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1.
Nat Commun ; 11(1): 1103, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107376

RESUMO

Lipid-protein complexes are the basis of pulmonary surfactants covering the respiratory surface and mediating gas exchange in lungs. Cardiolipin is a mitochondrial lipid overexpressed in mammalian lungs infected by bacterial pneumonia. In addition, increased oxygen supply (hyperoxia) is a pathological factor also critical in bacterial pneumonia. In this paper we fabricate a micrometer-size graphene-based sensor to measure oxygen permeation through pulmonary membranes. Combining oxygen sensing, X-ray scattering, and Atomic Force Microscopy, we show that mammalian pulmonary membranes suffer a structural transformation induced by cardiolipin. We observe that cardiolipin promotes the formation of periodic protein-free inter-membrane contacts with rhombohedral symmetry. Membrane contacts, or stalks, promote a significant increase in oxygen gas permeation which may bear significance for alveoli gas exchange imbalance in pneumonia.


Assuntos
Cardiolipinas/metabolismo , Grafite/química , Bicamadas Lipídicas/metabolismo , Oxigênio/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Humanos , Microscopia de Força Atômica/instrumentação , Microscopia Confocal/instrumentação , Microtecnologia/instrumentação , Pneumonia Bacteriana/fisiopatologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/ultraestrutura , Troca Gasosa Pulmonar/fisiologia , Espalhamento a Baixo Ângulo , Transistores Eletrônicos , Difração de Raios X/instrumentação
2.
Life Sci ; 242: 117213, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31881228

RESUMO

Acute respiratory distress syndrome (ARDS) is a multifactorial, inflammatory lung injury disease with high morbidity and mortality. However, the underlying pathogenic mechanism remains unknown. In this study, lipopolysaccharide (LPS)-stimulated alveolar epithelial cells were used to mimic the inflammatory pathogenesis of ARDS in vitro. We here investigated the role of miR-424 in LPS-stimulated alveolar epithelial cells and found it to be substantially downregulated. Overexpression of miR-424 inhibited apoptosis and inflammation in LPS-stimulated alveolar epithelial cells, and the miR-424 inhibitor exhibited the opposite effect. A bioinformatic analysis revealed a potential binding site of miR-424 in the 3'-UTR of fibroblast growth factor 2 (FGF2). A luciferase reporter assay suggested that miR-424 targeted FGF2 in alveolar epithelial cells. The level of FGF2 protein was inhibited by miR-424 mimic, whereas was significantly upregulated after miR-424 suppression in LPS-stimulated alveolar epithelial cells. MiR-424 also exhibited the protective role in LPS-induced apoptosis and inflammation by directly targeting FGF2 via the NF-κB pathway. In conclusion, our results demonstrate that miR-424 had a protective role in LPS-induced apoptosis and inflammation of alveolar epithelial cells by targeting FGF2 via regulating NF-κB pathway. This might contribute novel evidence to help identify a therapeutic target for treating ARDS.


Assuntos
Células A549/metabolismo , Apoptose/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/fisiologia , Inflamação/fisiopatologia , Lipopolissacarídeos/farmacologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais , Células A549/fisiologia , Apoptose/fisiologia , Western Blotting , Fator 2 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Humanos , Inflamação/metabolismo , MicroRNAs/fisiologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , Transdução de Sinais/fisiologia
3.
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
4.
Eur J Histochem ; 63(3)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31505925

RESUMO

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.


