Differentiation Of Respiratory Epithelial Cells Research Articles

The Modulation of Respiratory Epithelial Cell Differentiation by the Thickness of an Electrospun Poly-ε-Carprolactone Mesh Mimicking the Basement Membrane.

The topology of the basement membrane (BM) affects cell physiology and pathology, and BM thickening is associated with various chronic lung diseases. In addition, the topology of commercially available poly (ethylene terephthalate) (PET) membranes, which are used in preclinical in vitro models, differs from that of the human BM, which has a fibrous and elastic structure. In this study, we verified the effect of BM thickness on the differentiation of normal human bronchial epithelial (NHBE) cells. To evaluate whether the thickness of poly-ε-carprolactone (PCL) mesh affects the differentiation of NHBE cells, cells were grown on thin- (6-layer) and thick-layer (80-layer) meshes consisting of electrospun PCL nanofibers using an air-liquid interface (ALI) cell culture system. It was found that the NHBE cells formed a normal pseudostratified epithelium composed of ciliated, goblet, and basal cells on the thin-layer PCL mesh; however, goblet cell hyperplasia was observed on the thick-layer PCL mesh. Differentiated NHBE cells cultured on the thick-layer PCL mesh also demonstrated increased epithelial-mesenchymal transition (EMT) compared to those cultured on the thin-layer PCL mesh. In addition, expression of Sox9, nuclear factor (NF)-κB, and oxidative stress-related markers, which are also associated with goblet cell hyperplasia, was increased in the differentiated NHBE cells cultured on the thick-layer PCL mesh. Thus, the use of thick electrospun PCL mesh led to NHBE cells differentiating into hyperplastic goblet cells via EMT and the oxidative stress-related signaling pathway. Therefore, the topology of the BM, for example, thickness, may affect the differentiation direction of human bronchial epithelial cells.

Decreased Expression of Pulmonary Homeobox NKX2.1 and Surfactant Protein C in Developing Lungs That Over-Express Receptors for Advanced Glycation End-Products (RAGE).

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobin superfamily prominently expressed by lung epithelium. Previous experiments demonstrated that over-expression of RAGE by murine alveolar epithelium throughout embryonic development causes neonatal lethality coincident with significant lung hypoplasia. In the current study, we evaluated the expression of NKX2.1 (also referred to as TTF-1), a homeodomain-containing transcription factor critical for branching morphogenesis, in mice that differentially expressed RAGE. We also contextualized NKX2.1 expression with the abundance of FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation and surfactant protein expression. Conditional RAGE over-expression was induced in mouse lung throughout gestation (embryonic day E0-18.5), as well as during the critical saccular period of development (E15.5-18.5), and analyses were conducted at E18.5. Histology revealed markedly less lung parenchyma beginning in the canalicular stage of lung development and continuing throughout the saccular period. We discovered consistently decreased expression of both NKX2.1 and FoxA2 in lungs from transgenic (TG) mice compared to littermate controls. We also observed diminished surfactant protein C in TG mice, suggesting possible hindered differentiation and/or proliferation of alveolar epithelial cells under the genetic control of these two critical transcription factors. These results demonstrate that RAGE must be specifically regulated during lung formation. Perturbation of epithelial cell differentiation culminating in respiratory distress and perinatal lethality may coincide with elevated RAGE expression in the lung parenchyma.

Establishing Bipotential Human Lung Organoid Culture System and Differentiation to Generate Mature Alveolar and Airway Organoids.

A robust in vitro model of the human respiratory epithelium, including the alveolar and the airway epithelium, is essential for understanding the biology and pathology of the human respiratory system. We previously described a protocol to derive human lung organoids from primary lung tissues. We now describe a protocol to induce bidirectional differentiation to generate mature alveolar or airway organoids. The lung organoids are consecutively expanded for over one year with high stability, while the differentiated alveolar and airway organoids morphologically and functionally simulate the human alveolar and airway epithelium to a near-physiological level. Thus, we establish a robust organoid culture system of the entire human respiratory epithelium, the first two-phase bipotential organoid culture system that enables long-term expansion and bidirectional differentiation of respiratory epithelial cells. The long-term expandable lung organoids and differentiated organoids generate a stable and renewable source of respiratory epithelial cells, enabling scientists to reconstruct and expand the human respiratory epithelium in culture dishes. The respiratory organoid system provides a unique and physiologically active in vitro model of the human respiratory epithelium for various applications, including studying respiratory viral infection, disease modeling, drug screening, and pre-clinical testing. Graphical abstract.

