RESUMEN
OBJECTIVES: Extensive reactive oxygen and nitrogen species (also reactive species) production is a mechanism involved in abdominal aortic aneurysm (AAA) development. White blood cells (WBCs) are a known source of reactive species. Their production may be decreased by statins, thereby reducing the AAA growth rate. Reactive species production in circulating WBCs of AAA patients and the effect of statins on their production was investigated. METHODS: This observational study investigated reactive species production in vivo and ex vivo in circulating WBCs of AAA patients, using venous blood from patients prior to elective AAA repair (n = 34; 18 statin users) and from healthy volunteers (n = 10). Reactive species production was quantified in circulating WBCs using immunofluorescence microscopy: nitrotyrosine (footprint of peroxynitrite, a potent reactive nitrogen species) in snap frozen blood smears; mitochondrial superoxide and cytoplasmic hydrogen peroxide (both reactive oxygen species) by live cell imaging. Neutrophils, lymphocytes, and monocytes were examined individually. RESULTS: In AAA patients using statins, the median nitrotyrosine level in neutrophils was 646 (range 422-2059), in lymphocytes 125 (range 74-343), and in monocytes 586 (range 291-663). Median levels in AAA patients not using statins were for neutrophils 928 (range 552-2095, p = .03), lymphocytes 156 (101-273, NS), and for monocytes 536 (range 535-1635, NS). The statin dose tended to correlate negatively with nitrotyrosine in neutrophils (Rs -0.32, p = .06). The median levels in controls were lower for neutrophils 466 (range 340-820, p < .01) and for monocytes 191 (range 102-386, p = .03), but similar for lymphocytes 99 (range 82-246) when compared to the AAA patients. There were no differences in mitochondrial superoxide and cytoplasmic hydrogen peroxide between statin and non-statin users within AAA patients. CONCLUSIONS: It was found that the peroxynitrite footprint in circulating neutrophils and monocytes of AAA patients is higher than in controls. AAA patients treated with statins had a lower peroxynitrite footprint in neutrophils than non-statin users.
Asunto(s)
Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Neutrófilos/efectos de los fármacos , Ácido Peroxinitroso/sangre , Adulto , Anciano , Anciano de 80 o más Años , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/diagnóstico , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Peróxido de Hidrógeno/sangre , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Neutrófilos/metabolismo , Tirosina/análogos & derivados , Tirosina/sangreRESUMEN
Successful stem cell therapy after acute myocardial infarction (AMI) is hindered by lack of engraftment of sufficient stem cells at the site of injury. We designed a novel technique to overcome this problem by assembling stem cell-microbubble complexes, named 'StemBells'. StemBells were assembled through binding of dual-targeted microbubbles (~3µm) to adipose-derived stem cells (ASCs) via a CD90 antibody. StemBells were targeted to the infarct area via an ICAM-1 antibody on the microbubbles. StemBells were characterized microscopically and by flow cytometry. The effect of ultrasound on directing StemBells towards the vessel wall was demonstrated in an in vitro flow model. In a rat AMI-reperfusion model, StemBells or ASCs were injected one week post-infarction. A pilot study demonstrated feasibility of intravenous StemBell injection, resulting in localization in ICAM-1-positive infarct area three hours post-injection. In a functional study five weeks after injection of StemBells cardiac function was significantly improved compared with controls, as monitored by 2D-echocardiography. This functional improvement neither coincided with a reduction in infarct size as determined by histochemical analysis, nor with a change in anti- and pro-inflammatory macrophages. In conclusion, the StemBell technique is a novel and feasible method, able to improve cardiac function post-AMI in rats.
