RESUMEN
Phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with putative anti-inflammatory potential. However, studies comparing PS and PG are rare and were mainly conducted with phospholipid-dispersions of large size and broad distributions. Thus, we prepared small-sized PS- and PG-loaded liposomes exhibiting narrow distribution, and additionally studied the impact of liposome-pegylation on the reduction of the TNFα-production caused by the PS- and PG-liposomes. These PS- and PG-containing nanodispersions had a small size around 100nm and a narrow distribution (PDI<0.1). The liposome-dispersions showed no toxicity in NHDF- and 3T3-cells and virtually no hemolytic activity. They decreased the TNFα-production of LPS-(lipopolysaccharide)-stimulated mouse peritoneal macrophages in vitro. PG-liposomes always decreased the TNFα-levels more potently than PS-liposomes. Pegylation of PS- and PG-liposomes caused different Zeta potentials, but did not change biological activity. The results of the current study indicate a high potential of the tested formulations for phospholipid-based anti-inflammatory therapies.
Asunto(s)
Macrófagos Peritoneales/metabolismo , Nanopartículas , Fosfatidilgliceroles , Fosfatidilserinas , Células 3T3 , Animales , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Liposomas , Macrófagos Peritoneales/patología , Ratones , Nanopartículas/química , Nanopartículas/uso terapéutico , Fosfatidilgliceroles/química , Fosfatidilgliceroles/farmacología , Fosfatidilserinas/química , Fosfatidilserinas/farmacologíaRESUMEN
Inflammation, ischemia or the microenvironment of solid tumors is often accompanied by a reduction of extracellular pH (acidosis) that stresses the cells and acts on cellular signaling and transcription. The effect of acidosis on the expression of various inflammatory markers, on functional parameters (migration, phagocytic activity) and on signaling pathways involved was studied in monocytic cells and macrophages. In monocytic cell lines acidosis led to a reduction in expression of most of the inflammatory mediators, namely IL-1ß, IL-6, TNF-α, MCP-1, COX-2 and osteopontin. In primary human monocytes MCP-1 and TNF-α were reduced but COX-2 and IL-6 were increased. In RAW264.7 macrophage cell line IL-1ß, COX-2 and iNOS expression was increased, whereas MCP-1 was reduced similar to the effect in monocytic cells. For primary human monocyte-derived macrophages the regulation of inflammatory markers by acidosis depended on activation state, except for the acidosis-induced downregulation of MCP-1 and TNF-α. Acidosis affected functional immune cell behavior when looking at phagocytic activity which was increased in a time-dependent manner, but cellular motility was not changed. Neither ERK1/2 nor CREB signaling was stimulated by the reduction of extracellular pH. However, p38 was activated by acidosis in RAW264.7 cells and this activation was critical for the induction of IL-1ß, COX-2 and iNOS expression. In conclusion, acidosis may impede the recruitment of immune cells, but fosters inflammation when macrophages are present by increasing the level of COX-2 and iNOS and by functionally forcing up the phagocytic activity.
Asunto(s)
Acidosis/inmunología , Mediadores de Inflamación/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Acidosis/complicaciones , Animales , Células Cultivadas , Quimiocina CCL2/inmunología , Ciclooxigenasa 2/inmunología , Humanos , Inflamación/complicaciones , Ratones , Óxido Nítrico Sintasa de Tipo II/inmunología , Fagocitosis , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
AIMS: To determine the interaction between HRV and inflammation and their association with cardiovascular/all-cause mortality in the general population. METHODS AND RESULTS: Subjects of the CARLA study (n = 1671; 778 women, 893 men, 45-83 years of age) were observed for an average follow-up period of 8.8 years (226 deaths, 70 cardiovascular deaths). Heart rate variability parameters were calculated from 5-min segments of 20-min resting electrocardiograms. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and soluble tumour necrosis factor-alpha receptor type 1 (sTNF-R1) were measured as inflammation parameters. The HRV parameters determined included the standard deviation of normal-to-normal intervals (SDNN), the root-mean-square of successive normal-interval differences (RMSSD), the low- and high-frequency (HF) power, the ratio of both, and non-linear parameters [Poincaré plot (SD1, SD2, SD1/SD2), short-term detrended fluctuation analysis]. We estimated hazard ratios by using covariate-adjusted Cox regression for cardiovascular and all-cause mortality incorporating an interaction term of HRV/inflammation parameters. Relative excess risk due to interactions (RERIs) were computed. We found an interaction effect of sTNF-R1 with SDNN (RERI: 0.5; 99% confidence interval (CI): 0.1-1.0), and a weaker effect with RMSSD (RERI: 0.4; 99% CI: 0.0-0.9) and HF (RERI: 0.4; 99% CI: 0.0-0.9) with respect to cardiovascular mortality on an additive scale after covariate adjustment. Neither IL-6 nor hsCRP showed a significant interaction with the HRV parameters. CONCLUSION: A change in TNF-α levels or the autonomic nervous system influences the mortality risk through both entities simultaneously. Thus, TNF-α and HRV need to be considered when predicating mortality.
Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Enfermedades Cardiovasculares/fisiopatología , Frecuencia Cardíaca , Corazón/inervación , Inflamación/fisiopatología , Anciano , Anciano de 80 o más Años , Proteína C-Reactiva/análisis , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/mortalidad , Causas de Muerte , Electrocardiografía , Femenino , Alemania , Humanos , Inflamación/sangre , Inflamación/diagnóstico , Inflamación/mortalidad , Mediadores de Inflamación/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Receptores Tipo I de Factores de Necrosis Tumoral/sangre , Factores de Riesgo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
In critically ill patients regulation of heart-rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization-activated cyclic nucleotide-gated cation-(HCN)-channels may interfere with heart-rate regulation. This study analyzes the effect of LPS, the HCN-channel blocker ivabradine or Ca(2+) -channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart-rate of adult CD1-mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 µg/mL) reduced the beating-rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating-rate, indicating interaction of both compounds with HCN-channels, while coincubation with Ca(2+) -channel blockers and LPS caused additive beating-rate reduction. In CD1-mice (containing an active autonomic-nervous-system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart-rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart-rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN-channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN-channels for sympathetic activation (tachycardic-effect), and in parallel directly inhibits channel activity (bradycardic-effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Animales , Benzazepinas/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Ivabradina , Masculino , Ratones , Ratas , Sistema Nervioso Simpático/fisiopatología , Taquicardia/inducido químicamente , Taquicardia/metabolismo , Taquicardia/fisiopatologíaRESUMEN
Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that ß-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Lipopolisacáridos/metabolismo , Microdominios de Membrana/metabolismo , Colesterol/metabolismo , Fenómenos Electrofisiológicos , Glicosilación , Células HEK293 , Frecuencia Cardíaca/fisiología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Lipopolisacáridos/química , Microdominios de Membrana/química , Microdominios de Membrana/efectos de los fármacos , Insuficiencia Multiorgánica/fisiopatología , Técnicas de Placa-Clamp , Canales de Potasio/genética , Canales de Potasio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Sepsis/fisiopatología , beta-Ciclodextrinas/farmacologíaRESUMEN
Emerging and re-emerging infections are a global threat driven by the development of antimicrobial resistance due to overuse of antimicrobial agents and poor infection control practices. Implantable devices are particularly susceptible to such infections due to the formation of microbial biofilms. Furthermore, the introduction of implants into the body often results in inflammation and foreign body reactions. The antimicrobial and anti-inflammatory properties of gallium (Ga) have been recognized but not yet utilized effectively to improve implantable device integration. Furthermore, defensin (De, hBD-1) has potent antimicrobial activity in vivo as part of the innate immune system; however, this has not been demonstrated as successfully when used in vitro. Here, we combined Ga and De to impart antimicrobial activity and anti-inflammatory properties to polymer-based implantable devices. We fabricated polylactic acid films, which were modified using Ga implantation and subsequently functionalized with De. Ga-ion implantation increased surface roughness and increased stiffness. Ga implantation and defensin immobilization both independently and synergistically introduced antimicrobial activity to the surfaces, significantly reducing total live bacterial biomass. We demonstrated, for the first time, that the antimicrobial effects of De were unlocked by its surface immobilization. Ga implantation of the surface also resulted in reduced foreign body giant cell formation and expression of proinflammatory cytokine IL-1ß. Cumulatively, the treated surfaces were able to kill bacteria and reduce inflammation in comparison to the untreated control. These innovative surfaces have the potential to prevent biofilm formation without inducing cellular toxicity or inflammation, which is highly desired for implantable device integration.
