RESUMEN
BACKGROUND: Ischaemia-reperfusion injury is inevitable during renal transplantation and can lead to delayed graft function and primary non-function. Preconditioning, reconditioning and postconditioning with argon and xenon protects against renal ischaemia-reperfusion injury in rodent models. The hypothesis that postconditioning with argon or xenon inhalation would improve graft function in a porcine renal autotransplant model was tested. METHODS: Pigs (n = 6 per group) underwent left nephrectomy after 60 min of warm ischaemia (renal artery and vein clamping). The procured kidney was autotransplanted in a separate procedure after 18 h of cold storage, immediately after a right nephrectomy. Upon reperfusion, pigs were randomized to inhalation of control gas (70 per cent nitrogen and 30 per cent oxygen), argon (70 per cent and 30 per cent oxygen) or xenon (70 per cent and 30 per cent oxygen) for 2 h. The primary outcome parameter was peak plasma creatinine; secondary outcome parameters included further markers of graft function (creatinine course, urine output), graft injury (aspartate aminotransferase, heart-type fatty acid-binding protein, histology), apoptosis and autophagy (western blot, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining), inflammatory mediators and markers of cell survival/growth (mRNA and tissue protein quantification), and animal survival. Results are presented as median (i.q.r.). ANOVA and Kruskal-Wallis tests were used where indicated. RESULTS: Peak plasma creatinine levels were similar between the groups: control 20·8 (16·4-23·1) mg/dl, argon 21·4 (17·1-24·9) mg/dl and xenon 19·4 (17·5-21·0) mg/dl (P = 0·607). Xenon was associated with an increase in autophagy and proapoptotic markers. Creatinine course, urine output, injury markers, histology, survival and inflammatory mediators were not affected by the intervention. CONCLUSION: Postconditioning with argon or xenon did not improve kidney graft function in this experimental model. Surgical relevance Ischaemia-reperfusion injury is inevitable during renal transplantation and can lead to delayed graft function and primary non-function. Based on mainly small animal experiments, noble gases (argon and xenon) have been proposed to minimize this ischaemia-reperfusion injury and improve outcomes after transplantation. The hypothesis that postconditioning with argon or xenon inhalation would improve graft function was tested in a porcine kidney autotransplantation model. The peak plasma creatinine concentration was similar in the control, argon and xenon groups. No other secondary outcome parameters, including animal survival, were affected by the intervention. Xenon was associated with an increase in autophagy and proapoptotic markers. Despite promising results in small animal models, postconditioning with argon or xenon in a translational model of kidney autotransplantation was not beneficial. Clinical trials would require better results.
Asunto(s)
Argón/farmacología , Supervivencia de Injerto/efectos de los fármacos , Poscondicionamiento Isquémico/métodos , Trasplante de Riñón/efectos adversos , Xenón/farmacología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Femenino , Poscondicionamiento Isquémico/efectos adversos , Riñón/fisiopatología , Riñón/cirugía , Pruebas de Función Renal/métodos , Trasplante de Riñón/métodos , Modelos Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/prevención & control , Tasa de Supervivencia , Porcinos , Trasplante Autólogo/efectos adversos , Trasplante Autólogo/métodosAsunto(s)
Angiotensina II , Aterosclerosis/metabolismo , Cardiomegalia/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/patología , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Apolipoproteínas E/metabolismo , Modelos Animales de Enfermedad , Fibrilina-1/metabolismo , Ratones , Tamaño de los Órganos , Fragmentos de Péptidos/metabolismo , Resultado del TratamientoRESUMEN
We recently reported that apolipoprotein E (ApoE)-deficient mice with a mutation in the fibrillin-1 gene (ApoE(-/-)Fbn1(C1039G+/-)) develop accelerated atherosclerosis with enhanced inflammation, atherosclerotic plaque rupture, myocardial infarction and sudden death. In the brain, fibrillin-1 functions as an attachment protein in the basement membrane, providing structural support to the blood-brain barrier (BBB). Here, we investigated whether fibrillin-1 impairment affects the permeability of the BBB proper and the blood-cerebrospinal fluid barrier (BCSFB), and whether this leads to the accelerated accumulation of lipids (xanthomas) in the brain. ApoE(-/-) (n=61) and ApoE(-/-)Fbn1(C1039G+/-) (n=73) mice