RESUMEN
BACKGROUND: In the human lung, epithelial progenitor cells in the airways give rise to the differentiated pseudostratified airway epithelium. In mice, emerging evidence confers a progenitor function to cytokeratin 5 (KRT5(+)) or cytokeratin 14 (KRT14(+))-positive basal cells of the airway epithelium. Little is known, however, about the distribution of progenitor subpopulations in the human lung, particularly about aberrant epithelial differentiation in lung disease, such as idiopathic pulmonary fibrosis (IPF). METHODS: Here, we used multi-color immunofluorescence analysis to detect and quantify the distribution of airway epithelial progenitor subpopulations in human lungs obtained from healthy donors or IPF patients. RESULTS: In lungs from both, healthy donors and IPF patients, we detected KRT5(+)KRT14(-), KRT5(-)KRT14(+) and KRT5(+)KRT14(+) populations in the proximal airways. KRT14(+) cells, however, were absent in the distal airways of healthy lungs. In IPF, we detected a dramatic increase in the amount of KRT5(+) cells and the emergence of a frequent KRT5(+)KRT14(+) epithelial population, in particular in distal airways and alveolar regions. While the KRT14(-) progenitor population exhibited signs of proper epithelial differentiation, as evidenced by co-staining with pro-SPC, aquaporin 5, CC10, or MUC5B, the KRT14(+) cell population did not co-stain with bronchial/alveolar differentiation markers in IPF. CONCLUSIONS: We provide, for the first time, a quantitative profile of the distribution of epithelial progenitor populations in human lungs. We show compelling evidence for dysregulation and aberrant differentiation of these populations in IPF.
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Células Progenitoras Endoteliales/patología , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Acuaporina 5/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Separación Celular , Células Cultivadas , Células Progenitoras Endoteliales/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Queratina-14/metabolismo , Queratina-5/metabolismo , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Mucina 5B/metabolismo , FenotipoRESUMEN
Chronic lung allograft dysfunction (CLAD) is a limiting factor for long-term survival in lung transplant recipients. Donor-specific human leukocyte antigen (HLA)-antibodies (DSA) have been suggested as potential risk factors for CLAD. However, their impact on clinical outcome following lung transplantation remains controversial. We performed a single-center study of 120 lung transplant recipients transplanted between 2006 and 2011. Patient sera were investigated before and after transplantation. The sera were screened by means of Luminex(®) technology (Luminex Inc., Austin, TX, USA) for IgG-HLA-class I and class II antibodies (ab). Using single antigen beads, DSA were identified and correlated retrospectively with clinical parameters. After transplantation 39 out of 120 patients (32.5%) were positive for HLA-ab. The incidence of de novo DSA formation was 27 of 120 patients (22.5%). Eleven of 27 (41%) of de novo DSA-positive patients developed BOS compared to 13 of 93 (14%) DSA-negative patients (p = 0.002). Furthermore, the generation of de novo DSA was independently associated with the development of BOS in multivariable analysis [hazard ration (HR) 2.5, 95% confidence interval (CI) 1.0-6.08; p = 0.046). Our results indicate that de novo DSA are associated with the development of BOS after lung transplantation. Monitoring of HLA-ab after transplantation is useful for identifying high-risk patients and offers an opportunity for early therapeutic intervention.
Asunto(s)
Anticuerpos/inmunología , Bronquiolitis Obliterante/inmunología , Antígenos HLA/inmunología , Adulto , Femenino , Humanos , Trasplante de Pulmón , Masculino , Persona de Mediana Edad , Donantes de TejidosRESUMEN
The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.