Asunto(s)
Antiinfecciosos , Galio , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Antiinflamatorios/farmacología , Biopelículas , Materiales Biocompatibles Revestidos/farmacología , Defensinas/farmacología , Galio/farmacología , Propiedades de SuperficieRESUMEN
Recently it was shown that lipopolysaccharide (LPS) impairs the pacemaker current in human atrial myocytes. It was speculated that reduced heart rate variability (HRV), typical of patients with severe sepsis, may partially be explained by this impairment. We evaluated the effect of various types of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) expressed in HEK293 cells, and on pacemaker channels in native murine sino-atrial node (SAN) cells, in order to determine the structure of LPS necessary to modulate pacemaker channel function. Application of LPS caused a robust inhibition of hHCN2-mediated current (I(hHCN2)) owing to a negative shift of the voltage dependence of current activation and to a reduced maximal conductance. In addition, kinetics of channel gating were modulated by LPS. Pro-inflammatory LPS-types lacking the O-chain did not reduce I(hHCN2), whereas pro-inflammatory LPS-types containing the O-chain reduced I(hHCN2). On the other hand, a detoxified LPS without inflammatory activity, but containing the O-chain reduced I(hHCN2). Similar observations were made in HEK293 cells expressing hHCN4 and in murine SAN cells. This mechanistic analysis showed the novel finding that the O-chain of LPS is required for reduction of HCN channel activity. In the clinical situation the observed modulation of HCN channels may slow down diastolic depolarization of pacemaker cells and, hence, influence heart rate variability and heart rate.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Lipopolisacáridos/farmacología , Moduladores del Transporte de Membrana/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/antagonistas & inhibidores , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Lipopolisacáridos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Factores de TiempoRESUMEN
Inflammation is a central element of atherogenesis. Innate pathways contribute to vascular inflammation. However, the initial molecular process(es) starting atherogenesis remain elusive. The various risk factors, represented by particular compounds (activators), may cause altered cellular functions in the endothelium (e.g. vascular endothelial cell activation or -dysfunction), in invading cells (e.g. inflammatory mediator production) or in local vessel wall cells (e.g. inflammatory mediators, migration), thereby triggering the innate inflammatory process. The cellular components of innate immunology include granulocytes, natural killer cells and monocytes. Among the molecular innate constituents are innate molecules, such as the toll-like receptors or innate cytokines. Interleukin-1 (IL-1) and IL-6 are among the innate cytokines. Cytokines are potent activators of a great number of cellular functions relevant to maintain or commove homeostasis of the vessel wall. Within the vessel wall, vascular smooth muscle cells (SMCs) can significantly contribute to the cytokine-dependent inflammatory network by: (i) production of cytokines, (ii) response to cytokines and (iii) cytokine-mediated interaction with invading leucocytes. The cytokines IL-1 and IL-6 are involved in SMC-leucocyte interaction. The IL-6 effects are proposed to be mediated by trans-signalling. Dysregulated cellular functions resulting from dysregulated cytokine production may be the cause of cell accumulation, subsequent low-density lipoprotein accumulation and deposition of extracellular matrix (ECM). The deposition of ECM, increased accumulation of leucocytes and altered levels of inflammatory mediators may constitute an 'innate-immunovascular-memory' resulting in an ever-growing response to anew invasion. Thus, SMC-fostered inflammation, promoted by invading innate cells, may be a potent component for development and acceleration of atherosclerosis.
