RESUMEN
During newborn lung injury, excessive activity of lysyl oxidases (LOXs) disrupts extracellular matrix (ECM) formation. Previous studies indicate that TGFß activation in the O2-injured mouse pup lung increases lysyl oxidase (LOX) expression. But how TGFß regulates this, and whether the LOXs generate excess pulmonary aldehydes are unknown. First, we determined that O2-mediated lung injury increases LOX protein expression in TGFß-stimulated pup lung interstitial fibroblasts. This regulation appeared to be direct; this is because TGFß treatment also increased LOX protein expression in isolated pup lung fibroblasts. Then using a fibroblast cell line, we determined that TGFß stimulates LOX expression at a transcriptional level via Smad2/3-dependent signaling. LOX is translated as a pro-protein that requires secretion and extracellular cleavage before assuming amine oxidase activity and, in some cells, reuptake with nuclear localization. We found that pro-LOX is processed in the newborn mouse pup lung. Also, O2-mediated injury was determined to increase pro-LOX secretion and nuclear LOX immunoreactivity particularly in areas populated with interstitial fibroblasts and exhibiting malformed ECM. Then, using molecular probes, we detected increased aldehyde levels in vivo in O2-injured pup lungs, which mapped to areas of increased pro-LOX secretion in lung sections. Increased activity of LOXs plays a critical role in the aldehyde generation; an inhibitor of LOXs prevented the elevation of aldehydes in the O2-injured pup lung. These results reveal new mechanisms of TGFß and LOX in newborn lung disease and suggest that aldehyde-reactive probes might have utility in sensing the activation of LOXs in vivo during lung injury.
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Aldehídos/metabolismo , Lesión Pulmonar/metabolismo , Pulmón/enzimología , Pulmón/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Aldehídos/química , Animales , Animales Recién Nacidos , Embrión de Mamíferos/patología , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Sondas Moleculares/metabolismo , Células 3T3 NIH , Proteína-Lisina 6-Oxidasa/genética , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Transducción de Señal , Proteínas Smad/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Fibrogenesis is the active production of extracellular matrix in response to tissue injury. In many chronic diseases persistent fibrogenesis results in the accumulation of scar tissue, which can lead to organ failure and death. However, no non-invasive technique exists to assess this key biological process. All tissue fibrogenesis results in the formation of allysine, which enables collagen cross-linking and leads to tissue stiffening and scar formation. We report herein a novel allysine-binding gadolinium chelate (GdOA), that can non-invasively detect and quantify the extent of fibrogenesis using magnetic resonance imaging (MRI). We demonstrate that GdOA signal enhancement correlates with the extent of the disease and is sensitive to a therapeutic response.
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Aminas/química , Quelantes/química , Imagen por Resonancia Magnética , Sondas Moleculares/química , Fibrosis Pulmonar/diagnóstico , Ácido 2-Aminoadípico/análogos & derivados , Ácido 2-Aminoadípico/química , Animales , Bleomicina , Gadolinio/química , Ratones , Conformación Molecular , Fibrosis Pulmonar/inducido químicamenteRESUMEN
Recent studies have suggested that brown adipose tissue (BAT) plays an important role in obesity, insulin resistance and heart failure. The characterization of BAT in vivo, however, has been challenging. No technique to comprehensively image BAT anatomy and function has been described. Moreover, the impact on BAT of the neuroendocrine activation seen in heart failure has only recently begun to be evaluated in vivo. The aim of this study was to use MRI to characterize the impact of heart failure on the morphology and function of BAT. Mice subjected to permanent ligation of the left coronary artery were imaged with MRI 6 weeks later. T2 weighted MRI of BAT volume and blood oxygen level dependent MRI of BAT function were performed. T2 * maps of BAT were obtained at multiple time points before and after administration of the ß3 adrenergic agonist CL 316 243 (CL). Blood flow to BAT