RESUMO
Critical limb ischemia (CLI) is associated with skeletal muscle damage. However, the pathophysiology of the muscle damage is poorly understood. Toll-like receptors (TLR) have been attributed to play a role in ischemia-induced tissue damage but their role in skeletal muscle damage in CLI is unknown. TLR2 and TLR6 expression was found to be upregulated in skeletal muscle of patients with CLI. In vitro, ischemia led to upregulation of TLR2 and TLR6 by myotubes, and activation of the downstream TLR signaling pathway. Ischemia-induced activation of the TLR signaling pathway led to secretion of the pro-inflammatory cytokine interleukin-6 and muscle apoptosis, which were abrogated by neutralising TLR2 and TLR6 antibodies. Our study demonstrates that TLR2 and TLR6 are upregulated in ischemic muscle and play a role in ischemia-induced muscle damage. Thus, manipulating the TLR pathway locally may be of potential therapeutic benefit.
Assuntos
Apoptose , Mediadores da Inflamação/metabolismo , Isquemia/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular , Estado Terminal , Feminino , Humanos , Interleucina-6/metabolismo , Isquemia/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
AIMS: A marked increase in HDL notwithstanding, the cholesterol ester transfer protein (CETP) inhibitor torcetrapib was associated with an increase in all-cause mortality in the ILLUMINATE trial. As underlying mechanisms remain elusive, the present study was designed to delineate potential off-target effects of torcetrapib. METHODS AND RESULTS: Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats were treated with torcetrapib (100 mg/kg/day; SHR-T and WKY-T) or placebo (SHR-P and WKY-P) for 3 weeks. Blood pressure transiently increased during the first 3 days of torcetrapib administration in SHRs and returned to baseline thereafter despite continued drug administration. Acetylcholine-induced endothelium-dependent relaxations of aortic rings were markedly impaired, and endothelial nitric oxide synthase (eNOS) mRNA and protein were down-regulated after 3 weeks of torcetrapib treatment in SHR (P < 0.0001, <0.01, and <0.05, resp. vs. SHR-P). Torcetrapib reduced NO release in cultured aortic endothelial cells (P < 0.01 vs. vehicle-treated cells) and increased generation of reactive oxygen species in aortas of SHR-T (P < 0.05, vs. SHR-P). Vascular reactivity to endothelin-1 (ET-1) and aortic ET-1 tissue content were increased in SHR-T (P < 0.05 vs. SHR-P). Importantly, the ET-1 receptor A/B (ET(A/B)) antagonist bosentan normalized endothelial function in SHR-T (P < 0.05). CONCLUSION: Torcetrapib induces a sustained impairment of endothelial function, decreases eNOS mRNA, protein as well as NO release, stimulates vascular ROS and ET production, an effect that is prevented by chronic ET(A/B)-receptor blockade. These unexpected off-target effects of torcetrapib need to be ruled out in the clinical development of novel CETP inhibitors, particularly before a large patient population at increased cardiovascular risk is exposed to these compounds.
