19 research outputs found
Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium
Background: Mechanisms of airway repair are poorly understood. It has been proposed that, following injury,
progenitor populations such as club cells (Clara) become undifferentiated, proliferate and re-differentiate to
re-epithelialise the airway. The exact phenotype of such cells during repair is unknown however. We hypothesised
that airway epithelial cells (AECs) undergo some degree of epithelial-mesenchymal transition (EMT) in order to
migrate over a denuded airway and effect re-epithelialisation. Furthermore, based on our previous findings that
BMP signalling is an early event in AECs following injury in vivo and that BMP4 down-regulates E-cadherin
expression and enhances migration in AECs in vitro, we hypothesised that BMPs could play a role in inducing such
a phenotypic switch.
Methods: Normal AECs were isolated from mouse lungs and analysed in a model of a disrupted epithelium. EMT
marker expression and BMP signalling were examined by immunofluorescence, Western blotting and RT-PCR.
Results: Following generation of a wound area, AECs at the wound edge migrated and acquired a mesenchymallike
morphology. E-cadherin expression was reduced in migrating cells while vimentin and α-smooth muscle actin
(α-SMA) expression was increased. Re-expression of membrane E-cadherin was subsequently observed in some cells
in the wound area following re-establishment of the monolayer. A transient increase in the incidence of nuclear
phosphorylated Smad1/5/8 was observed in migrating cells compared with confluent cells, indicating active BMP
signalling during migration. BMP antagonists noggin and gremlin inhibited cell migration, confirming the
involvement of BMP signalling in migration and indicating autocrine signalling, possibly involving BMP7 or BMP4
which were expressed in AECs. Exogenous BMP2, BMP4 and BMP7 induced a mesenchymal-like morphology in
AECs, enhanced the rate of cell migration and increased α-SMA protein expression in AECs.
Conclusions: Following disruption of an intact epithelium, migrating AECs at the wound edge acquire an EMT-like
phenotype involving altered expression of E-cadherin, vimentin and α-SMA. BMP signalling is involved in AEC
migration and is likely to mediate the switch towards an EMT-like phenotype by altering protein expression to
facilitate cell migration and wound closure. We propose therefore that acquisition of an EMT-like phenotype by
AECs is a normal aspect of wound repair. Furthermore, we suggest that diseases involving fibrosis may arise
because the EMT phase of repair is prolonged by chronic injury/inflammation, rather than being caused by it, as is
the current paradigm
Bone morphogenetic protein signalling in airway epithelial cells during regeneration
Mechanisms of lung regeneration after injury remain poorly understood. Bone morphogenetic protein 4
(BMP4) is critical for lung morphogenesis and regulates differentiation of the airway epithelium during
development, although its mechanism of action is unknown. The role of BMPs in adult lungs is unclear. We
hypothesised that BMP signalling is involved in regeneration of damaged adult airways after injury. Our aims
were to characterise the regeneration process in 1-nitronaphthalene (1-NN) injured airways, to determine if
and when BMP signalling is activated during this process and investigate the effects of BMP4 on normal adult
airway epithelial cells (AECs). Rats were injected with 50 mg/kg 1-NN and protein expression in AECs was
examined by Western blotting of lung lysis lavage, and by immunofluorescence, at 6, 24, 48 and 96 h post
injection. Expression of signalling molecules p-ERK-1, p-ERK-2 and p-Smad1/5/8 in AECs peaked at 6 h post
injection, coincident with maximal inflammation and prior to airway denudation which occurred at 24 h.
