RESUMEN
Humans interact with thousands of chemicals. This study aims to identify substances of emerging concern and in need of human health risk evaluations. Sixteen pooled human serum samples were constructed from 25 individual samples each from the National Institute of Environmental Health Sciences' Clinical Research Unit. Samples were analyzed using gas chromatography (GC) × GC/time-of-flight (TOF)-mass spectrometry (MS) in a suspect screening analysis, with follow-up confirmation analysis of 19 substances. A standard reference material blood sample was also analyzed through the confirmation process for comparison. The pools were stratified by sex (female and male) and by age (≤45 and >45). Publicly available information on potential exposure sources was aggregated to annotate presence in serum as either endogenous, food/nutrient, drug, commerce, or contaminant. Of the 544 unique substances tentatively identified by spectral matching, 472 were identified in females, while only 271 were identified in males. Surprisingly, 273 of the identified substances were found only in females. It is known that behavior and near-field environments can drive exposures, and this work demonstrates the existence of exposure sources uniquely relevant to females.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Pruebas Hematológicas , Femenino , Humanos , Masculino , Cromatografía de Gases y Espectrometría de Masas/métodos , Pruebas Hematológicas/métodos , Adulto , Persona de Mediana EdadRESUMEN
BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) carry significant morbidity and mortality. AECOPD treatment remains limited. High molecular weight hyaluronan (HMW-HA) is a glycosaminoglycan sugar, which is a physiological constituent of the lung extracellular matrix and has notable anti-inflammatory and hydrating properties. RESEARCH QUESTION: We hypothesized that inhaled HMW-HA will improve outcomes in AECOPD. METHODS: We conducted a single center, randomized, placebo-controlled, double-blind study to investigate the effect of inhaled HMW-HA in patients with severe AECOPD necessitating non-invasive positive-pressure ventilation (NIPPV). Primary endpoint was time until liberation from NIPPV. RESULTS: Out of 44 screened patients, 41 were included in the study (21 for placebo and 20 for HMW-HA). Patients treated with HMW-HA had significantly shorter duration of NIPPV. HMW-HA treated patients also had lower measured peak airway pressures on the ventilator and lower systemic inflammation markers after liberation from NIPPV. In vitro testing showed that HMW-HA significantly improved mucociliary transport in air-liquid interface cultures of primary bronchial cells from COPD patients and healthy primary cells exposed to cigarette smoke extract. INTERPRETATION: Inhaled HMW-HA shortens the duration of respiratory failure and need for non-invasive ventilation in patients with AECOPD. Beneficial effects of HMW-HA on mucociliary clearance and inflammation may account for some of the effects (NCT02674880, www.clinicaltrials.gov ).
Asunto(s)
Ácido Hialurónico/administración & dosificación , Mediadores de Inflamación/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Insuficiencia Respiratoria/tratamiento farmacológico , Insuficiencia Respiratoria/metabolismo , Adyuvantes Inmunológicos/administración & dosificación , Administración por Inhalación , Anciano , Anciano de 80 o más Años , Células Cultivadas , Método Doble Ciego , Femenino , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Tiempo de Internación/tendencias , Masculino , Persona de Mediana Edad , Peso Molecular , Proyectos Piloto , Contaminación por Humo de Tabaco/efectos adversosRESUMEN
BACKGROUND: Girls who are overweight/obese (OB) develop breast tissue but do not undergo menarche (the first menstrual period) significantly earlier than girls of normal weight (NW). It has been proposed that estrogen synthesized by adipose tissue may be contributory, yet OB do not have higher serum estrogen levels than NW matched on breast stage. We hypothesized that estrogen synthesized locally, in mammary fat, may contribute to breast development. This hypothesis would predict that breast development would be more advanced than other estrogen-sensitive tissues as a function of obesity and body fat. METHODS: Eighty premenarchal girls (26 OB, 54 NW), aged 8.2-14.7 years, underwent dual-energy x-ray absorptiometry to calculate percent body fat (%BF), Tanner staging of the breast, breast ultrasound for morphological staging, trans-abdominal pelvic ultrasound, hand x-ray (bone age, BA), a blood test for reproductive hormones, and urine collection to determine the vaginal maturation index (VMI), an index of estrogen exposure in urogenital epithelial cells. RESULTS: When controlling for breast morphological stage determined by ultrasound, %BF was not associated with serum estrogen or gonadotropin (LH and FSH) levels or with indices of systemic estrogen action (uterine volume, endometrial thickness, BA advancement, and VMI). Tanner breast stage did not correlate with breast morphological stage and led to misclassification of chest fatty tissue as breast tissue in some OB. CONCLUSIONS: These studies do not support the hypothesis that estrogen derived from total body fat or local (mammary) fat contributes to breast development in OB girls.