was studied after CL injection using the flow alternating inversion recovery (FAIR) approach. Excised BAT tissue was analyzed for lipid droplet content and for uncoupling protein 1 (UCP1) mRNA expression. BAT volume was significantly lower in heart failure (51 ± 1 mm(3) versus 65 ± 3 mm(3) ; p < 0.05), and characterized by a reduction in lipid globules and a fourfold increase in UCP1 mRNA (p < 0.05). CL injection increased BAT T2 * in healthy animals but not in mice with heart failure (24 ± 4% versus 6 ± 2%; p < 0.01), consistent with an increase in flow in control BAT. This was confirmed by a significant difference in the FAIR response in BAT in control and heart failure mice. Heart failure results in the chronic activation of BAT, decreased BAT lipid stores and decreased BAT volume, and it is associated with a marked decrease in ability to respond to acute physiological stimuli. This may have important implications for substrate utilization and overall metabolic homeostasis in heart failure. Copyright © 2016 John Wiley & Sons, Ltd.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/patología , Técnicas de Imagen Cardíaca/métodos , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/metabolismo , Imagen por Resonancia Magnética/métodos , Oxígeno/sangre , Tejido Adiposo Pardo/diagnóstico por imagen , Animales , Femenino , Insuficiencia Cardíaca/patología , Gotas Lipídicas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Oximetría/métodosRESUMEN
BACKGROUND: The arrangement of myofibers in the heart is highly complex and must be replicated by injected cells to produce functional myocardium. A novel approach to characterize the microstructural response of the myocardium to ischemia and cell therapy, with the use of serial diffusion tensor magnetic resonance imaging tractography of the heart in vivo, is presented. METHODS AND RESULTS: Validation of the approach was performed in normal (n=6) and infarcted mice (n=6) as well as healthy human volunteers. Mice (n=12) were then injected with bone marrow mononuclear cells 3 weeks after coronary ligation. In half of the mice the donor and recipient strains were identical, and in half the strains were different. A positive response to cell injection was defined by a decrease in mean diffusivity, an increase in fractional anisotropy, and the appearance of new myofiber tracts with the correct orientation. A positive response to bone marrow mononuclear cell injection was seen in 1 mouse. The response of the majority of mice to bone marrow mononuclear cell injection was neutral (9/12) or negative (2/12). The in vivo tractography findings were confirmed with histology. CONCLUSIONS: Diffusion tensor magnetic resonance imaging tractography was able to directly resolve the ability of injected cells to generate new myofiber tracts and provided a fundamental readout of their regenerative capacity. A highly novel and translatable approach to assess the efficacy of cell therapy in the heart is thus presented.
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Trasplante de Médula Ósea/métodos , Imagen de Difusión Tensora/métodos , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Isquemia Miocárdica/patología , Isquemia Miocárdica/terapia , Animales , Anisotropía , Modelos Animales de Enfermedad , Voluntarios Sanos , Imagenología Tridimensional/métodos , Ratones , Ratones Endogámicos C57BL , Miocardio/patologíaRESUMEN
OBJECTIVES: Cell death in lymphatic organs, such as the spleen, is in part responsible for immunosuppression and contributes to mortality during sepsis. An early and noninvasive detection of lymphoid cell death could thus have significant clinical implications. Here, we tested in vivo imaging of lymphoid cell death using a near-infrared annexin V (AV-750). DESIGN: Animal study. SETTING: Laboratory investigation. SUBJECTS: C57BL/6J wild-type and toll-like receptor 3 knockout mice. INTERVENTIONS: Mild and severe polymicrobial sepsis was induced with cecum ligation and puncture. Serum cytokines and acute kidney injury markers were tested by immunoassay and quantitative reverse transcription-polymerase chain reaction, respectively. Sepsis-induced lymphoid cell death was detected by fluorescent AV-750 accumulation in the thorax and abdomen (in vivo), in isolated organs (ex vivo), and in isolated cells (flow cytometry). Caspase-3 