Asunto(s)
Aterosclerosis/inmunología , Citocinas/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Músculo Liso Vascular/inmunología , Transducción de Señal/inmunología , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Modelos Inmunológicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Inflammatory pathways are involved in the development of atherosclerosis. Interaction of vessel wall cells and invading monocytes by cytokines may trigger local inflammatory processes. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are standard medications used in cardiovascular diseases. They are thought to have anti-inflammatory capacities, in addition to their lipid-lowering effects. We investigated the anti-inflammatory effect of statins in the cytokine-mediated-interaction-model of human vascular smooth muscle cells (SMC) and human mononuclear cells (MNC). In this atherosclerosis-related inflammatory model LPS (lipopolysaccharide, endotoxin), as well as high mobility group box 1 stimulation resulted in synergistic (i.e. over-additive) IL-6 (interleukin-6) production as measured in ELISA. Recombinant IL-1, tumour necrosis factor-α and IL-6 mediated the synergistic IL-6 production. The standard anti-inflammatory drugs aspirin and indomethacin (Indo) reduced the synergistic IL-6 production by 60%. Simvastatin, atorvastatin, fluvastatin or pravastatin reduced the IL-6 production by 53%, 50%, 64% and 60%, respectively. The inhibition by the statins was dose dependent. Combination of statins with aspirin and/or Indo resulted in complete inhibition of the synergistic IL-6 production. The same inhibitors blocked STAT3 phosphorylation, providing evidence for an autocrine role of IL-6 in the synergism. MNC from volunteers after 5 day aspirin or simvastatin administration showed no decreased IL-6 production, probably due to drug removal during MNC isolation. Taken together, the data show that anti-inflammatory functions (here shown for statins) can be sensitively and reproducibly determined in this novel SMC/MNC coculture model. These data implicate that statins have the capacity to affect atherosclerosis by regulating cytokine-mediated innate inflammatory pathways in the vessel wall.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Interleucina-6/metabolismo , Leucocitos Mononucleares/metabolismo , Miocitos del Músculo Liso/metabolismo , Aspirina/administración & dosificación , Aspirina/farmacología , Atorvastatina , Separación Celular , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Proteína HMGB1/metabolismo , Ácidos Heptanoicos/farmacología , Humanos , Indometacina/farmacología , Interleucina-1/farmacología , Interleucina-6/biosíntesis , Interleucina-6/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Fosforilación/efectos de los fármacos , Pirroles/farmacología , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT3/metabolismo , Simvastatina/administración & dosificación , Simvastatina/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Phosphatidylserine (PS) and phosphatidylglycerol (PG) are naturally occurring phospholipids (PL) with intrinsic anti-inflammatory properties. The therapeutic potential of PS and PG has not been extensively explored and the main focus had been directed towards PS- and PG-liposomes. In order to increase the formulation options, we explored whether mixed micelles (MM) could be an alternative to liposomes. Potential advantages of MM are their thermodynamic stability, small size and ease of manufacture. DOPS- and DOPG-enriched MM were obtained via a co-precipitation technique and physicochemical characterization was performed. The MM, approximately 10 nm in diameter, showed no toxicity on fibroblast cell lines in vitro and virtually no hemolytic activity. The MM suppressed the TNFα-production of mIFNγ/LPS-stimulated mouse peritoneal macrophages (MPM) in vitro similar to DOPS- and DOPG-liposomes. Therefore, DOPS- and DOPG-loaded MM are promising new options for the treatment of inflammatory diseases.
Asunto(s)
Fosfatidilgliceroles , Fosfatidilserinas , Animales , Antiinflamatorios/farmacología , Liposomas , Ratones , MicelasRESUMEN
Non-viral gene delivery in its current form is largely dependent upon the ability of a delivery vehicle to protect its cargo in the extracellular environment and release it efficiently inside the target cell. Also a simple delivery system is required to simplify a GMP conform production if a marketing authorization is striven for. This work addresses these problems. We have developed a synthetic polycationic peptide-mimicking amphiphile, namely DiTT4, which shows efficient transfection rates and good biocompatibility without the use of a co-lipid in the formulation. The lipid-nucleic acid complex (lipoplex) was characterized at the structural (electron microscopy), physical (laser Doppler velocimetry and atomic force microscopy) and molecular levels (X-ray scattering). Stability studies of the lipoplexes in the presence of serum and heparin indicated a stable formation capable of protecting the cargo against the extracellular milieu. Hemocompatibility studies (hemolysis, complement activation and erythrocyte aggregation) demonstrated the biocompatibility of the formulation for systemic administration. The transfection efficiency was assessed in vitro using the GFP assay and confocal laser scanning microscopy studies. With the chorioallantoic membrane model, an animal replacement model according to the 3R strategy (replacement, refinement, and reduction), initial in vivo experiments were performed which demonstrate fast and efficient transfection in complex tissues and excellent biocompatibility.
