A low rate of cell proliferation and reduced DNA uptake limit cationic lipid-mediated gene transfer to primary cultures of ciliated human airway epithelia

ABSTRACT The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FRα), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FRα on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FRα-blocking antibodies, we demonstrated FRα-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FRα was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

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Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00377.2003 ◽

2004 ◽

Vol 287 (2) ◽

pp. L428-L437 ◽

Cited By ~ 96

Author(s):

Hong Peng Jia ◽

Joel N. Kline ◽

Andrea Penisten ◽

Michael A. Apicella ◽

Theresa L. Gioannini ◽

...

Keyword(s):

Primary Cultures ◽

Airway Epithelial Cells ◽

Response To Treatment ◽

Airway Epithelial ◽

Airway Epithelia ◽

Human Airway ◽

Tnf Α ◽

Pulmonary Macrophages ◽

Well Differentiated ◽

Human Airway Epithelia

The expression of inducible antimicrobial peptides, such as human β-defensin-2 (HBD-2) by epithelia, comprises a component of innate pulmonary defenses. We hypothesized that HBD-2 induction in airway epithelia is linked to pattern recognition receptors such as the Toll-like receptors (TLRs). We found that primary cultures of well-differentiated human airway epithelia express the mRNA for TLR-4, but little or no MD-2 mRNA, and display little HBD-2 expression in response to treatment with purified endotoxin ± LPS binding protein (LBP) and soluble CD14. Expression of endogenous MD-2 by transduction of airway epithelial cells with an adenoviral vector encoding MD-2 or extracellular addition of recombinant MD-2 both increased the responses of airway epithelia to endotoxin + LBP and sCD14 by >100-fold, as measured by NF-κB-luciferase activity and HBD-2 mRNA expression. MD-2 mRNA could be induced in airway epithelia by exposure of these cells to specific bacterial or host products (e.g., killed Haemophilus influenzae, the P6 outer membrane protein from H. influenzae, or TNF-α + IFN-γ). These findings suggest that MD-2, either coexpressed with TLR-4 or secreted when produced in excess of TLR-4 from neighboring cells, is required for airway epithelia to respond sensitively to endotoxin. The regulation of MD-2 expression in airway epithelia and pulmonary macrophages may serve as a means to modify endotoxin responsiveness in the airway.

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Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia

Journal of Clinical Investigation ◽

10.1172/jci8858 ◽

2000 ◽

Vol 105 (5) ◽

pp. 589-596 ◽

Cited By ~ 52

Author(s):

Paola T. Drapkin ◽

Catherine R. O’Riordan ◽

Su Min Yi ◽

John A. Chiorini ◽

Jonathan Cardella ◽

...

Keyword(s):

Gene Transfer ◽

Plasminogen Activator ◽

Urokinase Plasminogen Activator ◽

Urokinase Plasminogen Activator Receptor ◽

Airway Epithelia ◽

Human Airway ◽

Plasminogen Activator Receptor ◽

Human Airway Epithelia

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KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response

Physiological Genomics ◽

10.1152/physiolgenomics.2001.6.2.81 ◽

2001 ◽

Vol 6 (2) ◽

pp. 81-89 ◽

Cited By ~ 23

Author(s):

LAWRENCE S. PRINCE ◽

PHILIP H. KARP ◽

THOMAS O. MONINGER ◽

MICHAEL J. WELSH

Keyword(s):

Keratinocyte Growth Factor ◽

Primary Cultures ◽

Airway Epithelia ◽

Altered Expression ◽

Human Airway ◽

Gene Transcripts ◽

Induced Genes ◽

Well Differentiated ◽

Cf Transmembrane Conductance Regulator ◽

Human Airway Epithelia

Keratinocyte growth factor (KGF) regulates several functions in adult and developing lung epithelia; it causes proliferation, stimulates secretion of fluid and electrolytes, enhances repair, and may minimize injury. To gain insight into the molecular processes influenced by KGF, we applied KGF to primary cultures of well-differentiated human airway epithelia and used microarray hybridization to assess the abundance of gene transcripts. Of 7,069 genes tested, KGF changed expression levels of 910. Earlier studies showed that KGF causes epithelial proliferation, and as expected, treatment altered expression of numerous genes involved in cell proliferation. We found that KGF stimulated transepithelial Cl−transport, but the number of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) transcripts fell. Although transcripts for ClC-1 and ClC-7 Cl−channels increased, KGF failed to augment transepithelial Cl−transport in CF epithelia, suggesting that KGF-stimulated Cl−transport in differentiated airway epithelia depends on the CFTR Cl−channel. Interestingly, KGF decreased transcripts for many interferon (IFN)-induced genes. IFN causes trafficking of Stat dimers to the nucleus, where they activate transcription of IFN-induced genes. We found that KGF prevented the IFN-stimulated trafficking of Stat1 from the cytosol to the nucleus, suggesting a molecular mechanism for KGF-mediated suppression of the IFN-signaling pathway. These results suggest that in addition to stimulating proliferation and repair of damaged airway epithelia, KGF stimulates Cl−transport and may dampen the response of epithelial cells to inflammatory mediators.