cleavage/activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were tested for apoptosis. MEASUREMENTS AND MAIN RESULTS: Severe sepsis induced marked apoptosis in the thymus, spleen, and liver as demonstrated by cleaved caspase-3 and an increase in caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. A significant increase in fluorescent AV-750 signal was seen in the thoracic and upper abdominal fields and associated with the severity of sepsis. The in vivo thoracic and abdominal AV-750 fluorescent signal was attributed to the thymus, liver, and spleen as determined by ex vivo imaging and highly correlated with the levels of cell death in thymocytes and splenocytes, respectively, as measured by flow cytometry. Compared with wild-type septic mice, toll-like receptor 3 septic mice had attenuated abdominal AV-750 fluorescent signal, reduced ex vivo fluorescence in the spleen, and decreased splenocyte cell death. CONCLUSIONS: In vivo AV-750 fluorescent imaging provides spatially resolved and organ-specific detection of lymphoid cell death during polymicrobial sepsis. The AV-750 fluorescent intensity in the thoracic and abdominal fields is associated with sepsis severity and well correlated with sepsis-induced cell death in the thymus and spleen, respectively.
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Bacteriemia/microbiología , Muerte Celular , Coinfección/microbiología , Linfocitos/citología , Análisis de Varianza , Animales , Apoptosis/fisiología , Bacteriemia/fisiopatología , Western Blotting , Caspasa 3/metabolismo , Coinfección/diagnóstico , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Etiquetado Corte-Fin in Situ/métodos , Linfocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
Heat-induced radiolabeling (HIR) yielded (89) Zr-Feraheme (FH) nanoparticles (NPs) that were used to determine NP pharmacokinetics (PK) by positron emission tomography (PET). Standard uptake values indicated a fast hepatic uptake that corresponded to blood clearance, and a second, slow uptake process by lymph nodes and spleen. By cytometry, NPs were internalized by circulating monocytes and monocytes inâ vitro. Using an IV injection of HIR (89) Zr-FH (rather than inâ vitro cell labeling), PET/PK provided a view of monocyte trafficking, a key component of the immune response.
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Calor , Nanopartículas del Metal/química , Monocitos/citología , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Circonio/farmacocinética , Animales , Ratones , Radioisótopos/química , Radioisótopos/farmacocinética , Radiofármacos/química , Distribución Tisular , Circonio/químicaRESUMEN
Idiopathic pulmonary fibrosis is a chronic, progressive, fibrosing interstitial pneumonia of unknown cause resulting in dyspnea and functional decline until death. There are currently no effective noninvasive tools to monitor disease progression and response to treatment. The objective of the present study was to determine whether molecular magnetic resonance imaging of the lung using a probe targeted to type I collagen could provide a direct, noninvasive method for assessment of pulmonary fibrosis in a mouse model. Pulmonary fibrosis was generated in mice by transtracheal instillation of bleomycin (BM). Six cohorts were imaged before and immediately after intravenous administration of molecular imaging probe: (1) BM plus collagen-targeted probe, EP-3533; (2) sham plus EP-3533; (3) BM plus nonbinding control probe, EP-3612; (4) sham plus EP-3612; (5) BM plus EP-3533 imaged early; and (6) BM plus EP-3533 imaged late. Signal-to-noise ratio (SNR) enhancement was quantified in the lungs and muscle. Lung tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline. BM-treated mice had 35% higher lung collagen than sham mice (P < 0.0001). The SNR increase in the lungs of fibrotic mice after EP-3533 administration was twofold higher than in sham animals and twofold higher than in fibrotic or sham mice that received control probe, EP-3612 (P < 0.0001). The SNR increase in muscle was similar for all cohorts. For EP-3533, we observed a strong, positive, linear correlation between lung SNR increase and hydroxyproline levels (r = 0.72). Collagen-targeted probe EP-3533-enhanced magnetic resonance imaging specifically detects pulmonary fibrosis in a mouse model of disease.