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Microburbujas , Infarto del Miocardio/terapia , Trasplante de Células Madre/métodos , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Administración Intravenosa , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Ecocardiografía , Corazón/diagnóstico por imagen , Corazón/fisiopatología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Proyectos Piloto , Ratas , Ratas Wistar , Sonicación , Células Madre/citología , Células Madre/metabolismoRESUMEN
In most pre-clinical animal studies investigating stem cell therapy in acute myocardial infarction (AMI), the administered stem cells are isolated from healthy donors. In clinical practice, however, patients who suffer from AMI will receive autologous cells, for example using adipose-derived stem cells (ASC). During AMI, inflammation is induced and we hypothesized that this might affect characteristics of ASC. To investigate this, ASC were isolated from rat adipose tissue 1 day (1D group, n = 5) or 7 days (7D group, n = 6) post-AMI, and were compared with ASC from healthy control rats (Control group, n = 6) and sham-operated rats (Sham 1D group, n = 5). We found that significantly fewer ASC were present 1 day post-AMI in the stromal vascular fraction (SVF), determined by a colony-forming-unit assay (p < 0.001 vs. Control and 7D). These data were confirmed by flow cytometry, showing fewer CD90-positive cells in SVF of the 1D group. When cultured, no differences were found in proliferation rate and cell size between the groups in the first three passages. Also, no difference in the differentiation capacity of ASC was found. In conclusion, it was shown that significantly fewer stem cells were present in the SVF 1 day post-AMI; however, the stem cells that were present showed no functional differences.
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Tejido Adiposo/citología , Infarto del Miocardio/patología , Células Madre/citología , Animales , Recuento de Células , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Masculino , Ratas Wistar , Células del Estroma/citologíaRESUMEN
Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.
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Tejido Adiposo/citología , Plaquetas/metabolismo , Sustitutos Sanguíneos/farmacología , Extractos Celulares/farmacología , Miocardio/patología , Suero/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Adulto , Anciano , Animales , Biomarcadores/metabolismo , Plaquetas/efectos de los fármacos , Bovinos , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo , Humanos , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
INTRODUCTION: Some studies reported an increased incidence of premature ventricular complexes (PVCs) during triggered myocardial contrast echocardiography (MCE) using high-intensity ultrasound destruction. Whether PVCs are also induced by real time MCE using low emission power, is unknown. The aim of the study was to assess the occurrence of arrhythmias during real time adenosine MCE in healthy volunteers and patients with stable coronary artery disease (CAD). METHODS: Fifty healthy volunteers and 26 patients with stable CAD underwent real time MCE using Sonovue and power pulse inversion (ATL 5000) at rest and during adenosine stress. The occurrence of premature atrial complexes (PAC) and PVCs was analyzed before and during MCE using ECG-tracings from videotapes. RESULTS: In healthy subjects, the occurrence of PVCs at baseline (0.04 +/- 0.23 PVCs/min) was similar at rest (0.04 +/- 0.23 PVCs/min, P = NS), and adenosine stress (0.03 +/- 0.14, P = NS). In CAD patients, the occurrence of PVCs at baseline was 0.30 +/- 0.76 PVC/min, compared to 0.29 +/- 0.74 at rest (P = NS), and 0.34 +/- 0.74 during adenosine stress (P = NS). The number of subjects demonstrating PVCs did not increase during MCE. The occurrence of PACs during MCE was not increased compared to baseline. CONCLUSION: Real time MCE using low emission power does not increase the occurrence of premature complexes in healthy volunteers or CAD patients.
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Adenosina , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Ecocardiografía/efectos adversos , Fosfolípidos , Hexafluoruro de Azufre , Complejos Prematuros Ventriculares/etiología , Adulto , Sistemas de Computación , Medios de Contraste , Prueba de Esfuerzo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Medición de Riesgo , Sensibilidad y Especificidad , Vasodilatadores , Complejos Prematuros Ventriculares/diagnósticoRESUMEN
BACKGROUND: Exercise training in pulmonary arterial hypertension (PH) is a promising adjunct to medical treatment. However, it is still unclear whether training is beneficial for all PH patients. We hypothesized that right ventricular adaptation plays a pivotal role in the response to training. METHODS AND RESULTS: Two different dosages of monocrotaline were used in rats to model stable PH with preserved cardiac output and progressive PH developing right heart failure. Two weeks after injection, PH was confirmed by echocardiography, and treadmill training was initiated. Rats were trained for 4 weeks unless manifest right heart failure developed earlier. At the end of the study protocol, all rats were functionally assessed by endurance testing, echocardiography, and invasive pressure measurements. Lungs and hearts were further analyzed in quantitative histomorphologic analyses. In stable PH, exercise training was well tolerated and markedly increased exercise endurance (from 25+/-3.9 to 62+/-3.9 minutes; P<0.001). Moreover, capillary density increased significantly (from 1.21+/-0.12 to 1.51+/-0.07 capillaries per cardiomyocyte; P<0.05). However, in progressive PH, exercise training worsened survival (hazard ratio, 2.7; 95% confidence interval, 1.1 to 14.2) and increased pulmonary vascular remodeling. In addition, training induced widespread leukocyte infiltration into the right ventricle (from 135+/-14 to 276+/-18 leukocytes per 1 mm(2); P<0.001). CONCLUSIONS: In our rat model, exercise training was found to be beneficial in stable PH but detrimental in progressive PH. Future studies are necessary to address the clinical implications of our findings.