Embryonic Over‐Expression Of RAGE In Mouse Lung Modulates Thyroid Transcription Factor (TTF)‐1 And Surfactant Expression

Receptors for advanced glycation end‐products (RAGE) are multi‐ligand cell surface receptors of the immunoglobin superfamily prominently expressed by lung epithelium. Previous experiments demonstrate that over‐expression of RAGE by murine alveolar epithelium throughout embryonic development causes neonatal lethality coincident with significant lung hypoplasia. In the current study, we evaluated the expression of TTF‐1, a homeodomain‐containing transcription factor critical for branching morphogeneis, in mice that differentially expressed RAGE. We also contexualized TTF‐1 expression with the abundance of FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation, and surfactant proteins. Conditional RAGE over‐expression was induced in mice throughout gestation (embryonic day (E)0‐18.5) as well as during the critical saccular period (E15.5‐18.5) of development and analyses were conducted on E18.5 lung tissue. Histology revealed marked loss of lung tissue beginning in the canalicular stage of lung development and continuing throughout the saccular period. We discovered consistently decreased expression for both TTF‐1 and FoxA2 in lungs from TG mice compared to age‐matched controls. We also clarified diminished surfactant protein abundance in TG mice, suggesting possible hindered differentiation and/or proliferation of alveolar epithelial cells under the genetic control of these two critical transcription factors. These results demonstrate that RAGE must be specifically regulated during lung formation and that perturbation of epithelial cell differentiation culminating in respiratory distress and perinatal lethality may coincide with elevated RAGE expression in the lung parenchyma.

Chitosan-hyaluronan: promotion of mucociliary differentiation of respiratory epithelial cells and development of olfactory receptor neurons

Developing a biomaterial that promotes regeneration of both respiratory epithelium (RE) and olfactory neuroepithelium (ON) improves the surgical outcome of endoscopic sinus surgery. Although chitosan (CS) inhibits mucociliary differentiation of RE, it has been reported to regenerate ON. In addition, hyaluronic acid (HA) has been demonstrated to promote regeneration of RE. Whether the composite CS + HA would simultaneously benefit RE and ON remains unexplored. Human nasal respiratory epithelial cells (RECs) and olfactory neuroepithelial cells (ONCs) are respectively obtained from the RE and the ON. They are cultured in vitro and divided into groups undergoing four treatments, control, CS, HA, and CS + HA and assessed by scanning electron microscope, immunocytochemistry, and Western blots following indicated growth conditions. RECs keep polygonal morphology with mucociliary differentiation in the CS + HA group. The levels of E-cadherin, zonula occludens-1, mucin 5AC, and forkhead box protein J1 are significantly higher in the CS + HA group than in the CS alone group. In addition, ONCs express lower cytokeratin 18 (CK18) and higher olfactory marker protein (OMP) in the CS + HA group than in HA alone group. ONCs express more signal transduction apparatuses, adenylate cyclase 3, in CS and CS + HA groups than in HA and controls. Chitosan-hyaluronan plays a part in promoting differentiation of ORNs and facilitating mucociliary differentiation of RECs. This composite is a promising biomaterial for the sinonasal application.

Flexible Endoscopic Spray Application of Respiratory Epithelial Cells as Platform Technology to Apply Cells in Tubular Organs.