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Imagen por Resonancia Magnética/métodos , Imagen Molecular/métodos , Fibrosis Pulmonar/diagnóstico , Fibrosis Pulmonar/metabolismo , Animales , Bleomicina/toxicidad , Colágeno/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Sonda Molecular , Sondas Moleculares/administración & dosificación , Fibrosis Pulmonar/inducido químicamenteRESUMEN
Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 µm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.
Asunto(s)
Cardiotoxicidad , Miocitos Cardíacos , Animales , Perros , Miocitos Cardíacos/metabolismo , Cardiotoxicidad/metabolismo , Autofagia , Doxorrubicina/farmacología , Doxorrubicina/metabolismo , Sirolimus/farmacologíaRESUMEN
BACKGROUND: Obese and pre-diabetic women have a higher risk for cardiovascular death than age-matched men with the same symptoms, and there are no effective treatments. We reported that obese and pre-diabetic female Zucker Diabetic Fatty (ZDF-F) rats recapitulate metabolic and cardiac pathology of young obese and pre-diabetic women and exhibit suppression of cardio-reparative AT2R. Here, we investigated whether NP-6A4, a new AT2R agonist with the FDA designation for pediatric cardiomyopathy, mitigate heart disease in ZDF-F rats by restoring AT2R expression. METHODS: ZDF-F rats on a high-fat diet (to induce hyperglycemia) were treated with saline, NP-6A4 (10 mg/kg/day), or NP-6A4 + PD123319 (AT2R-specific antagonist, 5 mg/kg/day) for 4 weeks (n = 21). Cardiac functions, structure, and signaling were assessed by echocardiography, histology, immunohistochemistry, immunoblotting, and cardiac proteome analysis. RESULTS: NP-6A4 treatment attenuated cardiac dysfunction, microvascular damage (-625%) and cardiomyocyte hypertrophy (-263%), and increased capillary density (200%) and AT2R expression (240%) (p < 0.05). NP-6A4 activated a new 8-protein autophagy network and increased autophagy marker LC3-II but suppressed autophagy receptor p62 and autophagy inhibitor Rubicon. Co-treatment with AT2R antagonist PD123319 suppressed NP-6A4's protective effects, confirming that NP-6A4 acts through AT2R. NP-6A4-AT2R-induced cardioprotection was independent of changes in body weight, hyperglycemia, hyperinsulinemia, or blood pressure. CONCLUSIONS: Cardiac autophagy impairment underlies heart disease induced by obesity and pre-diabetes, and there are no drugs to re-activate autophagy. We propose that NP-6A4 can be an effective drug to reactivate cardiac autophagy and treat obesity- and pre-diabetes-induced heart disease, particularly for young and obese women.
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Cardiomiopatías , Cardiopatías , Hiperglucemia , Estado Prediabético , Femenino , Ratas , Animales , Ratas Zucker , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/etiologíaRESUMEN
Innate immune response after transient ischemia is the most common cause of myocardial inflammation and may contribute to injury, yet the detailed signaling mechanisms leading to such a response are not well understood. Herein we tested the hypothesis that myocardial ischemia activates interleukin receptor-associated kinase-1 (IRAK-1), a kinase critical for the innate immune signaling such as that of Toll-like receptors (TLRs), via a mechanism that involves heat shock proteins (HSPs) and TLRs. Coronary artery occlusion induced a rapid myocardial IRAK-1 activation within 30 min in wild-type (WT), TLR2(-/-), or Trif(-/-) mice, but not in TLR4(def) or MyD88(-/-) mice. HSP60 protein was markedly increased in serum or in perfusate of isolated heart following ischemia/reperfusion (I/R). In vitro, recombinant HSP60 induced IRAK-1 activation in cells derived from WT, TLR2(-/-), or Trif(-/-) mice, but not from TLR4(def) or MyD88(-/-) mice. Both myocardial ischemia- and HSP60-induced IRAK-1 activation was abolished by anti-HSP60 antibody. Moreover, HSP60 treatment of cardiomyocytes (CMs) led to marked activation of caspase-8 and -3, but not -9. Expression of dominant-negative mutant of Fas-associated death domain protein or a caspase-8 inhibitor completely blocked HSP60-induced caspase-8 activation, suggesting that HSP60 likely activates an apoptotic program via the death-receptor pathway. In vivo, I/R-induced myocardial apoptosis and cytokine expression were significantly attenuated in TLR4(def) mice or in WT mice treated with anti-HSP60 antibody compared with WT controls. Taken together, the current study demonstrates that myocardial ischemia activates an innate immune signaling via HSP60 and TLR4, which plays an important role in mediating apoptosis and inflammation during I/R.