Asunto(s)
ADN/administración & dosificación , Lípidos/química , Transfección/métodos , Células A549 , Animales , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , ADN/química , ADN/farmacocinética , Técnicas de Transferencia de Gen , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ensayo de Materiales , Microscopía Confocal , PolielectrolitosRESUMEN
For the development of gene therapeutics for systemic administration several hurdles have to be overcome. In this article we screen the branched fatty acid lysine conjugate T14diLys, a newly designed cationic lipid for lipofection, regarding this problem. The structure and particle size of lipoplexes, prepared with lipid formulations which are based on these lipid as nucleic acid complexing agent, are investigated in absence and presence of serum. Nuclease digestion assays were performed to evaluate the protective characteristics of the lipid formulation for the complexed nucleic acid. Furthermore, the lipid formulation is investigated regarding the interaction with different serum proteins to get first insights into the protein corona formation. Another focus is set on the hemocompatibility using in vitro assays for hemolysis and complement activation and the irritation test at the chorion allantois membrane of the chicken embryo as in vivo model. Finally, preliminary transfection efficiency studies with cell culture models for cells which are assessable via systemic administration are performed to evaluate possibilities for future therapeutic applications of the new lipid formulations. Summarizing, T14diLys with the co-lipid DOPE can be used to prepare a lipoplex formulation which can be applied systemically and can be used to develop gene therapeutics for targeting endothelial cells, macrophages, or leucocytes.
Asunto(s)
ADN/química , Ácidos Grasos/química , Lípidos/química , Lisina/química , Animales , Supervivencia Celular , Células Cultivadas , Humanos , Células Jurkat , Liposomas/síntesis química , Liposomas/química , Ratones , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Monocytes and macrophages contribute to pathogenesis of various inflammatory diseases, including auto-inflammatory diseases, cancer, sepsis, or atherosclerosis. They do so by production of cytokines, the central regulators of inflammation. Isoprenylation of small G-proteins is involved in regulation of production of some cytokines. Statins possibly affect isoprenylation-dependent cytokine production of monocytes and macrophages differentially. Thus, we compared statin-dependent cytokine production of lipopolysaccharide (LPS)-stimulated freshly isolated human monocytes and macrophages derived from monocytes by overnight differentiation. Stimulated monocytes readily produced tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Statins did not alter cytokine production of LPS-stimulated monocytes. In contrast, monocyte-derived macrophages prepared in the absence of statin lost the capacity to produce cytokines, whereas macrophages prepared in the presence of statin still produced cytokines. The cells expressed indistinguishable nuclear factor-kB activity, suggesting involvement of separate, statin-dependent regulation pathways. The presence of statin was necessary during the differentiation phase of the macrophages, indicating that retainment-of-function rather than costimulation was involved. Reconstitution with mevalonic acid, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate blocked the retainment effect, whereas reconstitution of cholesterol synthesis by squalene did not. Inhibition of geranylgeranylation by GGTI-298, but not inhibition of farnesylation or cholesterol synthesis, mimicked the retainment effect of the statin. Inhibition of Rac1 activation by the Rac1/TIAM1-inhibitor NSC23766 or by Rac1-siRNA (small interfering RNA) blocked the retainment effect. Consistent with this finding, macrophages differentiated in the presence of statin expressed enhanced Rac1-GTP-levels. In line with the above hypothesis that monocytes and macrophages are differentially regulated by statins, the CD14/CD16-, merTK-, CX3CR1-, or CD163-expression (M2-macrophage-related) correlated inversely to the cytokine production. Thus, monocytes and macrophages display differential Rac1-geranylgeranylation-dependent functional capacities, that is, statins sway monocytes and macrophages differentially.
