RESUMEN
Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study, we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localize to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52 to 53, localized to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four-and-half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin, and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in the maintenance and/or regulation of sarcomeric organization and function.
Asunto(s)
Conectina , Proteínas con Dominio LIM , Proteínas Musculares , Miocitos Cardíacos , Proteínas del Tejido Nervioso , Proteínas Nucleares , Sarcómeros , Animales , Humanos , Ratones , Ratas , Conectina/metabolismo , Conectina/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Homeodominio LIM , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Miocitos Cardíacos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Sarcómeros/metabolismo , Factores de TranscripciónRESUMEN
BACKGROUND: There is compelling evidence implicating dysregulated inflammation in the mechanism of ventricular remodeling and heart failure (HF) after MI. The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2, encoded by Nfe2l2) is a promising target in this context since it impedes transcriptional upregulation of pro-inflammatory cytokines and is anti-inflammatory in various murine models. OBJECTIVES: We aimed to investigate the contribution of Nrf2 to the inflammatory response after experimental myocardial infarction (MI). METHODS: We subjected Nrf2-/- mice and wild type (WT) controls to permanent left coronary artery (LCA) ligation. The inflammatory response was investigated with fluorescence-activated cell sorting (FACS) analysis of peripheral blood and heart cell suspensions, together with qRT-PCR of infarcted tissue for chemokines and their receptors. To investigate whether Nrf2-mediated transcription is a dedicated function of leukocytes, we interrogated publicly available RNA-sequencing (RNA-seq) data from mouse hearts after permanent LCA ligation for Nrf2-regulated gene (NRG) expression. RESULTS: FACS analysis demonstrated a profoundly inflamed phenotype in the hearts of global Nrf2-/- mice as compared to WT mice after MI. Moreover, infarcted tissue from Nrf2-/- mice displayed higher expression of mRNA coding for inflammatory cytokines, chemokines, and their receptors, including IL-6, Ccl2, and Cxcr4. RNA-seq analysis showed upregulated NRG expression in WT mice after MI compared to naive mice, which was significantly higher in bioinformatically isolated CCR2+ cells. CONCLUSIONS: Taken together, the results suggest that Nrf2 signalling in leukocytes, and possibly CCR2+ monocytes and monocyte-derived cardiac resident macrophages, may be potential targets to prevent post-MI ventricular remodeling.
Asunto(s)
Infarto del Miocardio , Factor 2 Relacionado con NF-E2/metabolismo , Remodelación Ventricular , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Factor 2 Relacionado con NF-E2/genética , Remodelación Ventricular/fisiologíaRESUMEN
OBJECTIVE: The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. CONCLUSIONS: These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.
Asunto(s)
Fibroblastos/enzimología , Hipertensión/enzimología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasa 2/metabolismo , Comunicación Paracrina , Remodelación Vascular , Angiotensina II , Animales , Aorta/metabolismo , Aorta/patología , Aorta/fisiopatología , Presión Sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Factor 6 de Diferenciación de Crecimiento/genética , Factor 6 de Diferenciación de Crecimiento/metabolismo , Hipertensión/inducido químicamente , Hipertensión/genética , Hipertensión/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , NADPH Oxidasa 2/genética , Transducción de SeñalRESUMEN
Alterations in the metabolism of substrates such as glucose are integrally linked to the structural and functional changes that occur in the remodeling heart. Assessment of such metabolic changes under in vivo conditions would provide important insights into this interrelationship. We aimed to investigate glucose carbon metabolism in pressure-overload and volume-overload cardiac hypertrophy by using an in vivo [U-13C]glucose labeling strategy to enable analyses of the metabolic fates of glucose carbons in the mouse heart. Therefore, [U-13C]glucose was administered in anesthetized mice by tail vein infusion, and the optimal duration of infusion was established. Hearts were then excised for 13C metabolite isotopomer analysis by NMR spectroscopy. [U-13C]glucose infusions were performed in mice 2 wk following transverse aortic constriction (TAC) or aortocaval fistula (Shunt) surgery. At this time point, there were similar increases in left ventricular (LV) mass in both groups, but TAC resulted in concentric hypertrophy with impaired LV function, whereas Shunt caused eccentric hypertrophy with preserved LV function. TAC was accompanied by significant changes in glycolysis, mitochondrial oxidative metabolism, glucose metabolism to anaplerotic substrates, and de novo glutamine synthesis. In contrast to TAC, hardly any metabolic changes could be observed in the Shunt group. Taken together, in vivo [U-13C]glucose labeling is a valuable method to investigate the fate of nutrients such as glucose in the remodeling heart. We find that concentric and eccentric cardiac remodeling are accompanied by distinct differences in glucose carbon metabolism.NEW & NOTEWORTHY This study implemented a method for assessing the fate of glucose carbons in the heart in vivo and used this to demonstrate that pressure and volume overload are associated with distinct changes. In contrast to volume overload, pressure overload-induced changes affect the tricarboxylic acid cycle, glycolytic pathways, and glutamine synthesis. A better understanding of cardiac glucose metabolism under pathological conditions in vivo may provide new therapeutic strategies specific for different types of hemodynamic overload.