Assuntos
Acetilgalactosamina/metabolismo , Brônquios/citologia , Células Epiteliais/metabolismo , Galactose/metabolismo , Alvéolos Pulmonares/citologia , Animais , Brônquios/crescimento & desenvolvimento , Feminino , Histocitoquímica/métodos , Peroxidase do Rábano Silvestre/química , Neuraminidase/química , Lectinas de Plantas/química , Gravidez , Alvéolos Pulmonares/crescimento & desenvolvimento , Ratos Wistar , Proteínas de Soja/química
5.
Environ Sci Pollut Res Int ; 26(31): 31913-31923, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31489544

RESUMO

The chemical and cytotoxicity properties of fine particulate matter (PM2.5) at indoor and outdoor environment were characterized in Xi'an, China. The mass concentrations of PM2.5 in urban areas (93.29~96.13 µg m-3 for indoor and 124.37~154.52 µg m-3 for outdoor) were higher than suburban (68.40 µg m-3 for indoor and 96.18 µg m-3 for outdoor). The PM2.5 concentrations from outdoor environment due to fossil fuel combustion were higher than indoor environment. An indoor environment without central heating demonstrated higher organic carbon-to-elemental carbon (OC / EC) ratios and n-alkanes values that potentially attributed to residential coal combustion activities. The cell viability of human epithelial lung cells showed dose-dependent decrease, while nitric oxide (NO) and oxidative potential showed dose-dependent increase under exposure to PM2.5. The variations of bioreactivities could be possibly related to different chemical components from different sources. Moderate (0.4 < R < 0.6) to strong (R > 0.6) correlations were observed between bioreactivities and elemental carbon (EC)/secondary aerosols (NO3-, SO42-, and NH4+)/heavy metals (Ni, Cu, and Pb). The findings suggest PM2.5 is associated with particle induced oxidative potential, which are further responsible for respiratory diseases under chronic exposure.


Assuntos
Poluentes Atmosféricos/análise , Poluição do Ar em Ambientes Fechados/análise , Material Particulado/análise , Material Particulado/toxicidade , Aerossóis/análise , Poluentes Atmosféricos/química , Poluentes Atmosféricos/toxicidade , Carbono/análise , Linhagem Celular , China , Cidades , Relação Dose-Resposta a Droga , Monitoramento Ambiental , Células Epiteliais/efeitos dos fármacos , Calefação , Humanos , Tamanho da Partícula , Alvéolos Pulmonares/citologia , Estações do Ano , Testes de Toxicidade
6.
Respir Res ; 20(1): 192, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438948

RESUMO

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by alveolar epithelial disruption. Lipoxins (LXs), as so-called "braking signals" of inflammation, are the first mediators identified to have dual anti-inflammatory and inflammatory pro-resolving properties. METHODS: In vivo, lipoxinA4 was administrated intraperitoneally with 1 µg/per mouse after intra-tracheal LPS administration (10 mg/kg). Apoptosis, proliferation and epithelial-mesenchymal transition of AT II cells were measured by immunofluorescence. In vitro, primary human alveolar type II cells were used to model the effects of lipoxin A4 upon proliferation, apoptosis and epithelial-mesenchymal transition. RESULTS: In vivo, lipoxin A4 markedly promoted alveolar epithelial type II cells (AT II cells) proliferation, inhibited AT II cells apoptosis, reduced cleaved caspase-3 expression and epithelial-mesenchymal transition, with the outcome of attenuated LPS-induced lung injury. In vitro, lipoxin A4 increased primary human alveolar epithelial type II cells (AT II cells) proliferation and reduced LPS induced AT II cells apoptosis. LipoxinA4 also inhibited epithelial mesenchymal transition in response to TGF-ß1, which was lipoxin receptor dependent. In addition, Smad3 inhibitor (Sis3) and PI3K inhibitor (LY294002) treatment abolished the inhibitory effects of lipoxinA4 on the epithelial mesenchymal transition of primary human AT II cells. Lipoxin A4 significantly downregulated the expressions of p-AKT and p-Smad stimulated by TGF-ß1 in primary human AT II cells. CONCLUSION: LipoxinA4 attenuates lung injury via stimulating epithelial cell proliferation, reducing epithelial cell apoptosis and inhibits epithelial-mesenchymal transition.


Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Lipoxinas/uso terapêutico , Síndrome do Desconforto Respiratório do Adulto/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Células Cultivadas , Humanos , Injeções Intraperitoneais , Lipopolissacarídeos , Lipoxinas/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/uso terapêutico , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Síndrome do Desconforto Respiratório do Adulto/induzido quimicamente
7.
Elife ; 82019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31140976

RESUMO

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.


