RESUMEN
The combination of nanoparticles (NPs) and cell-penetrating peptide (CPP) represents a new opportunity to develop plasmid DNA (pDNA) delivery systems with desirable properties for lung delivery. In this study, poly(lactide-co-glycolide) (PLGA) NPs containing pDNA were formulated with and without CPP using a double-emulsion technique. NPs were characterized in regards of size, surface charge, release profile, pDNA encapsulation efficiency and pDNA integrity. Cellular uptake, intracellular trafficking, uptake mechanism and pDNA expression were assessed in both A549 and Beas-2B cells. Manufactured PLGA-NPs efficiently encapsulated pDNA with approximately 50% released in the first 24 h of incubation. Addition of CPP was essential to promote NP internalization in both cell lines, with 83.85 ± 1.2% and 96.76 ± 1.7% of Beas-2B and A549 cells, respectively, with internalized NP-DNA-CPP after 3 h of incubation. Internalization appears to occur mainly via clathrin-mediated endocytosis, with other pathways also being used by the different cell lines. An endosomal-escape mechanism seems to happen in both cell lines, and eGFP expression was observed in Beas-2B after 96 h of incubation. In summary, the NP-DNA-CPP delivery system efficiently encapsulated and protected pDNA structure and is being investigated as a promising tool for gene delivery to the lungs.
Asunto(s)
Péptidos de Penetración Celular/química , ADN/administración & dosificación , Nanopartículas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Células A549 , Línea Celular , Clatrina/metabolismo , Emulsiones , Endocitosis , Células Epiteliales , Técnicas de Transferencia de Gen , Humanos , Pulmón/citología , Pulmón/metabolismo , PlásmidosRESUMEN
PURPOSE: In this study, a cell penetrating peptide was used as an uptake enhancer for pDNA delivery to the lungs. METHODS: Polyplexes were prepared between pDNA and CPP. Intracellular delivery of pDNA was assessed in both alveolar (A549) and bronchial (Calu-3) epithelial cells. Aerosol delivery was investigated using a mesh nebulizer. RESULTS: Efficient intracellular delivery of pDNA occurs in both A549 and Calu-3 cells when delivered as polyplexes. Protection against nucleases and endosomal escape mechanism occurs when pDNA is formulated within the polyplexes. For aerosol delivery, 1% (w/v) mannitol was able to protect naked DNA structure during nebulization with a significant increase in fine particle fraction (particles <5 µm). The structure of polyplexes when delivered via a mesh nebulizer using 1% (w/v) mannitol could partially withstand the shear forces involved in aerosolization. Although some loss in functionality occurred after nebulization, membrane-associated fluorescence was observed in A549 cells. In Calu-3 cells mucus entrapment was a limiting factor for polyplex delivery. CONCLUSIONS: The presence of CPP is essential for efficient intracellular delivery of pDNA. The polyplexes can be delivered to lung epithelial cells using mesh nebulizer. The use of different excipients is essential for further optimization of these delivery systems.
Asunto(s)
ADN/administración & dosificación , Administración por Inhalación , Aerosoles , Células Epiteliales Alveolares/metabolismo , Transporte Biológico , Bronquios/metabolismo , Línea Celular , Supervivencia Celular , Péptidos de Penetración Celular/química , Liberación de Fármacos , Técnicas de Transferencia de Gen , Humanos , Pulmón/metabolismo , Nebulizadores y Vaporizadores , Conformación de Ácido Nucleico , Tamaño de la Partícula , Plásmidos , Propiedades de SuperficieRESUMEN
Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 µM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction.
Asunto(s)
Doxiciclina/uso terapéutico , Quinasa 2 de Adhesión Focal/efectos de los fármacos , Linfangioleiomiomatosis/etiología , Sirolimus/uso terapéutico , Esclerosis Tuberosa/tratamiento farmacológico , Quinasas Asociadas a rho/efectos de los fármacos , Animales , Doxiciclina/administración & dosificación , Linfangioleiomiomatosis/tratamiento farmacológico , Ratones , RatasRESUMEN
Asthma and chronic obstructive pulmonary disease (COPD) are highly prevalent respiratory diseases characterized by airway inflammation, airway obstruction and airway hyperresponsiveness. Whilst current therapies, such as ß-agonists and glucocorticoids, may be effective at reducing symptoms, they do not reduce disease progression. Thus, there is a need to identify new therapeutic targets. In this review, we summarize the potential of novel targets or tools, including anti-inflammatories, phosphodiesterase inhibitors, kinase inhibitors, transient receptor potential channels, vitamin D and protease inhibitors, for the treatment of asthma and COPD.
