Chloride channels regulate differentiation and barrier functions of the mammalian airway.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function

On the Role of Mechanics in Chronic Lung Disease.

Progressive airflow obstruction is a classical hallmark of chronic lung disease, affecting more than one fourth of the adult population. As the disease progresses, the inner layer of the airway wall grows, folds inwards, and narrows the lumen. The critical failure conditions for airway folding have been studied intensely for idealized circular cross-sections. However, the role of airway branching during this process is unknown. Here, we show that the geometry of the bronchial tree plays a crucial role in chronic airway obstruction and that critical failure conditions vary significantly along a branching airway segment. We perform systematic parametric studies for varying airway cross-sections using a computational model for mucosal thickening based on the theory of finite growth. Our simulations indicate that smaller airways are at a higher risk of narrowing than larger airways and that regions away from a branch narrow more drastically than regions close to a branch. These results agree with clinical observations and could help explain the underlying mechanisms of progressive airway obstruction. Understanding growth-induced instabilities in constrained geometries has immediate biomedical applications beyond asthma and chronic bronchitis in the diagnostics and treatment of chronic gastritis, obstructive sleep apnea and breast cancer

Assembly and functional analysis of an S/MAR based episome with the cystic fibrosis transmembrane conductance regulator gene

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease

The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in Aspergillus fumigatus sensitized mice.

BackgroundLipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models.MethodsLp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. β- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice.ResultsPAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released β-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice.ConclusionsWe conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge

Next generation bioengineering of lung tissue for transplantation

Lung transplantation is the only option for end-stage lung diseases, but organ shortage remains problematic. Generating lungs ex vivo could overcome shortages with current approaches being explored for lung tissue engineering utilizing a biologically derived, synthetic or hybrid scaffold which is seeded with cells and cultured ex vivo. Ideally, cells could be sourced from the transplant recipient and thus are conceptualized to reduce the long-term requirements for immunosuppressive drugs and the risk for rejection. Progenitor cell populations can be controlled more easily than induced pluripotent stem cells (IPSCs), with lower risk of tumour formation. However, as presented in this thesis, progenitor cells can be deranged in diseased lungs such as for example idiopathic pulmonary fibrosis (IPF) and are therefore unlikely candidates to generate healthy tissue. In addition to healthy cells, scaffolds with attributes known to be pro-regenerative are required to generate healthy tissues. In the thesis it is shown that both scaffold and cell age plays a role in the regenerative capacity of a tissue. From this it is clear that to generate a healthy tissue or organ, it is critical to find the appropriate scaffold and cell type. Additionally, bioengineering manufacturing methods that generate reproducible, custom-made, high resolution constructs using cytocompatible materials are ideal for tissue engineering approaches. One such method which is compatible with the criteria above and that has emerged in recent years is 3D printing. 3D printing or bioprinting (when cells are printed) can generate custom structures relevant for human lungs. In this thesis, potential bioinks for bioprinting lung tissue are investigated. A tissue-specific hybrid bioink consisting of alginate, reinforced with extracellular matrix from decellularized lung tissue (rECM) was used to 3D bioprint human airways comprised of regionally specified primary cells which remained patent over time. The biocompatibility and vascularisation of rECM hydrogels was investigated in both T-cell immunodeficient mice mimicking the clinical scenario and immunocompetent mice. Bioprinted rECM hydrogels support the formation of an intact vascular network throughout the full thickness of the graft, comprised of both large and small size blood vessels and integrate well in the surrounding tissue

Absolute electrical impedance tomography (aEIT) guided ventilation therapy in critical care patients: simulations and future trends