Assuntos
Anticolesterolemiantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Hipertensão/fisiopatologia , Quinolinas/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Aorta/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Bosentana , Células Cultivadas , Relação Dose-Resposta a Droga , Antagonistas dos Receptores de Endotelina , Endotelina-1/metabolismo , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espécies Reativas de Oxigênio/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Sulfonamidas/farmacologia , Superóxidos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Studies were carried out to test the hypothesis that administration of a glucocorticoid Type II receptor antagonist, mifepristone (RU38486), just prior to withdrawal from chronic alcohol treatment, would prevent the consequences of the alcohol consumption and withdrawal in mice. MATERIALS AND METHODS: The effects of administration of a single intraperitoneal dose of mifepristone were examined on alcohol withdrawal hyperexcitability. Memory deficits during the abstinence phase were measured using repeat exposure to the elevated plus maze, the object recognition test, and the odor habituation/discrimination test. Neurotoxicity in the hippocampus and prefrontal cortex was examined using NeuN staining. RESULTS: Mifepristone reduced, though did not prevent, the behavioral hyperexcitability seen in TO strain mice during the acute phase of alcohol withdrawal (4 hours to 8 hours after cessation of alcohol consumption) following chronic alcohol treatment via liquid diet. There were no alterations in anxiety-related behavior in these mice at 1 week into withdrawal, as measured using the elevated plus maze. However, changes in behavior during a second exposure to the elevated plus maze 1 week later were significantly reduced by the administration of mifepristone prior to withdrawal, indicating a reduction in the memory deficits caused by the chronic alcohol treatment and withdrawal. The object recognition test and the odor habituation and discrimination test were then used to measure memory deficits in more detail, at between 1 and 2 weeks after alcohol withdrawal in C57/BL10 strain mice given alcohol chronically via the drinking fluid. A single dose of mifepristone given at the time of alcohol withdrawal significantly reduced the memory deficits in both tests. NeuN staining showed no evidence of neuronal loss in either prefrontal cortex or hippocampus after withdrawal from chronic alcohol treatment. CONCLUSIONS: The results suggest mifepristone may be of value in the treatment of alcoholics to reduce their cognitive deficits.
Assuntos
Consumo de Bebidas Alcoólicas/tratamento farmacológico , Glucocorticoides/antagonistas & inibidores , Antagonistas de Hormônios/uso terapêutico , Mifepristona/uso terapêutico , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Consumo de Bebidas Alcoólicas/efeitos adversos , Animais , Glucocorticoides/análise , Antagonistas de Hormônios/farmacologia , Masculino , Transtornos da Memória/complicações , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Mifepristona/farmacologia , Síndrome de Abstinência a Substâncias/complicações , Síndrome de Abstinência a Substâncias/psicologiaRESUMO
Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.
Assuntos
Artroplastia do Joelho , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Óxido Nítrico Sintase/análise , Osteoartrite do Joelho/cirurgia , Traumatismo por Reperfusão/enzimologia , Reperfusão/efeitos adversos , Torniquetes/efeitos adversos , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Isquemia/genética , Extremidade Inferior , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I/análise , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo III/análise , RNA Mensageiro/análise , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.
Assuntos
Acetilcisteína/farmacologia , Encéfalo/metabolismo , Endotelina-1/metabolismo , Endotélio Vascular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Encéfalo/citologia , Linhagem Celular , Endotelina-1/genética , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Fosforilação , Transporte Proteico , Ratos , Regulação para CimaRESUMO
Altered activity of retinal endothelin-1 (ET-1) and nitric oxide may play a causal role in the hemodynamic and histopathological changes of diabetic retinopathy. This study evaluated the therapeutic potential of long-term selective blockade of the ET-1(A) receptor (ETRA) to prevent the development of retinopathy in a genetic mouse model of nonobese type 1 diabetes (NOD). Mice with NOD that received subcutaneous implantation of insulin pellets and wild-type control mice were treated for 4 months with the selective ETRA antagonist LU208075 (30 mg/kg/day) via drinking water. At the end of the study, blood glucose levels were evaluated, and animals were anesthetized and perfused intracardially with FITC-labeled dextran. Retinas were removed and either fixed in formalin for confocal microscope evaluation of retinal vascular filling or transferred to RNALater for quantitative reverse transcriptase-polymerase chain reaction to evaluate expression of NOS-3, NOS-1, ET-1, ETRA, ETRB, and the angiogenic factor adrenomedullin. Compared with wild-type controls, expression of ET-1, ETRA, ETRB, and adrenomedullin in mice with NOD were markedly upregulated in the retinas of nontreated mice (cycle time values relative to GAPDH [deltaCt], 14.8 vs. 13.7, 18.57 vs. 17.5, 10.76 vs. 9.9, and 11.7 vs. 9.1, respectively). Mean integral fluorescence intensity (MIFI) of retinal vascular filling was reduced from normal values of 24 to 12.5 in nontreated animals. LU208075 treatment normalized the upregulated expression of ET-1 and adrenomedullin, as well as the deficit in MIFI, but did not affect the increased ETRA and ETRB expression or the elevated plasma glucose levels found in nontreated animals. NOS isoform expression was essentially unchanged. ETRA antagonists may provide a novel therapeutic strategy to slow or prevent progression of retinal microvascular damage and proliferation in patients for whom there is clear evidence of activation of the ET-1 system.