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Asma/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Animales , Antiasmáticos/uso terapéutico , Antiinflamatorios/uso terapéutico , Humanos , Inhibidores de Fosfodiesterasa/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Canales de Potencial de Receptor Transitorio/uso terapéutico , Vitamina D/uso terapéuticoRESUMEN
Airway smooth muscle (ASM) is the main regulator of bronchomotor tone. Extensive studies show that in addition to their physical property, human airway smooth muscle (ASM) cells can participate in inflammatory processes modulating the initiation, perpetuation, amplification, and perhaps resolution of airway inflammation. Upon stimulation or interaction with immune cells, ASM cells produce and secrete a variety of inflammatory cytokines and chemokines, cell adhesion molecules, and extracellular matrix (ECM) proteins. These released mediators can, in turn, contribute to the inflammatory state, airway hyperresponsiveness, and airway remodeling present in asthma. As our knowledge of ASM myocyte biology improves, novel bioactive factors are emerging as potentially important regulators of inflammation. This review provides an overview of our understanding of some of these molecules, identifies rising questions, and proposes future studies to better define their role in ASM cell modulation of inflammation and immunity in the lung and respiratory diseases.
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Inflamación/patología , Miocitos del Músculo Liso/metabolismo , Enfermedades Respiratorias/fisiopatología , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Animales , Asma/inmunología , Asma/fisiopatología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Humanos , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/fisiopatología , Músculo Liso/citología , Músculo Liso/inmunología , Músculo Liso/metabolismo , Miocitos del Músculo Liso/inmunología , Enfermedades Respiratorias/inmunologíaRESUMEN
Asthma is characterized in part by variable airflow obstruction and non-specific hyperresponsiveness to a variety of bronchoconstrictors, both of which are mediated by the airway smooth muscle (ASM). The ASM is also involved in the airway inflammation and airway wall remodeling observed in asthma. For all these reasons, the ASM provides an important target for the treatment of asthma. Several classes of drugs were developed decades ago which targeted the ASM - including ß-agonists, anti-cholinergics, anti-histamines and anti-leukotrienes - but no substantially new class of drug has appeared recently. In this review, we summarize the on-going work of several laboratories aimed at producing novel targets and/or tools for the treatment of asthma. These range from receptors and ion channels on the ASM plasmalemma, to intracellular effectors (particularly those related to cyclic nucleotide signaling, calcium-homeostasis and phosphorylation cascades), to anti-IgE therapy and outright destruction of the ASM itself.
Asunto(s)
Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Músculo Liso/efectos de los fármacos , Obstrucción de las Vías Aéreas/tratamiento farmacológico , Obstrucción de las Vías Aéreas/patología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Animales , Asma/fisiopatología , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/fisiopatología , Diseño de Fármacos , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Terapia Molecular Dirigida , Músculo Liso/metabolismoRESUMEN
The airway smooth muscle (ASM) plays an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD). ASM cells express a wide range of receptors involved in contraction, growth, matrix protein production and the secretion of cytokines and chemokines. Transforming growth factor beta (TGF-ß) is one of the major players in determining the structural and functional abnormalities of the ASM in asthma and COPD. It is increasingly evident that TGF-ß functions as a master switch, controlling a network of intracellular and autocrine signaling loops that effect ASM phenotype and function. In this review, the various elements that participate in non-canonical TGF-ß signaling, including MAPK, PI3K, WNT/ß-catenin, and Ca(2+), are discussed, focusing on their effect on ASM phenotype and function. In addition, new aspects of ASM biology and their possible association with non-canonical TGF-ß signaling will be discussed.
Asunto(s)
Asma/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Fenotipo , Transducción de SeñalRESUMEN
Transforming growth factor (TGF) ß1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFß1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFß1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFß1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110ß inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL-6, calponin or smooth muscle α-actin (α-SMA) expression. However, TGFß1 increased calponin and α-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFß1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFß1 induced production of the contractile proteins calponin and α-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling.
