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1.
Cell Mol Life Sci ; 80(3): 79, 2023 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-36867267

RESUMO

Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways. Here, we show that precursor NE cells start to develop in the SOX2 + SOX21 + airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1 + precursor cells early in development, and the number of mature cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells.


Assuntos
Células Neuroendócrinas , Fatores de Transcrição SOXB1 , Fatores de Transcrição SOXB2 , Animais , Camundongos , Peptídeo Relacionado com Gene de Calcitonina , Diferenciação Celular/genética , Epitélio , Hiperplasia , Células Neuroendócrinas/citologia , Células Neuroendócrinas/metabolismo
2.
J Biol Chem ; 298(2): 101574, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35007536

RESUMO

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.


Assuntos
Dexametasona , Mutação Puntual , Receptores de Glucocorticoides , Animais , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Ligantes , Camundongos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo
3.
Lab Invest ; 103(11): 100233, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37567389

RESUMO

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.


Assuntos
Síndrome da Persistência do Padrão de Circulação Fetal , Alvéolos Pulmonares/anormalidades , Recém-Nascido , Humanos , Síndrome da Persistência do Padrão de Circulação Fetal/diagnóstico , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Relevância Clínica , Alvéolos Pulmonares/patologia , Fatores de Transcrição Forkhead/genética
4.
Prenat Diagn ; 43(10): 1296-1309, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37539818

RESUMO

Pathophysiological studies have shown that pulmonary vascular development is impaired in fetuses with a congenital diaphragmatic hernia (CDH), leading to a simplified vascular tree and increased vascular resistance. Multiple studies have described prenatal ultrasound parameters for the assessment of the pulmonary vasculature, but none of these parameters are used in daily clinical practice. We provide a comprehensive review of the literature published between January 1990 and February 2022 describing these parameters, and aim to explain the clinical relevance of these parameters from what is known from pathophysiological studies. Prenatal detection of a smaller diameter of the contralateral (i.e. contralateral to the diaphragmatic defect) first branch of the pulmonary artery (PA), higher pulsatility indices (PI), higher peak early diastolic reverse flow values, and a lower vascularization index seem of added value for the prediction of survival and, to a lesser extent, morbidity. Integration within the routine evaluation is complicated by the lack of uniformity of the methods used. To address the main components of the pathophysiological changes, we recommend future prenatal studies in CDH with a focus on PI values, PA diameters and pulmonary vascular branching.


Assuntos
Hérnias Diafragmáticas Congênitas , Gravidez , Feminino , Humanos , Hérnias Diafragmáticas Congênitas/diagnóstico por imagem , Ultrassonografia Pré-Natal/métodos , Pulmão , Artéria Pulmonar/diagnóstico por imagem , Feto
5.
Int J Mol Sci ; 23(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36361852

RESUMO

The lung originates from the ventral foregut and develops into an intricate branched structure of airways, alveoli, vessels and support tissue. As the lung develops, cells become specified and differentiate into the various cell lineages. This process is controlled by specific transcription factors, such as the SRY-related HMG-box genes SOX2 and SOX21, that are activated or repressed through intrinsic and extrinsic signals. Disturbances in any of these processes during the development of the lung may lead to various pediatric lung disorders, such as Congenital Diaphragmatic Hernia (CDH), Congenital Pulmonary Airway Malformation (CPAM) and Broncho-Pulmonary Dysplasia (BPD). Changes in the composition of the airways and the alveoli may result in reduced respiratory function and eventually lead to chronic lung disorders. In this concise review, we describe different intrinsic and extrinsic cellular processes required for proper differentiation of the epithelium during development and regeneration, and the influence of the microenvironment on this process with special focus on SOX2 and SOX21.