Asunto(s)
Tejido Adiposo/metabolismo , Mama/metabolismo , Desarrollo Infantil/fisiología , Estrógenos/metabolismo , Sobrepeso/metabolismo , Maduración Sexual/fisiología , Absorciometría de Fotón , Tejido Adiposo/crecimiento & desarrollo , Adolescente , Mama/crecimiento & desarrollo , Niño , Femenino , Encuestas Epidemiológicas , Humanos , Menarquia , North Carolina/epidemiología , Sobrepeso/epidemiología , Vagina/citologíaRESUMEN
Cerium dioxide nanoparticles (nanoceria), currently used as catalysts including additives to diesel fuel, also present potential as a novel therapeutic agent for disorders involving oxidative stress. However, little is known about the effects of nanoceria on primary human cells involved in the innate immune response. Here, we evaluate nanoceria effects on monocyte derived macrophages (MDMs) from healthy human subjects. Peripheral blood monocytes were isolated from healthy human volunteers. MDMs were obtained by maturing monocytes over a five-day period. MDMs were exposed to well-characterized nanoceria suspensions (0, 5, 10, 20 µg/mL) for 24 or 48 hours. We evaluated particle uptake, ultrastructural changes, cytotoxicity, and mitochondrial damage in MDMs through transmission electron microscopy (TEM), confocal imaging, flow cytometry, spectrometry, western blots, and immunofluorescence techniques. The role that intracellular concentration of nanoceria plays in the toxicity of MDMs was evaluated by 3D image analysis and compared to monocytes as a nanoceria sensitive cell model. Nanoceria failed to induce cytotoxicity in MDMs at the tested doses. Nanoceria-exposed MDMs showed no mitochondrial damage and displayed significant accumulation of anti-apoptotic proteins (Mcl-1 and Bcl-2) during the maturation process. TEM and confocal analyses revealed efficient uptake of nanoceria by MDMs, however 3D image analyses revealed lower nanoceria accumulation per unit cell volume in MDMs compared to monocytes. Taken together, our results suggest that mitochondrial protection and reduced volume-corrected intracellular nanoparticle concentration account for the lower sensitivity of human MDMs to nanoceria.
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Cerio/toxicidad , Macrófagos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Nanopartículas/toxicidad , Adulto , Células Cultivadas , Cerio/farmacocinética , Humanos , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas/metabolismoRESUMEN
Multiwalled carbon nanotubes (MWCNTs) have seen increasing application in consumer products over the past decade, resulting in an increasing risk of human exposure. While numerous toxicological studies have been performed using acute high doses of various carbonaceous nanomaterials, the effects of longer-term, low doses of MWCNTs remain relatively unexplored. This study examined bronchoscopy-derived healthy human bronchial epithelial cells exposed in submerged culture to noncytotoxic doses of MWCNTs over 7 days. Under these conditions, doses as low as 3 µg/mL caused altered cell morphology, superficially resembling fibroblasts. Electrical impedance of the epithelial monolayer was greatly reduced following MWCNT exposure. However, Western blot and polymerase chain reaction showed no elevated expression of the fibroblast markers, vimentin, α-smooth muscle actin, or fibronectin, indicating that a mechanism other than epithelial-mesenchymal transition may be responsible for the changes. Phalloidin and tubulin immunostaining showed disruption of the cytoskeleton, and confocal imaging showed a reduction of the tight junction proteins, zona occludens 1 and occludin. We propose that MWCNTs interfere with the cytoskeleton of the lung epithelium, which can result in a harmful reduction in barrier function over time, even at noncytotoxic doses.