Asunto(s)
Asma/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/metabolismo , Pulmón/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Asma/patología , Benzamidas/farmacología , Células Cultivadas , Cromonas/farmacología , Fosfatidilinositol 3-Quinasa Clase I , Proteínas Contráctiles/metabolismo , Dioxoles/farmacología , Femenino , Humanos , Interleucina-6/genética , Pulmón/citología , Masculino , Persona de Mediana Edad , Morfolinas/farmacología , Músculo Liso/citología , Fosfatidilinositol 3-Quinasas/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Quinazolinas/farmacología , Donantes de Tejidos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Recent evidence suggests that the rare and progressive lung disease lymphangioleiomyomatosis (LAM) is metastatic in nature. Dysfunction of the tumor suppressor genes tuberous sclerosis complex (TSC), in particular mutational inactivation of TSC2, enhances both cell proliferation and migration. Although substantial progress has been made in understanding the role of TSC2 in abnormal LAM cell proliferation and its pharmacological targeting, the mechanisms underlying the enhanced migratory capacity in LAM are not well understood. In this study, we examined the role of TSC2 in cell attachment, spreading, and migration, processes that contribute to the metastatic phenotype. Here we show that loss of TSC2 increased both the attachment and spreading of mouse embryonic fibroblasts to the extracellular matrix proteins collagen type I and fibronectin and that reexpression of TSC2 reduced these effects. Integrin-α1ß1 modulated cell migration with the ß1-subunit involved in cell attachment and spreading as shown by using functional blocking antibodies. Loss of TSC2 increased integrin-α1 expression, and inhibition of this integrin subunit reduced cell migration. The enhanced attachment and spreading were independent of the intracellular signaling pathways mammalian target of rapamycin complex 1 and Rho-associated kinase, as pharmacological inhibition with rapamycin or Y27632, respectively, was without effect. Together, these data demonstrate that TSC2 controls cell migration, attachment, and spreading through the α1ß1-integrin receptor and thus suggest a potential therapeutic target for the treatment of increased cell invasiveness in LAM.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Integrina alfa1beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatosis/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/citología , Leiomioma , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Mutantes , Complejos Multiproteicos , Invasividad Neoplásica/patología , Proteínas/metabolismo , Ratas , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR , Proteína 2 del Complejo de la Esclerosis Tuberosa , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
ß(2)-Adrenergic receptor (ß2AR) agonists induce airway relaxation via cAMP. Phosphodiesterase (PDE)s degrade and regulate cAMP, and in airway smooth muscle (ASM) cells PDE4D degrades cAMP. Long-acting ß(2)-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budesonide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)-ß(1), formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with ß2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Asma/patología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Etanolaminas/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Budesonida/farmacología , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Femenino , Fumarato de Formoterol , Glucocorticoides/farmacología , Humanos , Interleucina-6/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Receptores Adrenérgicos beta 2/metabolismo , Taquifilaxis , Adulto JovenRESUMEN
INTRODUCTION: Lymphangioleiomyomatosis (LAM) is a rare lung disease that is characterized by smooth muscle-like cell growth in the lungs. The current available oral treatment rapamycin slows down the disease progression but does not result in a cure. Rapamycin is also limited by its low bioavailability and dose-related adverse side effects. New treatments are, therefore, underway to investigate alternative targets and combination therapies for LAM. In recent years, much focus has been on the development of therapies based on inhaled nanotechnology using carriers to deliver drugs, as it is shown to improve drug solubility, local targeted treatment, and bioavailability. AREAS COVERED: This review, therefore, focuses on future prospective treatments for LAM using nanoparticles and lipid-based nanocarriers, including liposomes, solid lipid nanoparticles, micelles, and polymeric nanoparticles. It also investigates how nanoparticles' physicochemical factors such as size and charge can affect the treatment of both pulmonary and extrapulmonary LAM. EXPERT OPINION: Advanced clinical research is still needed to demonstrate the full potential and drive future commercialization of LAM treatments delivered via inhaled lipid nanobased formulations. If successful, the resultant effects will be seen in the improvement in the life expectancy and life quality of LAM patients.