Assuntos
Adenocarcinoma de Pulmão/patologia , Carcinógenos/metabolismo , Exposição Ambiental , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Camundongos , Alvéolos Pulmonares/citologia , Mucosa Respiratória/citologia , Fumar Tabaco/efeitos adversos
8.
Cell Physiol Biochem ; 52(5): 984-1002, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977984

RESUMO

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role in lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that αENaC mRNA expression is downregulated in part via posttranscriptional regulation of mRNA stability. In the present work, the role of the αENaC 3' untranslated region (3'UTR) in the regulation of mRNA stability was studied further. METHODS: Quantitative reverse transcription PCR (qRT-PCR) was performed to investigate the expression of αENaC in alveolar epithelial cells. The role of the αENaC 3'UTR was evaluated through sequential deletions. RNA affinity chromatography and mass spectrometry were achieved to investigate the nature of the proteins that could bind this sequence. The function of these proteins was assessed through knockdown and overexpression in vitro. RESULTS: First, we found that αENaC mRNA half-life was much shorter than expected when using a transcriptionally controlled plasmid expression system compared to Actinomycin D treatment. Sequential deletions of the αENaC 3'UTR revealed that the αENaC 3'UTR plays an important role in the modulation of αENaC mRNA stability, and that there is a complex stabilizing and destabilizing interplay between different regions of the 3'UTR that modulate this process. Finally, we identified RNA-binding proteins that interact with the αENaC 3'UTR and showed that Dhx36 and Tial1 are involved in the decrease in αENaC mRNA stability via the proximal region of its 3'UTR. CONCLUSION: Taken together, these findings indicate that the αENaC 3'UTR plays an important role in modulating transcript levels, and Dhx36 and Tial1 seem to be involved in posttranscriptional regulation of αENaC expression in alveolar epithelial cells.


Assuntos
Regiões 3' não Traduzidas , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/biossíntese , Regulação da Expressão Gênica , Alvéolos Pulmonares/metabolismo , Estabilidade de RNA , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células Epiteliais/citologia , Canais Epiteliais de Sódio/genética , Masculino , Alvéolos Pulmonares/citologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley
9.
Toxicol In Vitro ; 59: 228-237, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31002973

RESUMO

Biosoluble AES wools are increasingly used since considered not hazardous, however, few toxicity studies are available. We evaluated cytotoxic, genotoxic-oxidative and inflammatory effects of two differently soluble AES wools, AES1 (high MgO percentage) and AES2 (high CaO percentage), on alveolar (A549) and bronchial (BEAS-2B) cells. Fiber dimensions and dissolution in cell media were evaluated by SEM analysis. Cell viability, LDH release, direct/oxidative DNA damage (fpg-comet assay) and IL-6, IL-8 and TNF-α release (ELISA), were analysed after 24 h exposure to 2-200 µg/ml. On A549 cells AES1 induced LDH release, slight direct DNA damage and oxidative DNA damage with very high IL-6 release at 100 µg/ml; AES2 induced higher DNA damage than AES1 and slight oxidative DNA damage. On BEAS-2B cells we found direct DNA damage (higher for AES1) and slight oxidative DNA damage (associated to slight increased IL-6 and IL-8 release for AES1). The higher genotoxicity of more soluble AES2 on A549 cells could be explained by higher respirable fibers % and fiber number/µg found after 24 h in RPMI-medium at 100 µg/ml. The higher membrane damage, oxidative DNA damage and inflammation induced by AES1 in A549 cells could be due to the higher DLG and silica percentage. These findings suggest further investigations on AES toxicity.


