The Experts below are selected from a list of 453 Experts worldwide ranked by ideXlab platform
Dirk Adriaensen - One of the best experts on this subject based on the ideXlab platform.
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the pulmonary Neuroepithelial Body microenvironment a multifunctional unit in the airway epithelium
2021Co-Authors: Inge Brouns, Line Verckist, Isabel Pintelon, Jeanpierre Timmermans, Dirk AdriaensenAbstract:Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary Neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.
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selective activation and proliferation of a quiescent stem cell population in the Neuroepithelial Body microenvironment
Respiratory Research, 2018Co-Authors: Line Verckist, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Dirk AdriaensenAbstract:The microenvironment (ME) of Neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs. Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection. The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs. A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.
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selective gene expression analysis of the Neuroepithelial Body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
Respiratory Research, 2017Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Sofie Thys, Dirk AdriaensenAbstract:The pulmonary Neuroepithelial Body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.
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Selective gene expression analysis of the Neuroepithelial Body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
'Springer Science and Business Media LLC', 2017Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Sofie Thys, Dirk AdriaensenAbstract:Abstract Background The pulmonary Neuroepithelial Body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. Methods A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Results Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. Conclusions This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular
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selective activation and proliferation of a unique stem cell population in the Neuroepithelial Body microenvironment by transient acute lung injury
The FASEB Journal, 2016Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Carmen Rottiers, Dirk AdriaensenAbstract:The microenvironment (ME) of pulmonary Neuroepithelial bodies (NEBs) has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. Regardless the...
Paul J Kemp - One of the best experts on this subject based on the ideXlab platform.
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purinergic signaling in the pulmonary Neuroepithelial Body microenvironment unraveled by live cell imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract:Pulmonary Neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a “reporter-patching” method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y2 receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X3 receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary Neuroepithelial Body microenvironment unraveled by live cell imaging.
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purinergic signaling in the pulmonary Neuroepithelial Body microenvironment unraveled by live cell imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract:Pulmonary Neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a “reporter-patching” method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-...
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functional live cell imaging of the pulmonary Neuroepithelial Body microenvironment
American Journal of Respiratory Cell and Molecular Biology, 2008Co-Authors: Ian De Proost, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Daniela Riccardi, Paul J Kemp, Alfons B A Kroese, Dirk AdriaensenAbstract:Pulmonary Neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
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o2 sensing by airway chemoreceptor derived cells protein kinase c activation reveals functional evidence for involvement of nadph oxidase
Journal of Biological Chemistry, 2000Co-Authors: I Okelly, Chris Peers, Anthony Lewis, Paul J KempAbstract:Accumulating evidence suggests that Neuroepithelial bodies are airway O2 sensors. Recently, we have established the H-146 small cell lung carcinoma line as a suitable model to study the biochemical basis of Neuroepithelial Body cell chemotransduction. Here we explore the possibility that hypoxic modulation of K+ channels is intimately linked to activity of NADPH oxidase. Graded hypoxia caused graded inhibition of whole cell K+ currents, which correlated well with membrane depolarization. Pretreatment with the phorbol ester, 12-O-tetradecanoyl (TPA), inhibited K+ currents at all potentials. Although 4α-phorbol 12,13-didecanoate and TPA in the presence of bisindolylmaleimide were also able to depress K+ currents, only TPA could significantly ameliorate hypoxic depression of these currents. Thus, protein kinase C (PKC) activation modulates the sensitivity of these cells to changes inpO2. Furthermore, because the addition of H2O2, a downstream product of NADPH oxidase, could only activate K+ currents during hypoxia (when endogenous H2O2 production is suppressed), it appears likely that PKC modulates the affinity of NADPH oxidase for O2 potentially via phosphorylation of the p47phox subunit, which is present in these cells. These data show that PKC is an important regulator of the O2-transduction pathway and suggests that NADPH oxidase represents a significant component of the airway O2sensor.
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potential identification of the o2 sensitive k current in a human Neuroepithelial Body derived cell line
American Journal of Physiology-lung Cellular and Molecular Physiology, 1999Co-Authors: I Okelly, R H Stephens, Chris Peers, Paul J KempAbstract:Whole cell recording of H-146 cells revealed that the outward K+ current was completely inhibited by quinidine (IC50 approximately 17 microM). In contrast, maximal concentrations of 4-aminopyridine (4-AP; >/=10 mM) reversibly blocked only approximately 60% (IC50 approximately 1.52 mM). Ten millimolar 4-AP had no effect on the inhibition by hypoxia, which reduced current density from approximately 27 to approximately 13 pA/pF, whereas 1 mM quinidine abolished the hypoxic effect. In current clamp, 10 mM 4-AP depolarized the cell by approximately 18 mV and hypoxia caused further reversible depolarization of approximately 4 mV. One millimolar quinidine collapsed the membrane potential and abrogated any further hypoxic depolarization. RT-PCR revealed expression of the acid-sensitive, twin P domain K+ channel TASK but not of TWIK, TREK, or the known hypoxia-sensitive Kv2.1, which was confirmed by sequencing and further PCR with primers to the coding region of TASK. However, a reduction in extracellular pH had no effect on K+ current. Thus, although the current more closely resembles TWIK than TASK pharmacologically, structurally the reverse appears to be true. This suggests that a novel acid-insensitive channel related to TASK may be responsible for the hypoxia-sensitive K+ current of these cells.