were fed a Western-type diet (WD). After 14 weeks WD, a significantly higher permeability of the BBB was observed in ApoE(-/-)Fbn1(C1039G+/-) mice compared to age-matched ApoE(-/-) mice. This was accompanied by leukocyte infiltration, enhanced expression of pro-inflammatory cytokines, matrix metalloproteinases and transforming growth factor-ß, and by decreased expression of tight junction proteins claudin-5 and occludin. After 20 weeks WD, 83% of ApoE(-/-)Fbn1(C1039G+/-) mice showed xanthomas in the brain, compared to 23% of their ApoE(-/-) littermates. Xanthomas were mainly located in fibrillin-1-rich regions, such as the choroid plexus and the neocortex. Our findings demonstrate that dysfunctional fibrillin-1 impairs BBB/BCSFB integrity, facilitating peripheral leukocyte infiltration, which further degrades the BBB/BCSFB. As a consequence, lipoproteins can enter the brain, resulting in accelerated formation of xanthomas.
Asunto(s)
Apolipoproteínas E/deficiencia , Barrera Hematoencefálica/fisiopatología , Encefalopatías/patología , Encéfalo/patología , Proteínas de Microfilamentos/metabolismo , Xantomatosis/patología , Acrilamidas/metabolismo , Animales , Apolipoproteínas E/genética , Barrera Hematoencefálica/ultraestructura , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrilina-1 , Fibrilinas , Gadolinio/farmacocinética , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/metabolismo , Permeabilidad , Molécula 1 de Adhesión Celular Vascular/metabolismo , Xantomatosis/genética , beta-Alanina/análogos & derivados , beta-Alanina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: 7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects. EXPERIMENTAL APPROACH: Cultured rabbit aorta SMCs (passage 2-6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined. KEY RESULTS: NR uptake declined upon 18 h exposure to 25 microM 7-ketocholesterol (-41+/-3%, n=13), 100 microM fluvastatin (-59%) or 30-100 microM simvastatin (-28 to -74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10-30 microM, simvastatin 1-10 microM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10-100 microM, 70-545% increase) and simvastatin (3-100 microM 43-322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 microM was always inactive. CONCLUSIONS AND IMPLICATIONS: 7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/toxicidad , Cetocolesteroles/toxicidad , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Anexina A5/metabolismo , Autofagia/efectos de los fármacos , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática , Citometría de Flujo , Técnicas In Vitro , Microscopía Electrónica de Transmisión , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Miocitos del Músculo Liso/ultraestructura , NAD/metabolismo , NADP/metabolismo , Rojo Neutro , Plásmidos/genética , Conejos , Sales de Tetrazolio , TiazolesRESUMEN
Rats with experimental colitis suffer from impaired gastric emptying (GE). We previously showed that this phenomenon involves afferent neurons within the pelvic nerve. In this study, we aimed to identify the mediators involved in this afferent hyperactivation. Colitis was induced by trinitrobenzene sulfate (TNBS) instillation. We determined GE, distal front, and geometric center (GC) of intestinal transit 30 min after intragastric administration of a semiliquid Evans blue solution. We evaluated the effects of the transient receptor potential vanilloid type 1 (TRPV1) antagonists capsazepine (5-10 mg/kg) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 1-10 mg/kg) and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (150 microg/kg). To determine TRPV1 receptor antagonist sensitivity, we examined their effect on capsaicin-induced relaxations of isolated gastric fundus muscle strips. Immunocytochemical staining of TRPV1 and RT-PCR analysis of TRPV1 mRNA were performed in dorsal root ganglion (DRG) L6-S1. TNBS-induced colitis reduced GE but had no effect on intestinal motility. Capsazepine reduced GE in controls but had no effect in rats with colitis. At doses that had no effects in controls, BCTC and CGRP-(8-37) significantly improved colitis-induced gastroparesis. Capsazepine inhibited capsaicin-induced relaxations by 35% whereas BCTC completely abolished them. TNBS-induced colitis increased TRPV1-like immunoreactivity and TRPV1 mRNA content in pelvic afferent neuronal cell bodies in DRG L6-S1. In conclusion, distal colitis in rats impairs GE via sensitized pelvic afferent neurons. We provided pharmacological, immunocytochemical, and molecular biological evidence that this sensitization is mediated by TRPV1 receptors and involves CGRP release.