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Adaptación Fisiológica/fisiología , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/terapia , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/terapia , Condicionamiento Físico Animal/fisiología , Animales , Biopsia , Capilares/fisiología , Cateterismo Cardíaco , Gasto Cardíaco/fisiología , Circulación Coronaria/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ecocardiografía , Insuficiencia Cardíaca/diagnóstico , Hipertensión Pulmonar/inducido químicamente , Masculino , Monocrotalina/toxicidad , Miocarditis/fisiopatología , Resistencia Física/fisiología , Ratas , Ratas Wistar , Descanso , Tasa de SupervivenciaRESUMEN
The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.).
RESUMEN
Endothelial hyperpermeability is regulated by a myosin light chain-2 (MLC2) phosphorylation-dependent contractile mechanism. Thrombin is a potent inducer of hyperpermeability of cultured monolayers of endothelial cells (ECs) via Rho kinase-mediated MLC2-phosphorylation. The aim of the present study was to investigate the effects of thrombin on in situ endothelial morphology and barrier integrity. Cytoskeletal dynamics, regions of paracellular flux, and MLC2-phosphorylation of ECs were visualized by digital three-dimensional imaging microscopy of pressurized rat kidney arterioles. Myosin phosphatase targeting subunit (MYPT1)-phosphorylation was used as a surrogate marker for Rho kinase activity. Thrombin induced the formation of F-actin filaments in ECs in situ and rounding of the ECs in the absence of obvious formation of gaps between ECs. These changes were accompanied by an increase in MLC2 phosphorylation and a decrease in barrier integrity. In vitro analysis revealed that Rho kinase activity on F-actin filaments was associated with a contractile response that enhanced opening of the barrier. Rho kinase activity was not detectable on F-actin filaments induced by histamine, an inducer of a more transient hyperpermeability response. Inhibition of the myosin phosphatase mimicked the effects of thrombin on barrier function. The thrombin-induced changes in in situ MLC2 phosphorylation and barrier function were Rho kinase dependent. These data demonstrate a direct effect of thrombin on EC morphology and barrier integrity in intact microvessels. Furthermore, they establish an important contribution of enhanced Rho kinase activity to the development of prolonged but not transient types of endothelial barrier dysfunction.
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Endotelio Vascular/fisiología , Microcirculación/fisiología , Fosfatasa de Miosina de Cadena Ligera/fisiología , Trombina/farmacología , Quinasas Asociadas a rho/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Concanavalina A/farmacología , Endotelio Vascular/efectos de los fármacos , Humanos , Microcirculación/efectos de los fármacos , Ratas , Ratas Wistar , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/fisiologíaRESUMEN
BACKGROUND: We previously demonstrated the involvement of the Ca2+-independent protein kinase C-delta (PKC-delta) isoform in sevoflurane-induced cardioprotection against ischaemia and reperfusion (I/R) injury. Since sevoflurane is known to modulate myocardial Ca2+-handling directly, in this study we investigated the role of the Ca2+-dependent PKC-alpha isoform in sevoflurane-induced cardioprotective signalling in relation to reactive oxygen species (ROS), adenosine triphosphate-sensitive mitochondrial K+ (mitoK+(ATP)) channels, and PKC-delta. METHODS: Preconditioned (15 min 3.8 vol% sevoflurane) isolated rat right ventricular trabeculae were subjected to I/R, consisting of 40 min superfusion with hypoxic, glucose-free buffer, followed by normoxic glucose-containing buffer for 60 min. After reperfusion, contractile recovery was expressed as percentage of force development before I/R. The role of PKC-alpha, ROS, mitoK+(ATP) channels, and PKC-delta was established using the following pharmacological inhibitors: Go6976 (GO; 50 nM), n-(2-mercaptopropionyl)-glycine (MPG; 300 microM), 5-hydroxydecanoic acid sodium (5HD; 100 microM), and rottlerin (ROT; 1 microM). RESULTS: Preconditioning of trabeculae with sevoflurane improved contractile recovery after I/R [65 (3)% (I/R + SEVO) vs 47 (3)% (I/R); n = 8; P < 0.05]. This cardioprotective effect was attenuated in trabeculae treated with GO [42 (4)% (I/R + SEVO + GO); P > 0.05 vs (I/R)]. In sevoflurane-treated trabeculae, PKC-alpha translocated towards mitochondria, as shown by immunofluorescent co-localization analysis. GO and MPG, but not 5HD or ROT, abolished this translocation. CONCLUSIONS: Sevoflurane improves post-ischaemic contractile recovery via activation of PKC-alpha. ROS production, but not opening of mitoK+(ATP) channels, precedes PKC-alpha translocation towards mitochondria. This study shows the involvement of Ca2+-dependent PKC-alpha in addition to the well-established role of Ca2+-independent PKC isoforms in sevoflurane-induced cardioprotection.