Assuntos
Hérnias Diafragmáticas Congênitas , Pneumopatias , Humanos , Criança , Diferenciação Celular/genética , Fatores de Transcrição , Alvéolos Pulmonares , Pulmão , Pneumopatias/genética , Fatores de Transcrição SOXB1/genética
6.
Am J Physiol Lung Cell Mol Physiol ; 321(4): L775-L786, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34378410

RESUMO

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1) cellular composition, 2) response to cigarette smoke exposure, and 3) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.


Assuntos
Brônquios/citologia , Células Epiteliais/citologia , Organoides/citologia , Mucosa Respiratória/citologia , Fumaça/efeitos adversos , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Heme Oxigenase-1/metabolismo , Humanos , Recém-Nascido , Interleucina-13/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Receptores Notch/antagonistas & inibidores , Produtos do Tabaco/efeitos adversos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
7.
Hum Mol Genet ; 28(9): 1429-1444, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30566624

RESUMO

Bronchopulmonary dysplasia (BPD), characterized by alveoli simplification and dysmorphic pulmonary microvasculature, is a chronic lung disease affecting prematurely born infants. Pulmonary hypertension (PH) is an important BPD feature associated with morbidity and mortality. In human BPD, inflammation leads to decreased fibroblast growth factor 10 (FGF10) expression but the impact on the vasculature is so far unknown. We used lungs from Fgf10+/- versus Fgf10+/+ pups to investigate the effect of Fgf10 deficiency on vascular development in normoxia (NOX) and hyperoxia (HOX, BPD mouse model). To assess the role of fibroblast growth factor receptor 2b (Fgfr2b) ligands independently of early developmentaldefects, we used an inducible double transgenic system in mice allowing inhibition of Fgfr2b ligands activity. Using vascular morphometry, we quantified the pathological changes. Finally, we evaluated changes in FGF10, surfactant protein C (SFTPC), platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin 2 (α-SMA) expression in human lung samples from patients suffering from BPD. In NOX, no major difference in the lung vasculature between Fgf10+/- and control pups was detected. In HOX, a greater loss of blood vessels in Fgf10+/- lungs is associated with an increase of poorly muscularized vessels. Fgfr2b ligands inhibition postnatally in HOX is sufficient to decrease the number of blood vessels while increasing the level of muscularization, suggesting a PH phenotype. BPD lungs exhibited decreased FGF10, SFTPC and PECAM but increased α-SMA. Fgf10 deficiency-associated vascular defects are enhanced in HOX and could represent an additional cause of morbidity in human patients with BPD.


Assuntos
Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/patologia , Suscetibilidade a Doenças , Fator 10 de Crescimento de Fibroblastos/deficiência , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Animais , Biomarcadores , Displasia Broncopulmonar/metabolismo , Biologia Computacional/métodos , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Genótipo , Hipóxia , Pulmão/patologia , Camundongos , Mutação , Neovascularização Fisiológica/genética , Consumo de Oxigênio , Fosforilação , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais
8.
Pediatr Res ; 89(3): 518-525, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32413891

RESUMO

BACKGROUND: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with heterozygous variants in the FOXF1 gene or its regulatory region. Patients with ACD/MPV unnecessarily undergo invasive and expensive treatments while awaiting a diagnosis. The aim of this study was to reduce the time to diagnose ACD/MPV by developing a targeted next-generation sequencing (NGS) panel that detects FOXF1 variants. METHODS: A FOXF1-targeted NGS panel was developed for detection of mutations and large genomic alterations and used for retrospective testing of ACD/MPV patients and controls. Results were confirmed with Sanger sequencing and SNP array analysis. RESULTS: Each amplicon of the FOXF1-targeted NGS panel was efficiently sequenced using DNA isolated from blood or cell lines of 15 ACD/MPV patients and 8 controls. Moreover, testing of ACD/MPV patients revealed six novel and six previously described pathogenic or likely pathogenic FOXF1 alterations. CONCLUSION: We successfully designed a fast and reliable targeted genetic test to detect variants in the FOXF1 gene and its regulatory region in one run. This relatively noninvasive test potentially prevents unnecessary suffering for patients and reduces the use of futile and expensive treatments like extra-corporeal membrane oxygenation. IMPACT: FOXF1-targeted NGS potentially prevents ACD/MPV patients from unnecessary suffering and expensive treatments. FOXF1-targeted NGS potentially reduces the number of misdiagnosis in ACD/MPV patients. Retrospective testing of ACD/MPV patients using FOXF1-targeted NGS revealed six novel pathogenic or likely pathogenic variants.