Introduction: Inoperable airway stenoses are currently treated by placing stents. A major problem of covered stents is missing mucociliary clearance, which is caused by covering the native respiratory epithelium. By coating a stent with respiratory epithelium, this problem can be overcome. However, no methods are available for efficient endoscopic cell seeding.Methods: We designed a flexible endoscopic spraying device based on a bronchoscope and tested it with respiratory epithelial cells. With this device cells can also be applied in a thin layer of fibrin glue. We evaluated the survival rate directly after spray application with a live-dead staining and the long-term differentiation capacity with histology and electron microscopy. Furthermore, the random distribution of cells when applied in a tube was analyzed and the macroscopic and microscopic characteristics of the endoscopic spray were investigated using high-speed visualization.Results: Spray visualization revealed a polydisperse character of the spray with the majority of droplets larger than epithelial cells. Spray application does not influence the survival rate and differentiation of respiratory epithelial cells. After 4 weeks, cells built up a pseudostratified epithelial layer with cilia and goblet cells. When cells are applied in a thin layer of fibrin gel into a tube, a nearest neighbor index of 1.2 is obtained, which suggests a random distribution of the cells.Conclusions: This spraying device is a promising tool for application of various cell types onto stents or implants with high survival rates and homogeneous distribution as shown in this study for ovine respiratory epithelial cells. The system could also be used for cell therapy to locally apply cells to the diseased parts of hollow organs. For the first time, the fluid dynamics of a spray device for cells were examined to validate in vitro results.

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold.

Receptors for Advanced Glycation End‐Products (RAGE) are Transcriptionally Inhibited by FoxA2 in Diverse Pulmonary Cell Types

The receptor for advanced glycation end‐products (RAGE) has been developmentally identified on the membranes of ATI cells and has been implicated in the spreading and adherence that characterize the transitioning of ATII cells to ATI cells. We have previously demonstrated that TTF‐1 and Egr‐1, transcriptional regulators that function during lung development, regulate RAGE transcription. However, the precise pattern of transcription factors that limit RAGE expression to pulmonary‐specific cell types remained unclear. In the current study, transcriptional regulation of RAGE was assessed by transfection of ATII‐like alveolar epithelium (A‐549), bronchiolar epithelium (Beas2B), and macrophages (RAW 264.7) with a luciferase reporter containing 0.9‐kb of the mouse RAGE promoter. In addition to the reporter construct, each cell type was transfected with FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation. In each case, FoxA2 repressed RAGE transcription in vitro. Mutagenesis of a proximal promoter region revealed that FoxA2 directly interacts with the coding sequence for RAGE. These data show that FoxA2 may negatively regulate RAGE expression by respiratory epithelium during pulmonary development. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Fibrin Gel as Alternative Scaffold for Respiratory Tissue Engineering

Fibrin gel has proven a valuable scaffold for tissue engineering. Complex geometries can be produced by injection molding; it offers effective cell seeding and can be produced autologous. In order to evaluate its suitability for respiratory tissue engineering, we examined proliferation, functionality, and differentiation of respiratory epithelial cells on fibrin gel in comparison to culture on collagen-coated, microporous membranes. Respiratory epithelial cells formed a confluent layer by day 4, and proliferation showed no significant difference with respect to surface. Measurement of the transepithelial electrical resistance reflected the development of a confluent epithelial cell layer and the subsequent initiation of adequate ion-transfer processes. Appearance of ciliae could be detected at similar time points, and ciliary beating could be observed for cells on both surfaces. Histology and immunohistochemistry of cells grown on fibrin gel revealed the onset of adequate differentiation. As no significant differences in respiratory epithelial cells' proliferation, function, and differentiation could be observed between cells grown on fibrin gel compared to cells on a collagen-coated, microporous surface, we concluded that fibrin gel might prove a suitable scaffold for respiratory tissue engineering and merits further investigation to overcome the limitations associated with scaffolds currently in use.