Asunto(s)
Glucemia/metabolismo , Metabolismo Energético , Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Isótopos de Carbono , Ciclo del Ácido Cítrico , Modelos Animales de Enfermedad , Glucólisis , Hipertrofia Ventricular Izquierda/fisiopatología , Cinética , Ácido Láctico/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice. METHODS AND RESULTS: We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate. CONCLUSION: These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.
Asunto(s)
Diástole/fisiología , Ecocardiografía , Cardiopatías/diagnóstico por imagen , Cardiopatías/fisiopatología , Animales , Cardiomegalia/complicaciones , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Modelos Animales de Enfermedad , Cardiopatías/complicaciones , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Ratones Endogámicos C57BL , Variaciones Dependientes del Observador , Presión , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Volumen Sistólico , Sístole/fisiología , Tapsigargina/farmacologíaRESUMEN
BACKGROUND: Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. METHODS: We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. RESULTS: Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. CONCLUSIONS: We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.
Asunto(s)
Presión Sanguínea/fisiología , Células Endoteliales/enzimología , Hipertensión/enzimología , Glicoproteínas de Membrana/deficiencia , Células Mieloides/enzimología , NADPH Oxidasas/deficiencia , Angiotensina II/toxicidad , Animales , Presión Sanguínea/efectos de los fármacos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Células Endoteliales/efectos de los fármacos , Hipertensión/inducido químicamente , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/efectos de los fármacos , NADPH Oxidasa 2RESUMEN
BACKGROUND: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling. METHODS AND RESULTS: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT. CONCLUSIONS: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling.
Asunto(s)
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Infarto del Miocardio/etiología , Infarto del Miocardio/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Fibrosis , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Hemodinámica , Ratones , Ratones Transgénicos , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , NADPH Oxidasa 2 , Especificidad de Órganos/genética , Especies Reactivas de Oxígeno/metabolismo , Disfunción Ventricular Izquierda , Remodelación VentricularRESUMEN
BACKGROUND: Bone marrow transplantation (BMT) is commonly used in experimental studies to investigate the contribution of BM-derived circulating cells to different disease processes. During studies investigating the cardiac response to acute myocardial infarction (MI) induced by permanent coronary ligation in mice that had previously undergone BMT, we found that BMT itself affects the remodelling response. METHODS AND RESULTS: Compared to matched naive mice, animals that had previously undergone BMT developed significantly less post-MI adverse remodelling, infarct thinning and contractile dysfunction as assessed by serial magnetic resonance imaging. Cardiac rupture in male mice was prevented. Histological analysis showed that the infarcts of mice that had undergone BMT had a significantly higher number of inflammatory cells, surviving cardiomyocytes and neovessels than control mice, as well as evidence of significant haemosiderin deposition. Flow cytometric and histological analyses demonstrated a higher number of alternatively activated (M2) macrophages in myocardium of the BMT group compared to control animals even before MI, and this increased further in the infarcts of the BMT mice after MI. CONCLUSIONS: The process of BMT itself substantially alters tissue macrophage phenotype and the subsequent response to acute MI. An increase in alternatively activated macrophages in this setting appears to enhance cardiac recovery after MI.
Asunto(s)
Trasplante de Médula Ósea , Rotura Cardíaca/prevención & control , Macrófagos/patología , Infarto del Miocardio/patología , Recuperación de la Función , Animales , Vasos Coronarios , Diástole , Femenino , Rotura Cardíaca/metabolismo , Rotura Cardíaca/mortalidad , Rotura Cardíaca/patología , Hemosiderina/metabolismo , Ligadura , Activación de Macrófagos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Infarto del Miocardio/mortalidad , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fenotipo , Volumen Sistólico , Análisis de Supervivencia , SístoleRESUMEN
Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodeling. As Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesized that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. We generated a potentially novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2fl/flFoxP3Cre+ mice) and transplanted with hearts from CB6F1 donors. As compared with those of littermate controls, Nox2fl/flFoxP3Cre+ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates, and diminished cardiomyocyte necrosis and allograft fibrosis. Single-cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8+ T cell infiltration in Nox2-deficient recipients compared with Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8+CD25- T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR-214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy.
