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.

Mig-6 deficiency cooperates with oncogenic Kras to promote mouse lung tumorigenesis.

OBJECTIVES: Lung cancer is the leading cause of cancer related deaths worldwide and mutation activating KRAS is one of the most frequent mutations found in lung adenocarcinoma. Identifying regulators of KRAS may aid in the development of therapies to treat this disease. The mitogen-induced gene 6, MIG-6, is a small adaptor protein modulating signaling in cells to regulate the growth and differentiation in multiple tissues. Here, we investigated the role of Mig-6 in regulating adenocarcinoma progression in the lungs of genetically engineered mice with activation of Kras. MATERIALS AND METHODS: Using the CCSPCre mouse to specifically activate expression of the oncogenic KrasG12D in Club cells, we investigated the expression of Mig-6 in CCSPCreKrasG12D-induced lung tumors. To determine the role of Mig-6 in KrasG12D-induced lung tumorigenesis, Mig-6 was conditionally ablated in the Club cells by breeding Mig6f/f mice to CCSPCreKrasG12D mice, yielding CCSPCreMig-6d/dKrasG12D mice (Mig-6d/dKrasG12D). RESULTS: We found that Mig-6 expression is decreased in CCSPCreKrasG12D-induced lung tumors. Ablation of Mig-6 in the KrasG12D background led to enhanced tumorigenesis and reduced life expectancy. During tumor progression, there was increased airway hyperplasia, a heightened inflammatory response, reduced apoptosis in KrasG12D mouse lungs, and an increase of total and phosphorylated ERBB4 protein levels. Mechanistically, Mig-6 deficiency attenuates the cell apoptosis of lung tumor expressing KRASG12D partially through activating the ErbB4 pathway. CONCLUSIONS: In summary, Mig-6 deficiency promotes the development of KrasG12D-induced lung adenoma through reducing the cell apoptosis in KrasG12D mouse lungs partially by activating the ErbB4 pathway.

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.

Respiratory Tissue Engineering: Current Status and Opportunities for the Future.

Currently, lung disease and major airway trauma constitute a major global healthcare burden with limited treatment options. Airway diseases such as chronic obstructive pulmonary disease and cystic fibrosis have been identified as the fifth highest cause of mortality worldwide and are estimated to rise to fourth place by 2030. Alternate approaches and therapeutic modalities are urgently needed to improve clinical outcomes for chronic lung disease. This can be achieved through tissue engineering of the respiratory tract. Interest is growing in the use of airway tissue-engineered constructs as both a research tool, to further our understanding of airway pathology, validate new drugs, and pave the way for novel drug therapies, and also as regenerative medical devices or as an alternative to transplant tissue. This review provides a concise summary of the field of respiratory tissue engineering to date. An initial overview of airway anatomy and physiology is given, followed by a description of the stem cell populations and signaling processes involved in parenchymal healing and tissue repair. We then focus on the different biomaterials and tissue-engineered systems employed in upper and lower respiratory tract engineering and give a final perspective of the opportunities and challenges facing the field of respiratory tissue engineering.

Aldehyde dehydrogenase activity enriches for proximal airway basal stem cells and promotes their proliferation.

Both basal and submucosal gland (SMG) duct stem cells of the airway epithelium are capable of sphere formation in the in vitro sphere assay, although the efficiency at which this occurs is very low. We sought to improve this efficiency of sphere formation by identifying subpopulations of airway basal stem cells (ABSC) and SMG duct cells based on their aldehyde dehydrogenase (ALDH) activity. ALDH(hi) ABSCs and SMG duct cells were highly enriched for the population of cells that could make spheres, while the co-culture of ALDH(hi) differentiated cells with the ALDH(hi) ABSCs increased their sphere-forming efficiency. Specific ALDH agonists and antagonists were used to show that airway specific ALDH isozymes are important for ABSC proliferation. Pathway analysis of gene expression profiling of ALDH(hi) and ALDH(lo) ABSCs revealed a significant upregulation of the arachidonic acid (AA) metabolism pathway in ALDH(hi) ABSCs. We confirmed the importance of this pathway in the metabolism of proliferating ALDH(hi) ABSCs using bioenergetics studies as well as agonists and antagonists of the AA pathway. These studies could lead to the development of novel strategies for altering ABSC proliferation in the airway epithelium.

Isolation and in vitro characterization of basal and submucosal gland duct stem/progenitor cells from human proximal airways.

Basal cells and submucosal gland (SMG) duct cells have been isolated and shown to be stem/progenitor cell populations for the murine airway epithelium. However, methods for the isolation of basal and SMG duct cells from human airways have not been defined. We used an optimized two-step enzyme digestion protocol to strip the surface epithelium from tracheal specimens separate from SMG cells, and we then sorted the basal and duct stem/progenitors using fluorescence-activated cell sorting. We used nerve growth factor receptor, as well as a combination of CD166 and CD44, to sort basal cells and also used CD166 to isolate SMG duct cells. Sorted stem/progenitor cells were cultured to characterize their self-renewal and differentiation ability. Both basal and SMG duct cells grew into spheres. Immunostaining of the spheres showed mostly dense spheres with little to no central lumen. The spheres expressed cytokeratins 5 and 14, with some mucus- and serous-secreting cells. The sphere-forming efficiency and the rate of growth of the spheres varied widely between patient samples and correlated with the degree of hyperplasia of the epithelium. We found that only aldehyde dehydrogenase (ALDH)(hi) basal and duct cells were capable of sphere formation. Global inhibition of ALDH, as well as specific inhibition of the ALDH2 isoform, inhibited self-renewal of both basal and duct cells, thereby producing fewer and smaller spheres. In conclusion, we have developed methods to isolate basal and SMG duct cells from the surface epithelium and SMGs of human tracheas and have developed an in vitro model to characterize their self-renewal and differentiation.