Asunto(s)
Colitis/fisiopatología , Ganglios Espinales/metabolismo , Motilidad Gastrointestinal , Gastroparesia/etiología , Intestinos/inervación , Neuronas Aferentes/metabolismo , Transducción de Señal , Canales Catiónicos TRPV/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/farmacología , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Capsaicina/análogos & derivados , Capsaicina/farmacología , Colitis/inducido químicamente , Colitis/complicaciones , Colitis/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiopatología , Vaciamiento Gástrico , Motilidad Gastrointestinal/efectos de los fármacos , Gastroparesia/metabolismo , Gastroparesia/fisiopatología , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Intestinos/fisiopatología , Masculino , Relajación Muscular , Neuronas Aferentes/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Pirazinas/farmacología , Piridinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Transducción de Señal/efectos de los fármacos , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Factores de Tiempo , Ácido TrinitrobencenosulfónicoRESUMEN
BACKGROUND AND PURPOSE: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism. EXPERIMENTAL APPROACH: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis. KEY RESULTS: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin. CONCLUSIONS AND IMPLICATIONS: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.
Asunto(s)
Aterosclerosis/prevención & control , Retículo Endoplásmico/efectos de los fármacos , Macrófagos/efectos de los fármacos , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/fisiología , Animales , Apoptosis/efectos de los fármacos , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Macrófagos/patología , Macrófagos/ultraestructura , Ratones , Microscopía Electrónica , Molsidomina/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/metabolismo , Penicilamina/análogos & derivados , Penicilamina/metabolismo , Penicilamina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermina/análogos & derivados , Espermina/metabolismo , Espermina/farmacología , Tapsigargina/farmacología , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Tunicamicina/farmacologíaRESUMEN
MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.
Asunto(s)
Autofagia/fisiología , Macrófagos/fisiología , Fagocitos/fisiología , Anoicis/efectos de los fármacos , Anoicis/fisiología , Autofagia/efectos de los fármacos , Autofagia/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/fisiología , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Immunoblotting , Macrófagos/citología , Macrófagos/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Fagocitos/citología , Fagocitos/metabolismo , Fagocitosis/efectos de los fármacos , Fosfatidilserinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tamoxifeno/farmacología , Transcripción GenéticaRESUMEN
Macrophage activation in atherosclerotic plaques plays a role in plaque destabilization, rupture and subsequent atherothrombosis. Platelet phagocytosis that occurs within human atherosclerotic plaques can activate macrophages and it has been suggested that the platelet constituent amyloid precursor protein (APP) is involved. Recent studies show that amyloid beta (Abeta), a peptide extensively studied in Alzheimer's disease and that is cleaved from APP by beta- and gamma-secretase, and/or Abeta-like peptides are also present in human atherosclerotic plaques, in particular in activated, inducible nitric oxide synthase (iNOS) expressing perivascular macrophages that had phagocytized platelets. In vitro studies confirm that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to Abeta-like peptides, resulting in iNOS induction. In addition, non-steroidal anti-inflammatory drugs (NSAIDs) and HMG-CoA reductase inhibitors (statins), two classes of drugs reported to affect APP processing and Abeta formation in Alzheimer's disease, have