Asunto(s)
Aloinjertos/inmunología , Rechazo de Injerto/inmunología , Trasplante de Corazón , NADPH Oxidasa 2/genética , Linfocitos T Reguladores/inmunología , Aloinjertos/metabolismo , Aloinjertos/patología , Animales , Linfocitos T CD8-positivos/fisiología , Movimiento Celular , Proliferación Celular , Femenino , Fibrosis , Rechazo de Injerto/sangre , Interferón gamma/metabolismo , Isoanticuerpos/sangre , Masculino , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Miocitos Cardíacos/patología , Necrosis , Receptores CCR4/metabolismo , Receptores CCR8/metabolismo , Linfocitos T Reguladores/metabolismo , Trasplante Homólogo , Troponina I/sangreRESUMEN
AIMS: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. METHODS AND RESULTS: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 µm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A. CONCLUSION: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.
Asunto(s)
Hipertrofia Ventricular Izquierda/enzimología , Miocitos Cardíacos/enzimología , NADPH Oxidasa 4/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Derivación Arteriovenosa Quirúrgica , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Fibrosis , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/patología , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 4/genética , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteína S6 Ribosómica/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
ABSTRACT: Leukocyte Nox2 is recognized to have a fundamental microbicidal function in sepsis but the specific role of Nox2 in endothelial cells (EC) remains poorly elucidated. Here, we tested the hypothesis that endothelial Nox2 participates in the pathogenesis of systemic inflammation and hypotension induced by LPS. LPS was injected intravenously in mice with Tie2-targeted deficiency or transgenic overexpression of Nox2. Mice with Tie2-targeted Nox2 deficiency had increased circulating levels of TNF-α, enhanced numbers of neutrophils trapped in lungs, and aggravated hypotension after LPS injection, as compared to control LPS-injected animals. In contrast, Tie2-driven Nox2 overexpression attenuated inflammation and prevented the hypotension induced by LPS. Because Tie2-Cre targets both EC and myeloid cells we generated bone marrow chimeric mice with Nox2 deletion restricted to leukocytes or ECs. Mice deficient in Nox2 either in leukocytes or ECs had reduced LPS-induced neutrophil trapping in the lungs and lower plasma TNF-α levels as compared to control LPS-injected mice. However, the pronounced hypotensive response to LPS was present only in mice with EC-specific Nox2 deletion. Experiments in vitro with human vein or aortic endothelial cells (HUVEC and HAEC, respectively) treated with LPS revealed that EC Nox2 controls NF-κB activation and the transcription of toll-like receptor 4 (TLR4), which is the recognition receptor for LPS. In conclusion, these results suggest that endothelial Nox2 limits NF-κB activation and TLR4 expression, which in turn attenuates the severity of hypotension and systemic inflammation induced by LPS.
Asunto(s)
Células Endoteliales/fisiología , Endotoxemia/etiología , Hipotensión/etiología , Inflamación/etiología , NADPH Oxidasa 2/fisiología , Receptor Toll-Like 4/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Hydrodynamic gene delivery to the liver has potential as a safe and effective approach for clinical liver gene therapy. However, the simplicity of the technique in rodents - an intravenous injection - belies the theoretical and practical complexity for clinical application. A key issue is that outflow obstruction of the DNA solution from the liver is a critical factor for raising intrahepatic vascular pressure, which in turn provides the force to swell the liver and effect gene delivery. For conventional hydrodynamic gene delivery via tail vein injection, this outflow obstruction is provided naturally by the vascular resistance of the gut, spleen and pancreas. For regional hydrodynamic gene delivery to the liver, outflow obstruction to create a closed system requires surgical intervention, making it unlikely that minimally invasive techniques will be possible in the clinic. Intrinsic factors, in particular compliance (elasticity) of the liver are likely to be crucial in determining the degree of swelling for a given level of intrahepatic vascular pressure. Liver compliance is likely to be the major reason for the low level of hydrodynamic gene delivery in the pig model, and will influence the effectiveness of the approach in man, both in general and in different disease states.