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Chaperonina 60/inmunología , Inmunidad Innata , Isquemia Miocárdica/inmunología , Miocitos Cardíacos/inmunología , Transducción de Señal , Receptor Toll-Like 4/inmunología , Animales , Apoptosis , Caspasas/metabolismo , Línea Celular , Inflamación , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones , Ratones Noqueados , Isquemia Miocárdica/patologíaRESUMEN
BACKGROUND: The study of myofiber reorganization in the remote zone after myocardial infarction has been performed in 2D. Microstructural reorganization in remodeled hearts, however, can only be fully appreciated by considering myofibers as continuous 3D entities. The aim of this study was therefore to develop a technique for quantitative 3D diffusion CMR tractography of the heart, and to apply this method to quantify fiber architecture in the remote zone of remodeled hearts. METHODS: Diffusion Tensor CMR of normal human, sheep, and rat hearts, as well as infarcted sheep hearts was performed ex vivo. Fiber tracts were generated with a fourth-order Runge-Kutta integration technique and classified statistically by the median, mean, maximum, or minimum helix angle (HA) along the tract. An index of tract coherence was derived from the relationship between these HA statistics. Histological validation was performed using phase-contrast microscopy. RESULTS: In normal hearts, the subendocardial and subepicardial myofibers had a positive and negative HA, respectively, forming a symmetric distribution around the midmyocardium. However, in the remote zone of the infarcted hearts, a significant positive shift in HA was observed. The ratio between negative and positive HA variance was reduced from 0.96 ± 0.16 in normal hearts to 0.22 ± 0.08 in the remote zone of the remodeled hearts (p < 0.05). This was confirmed histologically by the reduction of HA in the subepicardium from -52.03° ± 2.94° in normal hearts to -37.48° ± 4.05° in the remote zone of the remodeled hearts (p < 0.05). CONCLUSIONS: A significant reorganization of the 3D fiber continuum is observed in the remote zone of remodeled hearts. The positive (rightward) shift in HA in the remote zone is greatest in the subepicardium, but involves all layers of the myocardium. Tractography-based quantification, performed here for the first time in remodeled hearts, may provide a framework for assessing regional changes in the left ventricle following infarction.
