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1.
Cell Physiol Biochem ; 53(6): 1015-1028, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31854953

RESUMO

BACKGROUND/AIMS: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. METHODS: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. RESULTS: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. CONCLUSION: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.


Assuntos
Antibacterianos/metabolismo , Esfingosina/metabolismo , Administração por Inalação , Animais , Antibacterianos/farmacologia , Brônquios/metabolismo , Brônquios/patologia , Ceramidas/análise , Humanos , Pulmão/patologia , Lisofosfolipídeos/análise , Espectrometria de Massas , Pseudomonas aeruginosa/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/análise , Esfingosina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Suínos , Porco Miniatura , Traqueia/metabolismo , Traqueia/patologia
2.
Toxicol Lett ; 316: 10-19, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476341

RESUMO

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 µg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.


Assuntos
Células Epiteliais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Tabaco/efeitos adversos , Traqueia/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Masculino , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos Sprague-Dawley , Medição de Risco , Fatores de Tempo , Técnicas de Cultura de Tecidos , Traqueia/metabolismo , Traqueia/ultraestrutura
3.
Pathol Res Pract ; 215(10): 152614, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31500927

RESUMO

BACKGROUND: Smoking cessation may help in the reversal of inflammation and damage caused by smoking. The endogenous annexin A1 (AnxA1) protein has anti-inflammatory effects which instigates the understanding of its role in the attenuation of inflammatory processes caused by smoking. MATERIAL AND METHODS: Wistar rats were exposed to cigarette smoke for 8 weeks. After the exposure period, one of the groups remained other 8 weeks in the absence of smoke. Animals not exposed to smoke were used as control. Blood, trachea and lungs were obtained for histopathological, immunohistochemical and biochemical analyses. RESULTS: Loss of cilia of the tracheal lining epithelium was found by smoke exposure, but smoking cessation led to recovery of the tracheal epithelium. Similarly, chronically exposed-to-smoke animals showed increased lymphocytes and macrophages in bronchoalveolar lavage and higher levels of glucose and gamma-GT in their blood. Reduction of lymphocytes, glucose and gamma-GT occurred after smoking cessation. In addition, IL-1ß, IL-6, IL-10, TNF-α and MCP-1 levels were elevated by smoke exposure. Smoking cessation significantly reduced the levels of IL-1ß, IL-6 and MCP-1 but increased the IL-10 concentration. Numerous mast cells and macrophages were observed in the lung of chronically exposed-to-smoke animals with reduction by smoking cigarette abstinence. AnxA1 increased expression and concomitant NF-κB reduction were found in the smoking cessation group. CONCLUSION: Our results showed that cigarette abstinence promoted partial recovery of the inflammatory process. The attenuation of the inflammatory profile may be associated with the overexpression of AnxA1 protein.


Assuntos
Anexina A1/metabolismo , Pulmão/metabolismo , Abandono do Hábito de Fumar , Fumar/metabolismo , Traqueia/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/patologia , Pulmão/patologia , Linfócitos/metabolismo , Macrófagos/metabolismo , Ratos , Ratos Wistar , Fumaça , Fumar/patologia , Traqueia/patologia
4.
Iran J Allergy Asthma Immunol ; 18(3): 320-331, 2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31522439

RESUMO

In this study we aimed to examine the relaxant effect of berberine, a compound extracted from a variety of herbs, on rat tracheal smooth muscle (TSM) and its possible mechanism(s). Cumulative concentrations of berberine (20, 65, 200 and 600 µg/mL) were added on pre-contracted TSM by methacholine or KCl in non-incubated or incubated tissues with atropine, chlorpheniramine, propranolol, diltiazem, glibenclamide, indomethacin, L-NG-nitro arginine methyl ester (L-NAME) and papaverine. The relaxant effects of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control and saline (1 mL) as negative control were also examined in non-incubated tissues. Berberine showed significant and concentration-dependent relaxant effects in non-incubated tissues contracted by KCl and methacholine (p<0.01 to p<0.001). There was no significant difference in the relaxant effects of berberine between non-incubated and incubated tissues with atropine, propranolol, diltiazem, glibenclamide, and papaverine. The relaxant effects of second concentrations of berberine in incubated tissues with L-NAME, its three lower concentration in incubated tissues with chlorpheniramine and its all concentrations in incubated tissues with indomethacin were significantly lower than non-incubated tissues (p<0.05 to p<0.001). The EC50 values of berberine in incubated tissues with chlorpheniramine was significantly higher than the non-incubated condition (p<0.05). Our findings reveal a relatively potent relaxant effect of berberine that is lower than the effect of theophylline. Proposed mechanisms for the relaxant effect of berberine are histamine (H1) receptor blockade, inhibition of cyclooxygenase pathways and/or nitric oxide formation.