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Bronquios/citología , Células Epiteliales/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Muerte Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Impedancia Eléctrica , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Nanotubos de Carbono/química , Uniones Estrechas/metabolismoRESUMEN
BACKGROUND: In vivo studies have demonstrated the ability of multi-walled carbon nanotubes (MWCNT) to induce airway remodeling, a key feature of chronic respiratory diseases like asthma and chronic obstructive pulmonary disease. However, the mechanism leading to remodeling is poorly understood. Particularly, there is limited insight about the role of airway epithelial injury in these changes. OBJECTIVES: We investigated the mechanism of MWCNT-induced primary human bronchial epithelial (HBE) cell injury and its contribution in inducing a profibrotic response. METHODS: Primary HBE cells were exposed to thoroughly characterized MWCNTs (1.5-24 µg/mL equivalent to 0.37-6.0 µg/cm2) and MRC-5 human lung fibroblasts were exposed to 1:4 diluted conditioned medium from these cells. Flow cytometry, ELISA, immunostainings/immunoblots and PCR analyses were employed to study cellular mechanisms. RESULTS: MWCNT induced NLRP3 inflammasome dependent pyroptosis in HBE cells in a time- and dose-dependent manner. Cell death and cytokine production were significantly reduced by antioxidants, siRNA to NLRP3, a caspase-1 inhibitor (z-WEHD-FMK) or a cathepsin B inhibitor (CA-074Me). Conditioned medium from MWCNT-treated HBE cells induced significant increase in mRNA expression of pro-fibrotic markers (TIMP-1, Tenascin-C, Procollagen 1, and Osteopontin) in human lung fibroblasts, without a concomitant change in expression of TGF-beta. Induction of pro-fibrotic markers was significantly reduced when IL-1ß, IL-18 and IL-8 neutralizing antibodies were added to the conditioned medium or when conditioned medium from NLRP3 siRNA transfected HBE cells was used. CONCLUSIONS: Taken together these results demonstrate induction of a NLRP3 inflammasome dependent but TGF-beta independent pro-fibrotic response after MWCNT exposure.
Asunto(s)
Células Epiteliales/patología , Fibroblastos/patología , Inflamación/patología , Nanotubos de Carbono/toxicidad , Fibrosis Pulmonar/patología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos , Inmunohistoquímica , Inflamación/inducido químicamente , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , SuspensionesRESUMEN
Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of the reactive sulfur trioxide ((â¢)SO3(-)), peroxymonosulfate ((-)O3SOO(â¢)), and sulfate (SO4(â¢-)) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions, and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of this study was to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage, possibly leading to (bi)sulfite-exacerbated allergic reactions.