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Camptotecina/análogos & derivados , Hígado/química , Modelos Animales , Piridonas/análisis , Quinazolinas/análisis , Administración Intravenosa , Administración Oral , Afatinib , Animales , Camptotecina/administración & dosificación , Camptotecina/análisis , Clorhidrato de Erlotinib , Técnicas In Vitro , Irinotecán , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Estructura Molecular , Piridonas/administración & dosificación , Quinazolinas/administración & dosificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Idiopathic pulmonary fibrosis is a fatal lung disease with a variable and unpredictable natural history and limited treatment options. Since publication of the ATS-ERS statement on IPF in the year 2000 diagnostic standards have improved and a considerable number of randomized controlled treatment trials have been published necessitating a revision. In the years 2006 - 2010 an international panel of IPF experts produced an evidence-based guideline on diagnosis and treatment of IPF, which was published in 2011. In order to implement this evidence-based guideline into the German Health System a group of German IPF experts translated and commented the international guideline, also including new publications in the field. A consensus conference was held in Bochum on December 3rd 2011 under the protectorate of the "Deutsche Gesellschaft für Pneumologie und Beatmungsmedizin (DGP)" and supervised by the "Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften" (AWMF). Most recommendations of the international guideline were found to be appropriate for the german situation. Based on recent clinical studies "weak negative" treatment recommendations for pirfenidone and anticoagulation were changed into "weak positive" for pirfenidone and "strong negative" for anticoagulation. Based on negative results from the PANTHER-trial the recommendation for the combination therapy of prednisone plus azathiorpine plus N-acetlycsteine was also changed into strong negative für patients with definite IPF. This document summarizes essential parts of the international IPF guideline and the comments and recommendations of the German IPF consensus conference.
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Antiinflamatorios/uso terapéutico , Fibrosis Pulmonar Idiopática/diagnóstico , Fibrosis Pulmonar Idiopática/terapia , Guías de Práctica Clínica como Asunto , Neumología/normas , Tomografía Computarizada por Rayos X/métodos , Alemania , Humanos , Fibrosis Pulmonar Idiopática/sangre , InternacionalidadRESUMEN
Respiratory diseases are one of the most important causes of mortality with tremendous costs for health care systems, not only in Germany, but worldwide. Up to now treatment options for most of these chronic diseases are limited. The German Ministry for Research and Education (BMBF) - following the example of the US National Institute of Health have supported the foundation of a German Centre for Lung Research (DZL) to speed up the development of preventive, diagnostic and therapeutic measures. Not only universities, but also non-university based research institutes are part of the DZL. To allow the translation from basic research experience into clinical practice to improve patient care, basic research orientated approaches will be combined with disease and patient focused approaches. The DZL is one of six German Centres for Health Care Research (neurological diseases, diabetes, cardiovascular diseases, infectious diseases, cancer, and lung diseases) for the optimisation of translational processes to overcome the burden of major diseases.
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Academias e Institutos/organización & administración , Investigación Biomédica/organización & administración , Trastornos Respiratorios/diagnóstico , Trastornos Respiratorios/terapia , Investigación Biomédica Traslacional/organización & administración , Alemania , Humanos , Centro RespiratorioRESUMEN
Pulmonary arterial hypertension (PAH) is a life-threatening disease characterised by vasoconstriction and remodelling of the pulmonary vasculature. The serotonin (5-hydroxytryptamine (5-HT)) pathway has been shown to play a major role in the pathogenesis of PAH, but pharmacological modulation of this pathway for treatment of PAH is, to date, at a pre-clinical level. Terguride is a 5-HT receptor (5-HTR) antagonist that is well tolerated and clinically approved for ovulation disorders. Immunohistochemistry against 5-HTR(2A/B) on human lungs revealed their localisation to the vascular smooth muscle layer and quantitative RT-PCR showed 5-HTR(2B) upregulation in pulmonary artery smooth muscle cells (PASMC) isolated from PAH patients. Proliferation and migration of cultured primary human PASMC were dose-dependently blocked by terguride. Therapeutic 5-HT signalling inhibition was 1) demonstrated in isolated, ventilated and perfused rat lungs and 2) by chronic terguride treatment of rats with monocrotaline (MCT)-induced pulmonary hypertension in a preventive or curative approach. Terguride inhibited proliferation of PASMCs and abolished 5-HT-induced pulmonary vasoconstriction. Chronic terguride treatment prevented dose-dependently the development and progression of MCT-induced PAH in rats. Thus, terguride represents a valuable novel therapeutic approach in PAH.