been evaluated for their capacity to inhibit macrophage activation evoked by platelet phagocytosis. Remarkably, the same NSAIDs reported to alter gamma-secretase activity in Alzheimer's disease also reduce macrophage activation after platelet phagocytosis and inhibit formation of Abeta-containing peptides. From the statins investigated (fluvastatin, atorvastatin, simvastatin, pravastatin, lovastatin and rosuvastatin) only fluvastatin and atorvastatin selectively inhibit macrophage activation after platelet phagocytosis, possibly through inhibition of Rho activity. Taken together, these new findings point to the involvement of platelet-derived APP in macrophage activation in atherosclerosis and suggest a biochemical link between atherosclerosis and Alzheimer's disease. Accordingly, drugs interfering with APP processing might have an impact on both diseases.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Aterosclerosis/metabolismo , Endopeptidasas/metabolismo , Enfermedad de Alzheimer/etiología , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas , Aterosclerosis/etiología , Humanos , Factores de RiesgoRESUMEN
BACKGROUND: Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. MATERIALS AND METHODS: The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. RESULTS: Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. CONCLUSION: Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking.
Asunto(s)
Arteriosclerosis/genética , ARN/genética , Especies Reactivas de Oxígeno/metabolismo , Estenosis Carotídea/genética , Humanos , Inmunohistoquímica/métodos , Oxidación-Reducción , Estrés Oxidativo/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 28S/genéticaRESUMEN
An increasing body of evidence from both animal models and human specimens suggests that apoptosis or programmed cell death is a major event in the pathophysiology of atherosclerosis. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture and thrombus formation. In this study, we have outlined some of our most recent results concerning initiation of apoptosis in atherosclerosis with a special focus on oxidative DNA and RNA damage. Furthermore, we provide a detailed picture of the pro- and anti-apoptotic genes/proteins that are involved during the initiation of cell death in atherosclerotic plaques by using a combination of established immunohistochemical stainings and recent molecular biology techniques. Our data suggest that smooth muscle cells and macrophages in atherosclerotic plaques express a different panel of apoptosis-related genes in response to proapoptotic stimuli. Although this may seem a promising starting-point for the development of anti-atherogenic drugs, it remains to be determined whether modulation of apoptosis can become a clinically important approach to influence plaque progression.
Asunto(s)
Apoptosis/genética , Arteriosclerosis/fisiopatología , Estrés Oxidativo/fisiología , Animales , Apoptosis/fisiología , Arteriosclerosis/patología , Caspasa 2 , Caspasas/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Regulación de la Expresión Génica , Humanos , ARN/metabolismo , Regulación hacia ArribaRESUMEN
An increasing body of evidence from both animal models and human specimens suggests that apoptosis or programmed cell death is a major event in the pathophysiology of atherosclerosis. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture and thrombus formation. Biochemical and genetic analyses of apoptosis provide an increasingly detailed picture of the intracellular signaling pathways involved. Nevertheless, it remains to be determined whether apoptosis can become a clinically important approach to modulate plaque progression. In this review, we have outlined some of the most recent results concerning apoptosis in atherosclerosis with a special focus on oxidized lipids, inflammation and therapeutic regulation of the apoptotic cell death process.
