RESUMEN
Experimental animal models to predict physiological responses to injury and stress in humans have inherent limitations. Therefore, the development of preclinical human models is of paramount importance. Ex vivo lung perfusion (EVLP) has typically been used to recondition donor lungs before transplantation. However, this technique has recently advanced into a model to emulate lung mechanics and physiology during injury. In the present study, we propose that the EVLP of diseased human lungs is a well-suited preclinical model for translational research on chronic lung diseases. Throughout this paper, we demonstrate this technique's feasibility in pulmonary arterial hypertension (PAH), idiopathic pulmonary fibrosis (IPF), emphysema, and non-disease donor lungs not suitable for transplantation. In this study, we aimed to perfuse the lungs for 6 h with the EVLP system. This facilitated a robust and continuous assessment of airway mechanics, pulmonary hemodynamics, gas exchange, and biochemical parameters. We then collected at different time points tissue biopsies of lung parenchyma to isolate RNA and DNA to identify each disease's unique gene expression. Thus, demonstrating that EVLP could successfully serve as a clinically relevant experimental model to derive essential insights into pulmonary pathophysiology and various human lung diseases.
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Circulación Extracorporea/métodos , Enfermedades Pulmonares/fisiopatología , Trasplante de Pulmón , Pulmón/fisiología , Preservación de Órganos/normas , Donantes de Tejidos/provisión & distribución , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , PerfusiónRESUMEN
BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.
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Anemia de Células Falciformes/complicaciones , Bilis/metabolismo , Colestasis/etiología , Insuficiencia Hepática/etiología , Hígado/patología , Adolescente , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Animales , Conductos Biliares Intrahepáticos/diagnóstico por imagen , Conductos Biliares Intrahepáticos/patología , Colestasis/patología , Colestasis/prevención & control , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Hemoglobina Falciforme/genética , Insuficiencia Hepática/patología , Insuficiencia Hepática/prevención & control , Humanos , Microscopía Intravital , Hígado/diagnóstico por imagen , Masculino , Ratones , Persona de Mediana Edad , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVE: Mesenchymal stem cells (MSC) have been shown to ameliorate the deleterious effects of bleomycin in murine models. However, the mechanism responsible for protection from pulmonary fibrosis by stem cell therapy is still poorly understood, especially in terms of endoplasmic reticulum (ER) stress. We hypothesized that during bleomycin-induced lung injury, markers of ER stress, specifically the activation of the unfolded protein response (UPR), increase during injury, resembling the kinetics of collagen deposition in the lung described for the bleomycin model. We aimed to elucidate the possible role of MSC in ER stress modulation. METHODS: To determine the kinetics of ER stress in aged mice, the expression of ER stress markers after bleomycin lung injury was measured in old mice at different time points (days 0, 3, 7, 14 and 21). To evaluate the consequences of systemic delivery of MSC on lung ER stress in the bleomycin model, we evaluated changes in body weight, lung histology and protein expression of ER stress markers. RESULTS: The level of expression of UPR transcription factor XBP-1 and its regulator BiP was elevated at day 7 and progressively increased up to day 21. MSC inhibited BiP expression in bleomycin-induced ER stress, attenuating ER stress via the protein kinase RNA-like ER kinase (PERK)-Nrf2 pathway. The expression levels of other ER stress markers were not perturbed by MSC. CONCLUSION: Our data suggest that MSC operate on ER stress via several pathways, but the PERK-Nrf2 pathway revealed to be the main functioning pathway in our bleomycin model.
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Estrés del Retículo Endoplásmico , Trasplante de Células Madre Mesenquimatosas , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/terapia , Respuesta de Proteína Desplegada , Animales , Bleomicina , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/fisiopatología , Proteína 1 de Unión a la X-Box/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-ß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15, and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.