Assuntos
Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Alvéolos Pulmonares/citologia , Silicatos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Citocinas/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Humanos , Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos
10.
EMBO J ; 38(12)2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31028085

RESUMO

Bronchioalveolar stem cells (BASCs) are a potential source for lung regeneration, but direct in vivo evidence for a multipotential lineage contribution during homeostasis and disease is critically missing, since specific genetic labeling of BASCs has not been possible. We developed a novel cell tracing approach based on intein-mediated assembly of newly engineered split-effectors, allowing selective targeting of dual-marker expressing BASCs in the mouse lung. RNA sequencing of isolated BASCs demonstrates that BASCs show a distinct transcriptional profile, characterized by co-expression of bronchiolar and alveolar epithelial genes. We found that BASCs generate the majority of distal lung airway cells after bronchiolar damage but only moderately contribute to cellular turnover under homeostatic conditions. Importantly, DTA-mediated ablation of BASCs compromised proper regeneration of distal airways. The study defines BASCs as crucial components of the lung repair machinery and provides a paradigmatic example for the detection and manipulation of stem cells that cannot be recognized by a single marker alone.


Assuntos
Células-Tronco Adultas/fisiologia , Alvéolos Pulmonares/citologia , Regeneração/fisiologia , Mucosa Respiratória/fisiologia , Células-Tronco Adultas/citologia , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Embrião de Mamíferos , Células HEK293 , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Mucosa Respiratória/citologia
11.
Bull Exp Biol Med ; 166(5): 637-640, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903504

RESUMO

In CBA mice infected with influenza viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05 in a dose of 10 MLD50, the mechanisms of death of pulmonary alveolocytes over 10 postinfection days were studied by light microscopy, immunohistochemistry, and morphometry. In mice infected with A/H1N1, alveolocytes died predominantly via necrosis, while apoptosis mostly employed the mitochondrial pathway. In mice infected with A/H5N1, apoptosis was the dominant mechanism of alveolocyte death proceeded via membrane receptor signaling followed by switching to FAS-mediated pathway via activation of FADD, the apoptotic signal transduction protein.


Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Pulmão/citologia , Alvéolos Pulmonares/virologia , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Infecções por Orthomyxoviridae/virologia , Alvéolos Pulmonares/citologia
12.
Biomed Pharmacother ; 113: 108748, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877881

RESUMO

Lung injury and inflammation are common characterizes in many pulmonary diseases. Alveolar epithelial cells, macrophages, pulmonary microvascular endothelial cells, and neutrophils, play crucial roles in lung injury and inflammation. They can secrete extracellular vesicles (EVs), involving exosomes, microvesicles, and apoptotic bodies. EVs are considered to be novel cell-cell communication tools, transferring diverse substances such as proteins, nucleic acid and lipids. Recently, the effects of EVs produced by pulmonary structural cells, immunoregulatory cells and stem cells on pulmonary diseases have attracted much attention. We summarized the recent findings of EV-related pathogeneses, therapies and diagnoses for lung injury and inflammation in this review.


Assuntos
Vesículas Extracelulares/metabolismo , Inflamação/patologia , Lesão Pulmonar/patologia , Animais , Comunicação Celular/fisiologia , Células Endoteliais/metabolismo , Exossomos/metabolismo , Humanos , Inflamação/diagnóstico , Inflamação/terapia , Lesão Pulmonar/diagnóstico , Lesão Pulmonar/terapia , Macrófagos/metabolismo , Alvéolos Pulmonares/citologia
13.
Monoclon Antib Immunodiagn Immunother ; 38(1): 18-24, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30802179

RESUMO

Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking. In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometric screening. One of the mAbs, PMab-213 (IgG2b, kappa), could specifically detect CHO/pPDPN cells through flow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was determined to be 2.1 × 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Alvéolos Pulmonares/citologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Citometria de Fluxo , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/química , Alvéolos Pulmonares/imunologia , Suínos/genética
15.
Hum Cell ; 32(2): 103-113, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30635859

RESUMO

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.