Asunto(s)
Agonistas de Dopamina/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Lisurida/análogos & derivados , Pulmón/efectos de los fármacos , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Adulto , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/patología , Lisurida/uso terapéutico , Pulmón/patología , Pulmón/fisiopatología , Trasplante de Pulmón , Masculino , Monocrotalina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , RatasRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterised by accumulation of activated (myo)fibroblasts and excessive extracellular matrix deposition. The enhanced accumulation of (myo)fibroblasts may be attributed, in part, to the process of transforming growth factor beta1 (TGFbeta1)-induced epithelial-mesenchymal transition (EMT), the phenotypic switching of epithelial to fibroblast-like cells. Although alveolar epithelial type II (ATII) cells have been shown to undergo EMT, the precise mediators and mechanisms remain to be resolved. The objective of this study is to investigate the role of SNAI transcription factors in the process of EMT and in IPF. METHODS: Using quantitative reverse transcription-PCR (RT-PCR), immunofluorescence, immunohistochemistry, western blotting, as well as gain- and loss-of-function studies and functional assays, the role of SNAI1 and SNAI2 in TGFbeta1-induced EMT in ATII cells in vitro was assessed; and the expression of SNAI transcription factors was analysed in experimental and human IPF in vivo. RESULTS: TGFbeta1 treatment increased the expression and nuclear accumulation of SNAI1 and SNAI2, in concert with induction of EMT in ATII cells. SNAI overexpression was sufficient to induce EMT, and small interfering RNA (siRNA)-mediated SNAI depletion attenuated TGFbeta1-induced ATII cell migration and EMT. SNAI expression was elevated in experimental and human IPF and localised to hyperplastic ATII cells in vivo. CONCLUSIONS: The results demonstrate that TGFbeta1-induced EMT in ATII cells is essentially controlled by the expression and nuclear translocation of SNAI transcription factors. Increased SNAI1 and SNAI2 expression in experimental and human IPF in vivo suggests that SNAI-mediated EMT may contribute to the fibroblast pool in idiopathic pulmonary fibrosis.
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Células Epiteliales/patología , Células Madre Mesenquimatosas/patología , Fibrosis Pulmonar/patología , Factores de Transcripción/fisiología , Adulto , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Proteínas de la Matriz Extracelular/farmacología , Femenino , Silenciador del Gen , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/fisiopatología , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease characterised by elevated blood pressure in the pulmonary circulation. Initial vasoconstriction, proliferation of pulmonary arterial smooth muscle cells (PASMC) and increased deposition of extracellular matrix (ECM) contribute to pathological remodelling of pulmonary arterioles in IPAH. Glycosaminoglycans (GAGs), components of the ECM, control cellular proliferation and differentiation, but their expression in IPAH remains elusive. In the present study, GAG expression was investigated in the lungs of patients with IPAH or control transplant donors, and expression and localisation of GAG-metabolising enzymes were analysed in vivo and in vitro. A significant increase in the expression of hyaluronic acid (HA) was detected in IPAH lungs, associated with increased hyaluronan synthase (Has)1 and decreased hyaluronoglucosaminidase 1 gene expression, as assessed by quantitative RT-PCR and Western blotting. HAS1 protein localised to PASMC in vivo and increased HA deposition was observed in remodelled pulmonary arteries in IPAH. Transforming growth factor-beta1, a profibrotic growth factor, led to increased HA secretion and HAS1 expression in primary PASMC. The results demonstrate an increased hyaluronic acid content in idiopathic pulmonary arterial hypertension lungs, associated with increased hyaluronan synthase 1 and decreased hyaluronoglucosaminidase 1 gene expression. Synergistic regulation of glycosaminoglycan-metabolising enzymes in favour of accumulation may, thus, regulate pathological vascular remodelling in idiopathic pulmonary arterial hypertension lungs.