Identification of an interleukin 13-induced epigenetic signature in allergic airway inflammation.

Epigenetic changes have been implicated in the pathogenesis of asthma. We sought to determine if IL13, a key cytokine in airway inflammation and remodeling, induced epigenetic DNA methylation and miRNAs expression changes in the airways in conjunction with its transcriptional gene regulation. Inducible expression of an IL13 transgene in the airways resulted in significant changes in DNA methylation in 177 genes, most of which were associated with the IL13 transcriptional signature in the airways. A large number of genes whose expression was induced by IL13 were found to have decreased methylation, including those involved in tissue remodeling (Olr1), leukocyte influx (Cxcl3, Cxcl5, CSFr2b), and the Th2 response (C3ar1, Chi3l4). Reciprocally, some genes whose expression was suppressed were found to have increased methylation (e.g. Itga8). In addition, miRNAs were identified with targets for lung development and Wnt signaling, amongst others. These results indicate that IL13 confers an epigenetic methylation and miRNA signature that accompanies its transcriptional program in the airways, which may play a critical role in airway inflammation and remodeling.

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic-ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self-renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell-based therapies for patients with airway diseases.

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 48h, 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 AECs.

Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.

Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury, and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots, and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady-state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer, and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)-expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in the persistence of K14+ cells in the airway epithelium in potentially premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis, and this predictive value was strongest in smokers, in which it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC.

Misconceptions about traumatic brain injury and recovery among nursing students.

OBJECTIVE: To identify potential misconceptions that nursing students have regarding traumatic brain injury (TBI) and recovery. DESIGN: Descriptive questionnaire. SETTING: University. MAIN MEASURE: Thirty-item questionnaire addressing knowledge about TBI and recovery. PARTICIPANTS: One hundred eight undergraduate students, 65 in the prenursing program and 43 nursing majors. RESULTS: In general, nursing students had a lower frequency of misconceptions than did university students in a prior study, and the more advanced nursing majors had a lower frequency of misconceptions than did the prenursing students. Substantial misconceptions did exist, though, especially concerning unconsciousness, amnesia, and recovery. CONCLUSIONS: Nursing students possess several misconceptions pertaining to TBI and recovery that have the potential to adversely affect assessment, treatment, and education of patients and family members. Education focusing on TBI and recovery should be included in the nursing curriculum and be a part of continuing education at the professional level.

Comparative evaluation of viral, nonviral and physical methods of gene delivery to normal and transformed lung epithelial cells.

Few studies have directly compared the efficiencies of gene delivery methods that target normal lung cells versus lung tumor cells. We report the first study directly comparing the efficiency and toxicity of viral [adeno-associated virus (AAV2, 5, 6) and lentivirus], nonviral (Effectene, SuperFect and Lipofectamine 2000) and physical [particle-mediated gene transfer (PMGT)] methods of gene delivery in normal mouse lung cells and in mouse adenocarcinoma cells. Lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein was the most efficient gene transfer method for normal mouse airway epithelial cells [25.95 (+/-3.57) %] whereas AAV6 was most efficient for MLE-12 adenocarcinoma cells [68.2 (+/-3.2) %]. PMGT was more efficient in normal mouse airway epithelial cells than AAV5, Lipofectamine 2000 and SuperFect. AAV5 displayed the lowest transfection efficiency at less than 10% in both cell types. PMGT was the only method that resulted in significant toxicity. In summary, for all of the gene delivery methods examined here, lung tumor cells were transfected more easily than normal lung cells. Lipofectamine 2000 is potentially highly selective for lung tumor cells whereas AAV6 and lentivirus vesicular stomatitis virus glycoprotein may be useful for gene delivery strategies that require targeting of both normal and tumor cells.

Generation of a Mig-6 conditional null allele.

Mitogen-inducible gene 6 (Mig-6) is a stress-induced gene that serves as a negative regulator of epidermal growth factor (EGF) signaling and acts as a tumor suppressor. Ablation of Mig-6 results in a significant percentage of embryo lethality as well as abnormalities in multiple tissues. To understand the physiological roles of Mig-6, a conditional null allele, Mig-6(f/f) was generated by introducing LoxP sites that flank exons 2 and 4. The Mig-6(f/f) allele was validated by generating recombined Mig-6(-/-) mice using the Zp3-Cre system. The conditional null allele was confirmed by assaying for Mig-6 gene expression in liver, lung, uterus, and skin. The recombined Mig-6(-/-) mice developed pathological changes, such as degenerative joint diseases and skin hyperplasia similar to the previously reported Mig-6 germline null allele. In addition, these mice also had enlarged uteri with endometrial hyperplasia. In summary, this Mig-6(f/f) mouse is a useful tool for the functional study of the Mig-6 gene in a tissue-specific fashion.