Asunto(s)
Citocinas/biosíntesis , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Benzamidas/farmacología , Diferenciación Celular/fisiología , Citocinas/inmunología , Humanos , Macrófagos/inmunología , Monocitos/inmunología , Prenilación/efectos de los fármacosRESUMEN
Interleukin-1 (IL-1) is a potent regulator of cardiovascular proliferation, apoptosis, contraction or production of inflammatory mediators. Thus, we investigated expression and function of IL-1 in cultured neonatal rat heart cells upon endotoxin stimulation. We show that cultured neonatal rat cardiomyocytes expressed IL-1alpha and IL-1beta mRNA. The cells expressed functional cell-associated IL-1 activity and a specific anti-IL-1alpha-antibody inhibited the activity. Biologically active IL-1alpha was present at the cell surface of the cardiomyocytes, as indicated in co-culture experiments. Immunohistochemistry showed IL-1alpha-staining of the neonatal cardiomyocytes. Although the cells also expressed IL-1beta mRNA, we did not detect IL-1beta in the supernatants of cultured cardiomyocytes by ELISA or in immunohistochemical staining. Furthermore, neonatal and adult rat heart tissues expressed IL-1alpha mRNA, whereas fetal, but not adult, human cardiac tissues expressed detectable IL-1alpha mRNA. In contrast, IL-1beta mRNA was present in rat and human fetal and adult samples. Furthermore, in patients with dilated or ischemic cardiomyopathy, we measured IL-1beta, but not IL-1alpha, mRNA. These results provide evidence for the presence of functionally active IL-1alpha on the cell surface of neonatal rat cardiomyocytes and may suggest a differential role of IL-1alpha in regulation of cellular functions during development, aging and disease in rat and human heart cells.
Asunto(s)
Endotoxinas/farmacología , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Salmonella , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/embriología , Humanos , Interleucina-1alfa/genética , Interleucina-1beta/genética , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Inflammation contributes to the pathogenesis of atherosclerosis. Proinflammatory cytokines, including interleukin-1 (IL-1), may be involved in the local inflammation occurring in the vessel wall. Vascular smooth muscle cells express the unprocessed IL-1beta precursor molecule. Invading leukocytes, such as monocytes or polymorphonuclear granulocytes (PMN) may activate the IL-1beta precursor during atherogenesis. Thus, we investigated the capacity of PMN to process IL-1beta and IL-18 precursors. Processing was analyzed using Western blot and bioassay for IL-1-activity was performed. As few as 80 to 400 PMN/mL detectably processed preIL-1beta. PMN also cleaved the caspase-1 substrate preIL-18. The preIL-1beta and preIL-18 cleavage products were located at the same apparent molecular weight as those resulting from cleavage by monocyte-derived caspase-1. PMN expressed caspase-1 mRNA and immunoreactive protein. The N-terminus of the preIL-1beta cleavage product expressed the sequence expected for caspase-1 cleavage. The cleavage product was active in the bioassay for IL-1 activity, and the caspase-1 inhibitor YVAD blocked processing. We have shown previously that SMC can block processing of preIL-1 by caspase-1. In contrast, SMC do not block processing of PARP by caspase-3. Here, we show that SMC also inhibited the PMN-mediated processing of recombinant and native preIL-1beta or preIL-18 depending on the cell number, whereas EC or fibroblasts did not block processing. Our results indicate that PMN can activate preIL-1beta in a caspase-1-like fashion. During inflammatory processes, PMN may activate preIL-1beta released from SMC, thereby altering IL-1-mediated cardiovascular functions, including contractility, apoptosis, and cytokine production.