Asunto(s)
Ácido Hialurónico/metabolismo , Hipertensión Pulmonar/metabolismo , Arteria Pulmonar/patología , Adulto , Diferenciación Celular , Femenino , Fibrosis , Regulación Enzimológica de la Expresión Génica , Glicosaminoglicanos/metabolismo , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Shroom is a PDZ-domain protein involved in the regulation and maintenance of cytoskeletal architecture by binding to actin. Hypertrophy and altered actin organisation of pulmonary arterial smooth muscle cells (PASMC) is a hallmark of pulmonary arterial hypertension (PAH). The aim of the present study was to localise and characterise Shroom expression in the lung in experimental and idiopathic PAH (IPAH). Shroom expression and localisation in hypoxia-induced PAH in mice and IPAH in humans, in vivo, as well as in primary PASMC, in vitro, was assessed by quantitative RT-PCR, immunofluorescence, laser-assisted microdissection and immunohistochemistry. Shroom localised exclusively to PASMC (both bronchial and vascular) in mouse and human lungs. Both in vivo and in primary PASMC, in vitro, Shroom exhibited spatially similar expression with alpha-smooth muscle actin (alpha-SMA). Shroom expression was significantly reduced in the mouse model of PAH, in primary murine PASMC exposed to hypoxia, and in primary PASMC isolated from patients with IPAH. The ratio between Shroom and alpha-SMA RNA expression further confirmed Shroom downregulation in both mouse and human PASMC. In summary, Shroom localises exclusively to pulmonary smooth muscle cells. Shroom downregulation in pulmonary arterial hypertension suggests a link between Shroom expression and pulmonary arterial smooth muscle cell hypertrophy in pulmonary arterial hypertension.
Asunto(s)
Citoesqueleto/metabolismo , Hipertensión Pulmonar/metabolismo , Proteínas de Microfilamentos/fisiología , Arteria Pulmonar/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Humanos , Hipertrofia , Hipoxia , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso/metabolismoRESUMEN
Smad6 and Smad7 are inhibitory Smad proteins, which are essential in the negative regulation of BMP and TGF-beta/activin-signalling. While Smad7 is ubiquitously expressed and inhibits BMP and TGF-beta signalling, Smad6 is expressed in a cell-specific fashion and preferentially inhibits BMP-signalling. Here, we identified in addition to the splice variant Smad6s, possessing a different N-terminus, a novel human Smad6 splice variant, termed Smad6B, in human prostatic and rodent testicular cell lines. Due to an alternative exon, the putative Smad6B protein exhibits a truncated C-terminus lacking the entire MH-2 domain and most parts of the linker region. While full-length Smad6 was expressed in almost all tissues and cell lines studied, expression of the splice variants Smad6s and Smad6B was more heterogenous. Species-specific sequence comparison revealed that Smad6 is found in most species, while Smad6s was found only in Homo sapiens and Pan troglodytes, and Smad6B was found only in Homo sapiens. In addition, Smad6 localisation was found in round spermatids of human testis. In conclusion, we propose that alternative splicing of Smad6s and Smad6B is restricted to higher mammals.
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Empalme Alternativo , Proteína smad6/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Pan troglodytes , Próstata/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteína smad6/genética , Testículo/metabolismoAsunto(s)
Enfermedades del Sistema Inmune/patología , Enfermedades Pulmonares/patología , Enfermedad Crónica , Ambiente , Epitelio/patología , Europa (Continente) , Fibrosis/patología , Predisposición Genética a la Enfermedad , Humanos , Sistema Inmunológico , Enfermedades del Sistema Inmune/complicaciones , Enfermedades Pulmonares/complicaciones , Portugal , Enfermedad Pulmonar Obstructiva Crónica/patología , Neumología/métodos , Neumología/organización & administración , Sociedades MédicasRESUMEN
Molecular mechanisms that regulate lung repair vs. progressive scarring in pulmonary fibrosis remain elusive. Interleukin (IL)-4 and IL-13 are pro-fibrotic cytokines that share common receptor chains including IL-13 receptor (R) α1 and are key pharmacological targets in fibrotic diseases. However, the roles of IL-13Rα1 in mediating lung injury/repair are unclear. We report dysregulated levels of IL-13 receptors in the lungs of bleomycin-treated mice and to some extent in idiopathic pulmonary fibrosis patients. Transcriptional profiling demonstrated an epithelial cell-associated gene signature that was homeostatically dependent on IL-13Rα1 expression. IL-13Rα1 regulated a striking array of genes in the lung following bleomycin administration and Il13ra1 deficiency resulted in exacerbated bleomycin-induced disease. Increased pathology in bleomycin-treated Il13ra1(-/-) mice was due to IL-13Rα1 expression in structural and hematopoietic cells but not due to increased responsiveness to IL-17, IL-4, IL-13, increased IL-13Rα2 or type 1 IL-4R signaling. These data highlight underappreciated protective roles for IL-13Rα1 in lung injury and homeostasis.