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Terapia Genética/métodos , Hígado/metabolismo , Animales , Vectores Genéticos/genética , Humanos , Modelos Animales , Transducción GenéticaRESUMEN
BACKGROUND: The mitochondrial unfolded protein response (UPRmt) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear. OBJECTIVES: The purpose of this study was to: 1) identify conditions that activate the UPRmt in the heart; and 2) study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function. METHODS: Cultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis. RESULTS: Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro-B-type natriuretic peptide). CONCLUSIONS: These results identify the induction of the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.
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Hemodinámica , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/fisiopatología , Apoptosis , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Ratones , Contracción Miocárdica/fisiología , Transducción de SeñalRESUMEN
BACKGROUND: Clinical application of hydrodynamic gene delivery to the liver requires the use of small volumes, an evaluation of the cardiovascular consequences of acute volume overload, and a better understanding of the intrahepatic vascular pressures driving gene delivery. Injection of DNA solution into the isolated segment of inferior vena cava (IVC) draining the hepatic veins is a potentially valuable low-volume approach. METHODS: Various volumes of DNA solution (pGL3 plasmid) were injected at 100 ml/min either systemically or into the isolated IVC segment in the DA rat. Arterial pressure, portal venous pressure, heart rate and electrocardiogram, in addition to reporter gene expression in the liver, were monitored. RESULTS: The 2% volume was > 10 000-fold more effective when delivered via the IVC segment than when given systemically, and as effective as 6% systemically. Isolation of the IVC segment caused profound falls in arterial pressure, with electrocardiogram signs of myocardial ischemia. On release of the IVC ties, without DNA infusion (no volume overload), arterial pressure recovered rapidly. However, with DNA infusion (volume overload) there was a brief recovery of arterial pressure, followed by complete heart block and fall in arterial pressure and pulse for several minutes. Portal venous pressure rose steeply to 30-33 mm Hg during the infusion. CONCLUSIONS: The IVC segment approach enables excellent gene delivery to the whole liver with small volumes, but causes severe cardiovascular disturbances in the rat. Portal venous pressures are slightly higher than in the mouse, and suggest functional outflow obstruction by the capillary bed of the intestines.
Asunto(s)
Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , ADN/administración & dosificación , Terapia Genética/métodos , Hígado/metabolismo , Vena Cava Inferior , Animales , Presión Sanguínea , Electrocardiografía , Terapia Genética/efectos adversos , Frecuencia Cardíaca , Infusiones Intravenosas/efectos adversos , Infusiones Intravenosas/métodos , Hígado/irrigación sanguínea , Masculino , Ratas , Ratas EndogámicasRESUMEN
The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II-induced (Ang II-induced) pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline, whereas Ang II-induced effector T cell (Teff) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of Ang II-induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 antibody depletion of Tregs. Mechanistically, Nox2-/y Tregs showed higher in vitro suppression of Teff proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on Ang II-induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling.
Asunto(s)
Angiotensina II/metabolismo , NADPH Oxidasa 2/metabolismo , Linfocitos T Reguladores/metabolismo , Remodelación Vascular/fisiología , Traslado Adoptivo , Angiotensina II/administración & dosificación , Angiotensina II/toxicidad , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Hipertensión/inmunología , Hipertensión/metabolismo , Hipertensión/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Cardiovasculares , Miocardio/inmunología , Miocardio/metabolismo , Miocardio/patología , NADPH Oxidasa 2/deficiencia , NADPH Oxidasa 2/genética , FN-kappa B/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Remodelación Vascular/efectos de los fármacos , Remodelación Vascular/inmunologíaRESUMEN
Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Homocisteína/metabolismo , Hepatopatías/prevención & control , Hígado/metabolismo , NADPH Oxidasas/fisiología , Animales , Betaína/metabolismo , Western Blotting , Células Cultivadas , Cisteína/metabolismo , Femenino , Glutatión/metabolismo , Células Hep G2 , Humanos , Técnicas para Inmunoenzimas , Hígado/efectos de los fármacos , Hígado/patología , Hepatopatías/etiología , Metionina/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 4 , Especies Reactivas de Oxígeno/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Transdifferentiation in vivo is an attractive option for autologous replacement of pancreatic ß cells in patients with type 1 diabetes. It has been achieved by adenoviral delivery of genes for transcription factors in the liver and pancreas of hyperglycaemic mice. However, these viral approaches are not clinically applicable. We used the hydrodynamic approach to deliver genes Pdx1, Ngn3 (Neurog3) and MafA singly and in combination to livers of normoglycaemic rats. Five expression plasmids were evaluated. Livers were removed 1, 3, 7, 14 and 28 days after gene delivery and assayed by quantitative PCR, semi-quantitative PCR and immunohistology. Functional studies on hyperglycaemic rats were performed. The highest and most sustained expression was from a CpG-depleted plasmid (pCpG) and a plasmid with an in-frame scaffold/matrix attachment region ((pEPI(CMV)). When Pdx1, Ngn3 and MafA were delivered together to normoglycaemic rats with these plasmids, insulin mRNA was detected at all time points and was ~50-fold higher with pCpG. Insulin mRNA content of livers at days 3 and 7 was equivalent to that of a pancreas, with scattered insulin-positive cells detected by immunohistology, but levels declined thereafter. Prohormone convertase 1/3 was elevated at days 3 and 7. In hyperglycaemic rats, fasting blood glucose was lower at days 1, 3 and 7 but not thereafter, and body weight was maintained to day 28. We conclude that hydrodynamic gene delivery of multiple transcription factors to rat liver can initiate transdifferentiation to pancreatic ß cells, but the process is reversible and probably requires more sustained transcription factor expression.
