Murine Tracheal and Nasal Septal Epithelium for Air–Liquid Interface Cultures: A Comparative Study

Woodworth, Bradford A.; Antunes, Marcelo B.; Bhargave, Geeta; Palmer, James N.; Cohen, Noam A.

doi:10.2500/ajr.2007.21.3068

Cited by 59 publications

(65 citation statements)

References 8 publications

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“…Primary lung epithelial cells were isolated from approximately 3-to 4-week-old mice and cultured as previously described (67,68). Briefly, lungs were digested in HBSS containing 10 mg/ml type II collagenase and 20 μg/ml DNase I at 37°C for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

Baek¹,

Bhang²,

Zaslavsky³

et al. 2013

J. Clin. Invest.

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Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1 -/-background. We found that oncogenic Kras-induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.

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Section: Methodsmentioning

confidence: 99%

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

Baek¹,

Bhang²,

Zaslavsky³

et al. 2013

J. Clin. Invest.

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“…The volume of fluid on apical surface of murine nasal epithelial cells depends on the balance between Na 1 reabsorption (mainly through ENaCs) and Cl 2 secretion (mainly through CFTR) (22 6C). These data indicated that IaI decreased ENaCs but not CFTR (or at least it decreased ENaCs to a much higher extent than CFTR), in agreement with our I sc measurements across ATII cell monolayers ( Figure 3).…”

Section: Iai Increased the Airway Surface Liquid Depth Overlying Primmentioning

confidence: 99%

“…Confluent monolayers of rat ATII cells (17) or murine (C57BL/6) nasal epithelial cells (22) were incubated with IaI (0.1 mg/ml) in 100 ml of Normal Ringers added on their apical side for 2 or 4 hours. Cells were mounted in Ussing chambers, and shortcircuit currents (I sc ) and transepithelial resistances were measured before and after addition of amiloride (10 mM), forskolin (10 mM), and glibenclamide (400 mM) in the apical compartments (21,23,24).…”

Section: Short-circuit Currents Across Atii Cellsmentioning

confidence: 99%

Inter-α-Inhibitor Blocks Epithelial Sodium Channel Activation and Decreases Nasal Potential Differences in ΔF508 Mice

Lazrak

Jurkuvenaite

Ness

et al. 2014

Am J Respir Cell Mol Biol

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Increased activity of lung epithelial sodium channels (ENaCs) contributes to the pathophysiology of cystic fibrosis (CF) by increasing the rate of epithelial lining fluid reabsorption. Intera-inhibitor (IaI), a serum protease inhibitor, may decrease ENaC activity by preventing its cleavage by serine proteases. High concentrations of IaI were detected in the bronchoalveolar lavage fluid (BALF) of children with CF and lower airway diseases. IaI decreased amiloride-sensitive (I ENaC ) but not cAMP-activated Cl 2 currents across confluent monolayers of rat ATII, and mouse nasal epithelial cells grew in primary culture by 45 and 25%, respectively. Changes in I ENaC by IaI in ATII cells were accompanied by increased levels of uncleaved (immature) surface a-ENaC. IaI increased airway surface liquid depth overlying murine nasal epithelial cells to the same extent as amiloride, consistent with ENaC inhibition. Incubation of lung slices from C57BL/6, those lacking phenylalanine at position 508 (ΔF508), or CF transmembrane conductance regulator knockout mice with IaI for 3 hours decreased the open probability of their ENaC channels by 50%. ΔF508 mice had considerably higher levels the amiloride-sensitive fractions of ENaC nasal potential difference (ENaC-NPD) than wild-type littermates and only background levels of IaI in their BALF. A single intranasal instillation of IaI decreased their ENaC-NPD 24 hours later by 25%. In conclusion, we show that IaI is present in the BALF of children with CF, is an effective inhibitor of ENaC proteolysis, and decreases ENaC activity in lung epithelial cells of ΔF508 mice.

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“…Primary cultures of airway epithelia have emerged as a powerful model to study epithelial cell biology and the impact of diseases on tissue function (18,22,39). Cells grown at the air-liquid interface (ALI) form a polarized, pseudostratified columnar epithelium that closely resembles the morphology of the in vivo surface epithelium of the conducting airways (2,18,24,33,63,64), providing an opportunity to study cell biology, disease pathogenesis, and treatments (66).…”

mentioning

confidence: 99%

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Ramachandran

Krishnamurthy

Jacobi

et al. 2013

American Journal of Physiology-Lung Cellular and Molecular Phys

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Ramachandran S, Krishnamurthy S, Jacobi AM, WohlfordLenane C, Behlke MA, Davidson BL, McCray PB, Jr. Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro. Am J Physiol Lung Cell Mol Physiol 305: L23-L32, 2013. First published April 26, 2013 doi:10.1152/ajplung.00426.2012.-Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-totransfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl Ϫ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses. primary airway epithelium; transfection; gene silencing; RNA interference; siRNA; air-liquid interface; porcine airway epithelial cells SMALL INTERFERING RNAs (siRNA) mediate posttranscriptional inhibition of targeted genes, offering a novel approach to achieve specific silencing in the airway epithelium. These approaches have broad applications for studies of cell biology, disease pathogenesis, and therapeutics. Primary cultures of airway epithelia have emerged as a powerful model to study epithelial cell biology and the impact of diseases on tissue function (18,22,39). Cells grown at the air-liquid interface (ALI) form a polarized, pseudostratified columnar epithelium that closely resembles the morphology of the in vivo surface epithelium of the conducting airways (2,18,24,33,63,64), providing an opportunity to study cell biology, disease pathogenesis, and treatments (66).Histological studies have shown that ALI cultures of primary airways grown for 4 wk or more form a well-differentiated epithelium resembling the in vivo architecture (12, 56). However, another study has observed that airway epithelia...

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Murine Tracheal and Nasal Septal Epithelium for Air–Liquid Interface Cultures: A Comparative Study

Cited by 59 publications

References 8 publications

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

Inter-α-Inhibitor Blocks Epithelial Sodium Channel Activation and Decreases Nasal Potential Differences in ΔF508 Mice

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

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