico/métodos , Acidente Vascular Cerebral/metabolismo , Regulação para Cima , Processamento Alternativo , Animais , Northern Blotting , Circulação Cerebrovascular , Modelos Animais de Doenças , Encefalite/genética , Encefalite/metabolismo , Encefalite/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Receptores de Hialuronatos/genética , Hialuronan Sintases , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/complicações , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Microcirculação/metabolismo , Microcirculação/patologia , Microglia/metabolismo , Microglia/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologiaRESUMO
The binding of Tyr-D-Arg(2)-Phe-sarcosine(Sar)(4) (TAPS), a proposed mu-opioid receptor-selective tetrapeptide analog of dermorphin to opioid receptors, was studied using selective binding assays for subtypes of mu-, delta- and kappa-opioid receptors. Subtype specific mu-opioid receptor binding was further characterized in the presence of sodium and guanosine nucleotides and the activity of TAPS in isolated guinea pig ileum was compared to other mu-opioid receptor-selective ligands. Further, the antinociceptive properties of TAPS following intrathecal (i.t.) administration in rats, as a model of spinal antinociception, were evaluated. The K(i)-values for TAPS at the mu(1)- and mu(2)-opioid receptor sites were 0.4 and 1.3 nM, respectively, suggesting high affinity binding to mu-opioid receptor binding sites with an increased selectivity to mu(1)-opioid receptor sites. The attenuated reduction of TAPS binding at the mu(2)-opioid receptor subtype in the presence of the stable guanosintriphosphate analog 5'-guanylylimidodiphosphate and sodium suggests a potential partial antagonist mode of action at this site.
Assuntos
Analgésicos/farmacologia , Naloxona/análogos & derivados , Oligopeptídeos/farmacologia , Receptores Opioides/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Catalepsia/induzido quimicamente , Relação Dose-Resposta a Droga , Estimulação Elétrica , Cobaias , Íleo/efeitos dos fármacos , Íleo/fisiologia , Técnicas In Vitro , Injeções Espinhais , Masculino , Contração Muscular/efeitos dos fármacos , Naloxona/farmacologia , Nociceptores/efeitos dos fármacos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos Opioides , Ventilação Pulmonar/efeitos dos fármacos , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Apoptosis as defined by contemporary science describes a form of cell death that involves discrete genetic and molecular programs, de novo protein expression and unique cellular phenotype. Evidence for the existence of apoptosis in the human heart has been reported in various cardiac diseases, including ischemic and non-ischemic heart failure, myocardial infarction and arrhythmias. Among the most potent stimuli that elicit cardiomyocyte apoptosis are: oxygen radicals (including NO), cytokines, (e.g., TNFalpha, FAS) neurohormonal factors (angiotension II), cardiotoxic drugs (e.g., doxorubicin) and mechanical, stretch situations. Several complex signal transduction pathways have been implicated in execution of cardiomyocyte apoptosis. Most prominent are: 1) Tyrosine kinase receptors (TRK) induced signaling involving stress or mitogen activated protein kinases (SAPK/MARK) and sphingolipids metabolites (ceramide); 2) G-protein coupled receptor (GPCR) signaling (Galphai, Galphaq) and 3) NF(K) B activation. Apoptosis of cardiac myocytes may contribute to progressive pump-failure, arrhythmias and cardiac remodeling. The recognition of diverse molecular targets associated with cardiomyocyte apoptosis provide new opportunities for pharmacologic manipulation, that may lead to discovery and development of therapeutic strategies for treatment of heart failure, arrhythmias and myocardial infarction.