While airways were re-epithelialised by 48 h, AEC proliferation peaked later at 96 h post 1-NN injection. In
vitro, BMP4 induced a mesenchymal-like morphology in normal AECs, downregulated E-cadherin expression
and increased migration in a wound closure assay. Thus, following acute injury, increased BMP signalling in
AECs coincides with inflammation and precedes airway denudation and re-epithelialisation. Our data indicate
that, similar to its role in controlling tissue architecture during development, BMP signalling regulates
regeneration of the airways following acute injury, involving downregulation of E-cadherin and induction of
migration in AEC
Delivery of nucleic acids to ex vivo porcine airways using electrospray
Aim of the Study: Nucleic acid-based therapies have the potential to provide clinically meaningful benefit across a wide spectrum of lung disease. However, in vivo delivery remains a challenge. Here we examined the feasibility of using electrospray to deliver nucleic acids to both porcine tracheal tissue sections and whole lung ex vivo. Materials and Methods: The effect of electrospray solution, emitter gauge, flow rate and voltage on plasmid DNA integrity was examined by analyzing supercoiled:open circle structure ratio by gel electrophoresis. Optimal parameters were used to deliver luciferase DNA and mRNA and siRNA-FITC to tracheal tissue sections. Luciferase mRNA was delivered to whole porcine lungs ex vivo using a catheter and bronchoscope system. Luciferase activity and fluorescence were analyzed by luminometry and microscopy respectively. Results: The incidence of DNA plasmid nicking was greatest in a low salt solution without ethanol compared with 1% and 20% ethanol with salt. From a range of emitters tested, a 32 gauge emitter produced the best supercoiled:open circle structure ratio, likely because less voltage was required to produce a stable electrospray with this emitter. Lower flow rates also showed a trend towards reduced DNA nicking. GFP DNA electrosprayed at 5 kV and 6 kV resulted in lower levels of GFP expression in A549 lung cells following lipofection compared with 3 kV and 4 kV. Optimised parameters of 20% ethanol solution, 32 gauge emitter, low flow rates and voltages of 3–5 kV, nucleic acid molecules were successful for delivery of luciferase DNA and mRNA as well as siRNA-FITC to porcine tracheal tissue sections and for delivery of luciferase mRNA to whole porcine lungs via bronchoscope. Conclusions: We report ex vivo delivery of nucleic acids to porcine lung tissue via electrospray and bronchoscopic electrospray delivery of nucleic acid to an ex vivo porcine lung model
Anatomy and Bronchoscopy of the Porcine Lung. A Model for Translational Respiratory Medicine
The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model
BMP4 induces an epithelial–mesenchymal transition-like response in adult airway epithelial cells
Bone morphogenetic proteins (BMPs) are critical morphogens and play key roles in epithelial–mesenchymal transitions
(EMTs) during embryogenesis. BMP4 is required for early mesoderm formation and also regulates morphogenesis and
epithelial cell differentiation in developing lungs. While, BMP signalling pathways are activated during lung inflammation in
adult mice, the role of BMPs in adult lungs remains unclear.We hypothesised that BMPs are involved in remodelling processes
in adult lungs and investigated effects of BMP4 on airway epithelial cells. BEAS-2B cell growth decreased in the presence of
BMP4. Cells acquired a mesenchymal-like morphology with downregulation of adherens junction proteins and increased cell
motility. Changes in extracellular matrix-related gene expression occurred with BMP4 treatment including upregulation of
collagens, fibronectin and tenascin C.We conclude that the activity of BMP4 in EMT during development is recapitulated in
adult airway epithelial cells and suggest that this activity may contribute to inflammation and fibrosis in vivo
Vector-free intracellular delivery by reversible permeabilization
Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent. Reversal of cell permeabilization is achieved by temporally and volumetrically controlling the contact of the target cells with this solution. Cells are seeded in conventional multi-well plates. Following removal of the supernatant, the cargo is mixed with the delivery solution and applied directly to the cells using an atomizer. After a short incubation period, permeabilization is halted by incubating the cells in a phosphate buffer saline solution that dilutes the ethanol and is non-toxic to the permeabilized cells. Normal culture medium is then added. The procedure lasts less than 5 min. With this method, proteins, mRNA, plasmid DNA and other molecules have been delivered to a variety of cell types, including primary cells, with low toxicity and cargo functionality has been confirmed in proof-of-principle studies. Co-delivery of different cargo types has also been demonstrated. Importantly, delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner. Unlike some other vector-free methods, adherent cells are addressed in situ without the need for detachment from their substratum. The method has also been adapted to address suspension cells. This delivery method is gentle yet highly reproducible, compatible with high throughput and automated cell-based assays and has the potential to enable a broad range of research, drug discovery and clinical applications
SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins
Bone morphogenetic protein (BMP) signaling is important for
correct lung morphogenesis, and there is evidence of BMP signaling
reactivation in lung diseases. However, little is known about BMP
signaling patterns in healthy airway homeostasis and inflammatory
airway disease and during epithelial repair. In this study, a rhesus
macaque (Macaca mulatta) model of allergic airway disease was used
to investigate BMP signaling throughout the airways in health,
disease, and regeneration. Stereologic quantification of
immunofluorescent images was used to determine the expression of
BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD)
1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient
was found along the airways of healthy juvenile rhesus macaques
(n = 3, P , 0.005). Membrane-localized BMPRIa expression was also
present in the epithelium of the healthy animals. After exposure to
house dust mite allergen and ozone, significant down-regulation of
nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals
were provided with a recovery period in filtered air, proliferating cell
nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa
expression were significantly increased in the epithelium of
conducting airways (P , 0.005). Furthermore, in the asthmatic
airways, altered BMPRIa localization was evident. Because of the
elevated eosinophil presence in these airways, we investigated the
effect of eosinophil-derived proteins on BMPRIa trafficking in
epithelial cells. Eosinophil-derived proteins (eosinophil-derived
neurotoxin, eosinophil peroxidase, and major basic protein) induced
transient nuclear translocation of membrane-bound BMPRIa. This
work mapping SMAD signaling in the airways of nonhuman
primates highlights a potential mechanistic relationship between
inflammatory mediators and BMP signaling and provides evidence
that basal expression of the BMP signaling pathway may be
important for maintaining healthy airways