Assuntos
Fatores de Transcrição Forkhead/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Anormalidades Múltiplas/genética , Variações do Número de Cópias de DNA , Análise Mutacional de DNA/métodos , Éxons/genética , Feminino , Fibroblastos/química , Duplicação Gênica , Humanos , Lactente , Recém-Nascido , Pulmão/química , Masculino , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Deleção de Sequência , Procedimentos Desnecessários
9.
Am J Physiol Lung Cell Mol Physiol ; 317(3): L317-L331, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268349

RESUMO

The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Hérnias Diafragmáticas Congênitas/patologia , Tretinoína/farmacologia , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Hérnias Diafragmáticas Congênitas/tratamento farmacológico , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Pericitos/efeitos dos fármacos , Pericitos/patologia , Transdução de Sinais/efeitos dos fármacos
10.
Am J Physiol Lung Cell Mol Physiol ; 315(2): L276-L285, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29745254

RESUMO

Patients with congenital diaphragmatic hernia (CDH) often suffer from severe pulmonary hypertension, and the choice of current vasodilator therapy is mostly based on trial and error. Because pulmonary vascular abnormalities are already present early during development, we performed a study to modulate these pulmonary vascular changes at an early stage during gestation. Pregnant Sprague-Dawley rats were treated with nitrofen at day 9.5 of gestation (E9.5) to induce CDH in the offspring, and subsequently, the phosphodiesterase-5 inhibitor sildenafil and/or the novel prostaglandin-I receptor agonist selexipag (active compound NS-304) were administered from E17.5 until E20.5. The clinical relevant start of the treatment corresponds to week 20 of gestation in humans, when CDH is usually detected by ultrasound. CDH pups showed increased density of air saccules that was reverted after the use of only sildenafil. The pulmonary vascular wall was thickened, and right ventricular hypertrophy was present in the CDH group and improved both after single treatment with sildenafil or selexipag, whereas the combination therapy with both compounds did not have additive value. In conclusion, antenatal treatment with sildenafil improved airway morphogenesis and pulmonary vascular development, whereas selexipag only acted positively on pulmonary vascular development. The combination of both compounds did not act synergistically, probably because of a decreased efficiency of both compounds caused by cytochrome- P450 3A4 interaction and induction. These new insights create important possibilities for future treatment of pulmonary vascular abnormalities in CDH patients already in the antenatal period of life.


Assuntos
Acetamidas/farmacologia , Hérnias Diafragmáticas Congênitas , Pulmão , Pirazinas/farmacologia , Citrato de Sildenafila/farmacologia , Animais , Quimioterapia Combinada , Hérnias Diafragmáticas Congênitas/tratamento farmacológico , Hérnias Diafragmáticas Congênitas/metabolismo , Hérnias Diafragmáticas Congênitas/patologia , Hérnias Diafragmáticas Congênitas/fisiopatologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Ratos , Ratos Sprague-Dawley
11.
Angiogenesis ; 21(4): 805-821, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29845518

RESUMO

AIMS: Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. METHODS AND RESULTS: Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G0/G1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca2+-mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. CONCLUSIONS: The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2.