Assuntos
Apoptose/efeitos dos fármacos , Cardiopatias/tratamento farmacológico , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Antagonistas de Receptores de Angiotensina , Apoptose/fisiologia , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Carvedilol , Fragmentação do DNA/efeitos dos fármacos , Cardiopatias/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Modelos Biológicos , Propanolaminas/farmacologia , Propanolaminas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
A humanized inhibitory anti-factor IX(a) antibody (SB 249417) has been compared to enoxaparin (Lovenox) in a rat model of arterial thrombosis. Pretreatment of rats with either SB 249417 (3.0 mg/kg, i. v.) or enoxaparin (30.0 mg/kg, i.v. or s.c.) resulted in comparable and significant reductions in thrombus formation. However, the efficacious dose of enoxaparin resulted in >30-fold increase in the aPTT over baseline, while the efficacious dose of SB 249417 prolonged the aPTT by only approximately 3-fold. Additionally, pretreatment with SB 249417 resulted in sustained blood flow and arterial patency throughout the experiment in >80% of rats treated. In contrast, <30% of rats pretreated with enoxaparin remained patent throughout the experiment. The data in this report indicate that the selective inhibition of factor IX(a) with the monoclonal antibody SB 249417 produces a superior antithrombotic profile to that of the low molecular weight heparin enoxaparin.
Assuntos
Anticorpos Monoclonais/farmacologia , Trombose das Artérias Carótidas/tratamento farmacológico , Doença das Coronárias/tratamento farmacológico , Enoxaparina/farmacologia , Fator IXa/imunologia , Fibrinolíticos/farmacologia , Animais , Anticorpos Monoclonais/uso terapêutico , Testes de Coagulação Sanguínea , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Trombose das Artérias Carótidas/induzido quimicamente , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Enoxaparina/normas , Enoxaparina/uso terapêutico , Fibrinolíticos/normas , Fibrinolíticos/uso terapêutico , Heparina de Baixo Peso Molecular/imunologia , Heparina de Baixo Peso Molecular/farmacologia , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Chemokines are potent proinflammatory and immune modulators. Increased expression of chemokines, eg, monocyte chemoattractant protein-1 (MCP-1), has recently been described in clinical and experimental heart failure. The present report is aimed at exploring the expression, localization, and binding site regulation of MCP-1, a member of the C-C chemokine family, in a rat model of volume-overload congestive heart failure (CHF). METHODS AND RESULTS: An aortocaval fistula was surgically created between the abdominal aorta and inferior vena cava. Rats with CHF were further subdivided into compensated and decompensated subgroups. Northern blot analysis and real-time quantitative polymerase chain reaction demonstrated upregulation of MCP-1 mRNA expression correlating with the severity of CHF (288+/-22, 502+/-62, and 826+/-138 copies/ng total RNA for sham, compensated, and decompensated animals, respectively; n=5, P:<0.05). MCP-1 protein was localized by immunohistochemistry in cardiomyocytes, vascular endothelium and smooth muscle cells, infiltrating leukocytes, and interstitial fibroblasts, and its intensity increased with severity of CHF. In addition, rats with CHF displayed a significant decrease of (125)I-labeled MCP-1 binding sites to myocardium-derived membranes (384.3+/-57.0, 181.3+/-8.8, and 123.3+/-14.1 fmol/mg protein for sham, compensated, and decompensated animals, respectively). CONCLUSIONS: Volume-overload CHF in rats is associated with alterations in the expression, immunohistochemical localization, and receptor binding of the MCP-1 chemokine in the myocardium. These changes were more pronounced in rats with decompensated CHF. The data suggest that activation of the MCP-1 system may contribute to the progressive cardiac decompensation and development of CHF in rats with aortocaval fistula.