Assuntos
Berberina/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Histamínicos H1/metabolismo , Transdução de Sinais , Animais , Berberina/química , Broncodilatadores/química , Broncodilatadores/farmacologia , Relação Dose-Resposta a Droga , Feminino , Masculino , Modelos Biológicos , Cloreto de Potássio/farmacologia , Ratos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
5.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261663

RESUMO

The up-regulation of heme oxygenase-1 (HO-1) is mediated through nicotinamaide adenine dinucleotide phosphate (NADPH) oxidases (Nox) and reactive oxygen species (ROS) generation, which could provide cytoprotection against inflammation. However, the molecular mechanisms of carbon monoxide-releasing molecule (CORM)-2-induced HO-1 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we found that pretreatment with CORM-2 attenuated the lipopolysaccharide (LPS)-induced intercellular adhesion molecule (ICAM-1) expression and leukocyte count through the up-regulation of HO-1 in mice, which was revealed by immunohistochemistrical staining, Western blot, real-time PCR, and cell count. The inhibitory effects of HO-1 by CORM-2 were reversed by transfection with HO-1 siRNA. Next, Western blot, real-time PCR, and promoter activity assay were performed to examine the HO-1 induction in HTSMCs. We found that CORM-2 induced HO-1 expression via the activation of protein kinase C (PKC)α and proline-rich tyrosine kinase (Pyk2), which was mediated through Nox-derived ROS generation using pharmacological inhibitors or small interfering ribonucleic acids (siRNAs). CORM-2-induced HO-1 expression was mediated through Nox-(1, 2, 4) or p47phox, which was confirmed by transfection with their own siRNAs. The Nox-derived ROS signals promoted the activities of extracellular signal-regulated kinase 1/2 (ERK1/2). Subsequently, c-Fos and c-Jun-activator protein-1 (AP-1) subunits-were up-regulated by activated ERK1/2, which turned on transcription of the HO-1 gene by regulating the HO-1 promoter. These results suggested that in HTSMCs, CORM-2 activates PKCα/Pyk2-dependent Nox/ROS/ERK1/2/AP-1, leading to HO-1 up-regulation, which suppresses the lipopolysaccharide (LPS)-induced airway inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Heme Oxigenase-1/metabolismo , Compostos Organometálicos/farmacologia , Traqueíte/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Heme Oxigenase-1/genética , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos ICR , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traqueia/citologia , Traqueia/metabolismo , Traqueíte/etiologia
6.
Int J Mol Sci ; 20(13)2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31262043

RESUMO

Interleukin-13 (IL-13) drives symptoms in asthma with high levels of T-helper type 2 cells (Th2-cells). Since tight junctions (TJ) constitute the epithelial diffusion barrier, we investigated the effect of IL-13 on TJ in human tracheal epithelial cells. We observed that IL-13 increases paracellular permeability, changes claudin expression pattern and induces intracellular aggregation of the TJ proteins zonlua occludens protein 1, as well as claudins. Furthermore, IL-13 treatment increases expression of ubiquitin conjugating E2 enzyme UBE2Z. Co-localization and proximity ligation assays further showed that ubiquitin and the proteasomal marker PSMA5 co-localize with TJ proteins in IL-13 treated cells, showing that TJ proteins are ubiquitinated following IL-13 exposure. UBE2Z upregulation occurs within the first day after IL-13 exposure. Proteasomal aggregation of ubiquitinated TJ proteins starts three days after IL-13 exposure and transepithelial electrical resistance (TEER) decrease follows the time course of TJ-protein aggregation. Inhibition of JAK/STAT signaling abolishes IL-13 induced effects. Our data suggest that that IL-13 induces ubiquitination and proteasomal aggregation of TJ proteins via JAK/STAT dependent expression of UBE2Z, resulting in opening of TJs. This may contribute to barrier disturbances in pulmonary epithelia and lung damage of patients with inflammatory lung diseases.