Asunto(s)
Radicales Libres/toxicidad , Hipersensibilidad/metabolismo , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Sulfitos/toxicidad , Radicales Libres/metabolismo , Humanos , Hipersensibilidad/etiología , Hipersensibilidad/patología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Peroxidasa/efectos de los fármacos , Peróxidos/química , Peróxidos/metabolismo , Peróxidos/toxicidad , Ésteres del Forbol/farmacología , Proteínas/metabolismo , Detección de Spin , Sulfatos/química , Sulfatos/metabolismo , Sulfatos/toxicidad , Sulfitos/metabolismo , Óxidos de Azufre/química , Óxidos de Azufre/metabolismo , Óxidos de Azufre/toxicidadRESUMEN
Cerium dioxide nanoparticles (CeO(2) NPs) have diversified industrial uses, and novel therapeutic applications are actively being pursued. There is a lack of mechanistic data concerning the effects of CeO(2) NPs on primary human cells. We aimed at characterizing the cytotoxic effects of CeO(2) NPs in human peripheral blood monocytes. CeO(2) NPs and their suspensions were thoroughly characterized, including using transmission electron microscopy (TEM), dynamic light scattering, and zeta potential analysis. Blood from healthy human volunteers was drawn through phlebotomy, and CD14+ cells were isolated. Cells were exposed to CeO(2) NPs (0.5-10 µg/mL) for 20 or 40 h, and mechanisms of cell injury were studied. TEM revealed that CeO(2) NPs are internalized by monocytes and are found either in vesicles or free in the cytoplasm. CeO(2) NP exposure leads to decrease in cell viability, and treated cells exhibit characteristic hallmarks of apoptosis (activation of Bax, loss of mitochondrial membrane potential, DNA fragmentation). CeO(2) NP toxicity is caused by mitochondrial damage and overexpression of apoptosis inducing factor, but is not due to caspase activation or reactive oxygen species production. Moreover, CeO(2) NP exposure leads to autophagy, which is further increased after pharmacological inhibition of tumor suppressor protein p53. Inhibition of autophagy partially reverses cell death by CeO(2) NPs. It is concluded that CeO(2) NPs are toxic to primary human monocytes at relatively low doses.
Asunto(s)
Cerio/toxicidad , Nanopartículas del Metal/toxicidad , Monocitos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Humanos , Técnicas In Vitro , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de Transmisión , Monocitos/metabolismo , Monocitos/patología , Nanotecnología , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: Cerium dioxide (CeO(2)) nanoparticles have potential therapeutic applications and are widely used for industrial purposes. However, the effects of these nanoparticles on primary human cells are largely unknown. The ability of nanoparticles to exacerbate pre-existing inflammatory disorders is not well documented for engineered nanoparticles, and is certainly lacking for CeO(2) nanoparticles. We investigated the inflammation-modulating effects of CeO(2) nanoparticles at noncytotoxic concentrations in human peripheral blood monocytes. METHODS: CD14(+) cells were isolated from peripheral blood samples of human volunteers. Cells were exposed to either 0.5 or 1 µg/mL of CeO(2) nanoparticles over a period of 24 or 48 hours with or without lipopolysaccharide (10 ng/mL) prestimulation. Modulation of the inflammatory response was studied by measuring secreted tumor necrosis factor-alpha, interleukin-1beta, macrophage chemotactic protein-1, interferon-gamma, and interferon gamma-induced protein 10. RESULTS: CeO(2) nanoparticle suspensions were thoroughly characterized using dynamic light scattering analysis (194 nm hydrodynamic diameter), zeta potential analysis (-14 mV), and transmission electron microscopy (irregular-shaped particles). Transmission electron microscopy of CD14(+) cells exposed to CeO(2) nanoparticles revealed that these nanoparticles were efficiently internalized by monocytes and were found either in vesicles or free in the cytoplasm. However, no significant differences in secreted cytokine profiles were observed between CeO(2) nanoparticle-treated cells and control cells at noncytotoxic doses. No significant effects of CeO(2) nanoparticle exposure subsequent to lipopolysaccharide priming was observed on cytokine secretion. Moreover, no significant difference in lipopolysaccharide-induced cytokine production was observed after exposure to CeO(2) nanoparticles followed by lipopolysaccharide exposure. CONCLUSION: CeO(2) nanoparticles at noncytotoxic concentrations neither modulate pre-existing inflammation nor prime for subsequent exposure to lipopolysaccharides in human monocytes from healthy subjects.