Assuntos
Retinopatia Diabética/prevenção & controle , Antagonistas do Receptor de Endotelina A , Endotelina-1/antagonistas & inibidores , Peptídeos/farmacologia , Vasodilatadores/farmacologia , Adrenomedulina , Animais , Glicemia/análise , Feminino , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Injeções Subcutâneas , Insulina/administração & dosagem , Insulina/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Fenilpropionatos/farmacologia , Piridazinas , Fatores de TempoRESUMO
Although experimental prevention studies have suggested therapeutic potential of endothelin (ET) antagonists for the treatment of heart failure, the results of clinical trials using ET antagonists on top of standard heart failure medications have been largely disappointing. This experimental study investigated the effects of chronic ET(A) receptor blockade in long-term survivors of myocardial infarction who had developed stable chronic heart failure in the absence of other treatments. Systolic blood pressure, heart rate, organ weights of the right atrium and ventricle, and the lungs were determined, and tissue ET-1 peptide levels were measured in cardiac tissue, lung, and aorta. The results show that chronic blockade of ET(A) receptors stabilizes systolic blood pressure and reverses the heart failure-induced weight increases of right heart chambers and lung. The changes observed occurred independently of tissue ET-1 concentrations and heart rate, suggesting mechanisms independent of local cardiac or pulmonary ET-1 synthesis, which are yet to be identified.
Assuntos
Antagonistas do Receptor de Endotelina A , Átrios do Coração/crescimento & desenvolvimento , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/crescimento & desenvolvimento , Isquemia Miocárdica/fisiopatologia , Animais , Pressão Sanguínea/fisiologia , Frequência Cardíaca/fisiologia , Masculino , Isquemia Miocárdica/etiologia , Tamanho do Órgão/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Endothelin regulates cytokine expression in vitro and in vivo. This study investigated the effects of chronic allograft rejection on hepatic endothelin-converting enzyme-1 (ECE-1) gene expression and endothelin-1 (ET-1) plasma clearance. Using the Lewis-F344 minor histocompatibility mismatch model of heterotopic cardiac transplantation, hepatic ECE-1 gene expression was measured by real-time polymerase chain reaction and host plasma clearance of ET-1 was measured 8 weeks after transplantation in the absence of immunosuppression. In animals undergoing allograft rejection, hepatic ECE-1 gene expression increased 2-fold (P < 0.05), whereas no effect of rejection on ET-1 clearance from plasma was observed. In summary, upregulation of ECE-1 gene expression occurs in the liver of the host during chronic allograft rejection. Because the liver represents both a key organ for cytokine production and for endothelin metabolism, increased hepatic ECE-1-mediated ET-1 synthesis may contribute to host responses and cytokine production during allograft rejection.
Assuntos
Ácido Aspártico Endopeptidases/genética , Regulação Enzimológica da Expressão Gênica , Rejeição de Enxerto , Fígado/enzimologia , Fígado/metabolismo , Metaloendopeptidases/genética , Animais , Ácido Aspártico Endopeptidases/biossíntese , Doença Crônica , Endotelina-1/farmacocinética , Enzimas Conversoras de Endotelina , Transplante de Coração , Metaloendopeptidases/biossíntese , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Fatores de Tempo , Transplante Homólogo , Regulação para CimaRESUMO
There is increasing evidence to suggest that chronic activation of the endothelin-1 system can lead to heterologous desensitization of the glucose-regulatory and mitogenic actions of insulin with subsequent development of glucose intolerance, hyperinsulinemia, impaired endothelial function and exacerbation of cardiovascular disease. Effects are mediated through a variety of mechanisms that include attenuation of key insulin signalling pathways and decreased tyrosine phosphorylation of insulin receptor substrates IRS-1, SHC and G alpha q/11. Other actions involve hemodynamic changes leading to reduced delivery of insulin and glucose to peripheral tissues as well as enhanced hepatic glycogenolysis, decreased glucose-transporter translocation and modulation of various adipokines that regulate insulin action. Overall the data suggest that ET-1 antagonists may provide an effective means of improving cardiac dysfunction and favourably influencing glucose tolerance in obese humans and patients with early insulin sensitivity where there is clear evidence for activation of the ET-1 system. Although most effects of ET-1 that modulate mechanisms leading to glucose intolerance appear to involve the ETA receptor subtype recent data indicates that combined ETA/ETB receptor antagonists may function as effectively as selective ETA blockers. Prospective trials are needed to assess whether ET-1 antagonists, either alone or in combination, are superior to other more conventional therapies such as insulin sensitizers and to evaluate effects of combined treatments on the development of insulin resistance and the progression of diabetes. Early screening of patients at risk for evidence of ET-1 activation would help to identify subjects who may benefit most from such treatment.