Asunto(s)
Linfangioleiomiomatosis , Nanopartículas , Humanos , Lípidos/uso terapéutico , Liposomas , Linfangioleiomiomatosis/tratamiento farmacológico , Sirolimus/uso terapéuticoRESUMEN
The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in the airway remodeling associated with asthma. The class IA PI3K isoforms are known to be activated by growth factors and cytokines. Because this pathway is a possible site of pharmacological intervention for treating the disease, it is important to know which isoforms contribute to this process. Therefore, we used a pharmacological approach to investigate the roles of the three class IA PI3K isoforms (p110α, p110ß, and p110δ) in airway remodeling using airway smooth muscle (ASM) cells derived from asthmatic subjects and ASM cells and lung fibroblasts from nonasthmatic subjects. These studies used the inhibitors N'-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylidene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide (PIK75) (which selectively inhibits p110α), 7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (TGX221) (which selectively inhibits p110ß), and 2-[(6-amino-9H-purin-9-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (IC87114) (which selectively inhibits p110δ). Cells were stimulated with transforming growth factor-ß (TGFß) and/or 10% fetal bovine serum in the presence or absence of inhibitor or vehicle control (dimethyl sulfoxide). PIK75, but not TGX221 or IC87114, attenuated TGFß-induced fibronectin deposition in all cell types tested. PIK75 and TGX221 each decreased secretion of vascular endothelial growth factor and interleukin-6 in nonasthmatic ASM cells and lung fibroblasts, whereas TGX221 was not as effective in asthmatic ASM cells. In addition, PIK75 decreased cell survival in TGFß-stimulated asthmatic, but not nonasthmatic, ASM cells. In conclusion, specific PI3K isoforms may play a role in pathophysiological events relevant to airway wall remodeling.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Sistema Respiratorio/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Western Blotting , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/enzimología , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Hidrazonas/farmacología , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Morfolinas/farmacología , Músculo Liso/citología , Músculo Liso/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Pirimidinonas/farmacología , Quinazolinas/farmacología , Sistema Respiratorio/citología , Especificidad por Sustrato , Sulfonamidas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
RATIONALE: Angiogenesis is a prominent feature of remodeling in asthma. Many proangiogenic factors are up-regulated in asthma, but little is known about levels of endogenous antiangiogenic agents. Collagen IV is decreased in the airway basement membrane in asthma. It has six alpha chains, of which the noncollagenous domain-1 domains have endogenous antiangiogenic properties. OBJECTIVES: To study the expression of the noncollagenous domain-1 of the alpha3 chain of collagen IV, tumstatin, in the airways of subjects with and without asthma and to examine the potential for tumstatin to regulate angiogenesis and inflammation. METHODS: We used immunohistochemistry and dot blots to examine the expression of tumstatin in bronchial biopsies, bronchoalveolar lavage fluid, and serum. We then used an in vitro angiogenesis assay and a murine model of allergic airways disease to explore tumstatin's biological function. MEASUREMENTS AND MAIN RESULTS: The level of tumstatin is decreased 18-fold in the airways of patients with asthma but not in subjects without asthma, including those with chronic obstructive pulmonary disease, cystic fibrosis, and bronchiectasis. In vitro, recombinant tumstatin inhibited primary pulmonary endothelial cell tube formation. In a mouse model of chronic allergic airways disease, tumstatin suppressed angiogenesis, airway hyperresponsiveness, inflammatory cell infiltration, and mucus secretion and decreased levels of vascular endothelial growth factor and IL-13. CONCLUSIONS: The observation that tumstatin is decreased in asthmatic airways and inhibits airway hyperresponsiveness and angiogenesis demonstrates the potential use of antiangiogenic agents such as tumstatin as a therapeutic intervention in diseases that are characterized by aberrant angiogenesis and tissue remodeling, such as asthma.