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Células Epiteliales Alveolares/metabolismo , Factor 15 de Diferenciación de Crecimiento/genética , Fibrosis Pulmonar Idiopática/genética , Transcriptoma , Anciano , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/patología , Animales , Bleomicina/administración & dosificación , Líquido del Lavado Bronquioalveolar/química , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/diagnóstico , Fibrosis Pulmonar Idiopática/mortalidad , Fibrosis Pulmonar Idiopática/fisiopatología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , TelómeroRESUMEN
Introduction: Bone marrow-derived multipotent adult progenitor cells (MAPCs) are adult allogeneic adherent stem cells currently investigated clinically for use in acute respiratory distress syndrome (ARDS). To date, there is no agreement on which is the best method for stem cells delivery in ARDS. Here, we compared the efficacy of two different methods of administration and biodistribution of MAPC for the treatment of ARDS in a sheep model. Methods: MAPC were labelled with [18F] fluoro-29-deoxy-D-glucose and delivered by endobronchial (EB) or intravenous route 1 hour after lipopolysaccharide infusion in sheep mechanically ventilated. PET/CT images were acquired to determine the biodistribution and retention of the cells at 1 and 5 hours of administration. Results: The distribution and retention of the MAPC was dependent on the method of cell administration. By EB route, PET images showed that MAPC remained at the site of administration and no changes were observed after 5 hours, whereas with intravenous route, the cells had broad biodistribution to different organs, being the lung the main organ of retention at 1 and 5 hours. MAPC demonstrated an equal effect on arterial oxygenation recovery by either route of administration. Conclusion: The EB or intravenous routes of administration of MAPC are both effective for the treatment of ARDS in an acute sheep model, and the effect of MAPC therapy is not dependent of parenchymal integration or systemic biodistribution.
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Células Madre Adultas/trasplante , Células Madre Multipotentes/trasplante , Síndrome de Dificultad Respiratoria/terapia , Animales , Bronquios , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Infusiones Intravenosas , Lipopolisacáridos/inmunología , Masculino , Tomografía Computarizada por Tomografía de Emisión de Positrones , Cultivo Primario de Células , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , Síndrome de Dificultad Respiratoria/inmunología , Ovinos , Resultado del TratamientoRESUMEN
Idiopathic pulmonary fibrosis (IPF) pathogenesis has been postulated to involve a variety of mechanisms associated with the aging process, including loss of protein homeostasis (proteostasis). Heat shock proteins are cellular chaperones that serve a number of vital maintenance and repair functions, including the regulation of proteostasis. Previously published data have implicated heat shock protein 70 (Hsp70) in the development of pulmonary fibrosis in animal models. We sought to identify alterations in Hsp70 expression in IPF lung. Hsp70 mRNA and protein were decreased in primary fibroblasts cultured from IPF versus normal donor lung tissue. In addition to cultured fibroblasts, Hsp70 expression was decreased in intact IPF lung, a stressed environment in which upregulation of protective heat shock proteins would be anticipated. In support of a mechanistic association between decreased Hsp70 and fibrosis, cultured primary lung fibroblasts deficient in Hsp70 secreted increased extracellular matrix proteins. Treatment of primary normal human lung fibroblasts in vitro with either of the profibrotic molecules IGFBP5 (insulin-like growth factor-binding protein 5) or transforming growth factor-ß1 downregulated Hsp70, suggesting Hsp70 is a downstream target in the fibrotic cascade. Hsp70-knockout mice subjected to an inhalational bleomycin model of pulmonary fibrosis demonstrated accelerated fibrosis versus wild-type control animals. We therefore conclude that reduced Hsp70 protein contributes to fibrosis and that interventions aimed at restoring normal expression of Hsp70 represent a novel therapeutic strategy for pulmonary fibrosis.
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Proteínas HSP70 de Choque Térmico/deficiencia , Fibrosis Pulmonar Idiopática/metabolismo , Espacio Intracelular/metabolismo , Envejecimiento/patología , Animales , Bleomicina , Fibroblastos/metabolismo , Fibroblastos/patología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP72/metabolismo , Humanos , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Pulmón/patología , Ratones , Fenotipo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.
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Bencilaminas/farmacología , Proteínas F-Box/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Perfusión/métodos , Piridinas/farmacología , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Adolescente , Adulto , Bencilaminas/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Proteínas F-Box/metabolismo , Femenino , Humanos , Lipopolisacáridos/toxicidad , Pulmón/patología , Masculino , Persona de Mediana Edad , Piridinas/uso terapéutico , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/patología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease for which age is the most important risk factor. Different mechanisms associated with aging, including stem cell dysfunction, have been described to participate in the pathophysiology of IPF. We observed an extrapulmonary effect associated with IPF: increase in cell senescence of bone marrow-derived mesenchymal stem cells (B-MSCs). METHODS: B-MSCs were obtained from vertebral bodies procured from IPF patients and age-matched normal controls. Cell senescence was determined by cell proliferation and expression of markers of cell senescence p16INK4A, p21, and ß-galactosidase activity. Mitochondrial function and DNA damage were measured. Paracrine induction of senescence and profibrotic responses were analyzed in vitro using human lung fibroblasts. The reparative capacity of B-MSCs was examined in vivo using the bleomycin-induced lung fibrosis model. RESULTS: In our study, we demonstrate for the first time that B-MSCs from IPF patients are senescent with significant differences in mitochondrial function, with accumulation of DNA damage resulting in defects in critical cell functions when compared with age-matched controls. Senescent IPF B-MSCs have the capability of paracrine senescence by inducing senescence in normal-aged fibroblasts, suggesting a possible link between senescent B-MSCs and the late onset of the disease. IPF B-MSCs also showed a diminished capacity to migrate and were less effective in preventing fibrotic changes observed in mice after bleomycin-induced injury, increasing illness severity and proinflammatory responses. CONCLUSIONS: We describe extrapulmonary alterations in B-MSCs from IPF patients. The consequences of having senescent B-MSCs are not completely understood, but the decrease in their ability to respond to normal activation and the risk of having a negative impact on the local niche by inducing inflammation and senescence in the neighboring cells suggests a new link between B-MSC and the onset of the disease.