Assuntos
Endotelina-1/antagonistas & inibidores , Resistência à Insulina , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Animais , Humanos , Síndrome Metabólica/tratamento farmacológico , Obesidade/tratamento farmacológicoRESUMO
BACKGROUND: In recent years, the assessment of the plasma aldosterone-to-renin ratio (ARR) has become an established screening method for the diagnosis of primary aldosteronism. Plasma renin activity (PRA) is usually measured to define ARR although, increasingly, renin concentration alone is often measured in clinical routine. OBJECTIVE: To determine the threshold of ARR using active renin concentration to screen for primary aldosteronism. DESIGN AND PARTICIPANTS: To determine the ARR threshold based on plasma immunoreactive renin concentration (irR), we measured plasma aldosterone concentration (PAC), irR and PRA in 36 hypertensive patients, nine thereof with adrenal adenoma, and compared ARRs calculated from irR and PRA, respectively. SETTING: Single-centre, hypertension clinic in a tertiary care hospital. RESULTS: PRA ranged from 0.41-14.9 ng/ml per h and irR from 1.1-72 ng/l. There was an excellent correlation between PRA and irR (r = 0.98, P < 0.0001) and between ARRPRA and ARRirR (r = 0.96, P < 0.0001). An ARRPRA > 750 pmol/l per ng/ml per h was previously found to be highly predictive of primary aldosteronism because 90% of the corresponding patients failed to suppress PAC upon saline infusion or fludrocortisone. The corresponding threshold value for ARRirR was 150 pmol/ng in our patients. Using these cut-offs, nine subjects had both increased ARRPRA and ARRirR while, in three patients, either ARRPRA or ARRirR were increased. The nine patients with increased ARRPRA and ARRirR also had PAC > 650 pmol/l. Only these patients had adrenal adenomas. CONCLUSIONS: The ARR threshold to screen for primary aldosteronism may be based on measurement of irR. An ARRirR > 150 pmol/ng may indicate primary aldosteronism.