Assuntos
Células Epiteliais/metabolismo , Interleucina-13/farmacologia , Junções Íntimas/metabolismo , Traqueia/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Janus Quinases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição STAT/metabolismo , Junções Íntimas/efeitos dos fármacos , Traqueia/citologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
7.
J Physiol Pharmacol ; 70(1)2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31172971

RESUMO

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Mucosa Respiratória/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
8.
Scand J Immunol ; 90(1): e12769, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31006127

RESUMO

Acinetobacter baumannii (A baumannii) is an emerging nosocomial pathogenic bacterium which leads to hospital infections. The increase in drug-resistant A baumannii strains makes it difficult to control by using common antibiotics. The development of effective vaccines is an alternative means to avoid A baumannii infections. In the present study, Balb/c mice were inoculated intratracheally with 30 µg of OmpK/Omp22 fusion protein alone or OmpK/Omp22 formulated with MF59 adjuvant. After two times of boosting at day 14 and 21, the antigen-specific antibody levels and the protective immunity against A baumannii challenge were evaluated. The results showed that the OmpK/Omp22 formulated with MF59 immunized mice produced much higher level of antigen-specific antibodies compared to mice immunized with OmpK/Omp22 alone (P < 0.01). Mice immunized with 30 µg of OmpK/Omp22 formulated with MF59 also provided more potent protection post-challenge, which showed lower bacterial loads in the blood and lung tissue, lower level of blood inflammatory cytokines and higher survival rate (83.3%) than mice immunized with OmpK/Omp22 alone (P < 0.001). In conclusion, this study demonstrated that OmpK/Omp22 fusion protein adjuvanted with MF59 induced superior immune response and better protection than OmpK/Omp22 alone through intratracheal inoculation in mice.


Assuntos
Infecções por Acinetobacter/imunologia , Acinetobacter baumannii/fisiologia , Adjuvantes Imunológicos , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Esqualeno/imunologia , Animais , Carga Bacteriana , Infecção Hospitalar , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Polissorbatos , Traqueia/metabolismo , Vacinação
9.
Dev Biol ; 451(2): 158-166, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30965042

RESUMO

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. In the trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with an additional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins and antimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals that regulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development of murine tracheal SMGs, with gland development arresting at the early bud stage in the absence of FGF10 signalling. As Fgf10 knockout mice are lethal at birth, inducible knockdown of Fgf10 at late embryonic stages was used to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygous Fgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. The reduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction in overall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both the number of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMG-dependent respiratory health.


Assuntos
Glândulas Exócrinas/embriologia , Fator 10 de Crescimento de Fibroblastos/metabolismo , Mucosa Respiratória/embriologia , Traqueia/embriologia , Animais , Cruzamentos Genéticos , Feminino , Fator 10 de Crescimento de Fibroblastos/deficiência , Fator 10 de Crescimento de Fibroblastos/genética , Masculino , Camundongos , Morfogênese , Muco/metabolismo , Traqueia/metabolismo
10.
Methods Mol Biol ; 1968: 183-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929215

RESUMO

Two-photon intravital imaging (2P-IVM) of the murine trachea is a powerful technique for real-time imaging of immune cell recruitment and trafficking during airborne pathogen infections. Neutrophils are an important component of the innate immune response that are able to rapidly infiltrate the airway mucosa in response to Streptococcus pneumoniae infection. Here we describe a protocol to visualize in vivo neutrophil extravasation and cell dynamics in the tracheal tissue of a S. pneumoniae-infected mouse using 2P-IVM. To perform this protocol, we infected and imaged the trachea of a lysozyme M green fluorescent protein (LysM-GFP) mouse, in which neutrophils express GFP. Additionally, we used a custom-designed platform, which allowed the intubation and fixation of the trachea after surgical exposition, and we injected intravenously a fluorescently labeled dextran solution to visualize the blood vessels.