Asunto(s)
Apoptosis/fisiología , Arteriosclerosis/fisiopatología , Inflamación/fisiopatología , Lipoproteínas LDL/metabolismo , Estrés Oxidativo/fisiología , Animales , Apoptosis/efectos de los fármacos , Células Espumosas/fisiología , Humanos , Metabolismo de los Lípidos , Lipoproteínas LDL/farmacología , TerapéuticaRESUMEN
OBJECTIVE: Progressive loss of cardiomyocytes is one of the most important pathogenic characteristics of heart failure. Apoptosis may be an important mode of cell death in heart failure but it must be demonstrated by multiple criteria and not just TUNEL staining alone. Previously, we and others have demonstrated that besides apoptosis other phenomena like active gene transcription can result in TUNEL positivity. Moreover, other types of cell death that are caspase-independent could be important in heart failure. This study examined the hypothesis whether TUNEL labeling parallels caspase activation. METHODS: Cardiac tissue of patients in the terminal stage of heart failure as a consequence of ischaemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) were studied. Embryonic mice hearts were used for positive control for detection of the classical apoptosis. RESULTS: In mice embryonic hearts we could clearly find apoptotic cell death detected by TUNEL labeling and immunohistochemistry for activated caspase-3. In heart failure, TUNEL-positive cardiomyocytes were negative for active caspase-3 but showed signs of active gene transcription (SC-35). However, autophagic cell death could be found in 0.3% of the cardiomyocytes. Autophagic cell death was demonstrated by granular cytoplasmic ubiquitin inclusions, an established marker of autophagocytosis in neurons. Interestingly, these autophagic cardiomyocytes were TUNEL and activated caspase-3 negative but were also negative for C9, a marker for necrosis. Western blot analysis confirmed that in cardiomyopathies no cleavage of caspase-3 and caspase-7 occurred. CONCLUSION: The present study demonstrates two fundamentally different situations of cell death in cardiac tissue. In embryonic mice, cardiomyocytes undergo caspase-dependent cell death. However, cardiomyocytes in heart failure show caspase-independent autophagic cell death rather than apoptotic cell death.
Asunto(s)
Cardiomiopatía Dilatada/patología , Isquemia Miocárdica/patología , Miocardio/patología , Animales , Apoptosis , Cardiomiopatía Dilatada/enzimología , Estudios de Casos y Controles , Caspasas/metabolismo , Muerte Celular , Fragmentación del ADN , Activación Enzimática , Corazón Fetal/enzimología , Corazón Fetal/patología , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Persona de Mediana Edad , Isquemia Miocárdica/enzimología , Miocardio/enzimología , ARN Nuclear Pequeño/metabolismoRESUMEN
Increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis. The oxidative environment is mainly created by the production of reactive oxygen species, which are assumed to mediate vascular tissue injury. Oxidative DNA damage resulting from free radical attack remains, however, a poorly examined field in atherosclerosis. Male New Zealand White rabbits were fed a cholesterol-rich diet (0.3%) for 24 weeks. The induced atherosclerotic plaques showed elevated levels of the DNA damage marker 7,8-dihydro-8-oxoguanine (8-oxoG) as demonstrated by immunohistochemistry. 8-oxoG immunoreactivity was found predominantly in the superficial layer of the plaque containing numerous macrophage-derived foam cells but not in the media or in arteries of age-matched control animals. Alkaline single-cell gel electrophoresis revealed that the number of DNA strand breaks was significantly higher in the plaque as compared with control samples of normolipemic animals. These changes were associated with the upregulation of DNA repair enzymes (poly[ADP-ribose] polymerase-1, p53, phospho-p53 [phosphorylated at Ser392], and XRCC1 [x-ray repair cross-complementing 1]). DNA strand breaks normalized after 4 weeks of dietary lipid lowering. However, a significant reduction of 8-oxoG immunoreactivity was only observed after a prolonged period of lipid lowering (12 to 24 weeks). Repair pathways started to decline progressively when cholesterol-fed animals were placed on a normal diet. In conclusion, oxidative DNA damage and increased levels of DNA repair, both associated with diet-induced hypercholesterolemia, are strongly reduced during dietary lipid lowering. These findings may provide a better insight into the benefits of lipid-lowering therapy on plaque stabilization.