Assuntos
Aldosterona/sangue , Hiperaldosteronismo/sangue , Hiperaldosteronismo/diagnóstico , Renina/sangue , Adenoma/sangue , Adenoma/metabolismo , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Idoso , Aldosterona/biossíntese , Limiar Diferencial , Feminino , Humanos , Hipertensão/sangue , Ensaio Imunorradiométrico , Masculino , Pessoa de Meia-Idade , Concentração OsmolarRESUMO
AIM: Endothelial dysfunction is a marker for development and progression of atherosclerosis. Statin therapy improves endothelial function in cardiovascular patients by reducing LDL-cholesterol and by pleiotropic effects. B-group vitamin supplementation restores endothelial function mainly by reducing homocysteine-induced oxidative stress. Thus, we evaluated the effect of rosuvastatin, B-group vitamins and their combination on endothelial function in high-risk cardiovascular patients. METHODS: Thirty-six patients with cardiovascular disease were randomly, double-blinded assigned to either rosuvastatin 10 mg (group R, n = 18) or vitamin supplementation consisting of folic acid 1 mg, vitamin B12 0.4 mg, and B6 10 mg (group V, n = 18) for 6 weeks. After 6 weeks all patients received rosuvastatin and vitamin supplementation in combination for additional 6 weeks. Endothelial function was assessed by flow-mediated vasodilation (FMD) at baseline and after 6- and 12-week treatment. RESULTS: At baseline, FMD, plasma lipids, vitamins, and homocysteine were comparable between both groups. After 6 weeks, FMD improved in both groups (from 4.4 ± 1.6 to 6.9 ± 1.4% group R, P= 0.0004 and from 4.9 ± 1.8 to 6.4 ± 1.8% group V, P= 0.0002). This improvement in FMD was mainly associated with a decrease of plasma lipids in group R and a decrease of homocysteine in group V. After 12 weeks, the combined therapy with rosuvastatin and vitamins further improved FMD to the normal range in 26/33 patients compared to 5/36 at baseline (P < 0.0001). CONCLUSIONS: In conclusion, both treatments, rosuvastatin and B-group vitamin supplementation, improved endothelial function in high-risk cardiovascular patients. The combination of both therapies had an additive effect on endothelial function suggesting different mechanisms of action.
Assuntos
Anticolesterolemiantes/uso terapêutico , Doenças Cardiovasculares/fisiopatologia , Endotélio/fisiologia , Homocisteína/antagonistas & inibidores , Vasodilatação/fisiologia , Idoso , Anticolesterolemiantes/efeitos adversos , Artéria Braquial/fisiologia , Método Duplo-Cego , Endotélio/efeitos dos fármacos , Determinação de Ponto Final , Feminino , Fluorbenzenos/uso terapêutico , Homocisteína/sangue , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente , Pirimidinas/uso terapêutico , Rosuvastatina Cálcica , Sulfonamidas/uso terapêutico , Vasodilatação/efeitos dos fármacos , Complexo Vitamínico B/efeitos adversos , Complexo Vitamínico B/sangue , Complexo Vitamínico B/uso terapêutico , Vitaminas/sangue , Vitaminas/uso terapêuticoRESUMO
Toll-like receptors (TLRs) are key receptors of the innate immune system which are expressed on immune and nonimmune cells. They are activated by both pathogen-associated molecular patterns and endogenous ligands. Activation of TLRs culminates in the release of proinflammatory cytokines, chemokines, and apoptosis. Ischaemia and ischaemia/reperfusion (I/R) injury are associated with significant inflammation and tissue damage. There is emerging evidence to suggest that TLRs are involved in mediating ischaemia-induced damage in several organs. Critical limb ischaemia (CLI) is the most severe form of peripheral arterial disease (PAD) and is associated with skeletal muscle damage and tissue loss; however its pathophysiology is poorly understood. This paper will underline the evidence implicating TLRs in the pathophysiology of cerebral, renal, hepatic, myocardial, and skeletal muscle ischaemia and I/R injury and discuss preliminary data that alludes to the potential role of TLRs in the pathophysiology of skeletal muscle damage in CLI.
RESUMO
Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1alpha in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1alpha using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1alpha stabilization or activity through different mechanisms. Stabilization of HIF-1alpha protein by hypoxia or CoCl(2) did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1alpha controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1alpha in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1alpha under normoxic conditions with physiological roles that differ from those induced by hypoxia.
Assuntos
Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Receptores Toll-Like/agonistas , Células Cultivadas , Células Dendríticas/citologia , Digoxina/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Endothelin-1 (ET-1) is mainly secreted by endothelial cells and acts as a potent vasoconstrictor. In addition ET-1 has also been shown to have pleiotropic effects on a variety of other systems including adaptive immunity. There are two main ET-1 receptors, ET(A) and ET(B), which have different tissue and functional distributions. Dendritic cells (DC) are pivotal antigen-presenting cells linking the innate with the adaptive immune system. DC are sentinels expressing pattern-recognition receptors, e.g. the toll-like receptors (TLR) for detecting danger signals released from pathogens or tissue injury. Here we show for the first time that stimulation of human monocyte-derived DC with exogenous as well as endogenous selective TLR4 and TLR2 agonists induces the production of ET-1 in a dose- and time-dependent manner. 'Alternative' activation of DC in the presence of 1alpha,25-dihydroxyvitamin D(3) results in a marked potentiation of the endothelin response, whereas prostaglandin E(2) or dexamethasone do not increase ET-1 production. Furthermore, chetomin, an inhibitor of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), prevents TLR-mediated secretion of ET-1. Surprisingly, stimulation of human monocytes with LPS does not lead to secretion of detectable amounts of ET-1. These results suggest a role of ET-1 as an important player in human DC biology and innate immunity in general.