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Cerio/farmacología , Nanopartículas del Metal/química , Monocitos/efectos de los fármacos , Análisis de Varianza , Supervivencia Celular , Células Cultivadas , Cerio/química , Cerio/farmacocinética , Citocinas/metabolismo , Histocitoquímica , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/efectos adversos , Monocitos/inmunología , Monocitos/patología , Nanomedicina , FagocitosisRESUMEN
The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite ((â¢)SO(3)(-)) and sulfate (SO(4)(â¢-)) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO(4)(â¢-)) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Radicales Libres/metabolismo , Activación Neutrófila , Neutrófilos/metabolismo , Sulfitos/metabolismo , Células Cultivadas , Humanos , Neutrófilos/enzimología , Oxidación-Reducción , Peroxidasa/antagonistas & inhibidores , Peroxidasa/metabolismo , Marcadores de SpinRESUMEN
We have studied nonsteroidal ligands of the human androgen receptor (hAR) and have shown elsewhere that when photoactivated by visible light they collide with O2 to yield singlet oxygens (1O2) in vitro. Here we report cell killing after brief light activation (405 nm) of 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ) in human prostate tumor cells. TDPQ/AR complexes were required for the death response because AR-positive LNCaP cells were killed, whereas AR-negative PC-3 cells were resistant. Excess dihydrotestosterone (DHT) blocked the TDPQ effect when the two were added together; irradiation of cells containing DHT alone had no effect. When LNCaP AR expression was suppressed using small interfering oligonucleotides targeting AR, photocytotoxicity was diminished. Conversely, stable transfection of hAR into PC-3 cells made the cells photosensitive to TDPQ. Similar results were obtained using a structural isomer of TDPQ, and also the synthetic steroidal AR ligand R1881. Cell death occurred via apoptosis as demonstrated by annexin V immunostaining, nuclear condensation, and caspase inhibition. Death involved oxidative stress, because it was prevented by addition of the antioxidant ascorbic acid during photoactivation. Detection of elevated levels of 8-hydroxy-2'-deoxyguanosine in nuclei of irradiated cells indicated oxidative DNA damage. Apoptosis spread into adjacent nonirradiated cells by direct cell-cell contacts, indicative of a bystander effect. Other photoactivatable ligands are described, implying a general method for ablation of cells bearing specific nuclear hormone receptors.
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Apoptosis/fisiología , Receptores Androgénicos/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Antagonistas de Receptores Androgénicos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Secuencia de Bases , Línea Celular Tumoral , Daño del ADN , Dihidrotestosterona/farmacología , Humanos , Ligandos , Masculino , Metribolona/farmacología , Metribolona/efectos de la radiación , Modelos Biológicos , Estrés Oxidativo , Fotobiología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Piridinas/farmacología , Piridinas/efectos de la radiación , Quinolonas/farmacología , Quinolonas/efectos de la radiación , ARN Interferente Pequeño/genética , Receptores Androgénicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The signal transducer and activator of transcription (Stat)-1 mediates growth arrest and apoptosis. We postulated that lung fibrosis characterized by excessive proliferation of lung fibroblasts would be enhanced in Stat1-deficient (Stat1-/-) mice. Two weeks after bleomycin aspiration (3 U/kg), Stat1-/- mice exhibited a more severe fibroproliferative response and significantly elevated total lung collagen compared to wild-type mice. Growth factors [epidermal growth factor (EGF) or platelet-derived growth factor (PDGF)] enhanced [3H]thymidine uptake in lung fibroblasts isolated from Stat1-/- mice compared to wild-type mice. Interferon (IFN)-gamma, which signals growth arrest via Stat1, inhibited EGF- or PDGF-stimulated mitogenesis in wild-type fibroblasts but enhanced [3H]thymidine uptake in Stat1-/- fibroblasts. Moreover, IFN-gamma treatment in the absence of growth factors induced a concentration-dependent increase in [3H]thymidine uptake in Stat1-/- but not wild-type fibroblasts. Mitogen-activated protein kinase (ERK-1/2) phosphorylation in response to PDGF or EGF did not differ among Stat1-/- and wild-type fibroblasts. However, Stat3 phosphorylation induced by PDGF, EGF, or IFN-gamma increased twofold in Stat1-/- fibroblasts compared to wild-type fibroblasts. Our findings indicate that Stat1-/- mice are more susceptible to bleomycin-induced lung fibrosis than wild-type mice due to 1) enhanced fibroblast proliferation in response to growth factors (EGF and PDGF), 2) stimulation of fibroblast growth by a Stat1-independent IFN-gamma signaling pathway, and 3) increased activation of Stat3.