Asunto(s)
Diferenciación Celular/genética , Diabetes Mellitus Tipo 1/terapia , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Células Secretoras de Insulina/citología , Hígado/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diabetes Mellitus Experimental/terapia , Proteínas de Homeodominio/genética , Hiperglucemia/terapia , Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Hígado/fisiología , Factores de Transcripción Maf de Gran Tamaño/genética , Masculino , Proteínas del Tejido Nervioso/genética , Páncreas/citología , Páncreas/fisiología , Plásmidos/genética , Ratas , Ratas Endogámicas , Transactivadores/genética , Transcripción Genética/genéticaRESUMEN
Hydrodynamic gene delivery to the liver is a promising approach for liver gene therapy in the clinic, but levels of gene expression in larger species have been much less than in rodents. The development of surgical techniques for pressurizing individual liver segments and the establishment of whether hepatic vascular anatomy in fact permits pressurization of individual segments are critical issues that need to be addressed. We have evaluated these issues using hydrodynamic delivery to individual segments of the pig liver, via branches of both portal and hepatic veins. Our objective was to develop surgical techniques that achieve elevated vascular pressures within individual liver segments with small volumes, but without interruption of portal blood flow or reduction in venous return to the heart. We report that, without specific surgical interventions to obstruct outflow of DNA solution from the targeted liver segment, little or no increase in intrahepatic vascular pressure occurs. We demonstrate, for the first time, that selective pressurization of individual liver segments is possible without compromising portal venous flow or venous return to the heart. Thus, hydrodynamic gene delivery to individual liver segments is technically achievable in a clinical setting, but will require open abdominal surgery rather than minimally invasive techniques.
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Técnicas de Transferencia de Gen , Hidrodinámica , Hígado/fisiología , Hígado/cirugía , Animales , Presión Sanguínea , ADN/administración & dosificación , Femenino , Fluoroscopía , Expresión Génica , Terapia Genética , Luciferasas/análisis , Luciferasas/genética , Plásmidos/metabolismo , Vena Porta/metabolismo , PorcinosRESUMEN
Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.
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Expresión Génica , Genes Reporteros , Mucosa Intestinal/metabolismo , Lactasa/metabolismo , beta-Galactosidasa/genética , Animales , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Humanos , Lactasa/administración & dosificación , Lactasa/genética , Hígado/metabolismo , Ratones , Regiones Promotoras Genéticas , Ratas , Transfección , beta-Galactosidasa/metabolismoRESUMEN
Xenogeneic anti-lymphocyte serum (ALS) remains a major reagent for immunosuppression in clinical practice, but mechanisms of action and risks of opportunistic infection have not been considered in the context of innate immunity and its role in immune responsiveness. Rabbit anti rat ALS was administered intraperitoneally. Blood was taken for flow cytometry to establish absolute counts of leucocyte subsets. Tissues were harvested for immunohistology to evaluate interstitial dendritic cells and tissue macrophages. At day 2 of ALS therapy, T cells are completely depleted, as anticipated. B cells are undiminished and form approximately 90% of blood leucocytes. Monocytes and natural killer (NK) cells are substantially (approximately 80%), but not completely, depleted, and there is a trend for diminished numbers of putative dendritic cells. Neither interstitial dendritic cells nor tissue macrophages in heart are affected. The results at day 7 were very similar to day 2. Substantial depletion of blood monocytes and NK cells might attenuate the innate immune system, and represent a possible supplementary mechanism (in addition to T cell depletion) for suppression of rejection. It might be of particular importance in reducing defences against infections. Monitoring these parameters could be of clinical value.