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1.
Vet J ; 254: 105405, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31836172

RESUMO

Published studies vary as to whether epithelial cells are included in differential counts for tracheal wash (TW) and bronchoalveolar lavage (BAL) cytology in horses. The aim of this study was to determine whether inclusion or exclusion of epithelial cells affects interpretation of airway cytology. Using criteria of >20% TW neutrophils, >10% BAL neutrophils and/or >5% BAL mast cells to indicate airway inflammation, there was a change in categorisation from 'normal' to 'abnormal' in 21%, 4% and 8% horses, respectively, when epithelial cells were excluded from differential counts. It is recommended that future equine respiratory research studies explicitly state whether epithelial cells are included or excluded in differential counts. A consensus on epithelial cell inclusion during cytology reporting is required.


Assuntos
Brônquios/citologia , Lavagem Broncoalveolar/veterinária , Células Epiteliais/citologia , Cavalos/anatomia & histologia , Alvéolos Pulmonares/citologia , Traqueia/citologia , Animais , Contagem de Células/veterinária , Feminino , Masculino , Estudos Retrospectivos
2.
Mater Sci Eng C Mater Biol Appl ; 105: 110142, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546345

RESUMO

Commonly reported decellularization protocols for trachea may take up from several weeks to months in order to remove the cellular materials. Two years ago, we significantly reduced the time of decellularization trachea process using trypsin. Despite the positive outcome, the protocol was useful to produce 5 cm graft length, an unsuitable length graft for most patients with tracheal disorders. In this work we improved the decellularization procedure for longer sections up to 10 cm without considerable extension in the necessary time process (2 weeks). Herein, for the first time, we completely describe and characterize the process for pig tracheal bioactive scaffolds. Histological and molecular biology analysis demonstrated effective removal of cellular components and nuclear material, which was also confirmed by the Immunohistochemical (IHC) analysis of the major histocompatibility complexes (MHCs) and DNA stain by 4'-6-diamidino-2-phenylindole (DAPI). The images and data obtained from scanning electron microscopy (SEM) and thermal analysis showed conservation of the hierarchical structures of the tracheal extracellular matrix (ECM), the biomechanical tests showed that decellularization approach did not lead to a significant alteration on the mechanical properties. In this paper, we demonstrate that the proposed cyclical-decellularization protocol allowed us to obtain a non-immunological 10 cm natural tracheal scaffold according to the in vivo immunological assessment. Furthermore, the recellularization of the matrix was successfully achieved by demonstrating first-stage cellular differentiation from stem cells to chondrocytes expressed by the SOX9 transcription factor; this organ-engineered tracheal matrix has the potential to act as a suitable template for organ regeneration.


Assuntos
Engenharia Tecidual/métodos , Tecidos Suporte/química , Traqueia/citologia , Animais , Fenômenos Biomecânicos , Fenômenos Biofísicos , Matriz Extracelular/química , Humanos , Masculino , Camundongos , Suínos , Traqueia/ultraestrutura , Fator de Necrose Tumoral alfa/metabolismo
3.
Vet Microbiol ; 235: 80-85, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282382

RESUMO

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.


Assuntos
Coinfecção/veterinária , Células Epiteliais , Interações Microbianas , Infecções por Pasteurella/veterinária , Infecções por Vírus Respiratório Sincicial/veterinária , Animais , Complexo Respiratório Bovino/microbiologia , Complexo Respiratório Bovino/virologia , Brônquios/citologia , Brônquios/microbiologia , Brônquios/virologia , Bovinos , Linhagem Celular , Células Cultivadas , Coinfecção/microbiologia , Coinfecção/virologia , Citocinas/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/virologia , Pulmão/citologia , Pulmão/microbiologia , Pulmão/virologia , Infecções por Pasteurella/virologia , Pasteurella multocida/genética , Pasteurella multocida/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/microbiologia , Vírus Sincicial Respiratório Bovino/genética , Vírus Sincicial Respiratório Bovino/isolamento & purificação , Traqueia/citologia , Traqueia/microbiologia , Traqueia/virologia
4.
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
5.
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
6.
Virology ; 534: 132-142, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31255797