Asunto(s)
Pulmón/patología , Fibrosis Pulmonar/patología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/fisiología , Animales , Bleomicina/toxicidad , Western Blotting , Proliferación Celular , Células Cultivadas , Colágeno/análisis , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/patología , Inhibidores de Crecimiento/farmacología , Hidroxiprolina/análisis , Interferón gamma/farmacología , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/fisiopatología , Factor de Transcripción STAT3/metabolismo , Timidina/metabolismoRESUMEN
In cystic fibrosis and chronic bronchitis, airways are chronically injured by exposure to neutrophil elastase (NE). We sought to identify factors required for epithelial repair following NE exposure. Normal human bronchial epithelial cells were treated with NE (50 nM, 22 h) or control vehicle. Following NE treatment, we found a marked and sustained decrease in epithelial proliferation as detected by Ki67 immunostaining. 3H-thymidine incorporation was also initially depressed but increased over 72 h in NE-treated cells, which suggests that DNA synthesis constitutes an early repair process following NE exposure. We hypothesized that ErbB2 receptor tyrosine kinase, a regulator of cancer cell proliferation, was required for epithelial DNA synthesis following NE exposure. Immediately following NE treatment, by flow cytometry analysis, we found a decrease in ErbB2 surface expression. Protein levels of the full-length 185 kD ErbB2 receptor significantly decreased following NE treatment and smaller ErbB2-positive bands, ranging in size from 23 to 40 kD, appeared, which suggests that NE caused ErbB2 degradation. By real-time RT-PCR analysis, we found no change in ErbB2 mRNA expression following NE treatment, which suggests that changes in ErbB2 protein levels were regulated at the post-translational level. Following NE treatment, full-length 185 kD ErbB2 levels increased to pretreatment levels, correlating with the increase in thymidine incorporation during the same time period. Importantly, inhibition of ErbB2 activity with AG825 (5 microM) or Herceptin (3.1 microM), an ErbB2-neutralizing antibody, blocked thymidine incorporation only in NE-treated cells. These results suggest ErbB2 is a critical factor for epithelial recovery following NE exposure.
Asunto(s)
Bronquios/efectos de los fármacos , Elastasa de Leucocito/toxicidad , Receptor ErbB-2/fisiología , Benzotiazoles , Bronquios/citología , Bronquios/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Receptor ErbB-2/antagonistas & inhibidores , Tirfostinos/farmacologíaRESUMEN
Peribronchiolar fibrosis is a prominent feature of airway remodeling in asthma and involves fibroblast growth and collagen deposition. Interleukin-13 (IL-13), a T-helper 2 cytokine, is a key mediator of airway remodeling in asthma, yet the mechanism through which IL-13 promotes fibroblast growth has not been investigated. In this study, we show that IL-13 stimulates the mitogenesis of mouse, rat, and human lung fibroblasts through release of a soluble mitogen that we identified as PDGF-AA. The IL-13-induced growth of human lung fibroblasts was attenuated by an anti-PDGF-AA neutralizing antibody, and IL-13 stimulated human lung fibroblasts to secrete PDGF-AA. Fibroblasts derived from mouse embryos possessing the lethal Patch mutation, which lack the PDGF-Ralpha, showed no mitogenic response to IL-13. However, Patch cells did exhibit IL-13-induced STAT-6 phosphorylation. Stable transfection of the PDGF-Ralpha into Patch cells restored the growth response to PDGF-AA and IL-13. Through the use of lung fibroblasts from STAT-6-deficient mice, we showed that IL-13-induced PDGF-AA release is STAT-6 dependent, but PDGF-AA-induced growth is STAT-6 independent. Finally, we showed that IL-1beta enhanced IL-13-induced mitogenesis of rat lung fibroblasts through up-regulation of the PDGF-Ralpha. Our findings indicate that IL-13 acts in synergy with IL-1beta to stimulate growth by coordinately up-regulating PDGF-AA and the PDGF-Ralpha, respectively.