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Imagen de Difusión Tensora , Ventrículos Cardíacos/patología , Infarto del Miocardio/diagnóstico , Miocardio/patología , Miofibrillas/patología , Remodelación Ventricular , Animales , Modelos Animales de Enfermedad , Ventrículos Cardíacos/fisiopatología , Humanos , Interpretación de Imagen Asistida por Computador , Imagenología Tridimensional , Modelos Estadísticos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Valor Predictivo de las Pruebas , Ratas , Reproducibilidad de los Resultados , OvinosRESUMEN
The diagnostic utility of the apoptosis-sensing nanoparticle (NP), AnxCLIO-Cy5.5, is well established. Here we sought to define the pathophysiological impact of the nanoparticle (NP) on apoptotic cells. Confocal microscopy showed that AnxCLIO-Cy5.5 remained bound to apoptotic cell membranes for 3 hours but by 7 hours had become completely internalized. AnxCLIO-Cy5.5 exposure did not impact energetics, metabolism or caspase-3 activity in apoptotic cells. Gene expression in cells exposed to AnxCLIO-Cy5.5 did not reveal upregulation of pro-inflammatory or cell-death pathways. Moreover, exposure to AnxCLIO-Cy5.5 decreased the frequency of membrane rupture of early apoptotic cells. Similarly, in mice exposed to 1 hour of ischemia -reperfusion, the injection of AnxCLIO-Cy5.5 at the onset of reperfusion reduced infarct size/area at risk by 16.2%. Our findings suggest that AnxCLIO-Cy5.5 may protect apoptotic cells by stabilizing their cell membranes and has the potential to become a theranostic agent, capable of both identifying and salvaging early apoptotic cells. From the Clinical Editor: This study demonstrates that AnxCLIO-Cy5.5 nanoparticles may protect apoptotic cells by cell membrane stabilization and have the potential to become a "theranostic agent" capable of identifying and salvaging early apoptotic cells.
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Anexinas/farmacología , Apoptosis/efectos de los fármacos , Carbocianinas/farmacología , Nanopartículas/química , Sustancias Protectoras/farmacología , Animales , Apoptosis/genética , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Metabolismo Energético/efectos de los fármacos , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Daño por Reperfusión/patologíaRESUMEN
Easy to find: magnetic nanoparticles bearing fluorochromes (red) that intercalate with DNA (green) form microaggregates with DNA generated by the polymerase chain reaction (PCR). These aggregates can be detected at low cycle numbers by magnetic resonance (MR).
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ADN/metabolismo , Colorantes Fluorescentes/química , Imagen por Resonancia Magnética , Magnetismo , Nanopartículas/química , Reacción en Cadena de la Polimerasa , Apoptosis , Óxido Ferrosoférrico/metabolismo , Fluorescencia , Colorantes Fluorescentes/metabolismo , Hematínicos/metabolismo , Sustancias Intercalantes/farmacología , Compuestos Orgánicos/metabolismoRESUMEN
Although thrombotic thrombocytopenic purpura (TTP) is rarely seen in pediatric patients, failure to recognize this condition often leads to severe consequences and poor outcomes. Classic features of TTP include thrombocytopenia, microangiopathic hemolytic anemia, acute kidney injury, fever, and central nervous system involvement. However, patients suffering from this condition may not present with all of the symptoms simultaneously. Therefore, it is of utmost importance for healthcare providers to have a high index of suspicion. Laboratory investigations may reveal the presence of schistocytes on peripheral blood smear, negative Coombs test, high lactate dehydrogenase levels and severely low platelet counts. The etiology of TTP is mainly due to insufficient cleavage of the large multimers of von Willebrand factor (vWF) secondary to decreased activity of ADAMTS13 (a disintegrin and metalloprotease with Thrombospondin type 1 repeats, member 13). TTP can be broadly classified into familial TTP (Upshaw Schulman syndrome) and non-familial TTP. Familial TTP is due to a congenital deficiency of ADAMTS13. Its mainstay of therapy is initiation of plasmapheresis during the acute phase, followed by regular fresh frozen plasma (FFP) infusions. Alternatively, non-familial TTP is due to a decrease in ADAMTS13 activity secondary to the presence of anti-ADAMTS13 antibodies. Once again, the primary treatment is plasmapheresis; however, recent anecdotal data also supports the use of rituximab in select cases.