The balance between the expressions of hASH1 and HES1 differs between large cell neuroendocrine carcinoma and small cell carcinoma of the lung

To clarify the biological differences between small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), we investigated the expression of two bHLH type transcription factors, human achaete-scute homolog 1 (hASH1) and hairy/enhancer of split 1 (HES1), which positively and negatively regulate the neuroendocrine differentiation of respiratory epithelial cells, respectively. Eighty-eight formalin-fixed and paraffin-embedded pulmonary carcinomas (32 SCLC, 32 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas) and 14 SCLC and 1 LCNEC derived cell lines were used. hASH1 and HES1 mRNA were detected using a highly sensitive in situ hybridization method with digoxigenin-labeled cRNA probes and biotinylated tyramide. The staining results were scored from 0 to 12 by multiplying the staining intensity by the percentage of positive tumor cells. The mean staining score of hASH1 mRNA was significantly higher in SCLC than in LCNEC (p<0.01); conversely, that of HES1 mRNA was lower in SCLC than in LCNEC (p<0.01). These findings reveal that SCLC more strongly expresses the neuroendocrine phenotype, while LCNEC shows characteristics more similar to the ciliated epithelium phenotype, suggesting that the biological characteristics of these two tumors are different.

Ectopic respiratory epithelial cell differentiation in bronchiolised distal airspaces in idiopathic pulmonary fibrosis

BackgroundBronchiolisation of distal airspaces is an unexplained feature of idiopathic pulmonary fibrosis (IPF). The authors sought to identify mechanisms driving the differentiation of mucus cells during the bronchiolisation process.MethodsPathways governing...

Regulation of ciliary differentiation of human respiratory epithelial cells by the receptor for hyaluronan-mediated motility on hyaluronan-based biomaterials

Selecting a scaffold that facilitates ciliary differentiation of respiratory epithelial cells (RECs) is crucial in developing tissue engineered respiratory epithelium. Hyaluronan derivative membranes, consisting of an esterified form of hyaluronan (HYAFF ®), have been proved to promote ciliary differentiation of RECs in the presence of retinoic acid (RA). However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. In addition to investigating the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA, this study elucidates the role of the receptor for hyaluronan-mediated motility (RHAMM) in promoting ciliary differentiation of RECs. Analytical results of culturing RECs on collagen and HYAFF indicate that only HYAFF can increase the ciliary differentiation of RECs under RA-free conditions. The expression level of RHAMM mRNA of RECs more significantly decreases on collagen than that on HYAFF without RA. Therefore, by using lentiviral vector-based short hairpin RNA targeting RHAMM, the study further reveals that knockdown of RHAMM obviously inhibits the ciliary differentiation of RECs on collagen with RA and on HYAFF with/without RA. In addition to demonstrating that hyaluronan-based biomaterials partially “replace” RA in the ciliary differentiation of RECs, which is regulated by RHAMM, this study establishes that RHAMM regulates the ciliary differentiation-promoting effect of RA on RECs.

Increased mucociliary differentiation of human respiratory epithelial cells on hyaluronan-derivative membranes

The selection of a scaffold to facilitate mucociliary differentiation of respiratory epithelial cells (RECs) is crucial in the development of tissue engineering of respiratory epithelium. However, how the differentiation of RECs is influenced by the biomaterials has never been thoroughly explored. Previously, hyaluronan derivatives were considered as unsuitable biomaterials for culture of respiratory epithelium. In contrast, this study demonstrates that the membranous scaffolds made from benzyl esters of hyaluronic acids are capable of providing a more preferential environment for human RECs than conventionally used collagen-based scaffolds. The proliferation and mucociliary differentiation of RECs were examined by MTT assays, scanning electron microscopy, immunofluorescence, immunoblotting and gene expression. The percentage of ciliated cells in cultured RECs increased from 12.4% on collagen to 20.4% on hyaluronan-derivative membranes with a pseudostratified polarized layer that closely resembled the composition of the native epithelium. The expression levels of MUC5AC and MUC5B mRNA were higher on hyaluronan-based scaffolds than those on collagen. The presence of a hyaluronan-binding domain, CD44 and the receptor for hyaluronan-mediated motility of RECs were also demonstrated. Accordingly, the mucociliary differentiation-promoting effect of hyaluronan-derivative membranes indicates that it may be applied to the tissue engineering of respiratory epithelium.