Asunto(s)
Interleucina-13/fisiología , Interleucina-1/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Animales , Becaplermina , División Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Medios de Cultivo Condicionados/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Humanos , Interleucina-1/genética , Interleucina-1/farmacología , Interleucina-13/genética , Interleucina-13/farmacología , Pulmón/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Sprague-Dawley , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/deficiencia , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Transcripción STAT6 , Transactivadores/fisiología , Factor de Crecimiento Transformador alfa/deficienciaRESUMEN
Vanadium compounds present in air pollution particulate matter activate signal transduction pathways in pulmonary cell types leading to pathological outcomes including aberrant cell proliferation, apoptosis, and cytokine expression. Vanadium has been proposed to activate transcription factors via the generation of hydrogen peroxide (H2O2). We investigated the mechanisms through which vanadium pentoxide (V2O5), the major form of vanadium released from the industrial burning of fuel oil, activated the signal transducer and activator of transcription (STAT)-1. V2O5-induced STAT-1 activation was blocked by catalase and N-acetyl-L-cysteine (NAC), suggesting vanadium-induced generation of H2O2. Surprisingly, however, V2O5 did not increase H2O2 levels released by rat lung myofibroblasts into cell culture supernatants. Instead, these quiescent myofibroblasts spontaneously released micromolar concentrations of H2O2, and the addition of V2O5 reduced H2O2 levels in cell culture supernatants within minutes. V2O5 suppressed H2O2 for as long as 24 h. Differences in the temporal activation of STAT-1 and p38 MAPK were observed following V2O5 or H2O2 treatment, and STAT-1 activation by V2O5 or H2O2 was attenuated by an inhibitor of the EGF receptor tyrosine kinase (AG1478) or p38 MAPK (SB203580). The phosphorylation of p38 MAPK by V2O5 was inhibited by NAC and catalase, yet the EGF receptor inhibitor AG1478 had no effect on V2O5-induced p38 MAPK activation. Collectively, our findings support the novel hypothesis that H2O2 spontaneously generated by myofibroblasts fuels vanadium-induced activation of STAT-1. Moreover, p38 MAPK and EGF receptor activation are required for V2O5-induced STAT-1 activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Peróxido de Hidrógeno/metabolismo , Pulmón/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transactivadores/metabolismo , Compuestos de Vanadio/farmacología , Acetilcisteína/farmacología , Animales , Carbono/efectos adversos , Catalasa/farmacología , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Pulmón/citología , Pulmón/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Factor de Transcripción STAT1 , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Vanadium pentoxide (V(2)O(5)) is a transition metal derived from the burning of petrochemicals that causes airway fibrosis and remodeling. Vanadium compounds activate many intracellular signaling pathways via the generation of hydrogen peroxide (H(2)O(2)) or other reactive oxygen species. In this study, we investigated the regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in human lung fibroblasts after V(2)O(5) treatment. V(2)O(5)-induced HB-EGF mRNA expression was abolished by N-acetyl-l-cysteine, suggesting an oxidant-mediated effect. Exogenous H(2)O(2) (>10 microM) mimicked the effect of V(2)O(5) in upregulating HB-EGF expression. Fibroblasts spontaneously released low levels of H(2)O(2) (1-2 microM), and the addition of V(2)O(5) depleted the endogenous H(2)O(2) pool within minutes. V(2)O(5) caused a subsequent increase of H(2)O(2) into the culture medium at 12 h. However, the burst of V(2)O(5)-induced H(2)O(2) occurred after V(2)O(5)-induced HB-EGF mRNA expression at 3 h, indicating that the V(2)O(5)-stimulated H(2)O(2) burst did not mediate HB-EGF expression. Either V(2)O(5) or H(2)O(2) activated ERK-1/2 and p38 MAP kinase. Inhibitors of the ERK-1/2 pathway (PD-98059) or p38 MAP kinase (SB-203580) significantly reduced either V(2)O(5)- or H(2)O(2)-induced HB-EGF expression. These data indicate that vanadium upregulates HB-EGF via ERK and p38 MAP kinases. The induction of HB-EGF is not related to a burst of H(2)O(2) in V(2)O(5) treated cells, yet the action of V(2)O(5) in upregulating HB-EGF is oxidant dependent and could be due to the reaction of V(2)O(5) with endogenous H(2)O(2).