Assuntos
Angiopoietina-1/metabolismo , Receptores Frizzled/deficiência , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Proteína Quinase C/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Transcrição Gênica , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização Wnt , Angiopoietina-1/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteína Quinase C/genética , Proteína Proto-Oncogênica c-ets-1/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
12.
Transgenic Res ; 27(1): 75-85, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29383478

RESUMO

Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to 'complex-specific' DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2). This tag was biotinylated by the bacterial birA protein and the resulting bioSox2 protein was used to identify associating partners of Sox2 at different phases of lung development in vivo (the Sox2 interactome). Homozygous bioSox2 mice are viable and fertile irrespective of the biotinylation of the bio tag, indicating that the bioSox2 gene is normally expressed and the protein is functional in all tissues. This suggests that partners of Sox2 are most likely able to associate with the bioSox2 protein. BioSox2 complexes were isolated with high affinity using streptavidin beads and analysed by MALDI-ToF mass spectrometry analysis. Several of the identified binding partners are already shown to have a respiratory phenotype. Two of these partners, Wdr5 and Tcf3, were validated to confirm their association in Sox2 complexes. This bioSox2 mouse model will be a valuable tool for isolating in vivo Sox2 complexes from different tissues.


Assuntos
Pulmão/embriologia , Camundongos Transgênicos , Fatores de Transcrição SOXB1/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biotinilação , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Respir Res ; 18(1): 187, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29115963

RESUMO

BACKGROUND: Patients with congenital diaphragmatic hernia (CDH) have structural and functional different pulmonary vessels, leading to pulmonary hypertension. They often fail to respond to standard vasodilator therapy targeting the major vasoactive pathways, causing a high morbidity and mortality. We analyzed whether the expression of crucial members of these vasoactive pathways could explain the lack of responsiveness to therapy in CDH patients. METHODS: The expression of direct targets of current vasodilator therapy in the endothelin and prostacyclin pathway was analyzed in human lung specimens of control and CDH patients. RESULTS: CDH lungs showed increased expression of both ETA and ETB endothelin receptors and the rate-limiting Endothelin Converting Enzyme (ECE-1), and a decreased expression of the prostaglandin-I2 receptor (PTGIR). These data were supported by increased expression of both endothelin receptors and ECE-1, endothelial nitric oxide synthase and PTGIR in the well-established nitrofen-CDH rodent model. CONCLUSIONS: Together, these data demonstrate aberrant expression of targeted receptors in the endothelin and prostacyclin pathway in CDH already early during development. The analysis of this unique patient material may explain why a significant number of patients do not respond to vasodilator therapy. This knowledge could have important implications for the choice of drugs and the design of future clinical trials internationally.


Assuntos
Hérnias Diafragmáticas Congênitas/metabolismo , Hérnias Diafragmáticas Congênitas/patologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Animais , Broncodilatadores/administração & dosagem , Feminino , Hérnias Diafragmáticas Congênitas/tratamento farmacológico , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Recém-Nascido , Masculino , Óxido Nítrico/administração & dosagem , Gravidez , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
14.
Am J Physiol Lung Cell Mol Physiol ; 311(4): L734-L742, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27521424

RESUMO

Patients with congenital diaphragmatic hernia (CDH) suffer from severe pulmonary hypertension attributable to altered development of the pulmonary vasculature, which is often resistant to vasodilator therapy. Present treatment starts postnatally even though significant differences in the pulmonary vasculature are already present early during pregnancy. We examined the effects of prenatal treatment with the phosphodiesterase-5 inhibitor sildenafil on pulmonary vascular development in experimental CDH starting at a clinically relevant time. The well-established, nitrofen-induced CDH rodent model was treated daily with 100 mg/kg sildenafil from day 17.5 until day 20.5 of gestation (E17.5-20.5). Importantly, this timing perfectly corresponds to the developmental stage of the lung at 20 wk of human gestation, when CDH is detectable by 2D-ultrasonography and/or MRI. At E21.5 pups were delivered by caesarean section and euthanized by lethal injection of pentobarbital. The lungs were isolated and subsequently analyzed using immunostaining, real-time PCR, and volume measurements. Prenatal treatment with sildenafil improved lung morphology and attenuated vascular remodeling with reduced muscularization of the smaller vessels. Pulmonary vascular volume was not affected by sildenafil treatment. We show that prenatal treatment with sildenafil within a clinically relevant period improves pulmonary vascular development in an experimental CDH model. This may have important implications for the management of this disease and related pulmonary vascular diseases in human.