Assuntos
Microscopia Intravital/métodos , Leucócitos/metabolismo , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/microbiologia , Traqueia/diagnóstico por imagem , Traqueia/metabolismo , Animais , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Neutrófilos/metabolismo , Streptococcus pneumoniae/patogenicidade
12.
J Perinat Med ; 47(4): 470-477, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-30789824

RESUMO

Background The pathogenesis of bronchopulmonary dysplasia (BPD) includes arrest of alveolar septation and enhanced fibrosis. We hypothesized that mesenchymal stromal cells (MSC) and transforming growth factor-ß1 (TGF-ß1) in tracheal aspirates of mechanically ventilated premature infants differ in BPD and non-BPD infants. Methods Tracheal aspirates were collected during the first week of life. Mononuclear cells were separated, cultured and immunophenotyped by flow cytometry. MSCs colony/cluster ratio was calculated as an index for dysplastic potentials. TGF-ß1 was assessed by enzyme-linked immunosorbent assay (ELISA). Setting: Neonatal intensive care unit. Patients Premature infants at risk for BPD. Results A total of 121 preterm infants were enrolled; 27 of them died and among the 94 survivors 23 infants had BPD. MSCs were identified in younger [gestational age (GA): 30.9±1.7 vs. 31.8±1.8, P=0.025] and smaller [birth weight (BW): 1.3±0.28 vs. 1.44±0.37 kg, P=0.04] infants with lower Apgar scores. The recovery rate of MSCs in BPD and non-BPD groups did not differ. BPD group had significantly smaller colony/cluster ratio compared to non-BPD (0.97 vs. 4.25, P=0.002). TGF-ß1 was significantly greater in BPD infants (4173.9±864.3 vs. 3705.8±540.5 pg/mL, P=0.021). Conclusion Infants with BPD had different MSCs morphology and greater TGF-ß1 expression. The pathogenesis for these morphological changes of resident lung MSCs needs further studying.


Assuntos
Displasia Broncopulmonar/diagnóstico , Células-Tronco Mesenquimais/patologia , Fator de Crescimento Transformador beta1/metabolismo , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Estudos Transversais , Diagnóstico Precoce , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Células-Tronco Mesenquimais/metabolismo , Traqueia/metabolismo , Traqueia/patologia
13.
Lymphat Res Biol ; 17(1): 19-29, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30648916

RESUMO

BACKGROUND: The fibroblast growth factor receptor (FGFR) family includes transmembrane receptors involved in a wide range of developmental and postdevelopmental biologic processes as well as a wide range of human diseases. In particular, FGFR3 has been implicated in the mechanism by which 9-cis retinoic acid (9-cisRA) induces lymphangiogenesis and improves lymphedema. The purpose of this study was to validate the efficacy of a novel small peptide FGFR3 inhibitor, peptide P3 (VSPPLTLGQLLS), and to elucidate the role of FGFR3 in 9-cisRA-induced lymphangiogenesis using this peptide. METHODS AND RESULTS: Peptide P3 effectively inhibited FGFR3 phosphorylation. In vitro, peptide P3-mediated FGFR3 inhibition did not decrease lymphatic endothelial cell (LEC) proliferation, migration, or tubule formation. However, peptide P3-mediated FGFR3 inhibition did block 9-cisRA-stimulated LEC proliferation, migration, and tubule formation. In vivo, peptide P3-mediated FGFR3 inhibition was sufficient to inhibit 9-cisRA-induced tracheal lymphangiogenesis. CONCLUSION: FGFR3 does not appear to be essential to nonpromoted LEC proliferation, migration, and tubule formation. However, FGFR3 may play a key role in LEC proliferation, migration, tubule formation, and postnatal in vivo lymphangiogenesis when pharmacologically induced by 9-cisRA. P3 may have the potential to be used as a precise regulatory control element for 9-cisRA-mediated lymphangiogenesis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Linfangiogênese/efeitos dos fármacos , Linfedema/genética , Oligopeptídeos/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Alitretinoína/antagonistas & inibidores , Alitretinoína/farmacologia , Sequência de Aminoácidos , Animais , Bioensaio , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Humanos , Linfangiogênese/genética , Linfedema/metabolismo , Linfedema/patologia , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/patologia
14.
PLoS Genet ; 15(1): e1007852, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645584