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Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer

Journal of Virology ◽

10.1128/jvi.74.8.3852-3858.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3852-3858 ◽

Cited By ~ 238

Author(s):

Joseph Zabner ◽

Michael Seiler ◽

Robert Walters ◽

Robert M. Kotin ◽

Wendy Fulgeras ◽

...

Keyword(s):

Gene Transfer ◽

Virus Type ◽

Target Cells ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway ◽

Human Airway Epithelia

ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

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Basolateral chloride current in human airway epithelia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00077.2007 ◽

2007 ◽

Vol 293 (4) ◽

pp. L991-L999 ◽

Cited By ~ 9

Author(s):

Omar A. Itani ◽

Fred S. Lamb ◽

James E. Melvin ◽

Michael J. Welsh

Keyword(s):

Cystic Fibrosis ◽

Signaling Pathways ◽

Basolateral Membrane ◽

Primary Cultures ◽

Cell Swelling ◽

Disulfonic Acid ◽

Airway Epithelia ◽

Human Airway ◽

Well Differentiated ◽

Human Airway Epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl− absorption. However, the pathways for basolateral Cl− exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl− conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl− conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl− current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl− > Br− >> I−. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca2+ and inducing cell swelling had no effect. The basolateral Cl− current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl− currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl− conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl− conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl− movement.

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An In Vitro Model of Differentiated Human Airway Epithelia: Methods for Establishing Primary Cultures

Epithelial Cell Culture Protocols ◽

10.1385/1-59259-185-x:115 ◽

2003 ◽

pp. 115-137 ◽

Cited By ~ 74

Author(s):

Philip H. Karp ◽

Thomas O. Moninger ◽

S. Pary Weber ◽

Tamara S. Nesselhauf ◽

Janice L. Launspach ◽

...

Keyword(s):

In Vitro Model ◽

Primary Cultures ◽

Airway Epithelia ◽

Human Airway ◽

Vitro Model ◽

Human Airway Epithelia

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1075. Goblet Cell Hyperplasia Inhibits Adenovirus but Not AAV5 Mediated Gene Transfer to Differentiated Human Airway Epithelia

Molecular Therapy ◽

10.1016/s1525-0016(16)43905-5 ◽

2002 ◽

Vol 5 (5) ◽

pp. S349

Keyword(s):

Gene Transfer ◽

Goblet Cell ◽

Cell Hyperplasia ◽

Airway Epithelia ◽

Goblet Cell Hyperplasia ◽

Human Airway ◽

Human Airway Epithelia

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The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00256.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. L25-L31 ◽

Cited By ~ 168

Author(s):

Alejandro A. Pezzulo ◽

Timothy D. Starner ◽

Todd E. Scheetz ◽

Geri L. Traver ◽

Ann E. Tilley ◽

...

Keyword(s):

Expression Profile ◽

Primary Cultures ◽

Liquid Interface ◽

Transcriptional Profile ◽

Surface Epithelium ◽

Airway Epithelia ◽

Human Airway ◽

Air Liquid Interface ◽

Human Airway Epithelia

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

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A Chimeric Type 2 Adenovirus Vector with a Type 17 Fiber Enhances Gene Transfer to Human Airway Epithelia

Journal of Virology ◽

10.1128/jvi.73.10.8689-8695.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8689-8695 ◽

Cited By ~ 65

Author(s):

Joseph Zabner ◽

Miguel Chillon ◽

Teresa Grunst ◽

Thomas O. Moninger ◽

Beverly L. Davidson ◽

...

Keyword(s):

Cystic Fibrosis ◽

Gene Transfer ◽

Recombinant Adenovirus ◽

Adenovirus Vector ◽

Adenovirus Type ◽

Wild Type ◽

Airway Epithelia ◽

Human Airway ◽

Human Airway Epithelia

ABSTRACT In studies of the genetic disease cystic fibrosis, recombinant adenovirus type 2 (Ad2) and Ad5 are being investigated as vectors to transfer cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia. However, earlier work has shown that human airway epithelia are resistant to infection by Ad2 and Ad5. Therefore, we examined the efficiency of other adenovirus serotypes at infecting airway epithelia. We found that several serotypes of adenoviruses, in particular, wild-type Ad17, infected a greater number of cells than wild-type Ad2. The increased efficiency of wild-type Ad17 could be explained by increased fiber-dependent binding to the epithelia. Therefore, we constructed a chimeric virus, Ad2(17f)/βGal-2, which is identical to Ad2/βGal-2 with the exception of having the fiber protein of Ad17 replace Ad2 fiber. This vector retained the increased binding and efficiency of gene transfer to well-differentiated human airway epithelia. These data suggest that inclusion of Ad17 fiber into adenovirus vectors may improve the outlook for gene delivery to human airway epithelia.

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