Asunto(s)
Arteriosclerosis/dietoterapia , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Grasas de la Dieta/farmacología , Estrés Oxidativo , Animales , Aorta/metabolismo , Aorta/patología , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Western Blotting , Colesterol/sangre , Colesterol/metabolismo , Colesterol/farmacología , Ensayo Cometa , ADN/metabolismo , ADN Ligasas/metabolismo , Dieta Aterogénica , Grasas de la Dieta/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Lípidos/sangre , Masculino , Conejos , Resultado del TratamientoRESUMEN
To develop better molecular genetic tools for the diploid yeast Candida albicans, the suitability of the MET15 gene as a visual selection marker was studied. Both MET15 alleles of C. albicans CAI-4 were isolated by functional complementation of a Saccharomyces cerevisiae strain lacking the MET15 gene. Growth of this complemented strain on Pb(2+)-containing medium was associated with a colour shift of brown into white colonies. The MET15 alleles of C. albicans were located on chromosome 4 by pulsed-field gel electrophoresis and Southern blotting. A met15-deficient strain of C. albicans CAI-4 was generated using the ura blaster technique. This strain showed a brown colony colour on Pb(2+)-containing medium, which corresponded with the colony colour of a S. cerevisiae strain lacking the MET15 gene. Unexpectedly, the met15-deficient strain of C. albicans still grew on methionine-depleted medium. However, this growth was severely delayed. In addition, complementation of this strain with an integrative or replicative plasmid containing either of the MET15 alleles resulted in the formation of white transformants on Pb(2+)-containing medium. These transformants grew very well on methionine-depleted medium. Colony sectoring was obtained with the replicative plasmid and not with the integrative one. This study demonstrates that the MET15 gene of C. albicans is suitable as a visual marker and therefore can be used to identify transformants and study plasmid stability. GenBank Accession Nos for MET15 nucleotide sequences are AF188273, AF188274 and AF188275.
Asunto(s)
Candida albicans/genética , Proteínas Fúngicas/genética , Complejos Multienzimáticos , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores , Candida albicans/enzimología , Candida albicans/crecimiento & desarrollo , Cisteína Sintasa , ADN Complementario/química , ADN Complementario/genética , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia de ADNRESUMEN
A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.
Asunto(s)
Clonación Molecular , Manosidasas/genética , Manosidasas/metabolismo , Trichoderma/enzimología , Secuencia de Aminoácidos , Aspergillus/enzimología , ADN Complementario , Manosidasas/química , Factor de Apareamiento , Datos de Secuencia Molecular , Penicillium/enzimología , Péptidos/genética , Pichia/enzimología , Pichia/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión , Proteínas Recombinantes , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.
Asunto(s)
Hemaglutininas Virales/inmunología , Inmunización , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/inmunología , Oxidorreductasas de Alcohol/genética , Amidohidrolasas/metabolismo , Animales , Vectores Genéticos/genética , Glicosilación , Hemaglutininas Virales/genética , Vacunas contra la Influenza/biosíntesis , Factor de Apareamiento , Ratones , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Péptidos/genética , Pichia/genética , Pichia/metabolismo , Polisacáridos/química , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Saccharomyces cerevisiae/genética , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Current influenza vaccines require repeated administration for long-term protection. Failure to develop broad-spectrum vaccines may be attributed to the chronic presentation of hypervariable, immunodominant epitopes displayed on the viral surface that keep the immune response somewhat fixed and limited by suppression of broadly neutralizing, low-titered antibodies. To test this hypothesis, we have attempted to dampen the immunogenicity of variable epitopes and potential immunodominant domains of the A/Victoria/3/75 (H3N2) neuraminidase by site-directed mutagenesis. The results suggest that the neuraminidase structure is extremely flexible, since many substitution combinations were tolerated, and constitute proof-of-principle that the antigenicity of this protein can be modulated to a large extent. However, mice immunized with neuraminidase mutants containing multiple amino acid substitutions showed a reduced protection rate against heterologous virus in comparison with the reference groups.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Neuraminidasa/inmunología , Vacunas Sintéticas/inmunología , Animales , Reacciones Cruzadas , Inmunización , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , SpodopteraRESUMEN
The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues. N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment. Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.