Asunto(s)
Asma/fisiopatología , Autoantígenos/fisiología , Bronquios/irrigación sanguínea , Hiperreactividad Bronquial/fisiopatología , Bronquitis/fisiopatología , Colágeno Tipo IV/fisiología , Neovascularización Patológica/fisiopatología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Animales , Asma/patología , Biopsia , Bronquios/patología , Hiperreactividad Bronquial/patología , Bronquitis/patología , Broncoscopía , División Celular/fisiología , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/patología , Células Endoteliales/fisiología , Eosinofilia/patología , Eosinofilia/fisiopatología , Femenino , Humanos , Interleucina-13/metabolismo , Pulmón/irrigación sanguínea , Pulmón/patología , Masculino , Ratones , Microscopía Fluorescente , Persona de Mediana Edad , Neovascularización Patológica/patología , Hipersensibilidad Respiratoria/patología , Hipersensibilidad Respiratoria/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto JovenRESUMEN
Rhinovirus (RV) infections are the major cause of asthma exacerbations in children and adults. Under normal circumstances, asthmatic airway obstruction improves spontaneously or characteristically briskly in response to inhaled beta(2)-adrenergic receptor (beta(2)AR) agonists. During virus-associated exacerbations, an impaired response to beta(2)AR agonists is observed; the reason for this is not known. The objective of this study was to determine the effect of RV infection on airway smooth muscle beta(2)AR function. The human cell line Beas-2B and primary human bronchial epithelial cells (HBECs) were infected with RV (multiplicity of infection = 1). After 1 or 5 days for primary and Beas-2B cells, respectively, cell culture supernatants were harvested, UV-irradiated to inactivate RV, and applied to human airway smooth muscle cells for 3 days to assess modifications of beta(2)AR function. RV conditioned medium from Beas-2B and HBECs decreased beta(2)AR agonist-induced cAMP by 50 and 65%, respectively (n = 5; P < 0.05). When cAMP was induced independently of the beta(2)AR using forskolin, no impairment was found. Using flow cytometry, we demonstrated that this decrease was likely the result of beta(2)AR desensitization because membrane but not total cell receptor beta(2)AR was decreased. Pretreatment of HBECs and Beas-2B cells but not human airway smooth muscle cells with the corticosteroids dexamethasone or fluticasone abolished virus-mediated beta(2)AR loss of function. This study shows that epithelial infection with RV induces a decrease of beta(2)AR function on airway smooth muscle cells, potentially explaining the clinical observation of loss of beta(2)AR agonist function during RV-induced asthma exacerbations.
Asunto(s)
Asma/complicaciones , Asma/virología , Receptores Adrenérgicos beta 2/metabolismo , Rhinovirus/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Bronquios/patología , Medios de Cultivo Condicionados/metabolismo , Células Epiteliales/citología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Infecciones por Picornaviridae/complicaciones , Infecciones por Picornaviridae/virologíaRESUMEN
Human bronchial epithelial (HBE) cells contribute to asthmatic airway inflammation by secreting cytokines, chemokines, and growth factors, including interleukin (IL)-6, IL-8 and transforming growth factor (TGF) ß1, all of which are elevated in asthmatic airways. This study examines the signaling pathways leading to TGFß1 induced IL-6 and IL-8 in primary HBE cells from asthmatic and non-asthmatic volunteers. HBE cells were stimulated with TGFß1 in the presence or absence of signaling inhibitors. IL-6 and IL-8 protein and mRNA were measured by ELISA and real-time PCR respectively, and cell signaling kinases by Western blot. TGFß1 increased IL-6, but inhibited IL-8 production in both asthmatic and non-asthmatic cells; however, TGF induced significantly more IL-6 in asthmatic cells. Inhibition of JNK MAP kinase partially reduced TGFß1 induced IL-6 in both cell groups. TGFß1 induced Smad2 phosphorylation, and blockade of Smad2/3 prevented both the TGFß1 modulated IL-6 increase and the decrease in IL-8 production in asthmatic and non-asthmatic cells. Inhibition of Smad2/3 also increased basal IL-8 release in asthmatic cells but not in non-asthmatic cells. Using CHIP assays we demonstrated that activated Smad2 bound to the IL-6, but not the IL-8 promoter region. We conclude that the Smad2/3 pathway is the predominant TGFß1 signaling pathway in HBE cells, and this is altered in asthmatic bronchial epithelial cells. Understanding the mechanism of aberrant pro-inflammatory cytokine production in asthmatic airways will allow the development of alternative ways to control airway inflammation.