Assuntos
Pulmão/embriologia , Organoides , Alvéolos Pulmonares , Mucosa Respiratória/crescimento & desenvolvimento , Animais , Apoproteínas/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Colágeno/farmacologia , Meios de Cultura/farmacologia , Combinação de Medicamentos , Fator 7 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/farmacologia , Laminina/farmacologia , Camundongos Endogâmicos ICR , Proteoglicanas/farmacologia , Alvéolos Pulmonares/citologia , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Selênio/farmacologia , Estimulação Química , Transferrina/farmacologia
16.
Vet Microbiol ; 229: 28-38, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30642596

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease with a significant impact on the pig industry. It is caused by PRRS virus (PRRSV), which predominantly infects and replicates in porcine pulmonary alveolar macrophages (PAMs). We pretreated PAMs with porcine interferon (IFN)-α to induce an antiviral state within the cells and subsequently infected them with highly pathogenic PRRSV. Changes in global gene expression in IFN-α-pretreated PAMs in response to PRRSV infection were determined by RNA-sequence analysis and confirmed by real-time PCR. We found that IRF7 and other antiviral interferon stimulating genes (ISG)s were suppressed by PRRSV infection. Further studies demonstrated that PRRSV could down-regulate the expression of IRF7 by the non-structure protein 7 (nsp7). In conclusion, PRRSV infection had a strong immunosuppressive effect of IFN. PRRSV nsp7 inhibits the expression of IRF7, thereby down-regulating the expression of IFN and downstream ISGs and facilitated the virus to replicate.


Assuntos
Fator Regulador 7 de Interferon/metabolismo , Interferon-alfa/farmacologia , Macrófagos/efeitos dos fármacos , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Alvéolos Pulmonares/citologia , Animais , Sequência de Bases , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Celular , Fator Regulador 7 de Interferon/genética , RNA/genética , RNA/metabolismo , Suínos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
17.
Monoclon Antib Immunodiagn Immunother ; 38(1): 30-36, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30681406

RESUMO

Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa), was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Alvéolos Pulmonares/citologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Citometria de Fluxo , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/química , Alvéolos Pulmonares/imunologia , Suínos
18.
Adv Exp Med Biol ; 1201: 261-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31898791

RESUMO

The respiratory system plays an essential role for human life. This system (like all others) undergoes physiological regeneration due to many types of stem cells found both in the respiratory tract itself and in the alveoli. The stem cell hierarchy is very extensive due to their variety in the lungs and is still not completely understood.The best described lung stem cells are alveolar type II cells, which as progenitor lung stem cells are precursors of alveolar type I cells, i.e., cells that perform gas exchange in the lungs. These progenitor stem cells, which reside in alveoli corners, express high levels of surfactant protein C (SFTPC). Despite the fact that type II pneumocytes occupy only 7-10% of the lung surface, there are almost twice as many as alveolar type I cells occupying almost 95% of the surface.Other stem cells making up the lung regenerative potential have also been identified in the lungs. Both endothelial, mesodermal, and epithelial stem cells are necessary for the lungs to function properly and perform their physiological functions.The lungs, like all other organs, undergo an aging process. As a result of this process, not only the total number of cells changes, the percentage of particular types of cells, but also their efficiency is reduced. With age, the proliferative potential of lung stem cells also decreases, not just their number. This brings about the need to increase the intensity of research in the field of regenerative medicine.


Assuntos
Pulmão/citologia , Células-Tronco/citologia , Humanos , Alvéolos Pulmonares/citologia , Medicina Regenerativa
19.
Exp Lung Res ; 44(4-5): 241-251, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30449218