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Anestésicos por Inhalación/farmacología , Precondicionamiento Isquémico Miocárdico/métodos , Éteres Metílicos/farmacología , Proteína Quinasa C-alfa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Calcio/fisiología , Activación Enzimática/efectos de los fármacos , Masculino , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Ratas , Ratas Wistar , Sevoflurano , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Técnicas de Cultivo de Tejidos , Translocación GenéticaRESUMEN
OBJECTIVE: Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin. Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity. METHODS AND RESULTS: Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr696-MYPT1) as a marker for Rho kinase activity, basal endothelial Rho kinase activity was observed at cell-cell contact sites, in vitro and in situ. Thrombin enhanced MYPT phosphorylation at F-actin stress fibers. Inhibition of basal Rho kinase activity for 24 hours or depletion of Rho kinase (ROCK-I and -II) by siRNA disrupted endothelial barrier integrity, opposite to the previously observed protection from the thrombin-enhanced endothelial permeability. This barrier dysfunction could not be explained by changes in RhoA, Rac1, eNOS, or apoptosis. Remarkably, basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin. CONCLUSIONS: Rho kinase has opposing activities in regulation of endothelial barrier function: (1) an intrinsic barrier-protective activity at the cell margins, and (2) an induced barrier-disruptive activity at contractile F-actin stress fibers. These findings may have implications for long-term antivascular leak therapy.
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Uniones Adherentes/fisiología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Endoteliales/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Citoesqueleto/fisiología , Humanos , ARN Interferente Pequeño , Venas Umbilicales , Quinasas Asociadas a rhoRESUMEN
BACKGROUND: Sevoflurane protects the myocardium against ischaemic injury through protein kinase C (PKC) activation, mitochondrial K+ATP-channel (mitoK+ATP) opening and production of reactive oxygen species (ROS). However, it is unclear whether the type of ischaemia determines the involvement of these signalling molecules. We therefore investigated whether hypoxia (HYP) or metabolic inhibition (MI), which differentially inhibit the mitochondrial electron transport chain (ETC), are comparable concerning the relative contribution of PKC, mitoK+ATP and ROS in sevoflurane-induced cardioprotection. METHODS: Rat right ventricular trabeculae were isolated and isometric contractile force (Fdev) was measured. Trabeculae were subjected to HYP (hypoxic glucose-free buffer; 40 min) or MI (glucose-free buffer, 2 mM cyanide; 30 min), followed by 60 min recovery (60 min). Contractile recovery (Fdev,rec) was determined at the end of the recovery period and expressed as a percentage of Fdev before hypoxia or MI, respectively. Chelerythrine (CHEL; 6 microM), 5-hydroxydecanoic acid sodium (100 microM) and n-(2-mercaptopropionyl)-glycine (MGP; 300 microM) were used to inhibit PKC, mitoK+ATP and ROS, respectively. RESULTS: Fdev,rec after HYP was reduced to 47 (3)% (P<0.001 vs control; n=5) whereas MI reduced Fdev,rec to 28 (5)% (P<0.001 vs control; n=5). A 15 min period of preconditioning with sevoflurane (3.8%) equally increased contractile recovery after HYP [76 (9)%; P<0.05 vs HYP] and MI [67 (8)%; P<0.01 vs MI]. Chelerythrine, 5-hydroxydecanoate and n-(2-mercaptopropionyl)-glycine abolished the protective effect of sevoflurane in both ischaemic models. Trabeculae subjected to HYP or MI did not demonstrate any increased apoptotic or necrotic markers. CONCLUSIONS: PKC, mitoK+ATP and ROS are involved in sevoflurane-induced cardioprotection after HYP or MI, suggesting that the means of mitochondrial ETC inhibition does not determine the signal transduction pathway for cardioprotection by anaesthetics.