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Asma/inmunología , Bronquios/inmunología , Células Epiteliales/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mucosa Respiratoria/inmunología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Adolescente , Adulto , Anciano , Asma/metabolismo , Sitios de Unión , Western Blotting , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Interleucina-6/genética , Interleucina-8/genética , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Persona de Mediana Edad , Fosforilación , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Transducción de Señal , Proteína Smad2/antagonistas & inhibidores , Proteína smad3/antagonistas & inhibidores , Regulación hacia Arriba , Adulto JovenRESUMEN
UNLABELLED: PPARgamma levels in asthma- and non-asthma-derived airway smooth muscle cells and PPARgamma activation-induced cell proliferation were investigated. In the presence of FBS, PPARgamma levels were higher in subconfluent asthma-derived cells but lower in confluent cells compared with non-asthma-derived. However, PPARgamma activation did not alter cell proliferation. BACKGROUND AND OBJECTIVE: Airway remodelling involves thickening of the airway smooth muscle (ASM) bulk. Proliferation of asthma-derived ASM cells is increased in vitro, but underlying mechanisms remain unknown. Peroxisome proliferators activated receptor-gamma (PPARgamma) regulates the cell cycle. It is suggested that PPARgamma agonists have anti-inflammatory effects, which may be valuable in the treatment of asthma, but information regarding their antiproliferative properties in ASM is lacking. Although corticosteroids reduce airway inflammation, in vitro they inhibit proliferation in only non-asthma ASM cells by reducing cyclin D1. We therefore investigated the effects of mitogenic stimulation (foetal bovine serum (FBS)), and a PPARgamma ligand (ciglitazone), on PPARgamma and cyclin D1 expression and proliferation of ASM cells. In addition, we examined the effects of ciglitazone on ASM cell proliferation. METHODS: We assessed PPARgamma and cyclin D1 mRNA and protein levels using quantitative PCR and immunoblotting. Cell proliferation was assessed using bromodeoxyuridine uptake. RESULTS: In the presence of 5% FBS, PPARgamma and cyclin D1 expression decreased over time in non-asthmatic cells but increased in asthmatic cells (compared with sub-confluent cells). FBS-induced proliferation of asthmatic cells increased at all time points, but occurred only at day 7 with non-asthmatic cells (compared with unstimulated time-matched control). Ciglitazone increased PPARgamma expression in both groups, but did not alter cell proliferation, while fluticasone increased PPARgamma protein only in asthmatic cells. CONCLUSIONS: Although in the presence of a mitogenic stimulus, PPARgamma was differentially expressed in asthma- and non-asthma-derived ASM; its expression was not related to the increased proliferation observed in asthmatic ASM.
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Asma/metabolismo , Asma/patología , Proliferación Celular , Ciclina D1/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , PPAR gamma/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Androstadienos/farmacología , Bronquios/patología , Broncodilatadores/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Fluticasona , Humanos , Masculino , Persona de Mediana Edad , Mitógenos/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , ARN Mensajero/metabolismo , Tiazolidinedionas/farmacología , Adulto JovenRESUMEN
Aim: Lymphangioleiomyomatosis is characterized by smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Oral rapamycin is restricted by low bioavailability approximately 15%. The aim of the present study is to systematically investigate the effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLN) surface charge on efficacy and penetration into the lymphatics. Materials & methods: Rapa-SLN formulations with different charge: neutral, positive and negative, were produced and assessed for their physicochemical particle characteristics and efficacy in vitro. Results: Negative Rapa-SLNs were significantly faster at entering the lymphatic endothelium and more potent at inhibiting lymphanigiogenesis compared with neutral and positive Rapa-SLNs. Conclusion: Negative Rapa-SLNs showed efficient lymphatic access and should therefore be investigated further as a treatment for targeting extrapulmonary lymphangioleiomyomatosis.
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Vasos Linfáticos , Nanopartículas , Administración Oral , Portadores de Fármacos , Lípidos , Pulmón , Tamaño de la Partícula , SirolimusRESUMEN
Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by uncontrolled growth of smooth muscle -like cells in the lungs that can spread via the lymphatic system to other parts of the body. The current treatment for LAM, oral rapamycin, is limited by its low oral bioavailability and side effects. This study aims to develop an inhaled formulation of rapamycin solid lipid nanoparticles (Rapa-SLNs) to avoid first-pass metabolism, increase invivo half-life and facilitate entry into the lymphatic system through the lungs. Rapa-SLNs were manufactured using a hot evaporation technique and freeze-dried overnight with 5% (w/v) mannitol and before being assessed further for particle characteristics and in vitro aerosol performance and release. The formulation's ability to penetrate through bronchial epithelial layer was evaluated using a Calu-3 cell model, while its ability to interfere with the LAM intracellular cascade was evaluated using Mouse Embryonic fibroblast (MEF) cells deficient for the tuberous sclerosis complex 2 (TSC2) and compared with rapamycin solution. Results showed that the Rapa- SLNs had the appropriate size (237.5⯱â¯1.8â¯nm), charge (-11.2), in vitro aerosol performance (MMAD=5.4⯱â¯0.4⯵m) and sustained release profile suitable for entry into the lymphatic system via the pulmonary route. Additionally, the nanoparticles were transported at a faster rate across the bronchial epithelial layer compared to free rapamycin solution. The formulation also showed similar mTOR (mammalian target of Rapamycin) inhibition properties compared to free rapamycin, and was able to significantly decrease the amount of proliferation in TSC2 negative MEF cells. This formulation is therefore a promising alternative treatment for LAM patients, as it could potentially reduce problems associated with low bioavailability and side effects of current oral treatment.