Asunto(s)
Fibrosis Pulmonar Idiopática/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Interleucina-13/inmunología , Lesión Pulmonar/inmunología , Adulto , Animales , Bleomicina , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Homeostasis/inmunología , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/patología , Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/deficiencia , Subunidad alfa1 del Receptor de Interleucina-13/genética , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Pulmón/inmunología , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/genética , Lesión Pulmonar/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/inmunología , Transducción de Señal , Transcripción GenéticaRESUMEN
The proteasome is a central regulatory hub for intracellular signaling by degrading numerous signaling mediators. Immunoproteasomes are specialized types of proteasomes involved in shaping adaptive immune responses, but their role in innate immune signaling is still elusive. Here, we analyzed immunoproteasome function for polarization of alveolar macrophages, highly specialized tissue macrophages of the alveolar lung surface. Classical activation (M1 polarization) of primary alveolar macrophages by LPS/IFNγ transcriptionally induced all three immunoproteasome subunits, low molecular mass protein 2 (LMP2), LMP7 and multicatalytic endopeptidase complex-like 1, which was accompanied by increased immunoproteasome activity in M1 cells. Deficiency of LMP7 had no effect on the LPS/IFNγ-triggered M1 profile indicating that immunoproteasome function is dispensable for classical alveolar macrophage activation. In contrast, IL-4 triggered alternative (M2) activation of primary alveolar macrophages was accompanied by a transcriptionally independent amplified expression of LMP2 and LMP7 and an increase in immunoproteasome activity. Alveolar macrophages from LMP7 knockout mice disclosed a distorted M2 profile upon IL-4 stimulation as characterized by increased M2 marker gene expression and CCL17 cytokine release. Comparative transcriptome analysis revealed enrichment of IL-4-responsive genes and of genes involved in cellular response to defense, wounding and inflammation in LMP7-deficient alveolar macrophages indicating a distinct M2 inflammation resolving phenotype. Moreover, augmented M2 polarization was accompanied by amplified AKT/STAT6 activation and increased RNA and protein expression of the M2 master transcription factor interferon regulatory factor 4 in LMP7(-/-) alveolar macrophages. IL-13 stimulation of LMP7-deficient macrophages induced a similar M2-skewed profile indicative for augmented signaling via the IL-4 receptor α (IL4Rα). IL4Rα expression was generally elevated only on protein but not RNA level in LMP7(-/-) alveolar macrophages. Importantly, specific catalytic inhibition with an LMP7-specific proteasome inhibitor confirmed augmented IL-4-mediated M2 polarization of alveolar macrophages. Our results thus suggest a novel role of immunoproteasome function for regulating alternative activation of macrophages by limiting IL4Rα expression and signaling.
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Cisteína Endopeptidasas/metabolismo , Macrófagos Alveolares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL17/análisis , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Factores Reguladores del Interferón/metabolismo , Interferón gamma/farmacología , Interleucina-13/farmacología , Interleucina-4/análisis , Lipopolisacáridos/toxicidad , Macrófagos Alveolares/citología , Macrófagos Alveolares/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Oligopéptidos/farmacología , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Mesoporous silica nanoparticles (MSNs) exhibit unique drug delivery properties and are thus considered as promising candidates for next generation nano-medicines. In particular, inhalation into the lungs represents a direct, non-invasive delivery route for treating lung disease. To assess MSN biocompatibility in the lung, we investigated the bioresponse of avidin-coated MSNs (MSN-AVI), as well as aminated (uncoated) MSNs, after direct application into the lungs of mice. We quantified MSN distribution, clearance rate, cell-specific uptake, and inflammatory responses to MSNs within one week after instillation. We show that amine-functionalized (MSN-NH2) particles are not taken up by lung epithelial cells, but induced a prolonged inflammatory response in the lung and macrophage cell death. In contrast, MSN-AVI co-localized with alveolar epithelial type 1 and type 2 cells in the lung in the absence of sustained inflammatory responses or cell death, and showed preferential epithelial cell uptake in in vitro co-cultures. Further, MSN-AVI particles demonstrated uniform particle distribution in mouse lungs and slow clearance rates. Thus, we provide evidence that avidin functionalized MSNs (MSN-AVI) have the potential to serve as versatile biocompatible drug carriers for lung-specific drug delivery.