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Anestésicos por Inhalación/farmacología , Precondicionamiento Isquémico Miocárdico/métodos , Éteres Metílicos/farmacología , Isquemia Miocárdica/etiología , Animales , Apoptosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Hipoxia/complicaciones , Masculino , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/prevención & control , Necrosis , Canales de Potasio/fisiología , Proteína Quinasa C/fisiología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sevoflurano , Transducción de Señal/efectos de los fármacos , Cianuro de Sodio , Técnicas de Cultivo de TejidosRESUMEN
In the present study, we addressed the interactions among ultrasound, microbubbles, and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H(2)O(2)) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H(2)O(2) and Ca(2+) influx. Ultrasound, in the absence or presence of SonoVue microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or 0.5 during 10 s. This was repeated every minute, for a total of five times. The production of H(2)O(2) was measured intracellularly with CM-H(2)DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca(2+) concentration with fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H(2)O(2) at MI 0.1 of 50% and MI 0.5 of 110% compared with control (P < 0.001). Furthermore, we found increases in intracellular Ca(2+) levels at both MI 0.1 and MI 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca(2+). In addition, in the presence of catalase, Ca(2+) influx immediately following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001). Finally, cell viability was not significantly affected, not even 24 h later. These results implicate a role for H(2)O(2) in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.
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Permeabilidad de la Membrana Celular/fisiología , Peróxido de Hidrógeno/metabolismo , Microburbujas , Animales , Calcio/metabolismo , Línea Celular , Supervivencia Celular/fisiología , Mioblastos/citología , Mioblastos/metabolismo , RatasRESUMEN
In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosis.
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Apoptosis/fisiología , Núcleo Celular/fisiología , Isquemia/fisiopatología , Glicoproteínas de Membrana/genética , Miocitos Cardíacos/fisiología , NADPH Oxidasas/genética , Animales , Línea Celular , Glicoproteínas de Membrana/biosíntesis , NADPH Oxidasa 2 , NADPH Oxidasas/biosíntesis , Ratas , Ratas Wistar , Transducción de Señal/fisiologíaRESUMEN
We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.
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Corazón/fisiología , Isquemia Miocárdica/fisiopatología , Miocitos Cardíacos/fisiología , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Western Blotting , Células Cultivadas , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Inmunohistoquímica , Técnicas In Vitro , Mioblastos/fisiología , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , RatasRESUMEN
The development of ultrasound contrast agents, containing encapsulated microbubbles, has increased the possibilities for diagnostic imaging. Ultrasound contrast agents are currently used to enhance left ventricular opacification, increase Doppler signal intensity, and in myocardial perfusion imaging. Diagnostic imaging with contrast agents is performed with low acoustic pressure using non-linear reflection of ultrasound waves by microbubbles. Ultrasound causes bubble destruction, which lowers the threshold for cavitation, resulting in microstreaming and increased permeability of cell membranes. Interestingly, this mechanism can be used for delivery of drugs or genes into tissue. Microbubbles have been shown to be capable of carrying drugs and genes, and destruction of the bubbles will result in local release of their contents. Recent studies demonstrated the potential of microbubbles and ultrasound in thrombolysis. In this article, we will review the recent advances of microbubbles as a vehicle for delivery of drugs and genes, and discuss possible therapeutic applications in thrombolysis.
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Microburbujas , Ultrasonografía Intervencional , Medios de Contraste/administración & dosificación , Enfermedad Coronaria/diagnóstico por imagen , Enfermedad Coronaria/terapia , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Terapia Genética , Humanos , Microesferas , Trombosis/diagnóstico por imagen , Trombosis/terapiaRESUMEN
Acute renal failure (ARF) necessitating renal replacement therapy is a common problem associated with high morbidity and mortality in the critically ill. Hypotension, followed by resuscitation, is the most common etiologic factor, mimicked by ischemia/reperfusion (I/R) in animal models. Although knowledge of the pathophysiology of ARF in the course of this condition is increasingly detailed, the intracellular and molecular mechanisms leading to ARF are still incompletely understood. This review aims at describing the role of cellular events and signals, including collapse of the cytoskeleton, mitochondrial and nuclear changes, in mediating cell dysfunction, programmed cell death (apoptosis), necrosis and others. Insight into the molecular pathways in the various elements of the kidney, such as vascular endothelium and smooth muscle and tubular epithelium leading to cell damage upon I/R will, hopefully, open new therapeutic modalities, to mitigate the development of ARF after hypotensive episodes and to promote repair and resumption of renal function once ARF has developed.