GP130-STAT3 Regulates Epithelial Cell Migration and Is Required for Repair of the Bronchiolar Epithelium

Following injury, bronchiolar cells undergo rapid squamous metaplasia, followed by proliferation and re-establishment of the complex columnar epithelium that is characteristic of the normal airway. Mechanisms that regulate the repair of bronchiolar epithelium are of considerable relevance for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. This study was designed to identify the role of the GP130-STAT3 signaling pathway during repair of the bronchiolar epithelium. STAT3 (signal transducer and activator of transcription 3) and GP130 (glycoprotein 130) were each selectively deleted from the pulmonary epithelial cells of transgenic mice in vivo, producing Stat3(Delta/Delta) and Gp130(Delta/Delta) mice, respectively. Airway injury was induced in adult mice by administration of naphthalene, a toxicant of nonciliated respiratory epithelial cells (Clara cells). Nuclear STAT3 staining was induced in bronchiolar epithelial cells following naphthalene-mediated injury in control (Stat3(flox/flox)) mice. Whereas nearly complete repair of the bronchiolar epithelium was observed in control mice within 13 days, restoration of cell shape, cell density, and the pattern of ciliated and nonciliated cells did not occur in the peripheral bronchioles of either Stat3(Delta/Delta) or Gp130(Delta/Delta) mice. Expression of dominant-negative STAT3 inhibited airway epithelial cell migration during repair in vitro; wild-type STAT3 expression activated such migration. In the present study, we show that GP130-STAT3 signaling functions in a cell-autonomous manner to restore cell shape and numbers required for repair of the bronchiolar epithelium following injury.

Sox17 influences the differentiation of respiratory epithelial cells

The Sry-related HMG box transcription factor, Sox17, is required for formation of definitive endoderm that gives rise to various organs, including thyroid, lung, liver, pancreas, and intestine. While expressed at high levels in the embryonic endoderm, Sox17 is also expressed in mature tissues, including the lung. Sox17 expression in respiratory epithelial cells was first detected in the fetal lung at embryonic day 18. Thereafter, Sox17 expression was restricted primarily to ciliated cells, suggesting its potential role in airway cell differentiation. When expressed in epithelial cells of the embryonic lung, Sox17 inhibited peripheral epithelial cell differentiation and disrupted branching morphogenesis. In vitro, Sox17 inhibited Sftpc and enhanced Foxj1 promoter activity, consistent with its expression in proximal airway cells. Conditional expression of Sox17 in peripheral respiratory epithelial cells of adult lung induced hyperplastic clusters of cells expressing increased levels of β-catenin and differentiation markers representing multiple proximal respiratory epithelial cell types. Sox17 prolonged survival and enhanced growth and differentiation of respiratory epithelial cells in vitro. Sox17 induced plasticity of respiratory epithelial cells, reprogramming alveolar cells into epithelial cells with characteristics more typical of the proximal airway. Sites of expression and the effects of Sox17 in vivo and in vitro are consistent with a role for Sox17 or other members of the Sox family of transcription factors in differentiation of the conducting airway epithelium.

A Possible Therapy for Smoke‐Induced Lung Injuries by Inhalation of All‐Trans Retinoic Acid in a Guinea Pig Model System

Smoking is known to be linked with various cardiovascular and pulmonary diseases. Cigarette smoke (CS) not only affects smokers but also contributes to the health problems of non-smokers. We have recently described a clinical relevant model system for mainstream (MS) and sidestream (SS) CS exposure in guinea pigs. Retinoic acid (RA) is necessary for the maintenance of respiratory epithelial cell differentiation in vivo and thus have prospects as preventive agents for some forms of cancer. Systemic applications of retinoids as therapy of smoke-induced lung injuries carry the risks of toxic side effects and may not accumulate in the target tissue. Therefore, we adapted a PARI LC Star nebulizer (Pari, Richmond, VA) to a tubical system, which allows the inhalation treatment of six guinea pigs at the same time. As a bolus, we applied a water-miscible formulation, consisting of Cremophor EL (ethoxylated castor oil), all-trans retinoic acid, butylated hydroxyl toluene (BHT) in PBS or without the retinoid as vehicle. Over a period of 5 weeks, 3 mL of this solution was nebulized for 20 minutes once a day. HPLC analysis of lung samples of sacrificed animals showed an increase in 13-cis retinoic acid of 23.85% (control) and 31.81% in MS animals. Other RA isomers could not be detected. (Supported by DAMD17-03-1-0352 & DAMD17-03-2-0054 grants)