Barry R Stripp - One of the best experts on this subject based on the ideXlab platform.
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molecular phenotype of airway side population cells
American Journal of Physiology-lung Cellular and Molecular Physiology, 2004Co-Authors: Adam Giangreco, Susan D Reynolds, Hongmei Shen, Barry R StrippAbstract:Lung epithelial-specific stem cells have been localized to discrete microenvironments throughout the adult conducting airway. Properties of these cells include pollutant resistance, multipotent differentiation, and infrequent proliferation. Goals of the present study were to use Hoechst 33342 efflux, a property of stem cells in other tissues, to purify and further characterize airway stem cells. Hoechst 33342 effluxing lung cells were identified as a verapamil-sensitive side population by flow cytometry. Lung side population cells were further subdivided on the basis of hematopoietic (CD45 positive) or nonhematopoietic (CD45 negative) origin. Nonhematopoietic side population cells were enriched for stem cell antigen-1 reactivity and expressed molecular markers specific to both airway and mesenchymal lineages. Analysis of the molecular phenotype of airway-derived side population cells indicates that they are similar to Neuroepithelial Body-associated variant Clara cells. Taken together, these data suggest that the nonhematopoietic side population isolated from lung is enriched for previously identified airway stem cells.
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terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction
American Journal of Pathology, 2002Co-Authors: Adam Giangreco, Susan D Reynolds, Barry R StrippAbstract:Cellular mechanisms contributing to renewal of terminal bronchioles remain poorly defined. Our previous studies identified pollutant-resistant Clara cell secretory protein (CCSP)-expressing stem cells that localize to the Neuroepithelial Body (NEB) and contribute to renewal of the proximal bronchiolar epithelium. However, activation of NEB-associated stem cells is unlikely to contribute to renewal of terminal bronchiolar epithelium because of the paucity of NEBs at this location. Goals of this study were to determine the location and properties of cells contributing to renewal of terminal bronchioles after Clara cell depletion. Pollutant-resistant CCSP-expressing cells were identified that localized to the bronchoalveolar duct junction (BADJ) and contribute to restoration of a phenotypically diverse epithelium. CCSP-expressing cells comprise the predominant proliferative population in initial terminal bronchiolar repair and include a population of label-retaining cells suggesting that they maintain characteristics of a stem cell population. Furthermore, immunohistochemical co-localization studies involving CCSP and the NEB-specific marker calcitonin gene-related peptide indicate that BADJ-associated CCSP-expressing stem cells function independently of NEB microenvironments. These studies identify a BADJ-associated, NEB-independent, CCSP-expressing stem cell population in terminal bronchioles and support the notion that regiospecific stem cell niches function to maintain epithelial diversity after injury.
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clara cell secretory protein expressing cells of the airway Neuroepithelial Body microenvironment include a label retaining subset and are critical for epithelial renewal after progenitor cell depletion
American Journal of Respiratory Cell and Molecular Biology, 2001Co-Authors: Kyung U Hong, Adam Giangreco, Susan D Reynolds, Cheryl M Hurley, Barry R StrippAbstract:Stem cells with potential to contribute to the re-establishment of the normal bronchiolar epithelium have not been definitively demonstrated. We previously established that Neuroepithelial bodies (NEBs) sequester regenerative cells that contribute to bronchiolar regeneration after selective chemical depletion of Clara cells, a major progenitor cell population. Two candidate stem cells were identified on the basis of proliferative potential after chemical ablation: a pollutant-resistant subpopulation of Clara cells that retain their expression of Clara cell secretory protein (CCSP) (variant CCSP-expressing [CE] cells or vCE cells) and calcitonin gene-related peptide (CGRP)–expressing pulmonary neuroendocrine cells (PNECs). In the present study, two populations of label-retaining cells were identified within the NEB: CGRP-expressing cells and a subpopulation of CE cells. To investigate contributions made by CE and CGRP-expressing cells to epithelial renewal, CE cells were ablated through acute administratio...