RESUMO

The chicken upper respiratory tract is the portal of entry for respiratory pathogens including avian influenza virus (AIV). There is a paucity of information about the role of airway epithelial cells in the induction of antiviral responses in the chicken trachea. A better understanding of the role of these cells in the initiation of innate responses may improve prophylactic or therapeutic strategies for control of viral infections. The present study aimed to characterize antiviral innate responses in chicken tracheal epithelial cells (cTECs) induced by TLR ligands. The results demonstrated that stimulation of cTECs with TLR ligands induced antiviral responses, and subsequently reduced the replication of AIV in cTECs. Additionally, stimulated cTECs were able to influence the function of other cells such as macrophages. Overall, these results provided evidence that cTECs mount antiviral responses after stimulation with TLR ligands through IRF7 and NF-κB signaling pathways, leading to activation of other cells, such as macrophages.


Assuntos
Células Epiteliais/imunologia , Vírus da Influenza A/fisiologia , Influenza Aviária/imunologia , Macrófagos/imunologia , Doenças das Aves Domésticas/imunologia , Traqueia/virologia , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/imunologia , Galinhas , Células Epiteliais/virologia , Imunidade Inata , Vírus da Influenza A/genética , Influenza Aviária/genética , Influenza Aviária/virologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Ligantes , Macrófagos/virologia , Poli I-C/imunologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Traqueia/citologia , Traqueia/imunologia
7.
Transplant Proc ; 51(5): 1611-1613, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31155202

RESUMO

INTRODUCTION/OBJECTIVE: Tracheal resection with primary reconstruction is the definitive treatment for many tracheal benign and malignant diseases. When primary resection is not deemed feasible as a result of the length of the stenosis, airway transplantation may become a solution. Tissue engineering offers an alternative way for creating tracheal substitutes. The development of tracheal allograft transplantation includes the decellularized tracheal scaffolds made of extracellular matrix that are seeded with the receptor's cells. Many protocols are used to obtain a decellularized scaffold. Most of them consist of cyclical physical-chemical steps with enzymes. This study proposes a protocol for decellularization based only in physical-chemical steps. METHODS: Decellularization of pig tracheal segments was carried out using a standardized protocol consisting of freezing and thawing, 10 cycles of agitation, exposure to sodium deoxycholate, and washing. The degree of decellularization was determined by quantifying residual DNA. We also analyzed the morphology under hematoxylin and eosin staining. RESULTS: Fourteen porcine tracheal segments were decellularized. All scaffolds obtained showed less than 2% of residual DNA (mean 20 ± 8 ng/mg) when compared to the fresh samples (mean 850 ± 123 ng/mg), P = .001. Morphological analysis showed that the epithelium and mixed glands were completely removed. It was possible to identify residual nuclei inside the cartilaginous rings (73.7 ± 12 × 26 ± 8 nuclei/field, P < .001). CONCLUSION: The protocol tested was able to provide effective decellularization of porcine tracheas.


Assuntos
Engenharia Tecidual/métodos , Tecidos Suporte , Traqueia , Animais , Masculino , Suínos , Traqueia/citologia
8.
PLoS One ; 14(6): e0217906, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31158257

RESUMO

Understanding the transcriptional pathways controlling tissue-specific gene expression is critical to unraveling the complex regulatory networks that underlie developmental mechanisms. Here, we assessed how the Drosophila crossveinless (cv) gene, that encodes a BMP-binding factor, is transcriptionally regulated in the developing embryonic tracheal system. We identify an upstream regulatory region of cv that promotes reporter gene expression in the tracheal precursors. We further demonstrate that this promoter region is directly responsive to the basic, helix-loop-helix-PAS domain factors Trachealess (Trh) and Tango (Tgo), that function to specify tracheal fate. Moreover, cv expression in embryos is lost in trh mutants, and the integrity of the Trh/Tgo binding sites are required for promoter-lacZ expression. These findings for the first time elucidate the transcriptional regulation of one member of a family of BMP binding proteins, that have diverse functions in animal development.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Traqueia/citologia , Fatores de Transcrição/metabolismo , Transcrição Genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Regiões Promotoras Genéticas/genética
9.
Int J Artif Organs ; 42(9): 500-507, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31081418