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Púrpura Trombocitopénica Trombótica/terapia , Proteínas ADAM/genética , Proteína ADAMTS13 , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Niño , Humanos , Plasmaféresis , Púrpura Trombocitopénica Trombótica/etiología , RituximabRESUMEN
Wound infection by multidrug-resistant (MDR) bacteria is a major disease burden. Systemic administration of broad-spectrum antibiotics colistin methanesulfonate (CMS) and vancomycin are the last lines of defense against deep wound infections by MDR bacteria. However, systemic administration of CMS and vancomycin are linked to life-threatening vital organ damage. Currently there are no effective topical application strategies to deliver these high molecular weight antibiotics across the stratum corneum. To overcome this difficulty, we tested if high molecular weight antibiotics delivered by Droplette micromist technology device (DMTD), a transdermal delivery device that generates a micromist capable of packaging large molecules, could attenuate deep skin tissue infections. Using green fluorescent protein-tagged E. coli and live tissue imaging, we show that (1) the extent of attenuation of deep-skin E. coli infection was similar when treated with topical DMTD- or systemic IP (intraperitoneal)-delivered CMS; (2) DMTD-delivered micromist did not spread the infection deeper; (3) topical DMTD delivery and IP delivery resulted in similar levels of vancomycin in the skin after a 2 h washout period; and (4) IP-delivered vancomycin was about 1000-fold higher in kidney and plasma than DMTD-delivered vancomycin indicating systemic toxicity. Thus, topical DMTD delivery of these antibiotics is a safe treatment for the difficult-to-treat deep skin tissue infections by MDR bacteria.
RESUMEN
Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.
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Autofagia , Colorantes Fluorescentes , Nanopartículas , Espectroscopía Infrarroja Corta , Animales , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Arginina/química , Autofagia/efectos de los fármacos , Carbocianinas/química , Catepsinas/química , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Colorantes Fluorescentes/química , Macrólidos/administración & dosificación , Macrólidos/farmacología , Imagen por Resonancia Magnética/métodos , Ratones , Nanopartículas/química , Espectroscopía Infrarroja Corta/métodosRESUMEN
Chronological aging as well as biological aging accelerated by various pathologies such as diabetes and obesity contribute to cardiovascular aging, and structural and functional tissue damage of the heart and vasculature. Cardiovascular aging in humans is characterized by structural pathologic remodeling including cardiac and vascular fibrosis, hypertrophy, stiffness, micro- and macro-circulatory impairment, left ventricular diastolic dysfunction precipitating heart failure with either reduced or preserved ejection fraction, and cardiovascular cell death. Cellular senescence, an important hallmark of aging, is a critical factor that impairs repair and regeneration of damaged cells in cardiovascular tissues whereas autophagy, an intracellular catabolic process is an essential inherent mechanism that removes senescent cells throughout life time in all tissues. Several recent reviews have highlighted the fact that all longevity treatment paradigms to mitigate progression of aging-related pathologies converge in induction of autophagy, activation of AMP kinase (AMPK) and Sirtuin pathway, and inhibition of mechanistic target of rapamycin (mTOR). These longevity treatments include health style changes such as caloric restriction, and drug treatments using rapamycin, the first FDA-approved longevity drug, as well as other experimental longevity drugs such as metformin, rapamycin, aspirin, and resveratrol. However, in the heart tissue, autophagy induction has to be tightly regulated since evidence show excessive autophagy results in cardiomyopathy and heart failure. Here we discuss emerging evidence for microRNA-mediated tight regulation of autophagy in the heart in response to treatment with rapamycin, and novel approaches to monitor autophagy progression in a temporal manner to diagnose and regulate autophagy induction by longevity treatments.