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conditional clara cell ablation reveals a self renewing progenitor function of pulmonary neuroendocrine cells
American Journal of Physiology-lung Cellular and Molecular Physiology, 2000Co-Authors: Susan D Reynolds, Adam Giangreco, Kyung U Hong, Gregory W Mango, Charanjeet Guron, Yasuo Morimoto, Barry R StrippAbstract:The Neuroepithelial Body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is...
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conditional clara cell ablation reveals a self renewing progenitor function of pulmonary neuroendocrine cells
American Journal of Physiology-lung Cellular and Molecular Physiology, 2000Co-Authors: Susan D Reynolds, Adam Giangreco, Kyung U Hong, Gregory W Mango, Charanjeet Guron, Yasuo Morimoto, Barry R StrippAbstract:The Neuroepithelial Body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for development of small cell lung carcinoma, mechanisms regulating expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. We have previously used a Clara cell-specific toxicant, naphthalene, to induce airway injury in mice and have demonstrated that naphthalene-resistant Clara cells, characterized by their expression of Clara cell secretory protein (CCSP), and PNE cells contribute to airway repair and associated hyperplasia of NEBs. This study was conducted to define the contribution of NEB-associated CCSP-expressing progenitor cells to PNE cell hyperplasia after Clara cell ablation. Transgenic (CCtk) mice were generated in which herpes simplex virus thymidine kinase was expressed within all CCSP-expressing cells of the conducting airway epithelium through the use of transcriptional regulatory elements from the mouse CCSP promoter. Chronic administration of ganciclovir (GCV) to CCtk transgenic mice resulted in selective ablation of CCSP-expressing cells within conducting airways. Proliferation and hyperplasia of PNE cells occurred in the absence of detectable proliferation among any other residual airway epithelial cell populations. These results demonstrate that PNE cells function as a self-renewing progenitor population and that NEB-associated Clara cells are not necessary for PNE cell hyperplasia.
Isabel Pintelon - One of the best experts on this subject based on the ideXlab platform.
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the pulmonary Neuroepithelial Body microenvironment a multifunctional unit in the airway epithelium
2021Co-Authors: Inge Brouns, Line Verckist, Isabel Pintelon, Jeanpierre Timmermans, Dirk AdriaensenAbstract:Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary Neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.
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selective activation and proliferation of a quiescent stem cell population in the Neuroepithelial Body microenvironment
Respiratory Research, 2018Co-Authors: Line Verckist, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Dirk AdriaensenAbstract:The microenvironment (ME) of Neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs. Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection. The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs. A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.
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selective gene expression analysis of the Neuroepithelial Body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
Respiratory Research, 2017Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Sofie Thys, Dirk AdriaensenAbstract:The pulmonary Neuroepithelial Body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.
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Selective gene expression analysis of the Neuroepithelial Body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
'Springer Science and Business Media LLC', 2017Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Sofie Thys, Dirk AdriaensenAbstract:Abstract Background The pulmonary Neuroepithelial Body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. Methods A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Results Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. Conclusions This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular
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selective activation and proliferation of a unique stem cell population in the Neuroepithelial Body microenvironment by transient acute lung injury
The FASEB Journal, 2016Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Carmen Rottiers, Dirk AdriaensenAbstract:The microenvironment (ME) of pulmonary Neuroepithelial bodies (NEBs) has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. Regardless the...
Jeanpierre Timmermans - One of the best experts on this subject based on the ideXlab platform.
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the pulmonary Neuroepithelial Body microenvironment a multifunctional unit in the airway epithelium
2021Co-Authors: Inge Brouns, Line Verckist, Isabel Pintelon, Jeanpierre Timmermans, Dirk AdriaensenAbstract:Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary Neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.
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selective activation and proliferation of a quiescent stem cell population in the Neuroepithelial Body microenvironment
Respiratory Research, 2018Co-Authors: Line Verckist, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Dirk AdriaensenAbstract:The microenvironment (ME) of Neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs. Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection. The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs. A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.
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selective gene expression analysis of the Neuroepithelial Body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
Respiratory Research, 2017Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Sofie Thys, Dirk AdriaensenAbstract:The pulmonary Neuroepithelial Body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.
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Selective gene expression analysis of the Neuroepithelial Body microenvironment in postnatal lungs with special interest for potential stem cell characteristics
'Springer Science and Business Media LLC', 2017Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Sofie Thys, Dirk AdriaensenAbstract:Abstract Background The pulmonary Neuroepithelial Body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. Methods A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Results Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. Conclusions This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular
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selective activation and proliferation of a unique stem cell population in the Neuroepithelial Body microenvironment by transient acute lung injury
The FASEB Journal, 2016Co-Authors: Line Verckist, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Carmen Rottiers, Dirk AdriaensenAbstract:The microenvironment (ME) of pulmonary Neuroepithelial bodies (NEBs) has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. Regardless the...