RESUMO

OBJECTIVE: To study the different concentrations of Triton X-100 and nuclease needed to remove cells from the tracheal matrix of rabbits and analyse their biocompatibility and cellular compatibility. METHODS: Fifty tracheas were harvested from donor New Zealand rabbits. Thirty tracheas were randomly divided into five groups (n = 6 each). The tracheas in group A were untreated and served as a control group, and those in groups B, C, D and E were treated with different concentrations of Triton X-100 (1%, 2%, 3% and 4%), respectively. The tracheas of the five groups were assessed by histological observation, scanning electron microscopy and mechanical evaluation. The remaining 20 donor tracheas, which were divided into a control group and an optimally decellularized group, were used for xenogeneic transplantation and cell seeding. RESULTS: Many epithelial cells and cartilage cells were observed in the tracheas of group A. There were fewer cartilage cells in the tracheas of groups C, D and E than in the tracheas of groups A and B under histological observation. In scanning electron microscopy, there were many ciliated epithelial cells in the tracheas of group A; in groups B and C, the ciliated epithelial cells disappeared, but the basement membrane was intact. The basement membranes were broken in the tracheas of groups D and E. Implanted decellularized tracheas showed good biocompatibility. Bone marrow mesenchymal stem cells grown in the decellularized tracheal matrix grew well. CONCLUSION: Decellularized tracheal matrix obtained from rabbits by 2% Triton X-100 may be suitable for the construction of tissue-engineered trachea because of its favourable morphological and biomechanical properties as well as its biocompatibility and cellular compatibly.


Assuntos
Engenharia Tecidual/métodos , Traqueia/citologia , Animais , Materiais Biocompatíveis , Bioprótese , Detergentes , Matriz Extracelular/transplante , Microscopia Eletrônica de Varredura , Octoxinol , Coelhos
10.
J Pharmacol Exp Ther ; 370(1): 104-110, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31068382

RESUMO

ß 2-Adrenoceptors (ß 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that ß 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of ß 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited ß 2AR production of cAMP in lipid raft domains without affecting ß 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.


Assuntos
Adenilil Ciclases/metabolismo , Brônquios/citologia , AMP Cíclico/biossíntese , Miócitos de Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Traqueia/citologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Humanos , Isoproterenol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos
11.
Eur J Pharmacol ; 853: 229-235, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30935895

RESUMO

Our previous study found that the anthelmintic drug niclosamide relaxed the constricted arteries and inhibited proliferation and migration of vascular smooth muscle cells. Here, we investigated the effect of niclosamide ethanolamine (NEN) on trachea function and the proliferation and migration of trachea smooth muscle cells. Isometric tension of trachea was recorded by multi-channel myograph system. The cell proliferation was detected by using BrdU cell proliferation assay. The cell migration ability was evaluated by using scratch assay. The protein level was measured by using western blot technique. Acute treatment with NEN dose-dependently relaxed acetylcholine chloride (Ach)- and High K+ physiological salt solution (KPSS)-induced constriction of mice trachea. Pre-treatment with NEN inhibited Ach- and KPSS-induced constriction of mice trachea. NEN treatment inhibited proliferation of human bronchial smooth muscle cells (HBSMCs), inhibited migration of HBSMCs and rat primary trachea smooth muscle cells. NEN treatment activated adenosine monophosphate activated protein kinase (AMPK) activity and inhibited signal transducer and activator of transcription 3 (STAT3) activity in HBSMCs. In conclusion, niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells, indicating that niclosamide might be a potential drug for chronic asthma treatment.