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Caspasa 1/biosíntesis , Interleucina-18/biosíntesis , Interleucina-1/biosíntesis , Músculo Liso Vascular/fisiología , Neutrófilos/metabolismo , Precursores de Proteínas/biosíntesis , Caspasa 1/genética , Inhibidores de Caspasas , Recuento de Células , Células Cultivadas , Endotelio Vascular/metabolismo , Fibroblastos/metabolismo , Humanos , Oligopéptidos/farmacología , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesisRESUMEN
Multiple organ dysfunction syndrome (MODS) is the failure of several organs after a trigger event. The mortality is high, at up to 70%. We hypothesize that autonomic dysfunction may substantially contribute to the development of MODS and speculate that there is an age dependence of autonomic dysfunction in MODS. A total of 90 consecutively admitted MODS patients were assigned to this study. Three variables of autonomic function were analyzed: heart rate variability (HRV), baroreflex sensitivity (BRS) and chemoreflex sensitivity (CRS). The patient cohort was divided into three age groups. The main finding was that BRS, CRS and almost all indices of HRV were attenuated in comparison to normal range data and there was no age dependence for HRV indices or CRS, but there was for BRS. In conclusion, autonomic function in MODS is attenuated. The influence of MODS on autonomic function overwhelms the age dependence of autonomic function observed in healthy subjects.
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Envejecimiento , Sistema Nervioso Autónomo/fisiopatología , Barorreflejo , Frecuencia Cardíaca , Insuficiencia Multiorgánica/fisiopatología , Consumo de Oxígeno , Retroalimentación , Femenino , Humanos , MasculinoRESUMEN
Myocardial depression in human sepsis was only unequivocally proven in the 1980s by the group of Parrillo, who used nuclear imaging techniques to measure heart volumes and function in intensive care patients. Heart failure in sepsis is frequently masked by a seemingly normal cardiac output. However, relative to the lowered systemic vascular resistance - resulting in a reduced afterload - cardiac outputs and ventricular ejection fractions are often not adequately enhanced. This septic cardiomyopathy (impairment of the heart within the scope of systemic sepsis) involves both the right and the left ventricles, and is potentially reversible. In response to volume substitution, the heart can be considerably enlarged. The cardiomyopathy is not primarily hypoxic in nature, but may be aggravated by ischemia. Autonomic dysfunction, documented by a reduced heart rate variability and impaired baroreflex and chemoreflex sensitivities, forms part of the disease entity. The severity of myocardial depression correlates with a poor prognosis. Noninfectious systemic inflammatory response syndrome can give rise to an analogous disease entity, namely, systemic inflammatory response syndrome cardiomyopathy.The etiology of septic cardiomyopathy is multifactorial. Several candidates with a potential pathogenetic impact on the heart were identified: bacterial toxins; cytokines and mediators including tumour necrosis factor-alpha, interleukin-1 and nitric oxide; cardiodepressant factors; oxygen reactive species; and catecholamines. Symptomatic treatment consists of volume substitution and catecholamine support; causal therapeutic approaches aiming at an interruption of the proinflammatory mediator cascades are being tested.
RESUMEN
OBJECTIVES: Cytokines contribute to the development of the systemic inflammatory response syndrome or multiple-organ failure frequently observed after cardiopulmonary bypass-supported cardiac surgery. To quantify the contribution of bypass-induced versus trauma-induced inflammatory response after coronary artery bypass grafting, we examined plasma cytokine levels in 120 patients with coronary artery disease who were treated with or without cardiopulmonary bypass-assisted procedures. METHODS: Patients were treated in accordance with one of the following protocols: (1) elective percutaneous coronary intervention without cardiopulmonary bypass (n = 69), (2) cardiopulmonary bypass-supported percutaneous coronary intervention (cardiopulmonary bypass-percutaneous coronary intervention; n = 10), and (3) cardiopulmonary bypass-supported coronary artery bypass grafting (cardiopulmonary bypass-coronary artery bypass grafting; n = 41). Cytokine levels (picograms/milliliter) were measured by enzyme-linked immunosorbent assay from plasma samples obtained at various time points. RESULTS: Interleukin-6 was measured in blood samples from all 3 patient populations. The maximum interleukin-6 level was 13.6 +/- 22.3 pg/mL in the percutaneous coronary intervention group, 170.4 +/- 165.4 pg/mL in the cardiopulmonary bypass-percutaneous coronary intervention group, and 640.3 +/- 285.7 pg/mL in the cardiopulmonary bypass-coronary artery bypass grafting group. Interleukin-6 levels were significantly different, and the 95% confidence intervals did not overlap. In the cardiopulmonary bypass-percutaneous coronary intervention group, bypass duration correlated well with interleukin-6 production ( r = 0.915; P < .001), whereas these parameters did not correlate in patients who underwent cardiopulmonary bypass-coronary artery bypass grafting ( r = 0.307; P = .054). CONCLUSIONS: These findings support the suggestion that surgical trauma and cardiopulmonary bypass contribute to the inflammatory response after cardiac surgery, although trauma may contribute to a higher degree.