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Lesión Renal Aguda/fisiopatología , Riñón/irrigación sanguínea , Daño por Reperfusión/fisiopatología , Actinas , Lesión Renal Aguda/prevención & control , Animales , Apoptosis/fisiología , Citoesqueleto/fisiología , Endotelio Vascular/fisiología , Tasa de Filtración Glomerular , Humanos , Túbulos Renales/fisiología , Necrosis/fisiopatología , Neutrófilos/fisiología , Especies Reactivas de Oxígeno , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Although gene therapy has great potential as a treatment for diseases, clinical trials are slowed down by the development of a safe and efficient gene delivery system. In this review, we will give an overview of the viral and nonviral vehicles used for drug and gene delivery, and the different nonviral delivery techniques, thereby focusing on delivery through ultrasound contrast agents. The development of ultrasound contrast agents containing encapsulated microbubbles has increased the possibilities not only for diagnostic imaging, but for therapy as well. Microbubbles have been shown to be able to carry drugs and genes, and destruction of the bubbles by ultrasound will result in local release of their contents. Furthermore, ligands can be attached so that they can be targeted to a specific target tissue. The recent advances of microbubbles as vehicles for delivery of drugs and genes will be highlighted.
RESUMEN
BACKGROUND: Recognition that hypertonic saline (HTS) modulates the inflammatory response has renewed interest in this agent for postinjury resuscitation. Changes in extracellular tonicity alter cell shape and are accompanied by cytoskeletal reorganization. Recent evidence suggests that cytoskeletal reorganization is critical for receptor-mediated signal transduction. We hypothesized that HTS-induced changes in the cytoskeleton interfere with cytotoxic signal transduction. METHODS: Isolated neutrophils (PMNs) were incubated in HTS (Na+ = 180 mmol/L) and activated with N-formyl-methionyl-leucyl-phenylalanine (receptor-mediated) or phorbol myristate (receptor independent). Actin polymerization was assessed by digital image microscopy and flow cytometry. PMN superoxide anion (O2-) production and p38 MAPK activation was measured by reduction of cytochrome c and Western blot. Pretreatment with cytochalasin B was used to disrupt HTS-induced actin reorganization. RESULTS: HTS inhibited receptor-mediated cytoskeletal reorganization and attenuated p38 MAPK activation and O2- production. HTS had no effect on receptor-independent O2- production. Cytoskeletal disruption (cytochalasin B) prevented HTS attenuation of receptor-mediated p38 MAPK activation. CONCLUSION: HTS attenuates the PMN cytotoxic response by interfering with intracellular signal transduction. Changes in the actin cytoskeleton appear to modulate receptor-mediated p38 MAPK signaling.