RESUMO

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Membrana/genética , Traqueia/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Polaridade Celular/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Mutação , Traqueia/metabolismo
15.
Biochem Biophys Res Commun ; 510(2): 205-210, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30691694

RESUMO

Postnatal organ-specific stem and progenitor cells are an attractive potential donor cell for tissue-engineering because they can be harvested autologous from the recipient and have sufficient potential to regenerate the tissue of interest with less risk for ectopic growth or tumor formation compared to donor cells from embryonic or fetal sources. We describe the generation of tissue-engineered larynx and trachea (TELT) from human and mouse postnatal organoid units (OU) as well as from human fetal OU. Mouse TELT contained differentiated respiratory epithelium lining large lumens, cartilage and smooth muscle. In contrast, human postnatal TE trachea, formed small epithelial lumens with rare differentiation, in addition to smooth muscle and cartilage. Human fetal TELT contained the largest epithelial lumens with all differentiated cell types as well as smooth muscle and cartilage. Increased epithelial cytokeratin 14 was identified in both human fetal and postnatal TELT compared to native trachea, consistent with regenerative basal cells. Cilia in TELT epithelium also demonstrated function with beating movements. While both human postnatal and fetal progenitors have the potential to generate TELT, there is more epithelial growth and differentiation from fetal progenitors, highlighting fundamental differences in these cell populations.


Assuntos
Epitélio/metabolismo , Laringe/fisiologia , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Traqueia/fisiologia , Animais , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Cílios/metabolismo , Células Epiteliais/metabolismo , Epitélio/embriologia , Receptores ErbB/metabolismo , Humanos , Interleucina-2/genética , Queratina-14/metabolismo , Laringe/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Músculo Liso/metabolismo , Organoides/metabolismo , Mucosa Respiratória/metabolismo , Traqueia/metabolismo
16.
Am J Physiol Lung Cell Mol Physiol ; 316(2): L385-L390, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30489155

RESUMO

Airway smooth muscle (ASM) cells express GABA A receptors (GABAARs), and previous reports have demonstrated that GABAAR activators relax ASM. However, given the activity of GABAARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.


Assuntos
Asma/tratamento farmacológico , Barreira Hematoencefálica/efeitos dos fármacos , GABAérgicos/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Cobaias , Humanos , Ligantes , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Receptores de GABA-A/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
17.
J Mol Cell Cardiol ; 127: 20-30, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30502350

RESUMO

BACKGROUND: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model. OBJECTIVE: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH. METHODS: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT + S2a, N = 8) or saline (MCT, N = 9). Age-matched rats served as controls (CTRL, N = 7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses. RESULTS: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P < .01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (P < .05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH. CONCLUSION: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.