Assuntos
Quimiocina CCL2/metabolismo , Insuficiência Cardíaca/metabolismo , Animais , Ligação Competitiva , Northern Blotting , Insuficiência Cardíaca/diagnóstico por imagem , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Miocárdio/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ensaio Radioligante , Ratos , Ratos Wistar , Ultrassonografia , Regulação para CimaRESUMO
OBJECTIVE: To develop an improved small animal experimental paradigm that more closely mimics human sepsis. DESIGN: Prospective, randomized, controlled animal study. SETTING: Medical school research laboratory. SUBJECTS: Male Sprague-Dawley rats (280-320 g). INTERVENTIONS: We monitored the hemodynamic, hematologic, and biochemical consequences of abdominal sepsis produced by intraperitoneal implantation of a fibrin clot containing Escherichia coli in conscious, antibiotic-treated, rats. MEASUREMENTS AND MAIN RESULTS: Similar to human sepsis, the implanted, infected clot (LD50 = 5-7 x 10(8) colony forming units/mL, n = 6) elevated cardiac index (>7% vs. sterile clot, p < .05, at 4 hrs), whereas mean arterial pressure and heart rate remained unaffected. The total peripheral resistance index and stroke volume index tended to decrease and increase, respectively. In contrast, an intravenous bolus injection of endotoxin (LD50 of E. coli lipopolysaccharide = 5.6 mg/kg, n = 7), the most commonly used sepsis model, induced profound hypodynamic responses manifested by a 27% decrease (vs. endotoxin vehicle, p < .01) in cardiac index, a 28% increase in the total peripheral resistance index (p < .01), and a 33% decrease in the stroke volume index (P < .01). The infectious peritonitis model also displayed dose-dependent thrombocytopenia (<61%, p < .05), leukopenia (<60%, p < .05), and mortality rate (50% at 5-7 x 10(8) colony forming units/mL, p < .05) with a minimally elevated serum tumor necrosis factor-alpha level (145 vs. 12 +/- 6 pg/mL in controls, p < .05). CONCLUSION: This rodent model of antibiotic-treated, intra-abdominal infection features key characteristics of clinical sepsis. Although the hyperdynamic response observed in septic patients undergoing resuscitation was not clearly elicited, this paradigm better mimics clinical sepsis compared with the commonly used endotoxin model. Thus, utilization of this paradigm may provide additional opportunities to explore mechanisms of sepsis and to examine novel therapeutics.
Assuntos
Modelos Animais de Doenças , Sepse/complicações , Sepse/fisiopatologia , Animais , Estado de Consciência , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
Three major mammalian mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury.
Assuntos
Apoptose/fisiologia , Coração/fisiopatologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Interleukin-1beta (IL-1beta) is a pleiotropic cytokine capable of inducing smooth muscle activation and leukocyte recruitment in restenosis and atherosclerosis. Our present study investigated the expression of IL-1beta, IL-1 receptor antagonist (IL-1ra), and IL-1 receptor (IL-1RI and IL-1RII) mRNA in carotid artery after balloon angioplasty using semiquantitative reverse transcription/polymerase chain reaction (RT/PCR) and Northern analysis. Time course studies revealed that IL-1beta mRNA was rapidly induced at 6 h (30-fold increase over control, P < 0.001) post balloon injury and diminished to basal levels at 24 h. The increased expression of IL-1ra mRNA was delayed, reaching a peak at 24 h (400-fold increase, P < 0.001) and sustained up to 14 days. The expression of IL-1RII mRNA was remarkably similar to that of IL-1beta, whereas the IL-1RI (the signaling receptor) mRNA expression was delayed (significantly induced at day 1; 14.2-fold increase, P < 0.01) post balloon injury. Immunohistochemical studies revealed a strong induction of IL-1beta in the area with actively proliferating and migrating smooth muscle cells (i.e., in the inner medial layer at day 1 and in neointima at day 14 after balloon injury). The differential but concomitant expression of IL-1beta, IL-1ra, IL-1RI, and IL-1RII mRNAs after balloon angioplasty suggests that each of these IL-1 system components may play a distinct role in neointima formation.