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Citoesqueleto/efectos de los fármacos , Citotoxicidad Inmunológica/fisiología , Neutrófilos/citología , Solución Salina Hipertónica/farmacología , Transducción de Señal/efectos de los fármacos , Actinas/fisiología , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos , Especies Reactivas de Oxígeno , Estallido Respiratorio/fisiología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The authors have previously shown that norepinephrine (NE) pretreatment attenuates Ca2+ overloading in cardiac rat trabeculae during metabolic inhibition, and improves contractile function during a subsequent recovery period. The present study investigated: (i) whether protection of sarcoplasmic reticulum (SR) function during metabolic inhibition (MI) is involved in the preconditioning-like effect of NE-pretreatment, and (ii) whether or not this process is PKC-dependent. A 15 min preincubation period was used with 1 micromol/l exogenous NE to precondition isolated, superfused rat trabeculae against contractile dysfunctioning following 40 min of MI in 2 mmol/l NaCN containing Tyrode (gassed with 95% O2/5% CO2; pH 7.4, 24 degrees C) without glucose at 1-Hz stimulation frequency. Contractile recovery was studied during a subsequent 60 min recovery period (RP) in glucose containing Tyrode at 0.2 Hz. Force and intracellular free calcium ([Ca2+]ii) were monitored throughout the experimental protocol. Pretreatment of trabeculae with NE (group NE) substantially diminished the Ca2+ rise from the onset of rigor development during MI, compared to preparations which were pretreated with NE, in the presence of specific PKC blocker chelerythrine (2 micromol/l; group NE+CHEL). After 40 min of MI, resting [Ca2+]i in group NE and NE+CHEL was increased to 0.50+/-0.03 and 2.08+/-0.20 micromol/l, respectively (P<0.05), whereas total intracellular ATP levels were similar in both groups (approximately 0.20 micromol/g dry wt). This corresponded with an increase in active force development (119%) and a decrease in twitch force relaxation time (77%) during subsequent RP in group NE, compared to pre-MI values of the same group. In contrast, a significant decrease in force recovery (54%) and an increase in twitch force relaxation time (123%) was observed in group NE+CHEL. Values for [Ca2+]i, contractile recovery, and twitch force relaxation time in untreated controls as well as CHEL preparations corresponded to those measured in the NE+CHEL group. Rapid cooling contractures (RCCs), which provide information on both SR-Ca2+ loading and Ca2+ re-uptake activity, revealed a 2-fold higher SR Ca2+ content during RP in group NE compared to controls and group NE+CHEL. In addition, kinetic analysis of the RCC rewarming spike (RWS) showed that this was accompanied by greater than a 28% increase in the maximum rate of RWS relaxation (-dF/dt/rws) in group NE compared to group NE+CHEL. The change of -dF/dt/rws in the NE group during RP following MI persisted after SR Ca2+-release channel blockade by ryanodine treatment (100 micromol/l), which suggests involvement of NE-induced, PKC-dependent protection of SR Ca2+-ATPase activity. The results of the present study point to an inverse relationship between the Ca2+ rise during MI and SR functioning, in which PKC appears to play a key role. It is concluded that the preconditioning-like effect of NE-pretreatment on contractile recovery is at least partly mediated by protection of SR function.
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Precondicionamiento Isquémico Miocárdico , Fibras Musculares Esqueléticas/efectos de los fármacos , Norepinefrina/uso terapéutico , Proteína Quinasa C/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Homeostasis , Masculino , Fibras Musculares Esqueléticas/metabolismo , Contracción Miocárdica/efectos de los fármacos , Ratas , Ratas Wistar , Rianodina/uso terapéutico , Retículo Sarcoplasmático/metabolismoRESUMEN
We have recently shown that norepinephrine (NE) pretreatment attenuates Ca2+ overloading in cardiac rat trabeculae during metabolic inhibition (MI) with NaCN (2 mmol/l), and improves contractile recovery during a subsequent recovery period (RP). In the present study, we investigated the effects of the continuous presence of NE (1 micro mol/l), i.e. before, during and after MI, on Ca2+ homeostasis maintenance and contractile recovery in the same model at 24 degrees C. In addition, we tested the effects of NE when only present in the rigor period during MI. The continuous presence of NE both before (30 min) and during MI (120 min)+RP (60 min) (group NE-I) significantly increased the proportion of trabeculae that resumed to contract during RP from 46+/-4% (mean+/-s.e.m.) in controls to 82+/-8%. The Ca2+ rise at the end of MI in failing control trabeculae (1.85+/-0.04 micro mol/l) was more than doubled compared to recovering control preparations (0.78+/-0.02 micro mol/l). However, the time-course of the Ca2+ rise during MI in recovering and failing NE-I preparations was similar, and eventually of the same magnitude as observed in failing control preparations (1.6+/-0. 02 and 1.85+/-0.07 micro mol/l, respectively). In contrast, when NE was present only in the rigor period during MI (group NE-II) the proportion of recovering preparations decreased significantly to 27+/-9%. Similar to the control group, recovering and failing preparations in group NE-II could be distinguished by a differential course in the Ca2+ rise during MI. The results show that when NE is present both before and during MI+RP, (i) recovery probability following MI is still improved, in spite of the deleterious effect on contractile recovery of the presence of NE in the rigor during MI, and (ii) there is no relationship between the magnitude of Ca2+ overload during MI and recovery probability during RP.