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Portadores de Fármacos/química , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Nanopartículas , Animales , Avidina , Línea Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Sistemas de Liberación de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Mediadores de Inflamación/metabolismo , Pulmón/citología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos BALB C , Nanomedicina , Nanopartículas/administración & dosificación , Nanopartículas/química , Nanopartículas/toxicidad , Dióxido de SilicioRESUMEN
BACKGROUND: Calcium channel blockers (CCB) of all subclasses: the dihydropyridines, benzothiazepines, and phenylalkylamines, at nanomolar concentrations, have been shown to up-regulate interleukin-6 (IL-6) mRNA. We investigated the underlying molecular mechanism responsible for IL-6 induction in response to the CCB amlodipine, diltiazem, and verapamil in primary human vascular smooth muscle cells (VSMC). METHODS AND RESULTS: All 3 CCB directly activated transcription of the human IL-6 gene in primary human VSMC in a time- and dose-dependent manner, as demonstrated by luciferase reporter gene assays using a 651-bp fragment of the human IL-6 gene promoter. Deletion analysis of the IL-6 promoter revealed that CCB inducible promoter activity was localized to a 160-bp fragment directly upstream of the transcriptional start site of the IL-6 gene. Known transcription factor consensus sequences within this fragment include a NF-IL6 and a NF-kappaB site. Site-directed mutagenesis suggested that both transcription factors had positive regulatory activity and cooperatively transmitted induction of the IL-6 gene by CCB. The data are confirmed by electrophoretic mobility shift analyses using nuclear extracts from CCB-stimulated and control primary VSMC. CCB of all subclasses increased DNA binding of NF-IL6 and NF-kappaB as early as 30 minutes after stimulation with the drugs. This effect was independent of intracellular calcium concentrations because calcium-free medium did not increase NF-IL6 or NF-kappaB activity. CONCLUSIONS: The results demonstrate that CCB of all 3 subclasses are capable of activating NF-IL6 and NF-kappaB. CCB may thus directly regulate cellular functions by affecting the activity of transcription factors independent of changes of intracellular calcium concentrations, an observation that is of interest considering the biological effects induced by CCB.
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Bloqueadores de los Canales de Calcio/farmacología , Proteínas de Unión al ADN/fisiología , Interleucina-6/genética , Músculo Liso Vascular/efectos de los fármacos , FN-kappa B/fisiología , Proteínas Nucleares/fisiología , Proteínas Potenciadoras de Unión a CCAAT , Calcio/metabolismo , Células Cultivadas , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Regiones Promotoras GenéticasRESUMEN
An imbalance between neutrophil-derived proteases and extracellular inhibitors is widely regarded as an important pathogenic mechanism for lung injury. Despite intense efforts over the last three decades, attempts to develop small-molecule inhibitors for neutrophil elastase have failed in the clinic. Here we discover an intrinsic self-cleaving property of mouse neutrophil elastase that interferes with the action of elastase inhibitors. We show that conversion of the single-chain (sc) into a two-chain (tc) neutrophil elastase by self-cleavage near its S1 pocket altered substrate activity and impaired both inhibition by endogenous α-1-antitrypsin and synthetic small molecules. Our data indicate that autoconversion of neutrophil elastase decreases the inhibitory efficacy of natural α-1-antitrypsin and small-molecule inhibitors, while retaining its pathological potential in an experimental mouse model. The so-far overlooked occurrence and properties of a naturally occurring tc-form of neutrophil elastase necessitates the redesign of small-molecule inhibitors that target the sc-form as well as the tc-form of neutrophil elastase.