Stage-specific regulation of respiratory epithelial cell differentiation by Foxa1

Foxa1 is a member of the winged helix family of transcription factors that is expressed in epithelial cells of the conducting airways and in alveolar type II cells of the lung. To determine the role of Foxa1 during lung morphogenesis, histology and gene expression were assessed in lungs from Foxa1-/- gene-targeted mice from embryonic day (E) 16.5 to postnatal day (PN) 13. Deletion of Foxa1 perturbed maturation of the respiratory epithelium at precise times during lung morphogenesis. While dilatation of peripheral lung saccules was delayed in Foxa1-/- mice at E16.5, sacculation was unperturbed later in development (E17.5-E18.5). At PN5, alveolarization was markedly delayed in Foxa1-/- mice; however, by PN13 lung histology was comparable to wild-type controls. Clara cell secretory protein (CCSP), prosurfactant protein (SP)-C, and SP-B protein content and immunostaining were decreased in Foxa1-/- mice between E16.5 and E18.5 but normalized after birth. Timing and sites of expression of thyroid transcription factor-1, Foxj1, and beta-tubulin were unaltered in lungs of Foxa1-/- mice. In vitro, Foxa1 regulated the activity of CCSP and SP-A, SP-B, SP-C, and SP-D promoters as assessed by luciferase reporter assays in HeLa, H441, and MLE15 cells. Although Foxa1 regulates respiratory epithelial differentiation and structural maturation of the lung at precise developmental periods, the delay in maturation is subsequently compensated at times to enable respiratory function and restore normal lung structure after birth.

β-Catenin regulates differentiation of respiratory epithelial cells in vivo

An activated form of beta-catenin [Catnb(Delta(ex3))] was expressed in respiratory epithelial cells of the developing lung. Although morphogenesis was not altered at birth, air space enlargement and epithelial cell dysplasia were observed in the early postnatal period and persisted into adulthood. The Catnb(Delta(ex3)) protein caused squamous, cuboidal, and goblet cell dysplasia in intrapulmonary conducting airways. Atypical epithelial cells that stained for surfactant pro protein C (pro-SP-C) and had morphological characteristics of alveolar type II cells were observed in bronchioles of the transgenic mice. Catnb(Delta(ex3)) inhibited expression of Foxa2 and caused goblet cell hyperplasia associated with increased staining for mucins and the MUC5A/C protein. In vitro, both wild type and activated beta-catenin negatively regulated the expression of the Foxa2 promoter. Catnb(Delta(ex3)) also caused pulmonary tumors in adult mice. Activation of beta-catenin caused ectopic differentiation of alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, demonstrating a critical role for the Wnt/beta-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung.

Inhibition of nonviral cationic liposome-mediated gene transfer into primary human respiratory cells by interferon-gamma.

The effect of interferon (IFN) gamma on cationic liposome-mediated gene transfer into primary respiratory epithelial cells was investigated. Treatment of primary respiratory epithelial cells with IFN-gamma resulted in a dose-dependent increase in the intermediate filament cytokeratin 13 and a decrease in cellular proliferation, indicating that respiratory cells underwent squamous differentiation. IFN-gamma pretreatment resulted in a dramatic inhibition of transfection efficiency mediated by a cationic liposome (DOTAP). Incubation of squamous nasal cells with DOTAP/DNA complexes for various periods at 4 degrees C and evaluation of luciferase levels suggested that IFN-gamma pretreatment inhibits complex binding to the cells. In primary nasal and bronchial cells cytofluorimetric analysis demonstrated that IFN-gamma reduces binding of FITC-labeled complexes. The data indicate that differentiation of respiratory epithelial cells to a squamous phenotype, which may occur in chronic respiratory diseases such as cystic fibrosis, induces a refractory condition to gene transfer by nonviral cationic liposomes.

Conditional Expression of Fibroblast Growth Factor-7 in the Developing and Mature Lung

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)(7)-cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta x (Teto)(7)-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.