Assuntos
Movimento Celular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Niclosamida/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Acetilcolina/farmacologia , Animais , Brônquios/citologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Potássio/farmacologia , Ratos , Traqueia/citologia , Vasoconstrição/efeitos dos fármacos
12.
Int Immunopharmacol ; 72: 459-466, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31035088

RESUMO

The inflammation-induced the excessive proliferation and migration of airway smooth muscle (ASM) cells in the airway wall contribute to airway remodeling in asthma pathogenesis. SET domain-containing lysine methyltransferase 7 (SETD7) has emerged as one of the key regulators of inflammation. Yet, the function of SETD7 in regulating inflammation-induced ASM cell proliferation and invasion remains unclear. In the present study, we aimed to investigate the function of SETD7 in regulating ASM cell proliferation and invasion induced by tumor necrosis factor (TNF)-α in vitro. Our results showed that SETD7 expression was upregulated in ASM cells stimulated with TNF-α. Silencing SETD7 significantly decreased TNF-α-induced ASM cell proliferation and migration, while SETD7 overexpression exhibited the opposite effect. Notably, silencing SETD7 decreased the activation of nuclear factor (NF)-κB and reduced the expression of CD38 induced by TNF-α. Blocking NF-κB activation significantly abrogated the promotional effect of SETD7 overexpression on CD38 expression. Moreover, overexpression of CD38 partially reversed the inhibitory effect of SETD7 silencing on TNF-α-induced ASM cell proliferation and migration. Overall, these results demonstrate that SETD7 regulates TNF-α-induced ASM cell proliferation and migration through modulation of NF-κB/CD38 signaling, suggesting a potential role of SETD7 in asthma airway remodeling.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Glicoproteínas de Membrana/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Traqueia/citologia , Animais , Asma/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Histona-Lisina N-Metiltransferase/genética , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
13.
Ann Otol Rhinol Laryngol ; 128(7): 585-594, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30832485

RESUMO

OBJECTIVES: In tracheal regeneration, the slow process of epithelialization is often a barrier to the stability and safety of the transplanted trachea. The aim of this study was to examine a new tracheal regeneration technique using organotypically cultured tissue composed of autologous cells. METHODS: Nine beagles were prepared. Chondrocytes from auricular cartilage and epithelial cells from buccal mucosa were isolated and cultured. Tissue-engineered cartilages were fabricated with chondrocytes at a density of 1 × 107 cells/mL (high-density group) and 1 × 106 cells/mL (low-density group). A fabricated epithelial cell sheet was laid on a poly(lactic-co-glycolic acid) block in atelocollagen gel containing the chondrocytes, and the organotypically cultured tissues were transplanted into a partially resected trachea. The control group had only the block transplanted. RESULTS: The tissue-engineered cartilages in the high-density group contained many viable chondrocytes and many cartilage matrices. The low-density group had abundant collagen fibers and no chondrocytes. Tracheal endoscopy revealed no deformation or atrophy at the transplant site in the high-density group. Histologically, partially hyaline cartilages covered with epithelium and lamina propria were found in the high-density group but not in the low-density and control groups. CONCLUSIONS: Stable tracheal regeneration was achieved using organotypically cultured tissue fabricated with autologous high-density chondrocytes and epithelial cells.


Assuntos
Condrócitos/citologia , Células Epiteliais/citologia , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/métodos , Traqueia/citologia , Animais , Materiais Biocompatíveis , Condrócitos/transplante , Colágeno , Cães , Cartilagem da Orelha/citologia , Células Epiteliais/transplante , Mucosa Bucal/citologia , Técnicas de Cultura de Órgãos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Regeneração , Tecidos Suporte , Traqueia/transplante , Transplante Autólogo
14.
J Toxicol Sci ; 44(3): 155-165, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30842368