Asunto(s)
Factor de Crecimiento Epidérmico/genética , Fibroblastos/enzimología , Pulmón/citología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Vanadio/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Peróxido de Hidrógeno/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fibrosis Pulmonar/metabolismo , ARN Mensajero/análisis , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Asma/complicaciones , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Interleucina-13/farmacología , Pulmón/efectos de los fármacos , Miofibrillas/efectos de los fármacos , Fibrosis Pulmonar/etiología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Animales , Asma/fisiopatología , Modelos Animales de Enfermedad , Fibroblastos/fisiología , Técnicas In Vitro , Pulmón/fisiopatología , Miofibrillas/fisiología , Fibrosis Pulmonar/fisiopatología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Myofibroblast proliferation is a central feature of pulmonary fibrogenesis. Several growth factors, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), stimulate myofibroblast growth by activating extracellular signal regulated kinases 1 and 2 (ERK1/2). In this report, we demonstrate that PDGF-BB and EGF also activate the p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 activity with the pyridinylimidazole compound SB203580 enhanced both PDGF-BB and EGF-stimulated DNA synthesis in rat lung myofibroblasts. ERK1/2 phosphorylation in response to either PDGF-BB or EGF treatment was significantly increased by pretreatment of cells with SB203580. We also demonstrated that ERK1/2-induced phosphorylation of PHAS-1 substrate was enhanced by inhibition of p38 MAP kinase with SB203580. However, SB203580 did not significantly increase growth factor-induced activation of MEK, the upstream kinase that phosphorylates ERK1/2. p38 MAP kinase was co-immunoprecipitated with ERK-1/2 following growth factor stimulation. Collectively, these data demonstrate that p38 MAP kinase activation negatively regulates PDGF- and EGF-mediated growth responses by directly interacting with ERK1/2 and suppressing its phosphorylation.
Asunto(s)
Fibroblastos/citología , Fibroblastos/enzimología , Sustancias de Crecimiento/farmacología , Pulmón/citología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Imidazoles/farmacología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/análisis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/análisis , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/farmacología , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The cyclooxygenase (COX)-2 enzyme has been implicated as an important mediator of pulmonary fibrosis. In this study, the lung fibrotic responses were investigated in COX-1 or COX-2-deficient (-/-) mice following vanadium pentoxide (V(2)O(5)) exposure. Lung histology was normal in saline-instilled wild-type and COX-deficient mice. COX-2(-/-), but not COX-1(-/-) or wild-type mice, exhibited severe inflammatory responses by 3 days following V(2)O(5) exposure and developed pulmonary fibrosis 2 weeks post-V(2)O(5) exposure. Western blot analysis and immunohistochemistry showed that COX-1 protein was present in type 2 epithelial cells, bronchial epithelial cells, and airway smooth muscle cells of saline or V(2)O(5)-exposed wild-type and COX-2(-/-) mice. COX-2 protein was present in Clara cells of wild-type and COX-1(-/-) terminal bronchioles and was strongly induced 24 hours after V(2)O(5) exposure. Prostaglandin (PG) E(2) levels in the bronchoalveolar lavage (BAL) fluid from wild-type and COX-1(-/-) mice were significantly up-regulated by V(2)O(5) exposure within 24 hours, whereas PGE(2) was not up-regulated in COX-2(-/-) BAL fluid. Tumor necrosis factor-alpha was elevated in the BAL fluid from all genotypes after V(2)O(5) exposure, but was significantly and chronically elevated in the BAL fluid from COX-2(-/-) mice above wild-type or COX-1(-/-) mice. These findings indicate that the COX-2 enzyme is protective against pulmonary fibrogenesis, and we suggest that COX-2 generation of PGE(2) is an important factor in resolving inflammation.