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Envejecimiento/patología , Senescencia Celular/genética , Fibrosis Pulmonar Idiopática/patología , Células Madre Mesenquimatosas/patología , Envejecimiento/genética , Animales , Bleomicina/toxicidad , Células de la Médula Ósea/patología , Proliferación Celular/genética , Daño del ADN/genética , Fibroblastos , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/genética , RatonesRESUMEN
The mechanisms of aging that are involved in the development of idiopathic pulmonary fibrosis (IPF) are still unclear. Although it has been hypothesized that the proliferation and activation of human lung fibroblasts (hLFs) are essential in IPF, no studies have assessed how this process works in an aging lung. Our goal was to elucidate if there were age-related changes on primary hLFs isolated from IPF lungs compared with age-matched controls. We investigated several hallmarks of aging in hLFs from IPF patients and age-matched controls. IPF hLFs have increased cellular senescence with higher expression of ß-galactosidase, p21, p16, p53, and cytokines related to the senescence-associated secretory phenotype (SASP) as well as decreased proliferation/apoptosis compared with age-matched controls. Additionally, we observed shorter telomeres, mitochondrial dysfunction, and upon transforming growth factor-ß stimulation, increased markers of endoplasmic reticulum stress. Our data suggest that IPF hLFs develop senescence resulting in a decreased apoptosis and that the development of SASP may be an important contributor to the fibrotic process observed in IPF. These results might change the existing paradigm, which describes fibroblasts as aberrantly activated cells, to a cell with a senescence phenotype.
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Envejecimiento/metabolismo , Senescencia Celular , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Adulto , Envejecimiento/patología , Línea Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citocinas/metabolismo , Femenino , Fibroblastos/patología , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Acute respiratory distress syndrome (ARDS) is the result of a wide variety of disorders, which can be associated with different clinical disorders or systemic diseases directly affecting the lungs. Currently, the only existing therapy is limited to supportive care. In a 6 hour pilot study, we analyzed the use of the combination of both stem cell and extracorporeal membrane oxygenation (ECMO) strategies to prevent or treat severe lung injury. A total of 11 sheep were used. Five sheep received Escherichia coli endotoxin as a control group (group 1). Three sheep that received E. coli endotoxin were treated with veno-venous ECMO support in group 2. In group 3, 3 sheep received a dose of clinical grade good manufacturing practice (GMP)-produced MultiPotent Adult Progenitor cells (MAPC) intratracheally after the end of the infusion of E. coli endotoxin, followed by ECMO support. The respiratory parameters by means of blood gas results, measurements of lung injury, inflammatory responses, and integrity of the alveolar capillary barrier after the infusion of these cells were analyzed. Our data suggest that the combination of ECMO and stem cell therapy showed better histopathologic changes with less inflammation. We believe that the combination of stem cells with the ECMO treatment may be useful in future studies investigating the diagnosis, treatment, and prevention of ARDS.