Assuntos
Células Dendríticas/imunologia , Endotelina-1/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Colecalciferol/análogos & derivados , Colecalciferol/farmacologia , Células Dendríticas/efeitos dos fármacos , Dexametasona/farmacologia , Dinoprostona/farmacologia , Dissulfetos/farmacologia , Endotelina-1/agonistas , Endotelina-1/antagonistas & inibidores , Glucocorticoides/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imunidade Inata , Alcaloides Indólicos/farmacologia , Lipopolissacarídeos/imunologia , Ocitócicos/farmacologia , Vitaminas/farmacologiaRESUMO
OBJECTIVE: Conventional harvesting of saphenous vein used for coronary artery bypass surgery induces a vasospasm that is overcome by high-pressure distension. Saphenous vein harvested with its cushion of perivascular tissue by a "no touch" technique does not undergo vasospasm and distension is not required, leading to an improved graft patency. The aim of this study is to investigate the effect of surgical damage and high-pressure distension on endothelial integrity and endothelial nitric oxide synthase expression and activity in saphenous vein harvested with and without perivascular tissue. METHODS: Saphenous veins from patients (n = 26) undergoing coronary artery bypass surgery were prepared with and without perivascular tissue. We analyzed the effect of 300 mm Hg distension on morphology and endothelial nitric oxide synthase/nitric oxide synthase activity using a combination of immunohistochemistry, Western blot analysis, reverse transcriptase polymerase chain reaction, and enzyme assay in distended (with and without perivascular tissue) compared with nondistended (with and without perivascular tissue) segments. RESULTS: Distension induced substantial damage to the luminal endothelium (assessed by CD31 staining) and vessel wall. Endothelial nitric oxide synthase expression and activity were significantly reduced by high-pressure distension and removal of, or damage to, perivascular tissue. The effect of distension was significantly less for those with perivascular tissue than for those without perivascular tissue in most cases. CONCLUSION: The success of the saphenous vein used as a bypass graft is affected by surgical trauma and distension. Veins removed with minimal damage exhibit increased patency rates. We show that retention of perivascular tissue on saphenous vein prepared for coronary artery bypass surgery by the "no touch" technique protects against distension-induced damage, preserves vessel morphology, and maintains endothelial nitric oxide synthase/nitric oxide synthase activity.
Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase/metabolismo , Veia Safena/enzimologia , Veia Safena/transplante , Coleta de Tecidos e Órgãos/métodos , Adulto , Idoso , Ponte de Artéria Coronária , Dilatação Patológica , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Veia Safena/metabolismo , Veia Safena/fisiologiaRESUMO
BACKGROUND: Chronic extrinsic denervation induced by small bowel transplantation (SBT) results in adrenergic hypersensitivity in rat ileum. This study evaluated the role of neuronal and/or muscular beta1-, beta2-, and beta3-adrenoceptor (AR) mechanisms on contractility. METHODS: Ileal longitudinal muscle strips from Lewis rats (n = 6 rats per group, 8 strips per rat): naïve controls (NC), 4 months after sham operation (SC) or after syngeneic orthotopic SBT were studied in vitro. Spontaneous contractile activity and dose responses (10(-8)-10(-4) mol) to isoprenaline (IP), a nonspecific beta-AR agonist were studied with or without selective antagonists (10(-5) mol), for beta1- (atenolol), beta2- (ICI 118551), or beta3- (SR 59230A) AR subtypes in the presence or absence of tetrodotoxin (TTX; 10(-6) mol; nerve blocker). RESULTS: pEC50 (neg log of EC50, which is the concentration where 50% of inhibition was observed) of IP was 7.2 +/- 0.2 (mean value +/- SEM) in SBT vs 6.3 +/- 0.1 in SC and 6.3 +/- 0.2 in NC (both P < .05 vs SBT), reflecting adrenergic hypersensitivity. Beta1- and beta2-AR blockade induced a TTX-sensitive right shift of the curve only in SBT and normalized pEC50 values from 7.2 +/- 0.2 to 6.4 +/- 0.1 and 7.2 +/- 0.2 to 6.6 +/- 0.1, respectively (P < .05). Beta3-AR blockade shifted the curve independent of the presence of TTX to the right in all groups (all P < .05). CONCLUSIONS: In rat ileum, adrenergic inhibition of contractility was dependent on muscular beta3-AR pathways, whereas posttransplant hypersensitivity was due to upregulated neuronal beta1- and beta2-AR mechanisms that were inactive before SBT.
Assuntos
Motilidade Gastrointestinal , Íleo/fisiopatologia , Íleo/transplante , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Agonistas Adrenérgicos beta/administração & dosagem , Agonistas Adrenérgicos beta/farmacologia , Animais , Relação Dose-Resposta a Droga , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/metabolismo , Técnicas In Vitro , Isoproterenol/administração & dosagem , Isoproterenol/farmacologia , Músculo Liso/metabolismo , Período Pós-Operatório , Ratos , Ratos Endogâmicos Lew , Tetrodotoxina/farmacologia , Transplante IsogênicoRESUMO
BACKGROUND/AIMS: It is postulated that nitric oxide (NO) is responsible for the hyperdynamic circulation of portal hypertension. Therefore, we investigated induction of fibrosis and hyperdynamic circulation in endothelial NO synthase knock-out (KO) mice. METHODS: Fibrosis was induced by bile duct ligation. Hemodynamic studies were performed after portal vein ligation. All studies were performed in wild-type (WT) and KO mice. RESULTS: Three to 4 weeks after bile duct ligation (BDL), both WT and KO groups had similar degrees of portal hypertension, 12 (9-14) and 11(8-15) mmHg, median (range), and liver function. Fibrosis increased from 0.0% in sham operated to 1.0 and 1.1% in WT and KO mice, respectively. Cardiac output was similar after portal vein ligation (20 and 17 ml/min in WT and KO mice, respectively). There was no difference in liver of mRNA for endothelin 1, inducible NO synthase (iNOS) and hem-oxygenase 1 (HO1); proteins of iNOS, HO1 and HO2; nor in endothelin A and B (EtA and EtB) receptor density between WT and KO mice after BDL. CONCLUSIONS: These results suggest that endothelial NO synthase is neither essential for the development of fibrosis and portal hypertension in bile duct ligated mice, nor for the hyperdynamic circulation associated with portal hypertension in the portal vein ligated mice.
Assuntos
Hipertensão Portal/etiologia , Cirrose Hepática/etiologia , Óxido Nítrico Sintase Tipo III/fisiologia , Animais , Ductos Biliares , Modelos Animais de Doenças , Endotelina-1/genética , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/genética , Hipertensão Portal/fisiopatologia , Ligadura , Fígado/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , RNA Mensageiro/análiseRESUMO
PURPOSE: To analyze how far an ischemic component might have been involved in optic neuritis. CASE REPORT: a 32-year-old man with symptoms characteristic for optic neuritis underwent extensive clinical, laboratory/serological and vascular examination for systemic associations and vascular involvement. RESULTS: The patient was found to have a temporary ocular blood flow dysregulation and increased plasma endothelin-1 levels which decreased after the acute phase of the optic nerve. CONCLUSIONS: We conclude that there might be an ischemic component in this patient with optic neuritis and hypothesize that this ischemic component is at least in part due to a temporarily increased endothelin-1 level.