RESUMO

Silver nanoparticles (AgNPs) are increasingly utilized in a number of applications. This study was designed to investigate AgNPs induced cytotoxicity, oxidative stress and apoptosis in rat tracheal epithelial cells (RTE). The RTE cells were treated with 0, 100 µg/L and 10,000 µg/L of the AgNPs with diameters of 10 nm and 100 nm for 12 hr. The cell inhibition level, apoptosis ratio, reactive oxygen species (ROS), malondialdehyde (MDA) and metallothionein (MT) content were determined. The mRNA expression of cytoc, caspase 3, and caspase 9 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, we also analyzed the cytoc, caspase 3, pro-caspase 3, caspase 9, and pro-caspase 9 protein expression by western blotting. Electric cell-substrate impedance sensing (ECIS) analysis showed that the growth and proliferation of RTE cells were significantly inhibited in a dose-dependent manner under AgNPs exposure. The cell dynamic changes induced by 10 nm AgNPs were more severe than that of the 100 nm AgNPs exposure group. The intracellular MT, ROS, and MDA content increased when the exposure concentration increased and size reduced, whereas Ca2+-ATPase activity and Na+/K+-ATPase activity changed inversely. The relative expression of protein of cytoc, caspase 3, and caspase 9 were upregulated significantly, which indicated that AgNPs induced apoptosis of RTE cells through the caspase-dependent mitochondrial pathway. Our results demonstrate that AgNPs caused obvious cytotoxicity, oxidative stress, and apoptosis in RTE cells, which promoted the releasing of cytochrome C and pro-apoptotic proteins into the cytoplasm to activate the caspase cascade and finally led to apoptosis.


Assuntos
Células Epiteliais/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traqueia/citologia
15.
Cell Prolif ; 52(3): e12598, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900363

RESUMO

OBJECTIVES: The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. MATERIALS AND METHODS: The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. RESULTS: With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. CONCLUSIONS: The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.


Assuntos
Engenharia Tecidual/métodos , Tecidos Suporte , Traqueia/citologia , Materiais Biocompatíveis/química , Fenômenos Biomecânicos , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Quitosana/química , Técnicas de Cocultura , Células Epiteliais/citologia , Fibroblastos/citologia , Gelatina/química , Humanos , Queratinócitos/citologia , Teste de Materiais , Microscopia Eletrônica de Varredura , Nanofibras/química , Nanofibras/ultraestrutura , Poliésteres/química , Tecidos Suporte/química , Traqueia/crescimento & desenvolvimento , Traqueia/fisiologia
16.
Pharmacol Rep ; 71(2): 225-232, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30785060

RESUMO

BACKGROUND: Muscarinic receptor antagonists are a usual treatment for chronic airway diseases, with increased bronchoconstriction, like asthma and chronic obstructive pulmonary disease. These diseases are usually accompanied by airway remodeling, involving airway smooth muscle cell (ASMC) proliferation. The purpose of this study was to examine the effect of the muscarinic receptor modulator gallamine on rabbit tracheal ASMC proliferation. METHODS: ASMCs were incubated with gallamine (1 nM-10 mM), atropine (1 fM-10 mM), and/or acetylcholine (1 nM-1 mM), in the presence or absence of FBS (1% or 10%). Cell proliferation was estimated by incorporation of radioactive thymidine, the Cell Titer AQueous One Solution method and cell number counting after Trypan blue exclusion. The mechanisms mediating cell proliferation were studied using the PI3K and MAPK inhibitors LY294002 (20 µM) and PD98059 (100 µM), respectively. Cell phenotype was studied by indirect immunofluorescence for α-actin, Myosin Heavy Chain and desmin. RESULTS: ASMC incubation with the muscarinic receptor allosteric modulator gallamine or the muscarinic receptor antagonist atropine increased methyl-[3H]thymidine incorporation and cell number in a dose-dependent manner. ASMC proliferation was mediated via PI3K and MAPK activation and was transient. Gallamine antagonized the mitogenic effect of 1% FBS. Furthermore, gallamine had a similar effect on contractile ASMCs, without synergizing with or affecting acetylcholine induced proliferation, or altering the percentage of ASMCs expressing contractile phenotype marker proteins. CONCLUSIONS: Gallamine, in the absence of any agonist, has a transient mitogenic effect on ASMCs, regardless of the cell phenotype, mediated by the PI3K and the MAPK signaling pathways.