Assuntos
Hérnias Diafragmáticas Congênitas/prevenção & controle , Inibidores da Fosfodiesterase 5/uso terapêutico , Citrato de Sildenafila/uso terapêutico , Remodelação Vascular/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Hérnias Diafragmáticas Congênitas/induzido quimicamente , Hérnias Diafragmáticas Congênitas/fisiopatologia , Pulmão/irrigação sanguínea , Pulmão/patologia , Exposição Materna , Troca Materno-Fetal , Éteres Fenílicos , Inibidores da Fosfodiesterase 5/farmacologia , Gravidez , Ratos Sprague-Dawley , Citrato de Sildenafila/farmacologia
15.
Respir Res ; 17: 44, 2016 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-27107715

RESUMO

Inspired by the increasing burden of lung associated diseases in society and an growing demand to accommodate patients, great efforts by the scientific community produce an increasing stream of data that are focused on delineating the basic principles of lung development and growth, as well as understanding the biomechanical properties to build artificial lung devices. In addition, the continuing efforts to better define the disease origin, progression and pathology by basic scientists and clinicians contributes to insights in the basic principles of lung biology. However, the use of different model systems, experimental approaches and readout systems may generate somewhat conflicting or contradictory results. In an effort to summarize the latest developments in the lung epithelial stem cell biology, we provide an overview of the current status of the field. We first describe the different stem cells, or progenitor cells, residing in the homeostatic lung. Next, we focus on the plasticity of the different cell types upon several injury-induced activation or repair models, and highlight the regenerative capacity of lung cells. Lastly, we summarize the generation of lung mimics, such as air-liquid interface cultures, organoids and lung on a chip, that are required to test emerging hypotheses. Moreover, the increasing collaboration between distinct specializations will contribute to the eventual development of an artificial lung device capable of assisting reduced lung function and capacity in human patients.


Assuntos
Órgãos Bioartificiais , Transplante de Pulmão/instrumentação , Pulmão/citologia , Pulmão/crescimento & desenvolvimento , Regeneração/fisiologia , Células-Tronco/citologia , Animais , Biomimética/instrumentação , Humanos , Respiração Artificial/instrumentação , Transplante de Células-Tronco/métodos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
16.
Am J Physiol Lung Cell Mol Physiol ; 308(2): L147-57, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25416379

RESUMO

Congenital diaphragmatic hernia (CDH) is a common life-threatening congenital anomaly resulting in high rates of perinatal death and neonatal respiratory distress. Some of the nonisolated forms are related to single-gene mutations or genomic rearrangements, but the genetics of the isolated forms (60% of cases) still remains a challenging issue. Retinoid signaling (RA) is critical for both diaphragm and lung development, and it has been hypothesized that subtle disruptions of this pathway could contribute to isolated CDH etiology. Here we used time series of normal and CDH lungs in humans, in nitrofen-exposed rats, and in surgically induced hernia in rabbits to perform a systematic transcriptional analysis of the RA pathway key components. The results point to CRPBP2, CY26B1, and ALDH1A2 as deregulated RA signaling genes in human CDH. Furthermore, the expression profile comparisons suggest that ALDH1A2 overexpression is not a primary event, but rather a consequence of the CDH-induced lung injury. Taken together, these data show that RA signaling disruption is part of CDH pathogenesis, and also that dysregulation of this pathway should be considered organ specifically.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Diafragma/embriologia , Hérnias Diafragmáticas Congênitas/metabolismo , Retinal Desidrogenase/biossíntese , Vitamina A/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Diafragma/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hérnias Diafragmáticas Congênitas/genética , Hérnias Diafragmáticas Congênitas/patologia , Humanos , Pulmão/embriologia , Masculino , Coelhos , Ratos , Retinal Desidrogenase/genética , Ácido Retinoico 4 Hidroxilase , Proteínas Celulares de Ligação ao Retinol/biossíntese , Proteínas Celulares de Ligação ao Retinol/genética , Transdução de Sinais/genética , Vitamina A/genética
17.
Ann Surg ; 262(6): 1130-40, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25563880