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Elastasa de Leucocito/metabolismo , Pulmón/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Péptidos/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/farmacología , Enfisema Pulmonar/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Citocinas/biosíntesis , Femenino , Expresión Génica , Células HEK293 , Humanos , Elastasa de Leucocito/administración & dosificación , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/química , Pulmón/enzimología , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neutrófilos/enzimología , Neutrófilos/patología , Péptidos/administración & dosificación , Péptidos/antagonistas & inhibidores , Péptidos/química , Proteolisis , Enfisema Pulmonar/inducido químicamente , Enfisema Pulmonar/enzimología , Enfisema Pulmonar/patología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Bibliotecas de Moléculas Pequeñas/farmacología , alfa 1-Antitripsina/farmacologíaRESUMEN
Changes in x-ray attenuating tissue caused by lung disorders like emphysema or fibrosis are subtle and thus only resolved by high-resolution computed tomography (CT). The structural reorganization, however, is of strong influence for lung function. Dark-field CT (DFCT), based on small-angle scattering of x-rays, reveals such structural changes even at resolutions coarser than the pulmonary network and thus provides access to their anatomical distribution. In this proof-of-concept study we present x-ray in vivo DFCTs of lungs of a healthy, an emphysematous and a fibrotic mouse. The tomographies show excellent depiction of the distribution of structural - and thus indirectly functional - changes in lung parenchyma, on single-modality slices in dark field as well as on multimodal fusion images. Therefore, we anticipate numerous applications of DFCT in diagnostic lung imaging. We introduce a scatter-based Hounsfield Unit (sHU) scale to facilitate comparability of scans. In this newly defined sHU scale, the pathophysiological changes by emphysema and fibrosis cause a shift towards lower numbers, compared to healthy lung tissue.
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Tomografía Computarizada por Rayos X/métodos , Animales , Femenino , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Enfermedades Pulmonares/diagnóstico por imagen , Enfermedades Pulmonares/patología , Ratones , Modelos AnimalesRESUMEN
FAST and Runx (CBFa) transcription factors, which are expressed during specific phases of embryogenesis and tissue patterning, bind directly to Smad proteins and integrate effects induced by various TGF-beta gene family members. The DNA binding sequences for FAST and Runx differ only minimally. The isoform Runx2 (previously termed CBFa1) is highly expressed by osteoblasts and regulates expression of the TGF-beta receptor I in these cells. Here we show that FAST-dependent transcription is endogenously restricted in osteoblasts but can be significantly enhanced by disruption of Runx2 expression. Native and synthetic Runx2 bind to both Runx and FAST binding sequences, whereas FAST-1 efficiently binds only to the FAST binding sequence. However, overexpression of FAST-1 potently suppresses TGF-beta receptor I gene expression in osteoblasts and thereby reduces TGF-beta activity independently of competing for Runx2 at the level of DNA binding. These results provide a new example of how nuclear factors associated with specific developmental states or tissue lineages may influence TGF-beta-dependent events in restricted ways.
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Receptores de Activinas Tipo I , Proteínas de Unión al ADN/fisiología , Proteínas de Neoplasias , Osteoblastos/metabolismo , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células COS , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Subunidades alfa del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica/fisiología , Osteoblastos/fisiología , Regiones Promotoras Genéticas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Eph receptors are a subfamily of receptor tyrosine kinases (RTKs), that are activated by ephrin ligands and appear to play important roles in axon guidance and cell migration during development of the nervous system. Over-expression or constitutive activation of Eph receptors has been linked with increased proliferation in various tumours. We have recently described lineage aberrant expression of EphA5 in primary human astrocytomas, glioblastomas and in the human glioblastoma U-118 MG cell line. A role for EphA5 expression in these tumours is not apparent, and we have investigated the cellular effects of EphA5 activation using the human glioblastoma U-118 MG cell line as a model. Immunofluorescent staining demonstrated cell surface expression of EphA5. Activation of the EphA5 receptor using an ephrin-A1 recombinant fusion protein resulted in tyrosine phosphorylation of EphA5 in a time-dependent manner. Exposure of U-118 MG glioblastoma cells to ephrin-A1 did not result in significant spontaneous or FCS-stimulated cell proliferation, though a marginal decrease was observed. This is in converse to the effects of Eph activation in other tumour cell lines, and is the first study to investigate EphA5 in glioblastoma cell lines.