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Angioplastia Coronaria con Balón , Puente Cardiopulmonar , Puente de Arteria Coronaria , Citocinas/sangre , Enfermedad Coronaria/cirugía , Procedimientos Quirúrgicos Electivos , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-10/sangre , Interleucina-6/sangre , Receptores de Lipopolisacáridos/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores Tipo I de Factores de Necrosis Tumoral/sangre , Receptores Tipo II del Factor de Necrosis Tumoral/sangre , Sialoglicoproteínas/sangre , Síndrome de Respuesta Inflamatoria Sistémica/sangreRESUMEN
Chronic inflammatory reactions hamper the use of biomaterials after implantation. Thus, the aim of the study was to develop a novel predictive in vitro macrophage/fibroblast co-culture model based on cell migration chambers that allows a timely and locally controlled interaction of both cell types to study the inflammatory responses of biomaterials in vitro. Here, self-assembled monolayers (SAMs) with different wettability and charge properties were used as model biomaterials on which co-cultures were established by use of fence chambers having internal and external compartments. This allowed establishing separated and mixed co-cultures of both cell types before and after removal of the chamber, respectively. The key advantages of this novel co-culture model included not only to establish a timely-resolved study of cytokine release, but also the ability to assess individual macrophage migration in both macrophage mono-cultures and co-cultures. All inflammatory reactions in terms of macrophage adhesion, macrophage migration, foreign body giant cell (FBGC) formation, ß1 integrin expression and pro-inflammatory cytokine production were found strongly surface property dependent. The results show that the hydrophobic CH3 surface caused the strongest inflammatory reactions, whereas the hydrophilic/anionic COOH surface caused the least inflammatory response, indicating low and high biocompatibility of the surfaces, respectively. Most importantly, we found that both macrophage motility and directional movement were increased in the presence of fibroblasts in co-cultures compared with macrophage mono-cultures. Overall, the novel co-culture system provides access to a range of parameters for studying inflammatory reactions and reveals how material surface properties affect the inflammatory responses.
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Materiales Biocompatibles/toxicidad , Bioensayo/instrumentación , Técnicas de Cocultivo/instrumentación , Fibroblastos/efectos de los fármacos , Inflamación/inducido químicamente , Macrófagos/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Citocinas/inmunología , Diseño de Equipo , Análisis de Falla de Equipo , Fibroblastos/inmunología , Humanos , Inflamación/inmunología , Inflamación/patología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Ensayo de Materiales/instrumentación , Pruebas de Toxicidad/instrumentaciónRESUMEN
In recent years, the pathophysiological concept of chronic heart failure (CHF) has changed from an isolated hemodynamic view to a more complex concept involving neuroendocrine and inflammatory pathways. New therapeutic strategies, such as beta-blocker therapy, are based on these new concepts and provide clinical evidence for a clinical benefit in patients with CHF. The survival benefit of beta-blocker therapy in CHF has been related to neurohumoral regulation. Thus, evidence evolved showing that following beta-blocker therapy cytokine levels in CHF patients are altered. We have shown that the levels of soluble TNF receptor type 2 correlated well with cAMP in leukocytes. Data from clinical studies in adult and infant CHF patients have demonstrated that beta-blocker therapy is accompanied by altered cytokine, cytokine antagonist, and/or soluble cytokine receptor levels. These alterations may result from a dysregulated interaction of beta-adrenergic pathways and the cytokine system, and are possibly related to cAMP-dependent regulation of the release or shedding of these mediators.