RESUMEN
Multiplexed imaging is essential for the evaluation of substrate utilization in metabolically active organs, such as the heart and brown adipose tissue (BAT), where substrate preference changes in pathophysiologic states. Optical imaging provides a useful platform because of its low cost, high throughput and intrinsic ability to perform composite readouts. However, the paucity of probes available for in vivo use has limited optical methods to image substrate metabolism. Here, we present a novel near-infrared (NIR) free fatty acid (FFA) tracer suitable for in vivo imaging of deep tissues such as the heart. Using click chemistry, Alexa Fluor 647 DIBO Alkyne was conjugated to palmitic acid. Mice injected with 0.05 nmol/g bodyweight of the conjugate (AlexaFFA) were subjected to conditions known to increase FFA uptake in the heart (fasting) and BAT [cold exposure and injection with the ß3 adrenergic agonist CL 316, 243(CL)]. Organs were subsequently imaged both ex vivo and in vivo to quantify AlexaFFA uptake. The blood kinetics of AlexaFFA followed a two-compartment model with an initial fast compartment half-life of 0.14 h and a subsequent slow compartment half-life of 5.2 h, consistent with reversible protein binding. Ex vivo fluorescence imaging after overnight cold exposure and fasting produced a significant increase in AlexaFFA uptake in the heart (58 ± 12%) and BAT (278 ± 19%) compared to warm/fed animals. In vivo imaging of the heart and BAT after exposure to CL and fasting showed a significant increase in AlexaFFA uptake in the heart (48 ± 20%) and BAT (40 ± 10%) compared to saline-injected/fed mice. We present a novel near-infrared FFA tracer, AlexaFFA, that is suitable for in vivo quantification of FFA metabolism and can be applied in the context of a low cost, high throughput, and multiplexed optical imaging platform.
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Tejido Adiposo Pardo/diagnóstico por imagen , Colorantes Fluorescentes/administración & dosificación , Corazón/diagnóstico por imagen , Microscopía Intravital/métodos , Imagen Óptica/métodos , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Animales , Línea Celular , Dioxoles/farmacología , Ácidos Grasos no Esterificados/metabolismo , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Fluorodesoxiglucosa F18 , Semivida , Corazón/efectos de los fármacos , Inyecciones Intravenosas , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Microscopía Fluorescente , Imagen Molecular/métodos , Miocardio/metabolismo , RatasRESUMEN
BACKGROUND: Substrate utilization in tissues with high energetic requirements could play an important role in cardiometabolic disease. Current techniques to assess energetics are limited by high cost, low throughput, and the inability to resolve multiple readouts simultaneously. Consequently, we aimed to develop a multiplexed optical imaging platform to simultaneously assess energetics in multiple organs in a high throughput fashion. METHODS AND RESULTS: The detection of 18F-Fluordeoxyglucose uptake via Cerenkov luminescence and free fatty acid uptake with a fluorescent C16 free fatty acid was tested. Simultaneous uptake of these agents was measured in the myocardium, brown/white adipose tissue, and skeletal muscle in mice with/without thoracic aortic banding. Within 5 weeks of thoracic aortic banding, mice developed left ventricular hypertrophy and brown adipose tissue activation with upregulation of ß3AR (ß3 adrenergic receptors) and increased natriuretic peptide receptor ratio. Imaging of brown adipose tissue 15 weeks post thoracic aortic banding revealed an increase in glucose (P<0.01) and free fatty acid (P<0.001) uptake versus controls and an increase in uncoupling protein-1 (P<0.01). Similar but less robust changes were seen in skeletal muscle, while substrate uptake in white adipose tissue remained unchanged. Myocardial glucose uptake was increased post-thoracic aortic banding but free fatty acid uptake trended to decrease. CONCLUSIONS: A multiplexed optical imaging technique is presented that allows substrate uptake to be simultaneously quantified in multiple tissues in a high throughput manner. The activation of brown adipose tissue occurs early in the onset of left ventricular hypertrophy, which produces tissue-specific changes in substrate uptake that may play a role in the systemic response to cardiac pressure overload.
Asunto(s)
Tejido Adiposo Pardo/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/diagnóstico , Imagen por Resonancia Cinemagnética/métodos , Tomografía de Emisión de Positrones/métodos , Tejido Adiposo Pardo/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Fluorodesoxiglucosa F18/farmacología , Ratones , Ratones Endogámicos C57BL , Fenotipo , Radiofármacos/farmacologíaRESUMEN
Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.