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Oxigenación por Membrana Extracorpórea/métodos , Trasplante de Células Madre Mesenquimatosas , Síndrome de Dificultad Respiratoria/terapia , Animales , Modelos Animales de Enfermedad , Proyectos Piloto , OvinosRESUMEN
Although different preclinical models have demonstrated a favorable role for bone marrow-derived mesenchymal stem cells (B-MSC) in preventing fibrosis, this protective effect is not observed with late administration of these cells, when fibrotic changes are consolidated. We sought to investigate whether the late administration of B-MSCs overexpressing microRNAs (miRNAs) let-7d (antifibrotic) or miR-154 (profibrotic) could alter lung fibrosis in a murine bleomycin model. Using lentiviral vectors, we transduced miRNAs (let-7d or miR-154) or a control sequence into human B-MSCs. Overexpression of let-7d or miR-154 was associated with changes in the mesenchymal properties of B-MSCs and in their cytokine expression. Modified B-MSCs were intravenously administered to mice at day 7 after bleomycin instillation, and the mice were euthanized at day 14 Bleomycin-injured animals that were treated with let-7d cells were found to recover quicker from the initial weight loss compared with the other treatment groups. Interestingly, animals treated with miR-154 cells had the lowest survival rate. Although a slight reduction in collagen mRNA levels was observed in lung tissue from let-7d mice, no significant differences were observed in Ashcroft score and OH-proline. However, the distinctive expression in cytokines and CD45-positive cells in the lung suggests that the differential effects observed in both miRNA mice groups were related to an effect on the immunomodulation function. Our results establish the use of miRNA-modified mesenchymal stem cells as a potential future research in lung fibrosis.
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Lesión Pulmonar/metabolismo , Lesión Pulmonar/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Transducción Genética , Animales , Biomarcadores/metabolismo , Bleomicina , Células de la Médula Ósea/citología , Colágeno/genética , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones Endogámicos C57BL , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Transfección , Pérdida de PesoRESUMEN
Asthma, a chronic inflammatory airway disease, is typified by high levels of TH2-cytokines and excessive generation of reactive nitrogen and oxygen species, which contribute to bronchial epithelial injury and airway remodeling. While immune function plays a major role in the pathogenesis of the disease, accumulating evidence suggests that altered cellular metabolism is a key determinant in the predisposition and disease progression of asthma. Further, several studies demonstrate altered mitochondrial function in asthmatic airways and suggest that these changes may be systemic. However, it is unknown whether systemic metabolic changes can be detected in circulating cells in asthmatic patients. Platelets are easily accessible blood cells that are known to propagate airway inflammation in asthma. Here we perform a bioenergetic screen of platelets from asthmatic and healthy individuals and demonstrate that asthmatic platelets show a decreased reliance on glycolytic processes and have increased tricarboxylic acid cycle activity. These data demonstrate a systemic alteration in asthma and are consistent with prior reports suggesting that oxidative phosphorylation is more efficient asthmatic individuals. The implications for this potential metabolic shift will be discussed in the context of increased oxidative stress and hypoxic adaptation of asthmatic patients. Further, these data suggest that platelets are potentially a good model for the monitoring of bioenergetic changes in asthma.
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Asma/sangre , Plaquetas/metabolismo , Glucólisis , Adulto , Asma/patología , Plaquetas/patología , Hipoxia de la Célula , Femenino , Humanos , Masculino , Mitocondrias/metabolismo , Estrés OxidativoRESUMEN
Hypoxia can be damaging either because cells are directly sensitive to low oxygen pressure in their local microenvironment and/or because they are exposed to circulating factors systemically secreted in response to hypoxia. The conventional hypoxia model, breathing hypoxic air, does not allow one to distinguish between these local and systemic effects. Here we propose and validate a model for differentially applying local and systemic hypoxic challenges in an animal. We used parabiosis, two mice sharing circulation by surgical union through the skin, and tested the hypothesis that when one of the parabionts breathes room air and the other one is subjected to hypoxic air, both mice share systemic circulation but remain normoxic and hypoxic, respectively. We tested two common hypoxic paradigms in 10 parabiotic pairs: continuous hypoxia (10% O2) mimicking chronic lung diseases, and intermittent hypoxia (40 s, 21% O2; 20 s, 5% O2) simulating sleep apnea. Arterial oxygen saturation and oxygen partial pressure at muscle tissue were measured in both parabionts. Effective cross-circulation was assessed by intraperitoneally injecting a dye in one of the parabionts and measuring blood dye concentration in both animals after 2 h. The results confirmed the hypothesis that tissues of the parabiont under room air were perfused with normally oxygenated blood and, at the same time, were exposed to all of the systemic mediators secreted by the other parabiont actually subjected to hypoxia. In conclusion, combination of parabiosis and hypoxic/normoxic air breathing is a novel approach to investigate the effects of local and systemic hypoxia in respiratory diseases.
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Hipoxia/fisiopatología , Oxígeno/sangre , Parabiosis , Animales , Modelos Animales de Enfermedad , Hipoxia/sangre , Ratones , RespiraciónRESUMEN
Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.