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Lípidos/administración & dosificación , Linfangioleiomiomatosis/tratamiento farmacológico , Nanopartículas/administración & dosificación , Sirolimus/administración & dosificación , Administración por Inhalación , Aerosoles/administración & dosificación , Animales , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular , Humanos , Pulmón/efectos de los fármacos , Linfangioleiomiomatosis/metabolismo , Ratones , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Esclerosis Tuberosa/metabolismoRESUMEN
Asthma is characterized by structural changes in the airways - airway remodelling. These changes include an increase in the bulk of the airway smooth muscle (ASM) and alterations in the profile of extracellular matrix (ECM) proteins in the airway wall. The mechanisms leading to airway remodelling are not well understood. ASM cells have the potential to play a key role in these processes through the production and release of ECM proteins. The ASM cells and ECM proteins are each able to influence the behaviour and characteristics of the other. The modified ECM profile in the asthmatic airway may contribute to the altered behaviour of the ASM cells, such responses to ECM proteins are modulated through the cell surface expression of integrin receptors. ASM cells from asthmatic individuals express different levels of some integrin subunits compared to nonasthmatic ASM cells, which have the potential to further influence their responses to the ECM proteins in the airways. ECM homeostasis requires the presence and activation of matrix metalloproteinases and their tissue inhibitors, which in turn modulate the interaction of the ASM cells and the ECM proteins. Furthermore, the complex interactions of the ASM cells and the ECM in the asthmatic airways and the role played by external stimuli, such as viral infections, to modulate airway remodelling are currently unknown. This review summarises our current understanding of the influence of the ECM on ASM function.
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Asma/patología , Bronquios/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Músculo Liso/patología , Sistema Respiratorio/patología , Asma/metabolismo , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Diferenciación Celular , Humanos , Integrinas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Modelos Biológicos , Células Musculares/metabolismo , Células Musculares/patología , Músculo Liso/metabolismo , Neumonía Viral/metabolismo , Fibrosis Pulmonar/metabolismo , Sistema Respiratorio/metabolismoRESUMEN
BACKGROUND: Integrin receptors signal to and from the extracellular matrix. Altered expression of the integrin receptors, such as the fibronectin receptor alpha(5)beta(1), might be implicated in extracellular matrix accumulation in airway remodeling in asthma. OBJECTIVE: We examined the effect of TGF-beta stimulation on integrin alpha(5)beta(1) expression and the role of alpha(5)beta(1) in fibronectin deposition and proliferation. METHODS: Integrin subunit alpha(5) and beta(1) expression in airway smooth muscle (ASM) from subjects with and without asthma was examined by means of PCR and flow cytometry. The effect of blocking alpha(5)beta(1) receptor on ASM proliferation to FBS was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. Cells were stimulated with TGF-beta in the presence or absence of extracellular signal-regulated kinase, phosphoinositide-3 kinase, or p38 inhibitors and antibodies to the alpha(5) and beta(1) subunits. The effect of blocking alpha(5)beta(1) receptor on fibronectin deposition was assessed by means of immunocytochemistry. RESULTS: Proliferation of ASM cells from asthmatic and nonasthmatic subjects was inhibited by blocking the fibronectin receptor subunit alpha(5)beta(1). TGF-beta-induced alpha(5)beta(1) was extracellular signal-regulated kinase dependent but not phosphoinositide-3 kinase or p38 dependent. Blockade of the alpha(5)beta(1) receptor inhibited TGF-beta-induced fibronectin matrix deposition. CONCLUSION: Through its increased expression by the profibrotic stimulus TGF-beta, integrin alpha(5)beta(1) might be important in regulating fibronectin deposition.