Assuntos
Aerossóis/administração & dosagem , Arritmias Cardíacas/terapia , Técnicas de Transferência de Genes , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/uso terapêutico , Traqueia/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/fisiopatologia , Conexina 43/metabolismo , Modelos Animais de Doenças , Terapia Genética , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Masculino , Canais de Potássio/genética , Canais de Potássio/metabolismo , /fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley
18.
Am J Physiol Lung Cell Mol Physiol ; 316(1): L105-L113, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30407863

RESUMO

We have previously reported that mice genetically deficient in the actin binding protein gelsolin exhibit impaired airway smooth muscle (ASM) relaxation. Primary cultured ASM cells from these mice demonstrate enhanced inositol triphosphate (IP3) synthesis and increased intracellular calcium in response to Gq-coupled agonists. We hypothesized that this was due to increased intracellular availability of unbound phosphatidylinositol 4,5-bisphosphate (PIP2), based on the fact that gelsolin contains a short peptide region that binds PIP2, presumably making it a less available substrate. We now questioned whether a peptide that corresponds to the PIP2 binding region of gelsolin could modulate ASM signaling and contraction. The 10 amino acid sequence of the gelsolin peptide within the PIP2-binding region was incubated with primary cultures of human ASM cells, and IP3 synthesis was measured in response to a Gq-coupled agonist. Gelsolin peptide-treated cells generated less IP3 under basal and bradykinin or acetylcholine (Gq-coupled) conditions. Acetylcholine-induced contractile force measured in isolated tracheal rings from mice and human tracheal muscle strips in organ baths was attenuated in the presence of the gelsolin peptide. The gelsolin peptide also attenuated methacholine-induced airway constriction in murine precision-cut lung slices. Furthermore, this peptide fragment delivered to the respiratory system of mice via nebulization attenuated subsequent methacholine-induced increases in airway resistance in vivo. The current study demonstrates that introduction of this small gelsolin peptide into the airway may be a novel therapeutic option in bronchoconstrictive diseases.


Assuntos
Broncoconstrição/efeitos dos fármacos , Gelsolina/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Peptídeos/farmacologia , Traqueia/metabolismo , Animais , Gelsolina/química , Humanos , Masculino , Camundongos , Músculo Liso/patologia , Peptídeos/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Traqueia/patologia
19.
J Leukoc Biol ; 105(3): 577-587, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30548974

RESUMO

Tracheitis secondary to placement of an endotracheal tube (ETT) is characterized by neutrophil accumulation in the tracheal lumen, which is generally associated with epithelial damage. Mitochondrial DNA (mtDNA), has been implicated in systemic inflammation and organ dysfunction following trauma; however, less is known about the effects of a foreign body on local trauma and tissue damage. We hypothesized that tracheal damage secondary to the ETT will result in local release of mtDNA at sufficient levels to induce TLR9 and NF-κB activation. In a swine model we compared the differences between uncoated, and chloroquine (CQ) and N-acetylcysteine (NAC) coated ETTs as measured by tracheal lavage fluids (TLF) over a period of 6 h. The swine model allowed us to recreate human conditions. ETT presence was characterized by neutrophil activation, necrosis, and release of proinflammatory cytokines mediated by TLR9/NF-κB induction. Amelioration of the tracheal damage was observed in the CQ and NAC coated ETT group as shown in tracheal tissue specimens and TLF. The role of TLR9/NF-κB dependent activity was confirmed by HEK-Blue hTLR9 reporter cell line analysis after coincubation with TLF specimens with predetermined concentrations of NAC or CQ alone or TLR9 inhibitory oligodeoxynucleotide (iODN). These findings indicate that therapeutic interventions aimed at preventing mtDNA/TLR9/NF-κB activity may have benefits in prevention of acute tracheal damage.


Assuntos
DNA Mitocondrial/genética , Intubação Intratraqueal , NF-kappa B/metabolismo , Receptor Toll-Like 9/metabolismo , Traqueia/metabolismo , Traqueia/patologia , Doença Aguda , Animais , Movimento Celular , Citocinas/metabolismo , Epitélio/metabolismo , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Suínos
20.
Am J Physiol Lung Cell Mol Physiol ; 316(1): L82-L93, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30284927

RESUMO

Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.


Assuntos
Luz , Relaxamento Muscular , Miócitos de Músculo Liso/metabolismo , Opsinas de Bastonetes/metabolismo , Traqueia/metabolismo , Animais , Humanos , Camundongos , Miócitos de Músculo Liso/citologia , Transdução de Sinais , Traqueia/citologia
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