RESUMO

Purpose/aim: Activated coagulation and reduced fibrinolysis in alveolar compartment are an important characteristics in acute respiratory distress syndrome (ARDS). Alveolar epithelial cell type II (AECII) participates in regulating the intra-alveolar abnormalities of coagulation and fibrinolysis mainly through adjusting the productions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and activated protein C (APC) in ARDS. NF-κB signal pathway may be involved in coagulation regulation in sepsis-induced ALI. The purpose of this study was to testify the hypothesis that NF-κB p65 (p65) knock-down would improve the abnormalities of coagulation and fibrinolysis mediated by lipopolysaccharide (LPS) stimulation in AECII. MATERIALS AND METHODS: p65 gene knock-down in AECII was achieved by small interfering RNA (siRNA) transfection. Rat AECII (RLE-6TN) with or without p65 gene knock-down were stimulated by LPS for 24 hours. And then cytolysate was used for TF, PAI-1 expression examination, and supernatant was collected for TF, PAI-1 and PC concentrations determination. Activation of NF-κB canonical pathway was simultaneously checked by western-blotting, RT-PCR and immunofluorescence respectively. RESULTS: TF, PAI-1 expressions in normal cells obviously increased under LPS stimulation with NF-κB canonical pathway activation represented by high levels of p65, p-p65, p-IκB with increased nuclear translocation of p-p65. Cells with NF-κB p65 knock-down, however, showed significant decreases in TF, PAI-1, p65, p-p65, p-IκB expressions following LPS stimulation with significant reduction in p-p65 nuclear translocation as compared to normal and siRNA control cells. The high concentrations of TF, PAI-1 and low level of APC in supernatant induced by LPS in normal cells were significantly reversed through p65 knock-down. CONCLUSIONS: The experimental findings demonstrate that NF-kB signaling pathway is involved in regulating the expressions of coagulation and fibrinolysis factors in LPS-stimulated AECII, which suggest that NF-kB signaling pathway may be a new target to correct intra-alveolar coagulation and fibrinolytic abnormalities in ARDS.


Assuntos
Células Epiteliais/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteína C/biossíntese , Alvéolos Pulmonares/citologia , Tromboplastina/metabolismo , Fator de Transcrição RelA/fisiologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Células Cultivadas , Fibrinólise/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/farmacologia , Proteína C/metabolismo , Ratos , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
20.
Proc Natl Acad Sci U S A ; 115(45): E10605-E10614, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30348760

RESUMO

Hox5 genes (Hoxa5, Hoxb5, Hoxc5) are exclusively expressed in the lung mesenchyme during embryogenesis, and the most severe phenotypes result from constitutive loss of function of all three genes. Because Hox5 triple null mutants exhibit perinatal lethality, the contribution of this paralogous group to postembryonic lung development is unknown. Intriguingly, expression of all three Hox5 genes peaks during the first 2 weeks after birth, reaching levels far exceeding those measured at embryonic stages, and surviving Hoxa5 single and Hox5 AabbCc compound mutants exhibit defects in the localization of alveolar myofibroblasts. To define the contribution of the entire Hox5 paralogous group to this process, we generated an Hoxa5 conditional allele to use with our existing null alleles for Hoxb5 and Hoxc5 Postnatally, mesenchymal deletion of Hoxa5 in an Hoxb5/Hoxc5 double-mutant background results in severe alveolar simplification. The elastin network required for alveolar formation is dramatically disrupted in Hox5 triple mutants, while the basal lamina, interstitial matrix, and fibronectin are normal. Alveolar myofibroblasts remain Pdgfrα+/SMA+ double positive and present in normal numbers, indicating that the irregular elastin network is not due to fibroblast differentiation defects. Rather, we observe that SMA+ myofibroblasts of Hox5 triple mutants are morphologically abnormal both in vivo and in vitro with highly reduced adherence to fibronectin. This loss of adhesion is a result of loss of the integrin heterodimer Itga5b1 in mutant fibroblasts. Collectively, these data show an important role for Hox5 genes in lung fibroblast adhesion necessary for proper elastin network formation during alveologenesis.


Assuntos
Adesão Celular , Elastina/metabolismo , Genes Homeobox , Miofibroblastos/citologia , Alvéolos Pulmonares/citologia , Alelos , Animais , Dimerização , Regulação da Expressão Gênica no Desenvolvimento , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Camundongos , Mutação , Miofibroblastos/metabolismo , Alvéolos Pulmonares/metabolismo
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