Assuntos
Proliferação de Células/efeitos dos fármacos , Trietiodeto de Galamina/farmacologia , Antagonistas Muscarínicos/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Acetilcolina/administração & dosagem , Acetilcolina/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Atropina/administração & dosagem , Atropina/farmacologia , Relação Dose-Resposta a Droga , Trietiodeto de Galamina/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Antagonistas Muscarínicos/administração & dosagem , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Coelhos , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Traqueia/citologia , Traqueia/efeitos dos fármacos
17.
Dev Biol ; 451(1): 79-85, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30735663

RESUMO

The terminal cells of the larval Drosophila tracheal system extend dozens of branched cellular processes, most of which become hollow intracellular tubes that support gas exchange with internal tissues. Previously, we undertook a forward genetic mosaic screen to uncover the pathways regulating terminal cell size, morphogenesis, and the generation and maintenance of new intracellular tubes. Our initial work identified several mutations affecting terminal cell size and branch number, and suggested that branch complexity and cell size are typically coupled but could be genetically separated. To deepen our understanding of these processes, we have further characterized and determined the molecular identities of mutations in the genes sprout, denuded and asthmatic, that had been implicated in our initial screen. Here we reveal the molecular identity of these genes and describe their function in the context of the TOR and Hippo pathways, which are widely appreciated to be key regulators of cell and organ size.


Assuntos
Mutação , Traqueia/embriologia , Animais , Tamanho Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/citologia , Larva/metabolismo , Morfogênese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Traqueia/citologia
18.
Virology ; 528: 152-163, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30616205

RESUMO

Influenza viruses are a group of respiratory pathogens that have evolved into four different types: A, B, C, and D. A common feature is that all four types are capable of replicating and transmitting among pigs. Here, we describe the development of isogenous cell culture system from the swine respiratory tract to study influenza viruses. Phenotypic characterization of swine primary nasal turbinate, trachea and lung cells revealed high expression of cytokeratin and demonstrated tissue site dependent expression of tight junction proteins. Furthermore, lectin binding assay on these cells demonstrated higher levels of Sia2-6Gal than Sia2-3Gal receptors and supported the replication of influenza A, B, C, and D viruses to appreciable levels at both 33 and 37 °C, but replication competence was dependent on virus type or temperature used. Overall, these swine primary respiratory cells showed epithelial phenotype, which is suitable for studying the comparative biology and pathobiology of influenza viruses.


Assuntos
Células Epiteliais/virologia , Pulmão/citologia , Orthomyxoviridae/fisiologia , Traqueia/citologia , Animais , Técnicas de Cultura de Células , Queratinas/genética , Pulmão/virologia , Fenótipo , Suínos , Proteínas de Junções Íntimas/genética , Traqueia/virologia , Replicação Viral
19.
Development ; 146(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30696710

RESUMO

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of ß-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.


Assuntos
Fator 10 de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Mucosa Respiratória/embriologia , Traqueia/embriologia , Via de Sinalização Wnt/fisiologia , Animais , Fator 10 de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Mutantes , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Mucosa Respiratória/citologia , Traqueia/citologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
20.
J Mech Behav Biomed Mater ; 90: 96-103, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30359857

RESUMO

Rapid development of tissue engineering technology provides new methods for tracheal cartilage regeneration. However, the current lack of an ideal scaffold makes engineering of trachea cartilage tissue into a three-dimensional (3-D) tubular structure a great challenge. Although a decellularized trachea matrix (DTM) has become a recognized scaffold for trachea cartilage regeneration, it is difficult for cells to detach from or penetrate the matrix because of its non-porous structure. To tackle these problems, a laser micropore technique (LMT) was applied in the current study to enhance trachea sample porosity, and facilitate decellularizing treatment and cell ingrowth. Furthermore, after optimizing LMT and decellularizing treatment parameters, LMT-treated DTM (LDTM) retained its natural tubular structure with only minor extracellular matrix damage. Moreover, compared with DTM, the current study showed that LDTM significantly improved the adherence rate of cells with perfect cell biocompatibility. Moreover, the optimal implantation cell density for chondrogenesis with LDTM was determined to be 1 × 108 cells/ml. Collectively, the results suggest that the novel LDTM is an ideal scaffold for trachea tissue engineering.


Assuntos
Lasers , Fenômenos Mecânicos , Tecidos Suporte , Traqueia/citologia , Animais , Cartilagem/metabolismo , Adesão Celular , Proliferação de Células , Porosidade , Coelhos
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