Assuntos
Angioplastia com Balão , Artérias Carótidas/metabolismo , Interleucina-1/biossíntese , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/biossíntese , Animais , Northern Blotting , Imuno-Histoquímica , Interleucina-1/genética , Cinética , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
We have established a focal preconditioning (PC) paradigm that produces significant and prolonged ischemic tolerance (IT) of the brain to subsequent permanent middle cerebral artery occlusion (MCAO). PC using 10 min of MCAO induces brain tolerance at 1-7 days of reperfusion that requires active protein synthesis. The protective protein(s) involved are unknown. In these studies the increased transcription and translation of the inducible 70-kDa heat shock protein (Hsp70) and the 27-kDa heat shock protein (Hsp27), and astrogliosis/glial fibrillary acidic protein (GFAP) were determined by Northern analysis and immunohistochemistry following PC. Cellular localization of proteins was determined by double labeling. PC produced no brain injury but did increase Hsp70 mRNA transiently at 6 h and increased Hsp27 mRNA later at 24 h for at least 5 days. Protein expression induced by PC exhibited a similar profile. Hsp70 protein was primarily expressed in neurons from 1 to 5 days post-PC throughout the PC cortex. Hsp27 protein expression was initiated later for a much longer period of time. A remarkable astroglyosis was verified with increased astrocytic Hsp27 from 1 to 7 days after PC. Gliosis with increased Hsp27 in the PC cortex was still present but reduced 4 weeks after PC. Therefore, PC that results in brain tolerance/neuroprotection increases neuronal Hsp70 in the PC cortex and activated astrocytic Hsp27 in the PC cortex in a temporal fashion associated with developing IT. The short duration of benign ischemia (PC) that produces IT produces a robust, long-lived cellular and protein synthetic response that extends throughout the entire cortex (i.e. well beyond the MCA perfusion territory). The resulting IT is associated with changes in astrocyte-activation that might provide increased support and protection from injury. Although both Hsp70 and Hsp27 may participate in the neuroprotection/brain tolerance induced by PC, the temporal expression patterns of these proteins indicate that they are not solely responsible for the tolerance to brain injury.
Assuntos
Astrócitos/metabolismo , Gliose/fisiopatologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Astrócitos/patologia , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Neurônios/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Traumatismo por Reperfusão/patologia , Fatores de TempoRESUMO
Apoptosis is a form of cell death that involves discrete genetic and molecular programs, de novo protein expression and a unique cellular phenotype. Evidence for the existence of apoptosis in the human heart has been reported in various cardiac diseases, including ischemic and non-ischemic heart failure, myocardial infarction and arrhythmias. Among the most potent stimuli that elicit cardiomyocyte apoptosis are: oxygen radicals (including NO), cytokines (FAS/TNF alpha-receptor signaling), stress conditions (chemical or physical, e.g., radiation), sphingolipid metabolites (ceramide) and autocoids, e.g., angiotensin II. Apoptosis of cardiac myocytes may contribute to progressive pump-failure, arrhythmias and cardiac remodeling. The recognition of numerous molecular targets associated with cardiomyocyte apoptosis may provide novel therapeutic strategies for diverse cardiac ailments, as recently suggested by pharmacologic studies in experimental animals. This review paper is aimed to highlight the role of protein kinase signaling pathways in apoptosis with special attention to the stress-activated protein kinases (SAPK) and mitogen-activated protein kinases (MAPK) systems.
Assuntos
Apoptose , Doenças Cardiovasculares/fisiopatologia , Sistema de Sinalização das MAP Quinases , Miocárdio/metabolismo , Estresse Oxidativo , Sistemas do Segundo Mensageiro , Antagonistas Adrenérgicos alfa/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Humanos , Fosfolipídeos/metabolismo , Proteínas Quinases/metabolismoRESUMO
The present study investigates intracellular enzymatic pathways involved in the elimination of reactive oxygen species in the left ventricular myocardium of 10 individuals without heart failure and 12 patients with end-stage heart failure due to idiopathic dilated cardiomyopathy. Left ventricular enzyme activities, mRNA and protein levels of the hydrogen peroxide scavenging enzymes catalase (CAT) and glutathione peroxidase (GPX), and the superoxide anion scavenging enzymes mitochondrial (Mn-SOD) and cytosolic (Cu/Zn-SOD) superoxide dismutases were measured. In failing myocardium, there was a significant decrease in CAT activity (4.83+/-0.32 U/mg v 6.59+/-0.52, P<0.01) despite unchanged mRNA expression and protein levels. GPX, Mn-SOD and Cu/Zn-SOD were similar concerning activity, mRNA and protein levels. As indirect free radical markers, similar levels of the products of lipid peroxidation, malondialdehyde and 4-hydroxy-alkenals, and similar tissue nitrotyrosin content were measured. The decrease in CAT activity appears to be a post-transcriptional mechanism. A decreased myocardial capacity to scavenge hydrogen peroxide might lead to a shift in the intracellular redox balance which potentially results in activation of redox sensitive signalling pathways. Direct reactive oxygen species mediated damage was not detected by the methods applied.