RESUMO

OBJECTIVE AND BACKGROUND: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. METHODS: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-ß2 in postnatal lung sections. We investigated miR-200b effects on TGF-ß signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. RESULTS: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-ß2 expression was lower in CDH lungs. miR-200b inhibited TGF-ß-induced SMAD signaling in cultures of human bronchial epithelial cells. CONCLUSIONS: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-ß/SMAD signaling.


Assuntos
Líquido Amniótico/metabolismo , Fetoscopia , Hérnias Diafragmáticas Congênitas/terapia , Pulmão/metabolismo , MicroRNAs/metabolismo , Traqueia/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Fetoscopia/métodos , Fetoscopia/mortalidade , Regulação da Expressão Gênica , Hérnias Diafragmáticas Congênitas/genética , Hérnias Diafragmáticas Congênitas/metabolismo , Hérnias Diafragmáticas Congênitas/mortalidade , Humanos , Recém-Nascido , Pulmão/anormalidades , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez
18.
Birth Defects Res C Embryo Today ; 102(4): 343-58, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25424472

RESUMO

Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease.


Assuntos
Pneumopatias/congênito , Artéria Pulmonar/anormalidades , Malformações Vasculares/etiologia , Humanos , Recém-Nascido , Pneumopatias/complicações , Artéria Pulmonar/fisiopatologia , Malformações Vasculares/fisiopatologia
19.
J Neurosci ; 33(41): 16146-57, 2013 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-24107947

RESUMO

Hearing impairment or vestibular dysfunction in humans often results from a permanent loss of critical cell types in the sensory regions of the inner ear, including hair cells, supporting cells, or cochleovestibular neurons. These important cell types arise from a common sensory or neurosensory progenitor, although little is known about how these progenitors are specified. Studies have shown that Notch signaling and the transcription factor Sox2 are required for the development of these lineages. Previously we and others demonstrated that ectopic activation of Notch can direct nonsensory cells to adopt a sensory fate, indicating a role for Notch in early specification events. Here, we explore the relationship between Notch and SOX2 by ectopically activating these factors in nonsensory regions of the mouse cochlea, and demonstrate that, similar to Notch, SOX2 can specify sensory progenitors, consistent with a role downstream of Notch signaling. However, we also show that Notch has a unique role in promoting the proliferation of the sensory progenitors. We further demonstrate that Notch can only induce ectopic sensory regions within a certain time window of development, and that the ectopic hair cells display specialized stereocilia bundles similar to endogenous hair cells. These results demonstrate that Notch and SOX2 can both drive the sensory program in nonsensory cells, indicating these factors may be useful in cell replacement strategies in the inner ear.


Assuntos
Diferenciação Celular/fisiologia , Orelha Interna/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Orelha Interna/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Transdução de Sinais/fisiologia
20.
Am J Respir Cell Mol Biol ; 51(2): 311-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24669837

RESUMO

Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.


Assuntos
Malformação Adenomatoide Cística Congênita do Pulmão/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Malformação Adenomatoide Cística Congênita do Pulmão/genética , Malformação Adenomatoide Cística Congênita do Pulmão/patologia , Células Epiteliais/patologia , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Células HEK293 , Humanos , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Fenótipo , Fosfoproteínas/genética , Fatores de Transcrição SOXB1/genética , Células-Tronco/patologia , Técnicas de Cultura de Tecidos , Transativadores/genética , Fatores de Transcrição/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Regulação para Cima
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