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Linfocitos T CD8-positivos/metabolismo , Rechazo de Injerto/etiología , Mediadores de Inflamación/metabolismo , Interleucina-17/metabolismo , Trasplante de Pulmón/efectos adversos , Pulmón/metabolismo , Neutrófilos/metabolismo , Neumonía/etiología , Proteínas de Dominio T Box/metabolismo , Enfermedad Aguda , Aloinjertos , Animales , Anticuerpos/farmacología , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Histocompatibilidad , Mediadores de Inflamación/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-17/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/inmunología , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Neumonía/prevención & control , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genéticaRESUMEN
Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.
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Matriz Extracelular/metabolismo , Pulmón/patología , Fenómenos Mecánicos , Andamios del Tejido , Envejecimiento/patología , Animales , Fenómenos Biomecánicos , Bleomicina/efectos adversos , Colágeno/metabolismo , Matriz Extracelular/efectos de los fármacos , Femenino , Fibrosis , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BLRESUMEN
Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.
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Adenosina Trifosfatasas/metabolismo , Anemia de Células Falciformes/metabolismo , Plaquetas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Activación Plaquetaria , Adulto , Estudios de Casos y Controles , Femenino , Hemólisis , Humanos , Masculino , Persona de Mediana Edad , ATPasas de Translocación de Protón Mitocondriales , Consumo de Oxígeno , Agregación Plaquetaria , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
INTRODUCTION: Acute respiratory distress syndrome (ARDS) is the most common cause of respiratory failure among critically ill subjects, sepsis and severe bacterial pneumonia being its most common causes. The only interventions that have proven beneficial are protective ventilation strategies and fluid conservation approaches. New therapies are needed to address this common clinical problem. Others and we have previously shown the beneficial effect of infusion of exogenous adult stem cells in different pre-clinical models of ARDS. METHODS: In the present study endotoxin was infused intravenously into 14 sheep from which 6 received different doses of adult stem cells by intrabronchial delivery to evaluate the effect of stem cell therapy. RESULTS: After administration of endotoxin, there was a rapid decline in oxygenation to hypoxemic values, indicative of severe-to-moderate ARDS. None of the animals treated with saline solution recovered to normal baseline values during the 6 hours that the animals were followed. In contrast, sheep treated with a dose of 40 million adult stem cells returned their levels of oxygen in their blood to baseline two hours after the cells were infused. Similarly, improvements in carbon dioxide (CO2) clearance, pulmonary vascular pressures and inflammation were observed and confirmed by histology and by the decrease in lung edema. CONCLUSIONS: We concluded that instillation of adult non-hematopoietic stem cells can diminish the impact of endotoxin and accelerate recovery of oxygenation, CO2 removal and inflammation in the ovine model, making the use of adult stem cells a real alternative for future therapies for ARDS.
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Células Madre Adultas/citología , Células de la Médula Ósea/citología , Trasplante de Médula Ósea/métodos , Síndrome de Dificultad Respiratoria/terapia , Trasplante de Células Madre/métodos , Animales , Modelos Animales de Enfermedad , Humanos , Lipopolisacáridos , Síndrome de Dificultad Respiratoria/inducido químicamente , OvinosRESUMEN
SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.
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Ubiquitina/química , Dominios Homologos src , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Ubiquitina/metabolismoRESUMEN
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism. Its activity is controlled by various types of signals, including growth factors, nutrients, and stresses. In this study, we show that changes in expression levels of two antiapoptotic proteins, Bcl-2 and Bcl-XL, also affect mTORC1 signaling activity. In cells overexpressing Bcl-XL, mTORC1 activity is increased and becomes less sensitive to growth factor or nutrient conditions. In contrast, reduction in expression levels of the two antiapoptotic proteins inhibits mTORC1 signaling activity. Our results suggest that the effect of Bcl-2 and Bcl-XL on mTORC1 is mediated by FKBP38, an inhibitor of mTORC1. The two proteins compete with mTORC1 for FKBP38 binding and hence alter mTORC1 activity. This study reveals a novel cross-talk between Bcl-2/XL and mTORC1 signaling, which is likely to contribute to cancer development.
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Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína bcl-X/metabolismo , Apoptosis , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
INTRODUCTION: The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. METHODS: A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6. RESULTS: Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1ß and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. CONCLUSIONS: We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic responses induced by endotoxemia by modulating the inflammatory response, mechanisms that do not involve engraftment or trans-differentiation of the cells. These observations may have important implications for the design of future cell therapies for ARDS.