Thoracic electrical impedance tomography (EIT) is a noninvasive, radiation-free monitoring technique whose aim is to reconstruct a cross-sectional image of the internal spatial distribution of conductivity from electrical measurements made by injecting small alternating currents via an electrode array placed on the surface of the thorax. The purpose of this paper is to discuss the fundamentals of EIT and demonstrate the principles of mechanical ventilation, lung recruitment, and EIT imaging on a comprehensive physiological model, which combines a model of respiratory mechanics, a model of the human lung absolute resistivity as a function of air content, and a 2-D finite-element mesh of the thorax to simulate EIT image reconstruction during mechanical ventilation. The overall model gives a good understanding of respiratory physiology and EIT monitoring techniques in mechanically ventilated patients. The model proposed here was able to reproduce consistent images of ventilation distribution in simulated acutely injured and collapsed lung conditions. A new advisory system architecture integrating a previously developed data-driven physiological model for continuous and noninvasive predictions of blood gas parameters with the regional lung function data/information generated from absolute EIT (aEIT) is proposed for monitoring and ventilator therapy management of critical care patients

Discrete cilia modelling with singularity distributions

We discuss in detail techniques for modelling flows due to finite and infinite arrays of beating cilia. An efficient technique, based on concepts from previous ‘singularity models’ is described, that is accurate in both near and far-fields. Cilia are modelled as curved slender ellipsoidal bodies by distributing Stokeslet and potential source dipole singularities along their centrelines, leading to an integral equation that can be solved using a simple and efficient discretisation. The computed velocity on the cilium surface is found to compare favourably with the boundary condition. We then present results for two topics of current interest in biology. 1) We present the first theoretical results showing the mechanism by which rotating embryonic nodal cilia produce a leftward flow by a ‘posterior tilt,’ and track particle motion in an array of three simulated nodal cilia. We find that, contrary to recent suggestions, there is no continuous layer of negative fluid transport close to the ciliated boundary. The mean leftward particle transport is found to be just over 1 μm/s, within experimentally measured ranges. We also discuss the accuracy of models that represent the action of cilia by steady rotlet arrays, in particular, confirming the importance of image systems in the boundary in establishing the far-field fluid transport. Future modelling may lead to understanding of the mechanisms by which morphogen gradients or mechanosensing cilia convert a directional flow to asymmetric gene expression. 2) We develop a more complex and detailed model of flow patterns in the periciliary layer of the airway surface liquid. Our results confirm that shear flow of the mucous layer drives a significant volume of periciliary liquid in the direction of mucus transport even during the recovery stroke of the cilia. Finally, we discuss the advantages and disadvantages of the singularity technique and outline future theoretical and experimental developments required to apply this technique to various other biological problems, particularly in the reproductive system

An in vitro hybrid method used to model realistic airway deposition and post-deposition behaviour for orally inhaled products

This research focuses on the development and validation of a new in vitro test method capable of characterising realistic airway deposition of inhaled pharmaceutical aerosols, and the events following deposition at the airway epithelia. An existing physical model of a medium-sized Virginia Commonwealth University (VCU) mouth throat (MT) and tracheal bronchial (TB) upper airway was modified and validated to integrate Calu-3 monolayers cells cultured onto Snapwell® inserts for the collection of deposited drug. The model was housed in a chamber and validation was carried out using two nebulised ciprofloxacin formulations: a controlled-released encapsulated liposomal solution termed “Ciprofloxacin for Inhalation” (CFI), and an un-encapsulated “free” solution termed “Free Ciprofloxacin for Inhalation” (FCI). Validation was carried out to ensure modifications to the airway model resulted in sufficient collection of drug for analysis, whilst ensuing minor changes to in vitro airway deposition behaviour. Studies were carried out to assess how experimental variables influenced in vitro drug permeation behaviour, i.e., choice of membrane material, formulation type and aerosol collection mechanism. Most importantly, the integrity of the barrier formation and tight junction functionality of the monolayers were assessed post airway deposition to evaluate if dynamic stresses caused from the airflow could affect in vivo drug transport behaviour. In conclusion, a novel hybrid in vitro test method has been extensively validated to successfully characterise realistic airway deposition of two different nebulised solutions of ciprofloxacin drug. It is hoped that this model can be used by others as a potential screening tool for head-to-head comparative studies without resorting to expensive clinical trials, as well as a foundation for the design of new inhalation platforms with improved performance efficiency and reproducibility

Modeling of flow generated sound in a constricted duct at low Mach number flow

Modelling flow and acoustics in a constricted duct at low Mach numbers is important for investigating many physiological phenomena such as phonation, generation of arterial murmurs, and pulmonary conditions involving airway obstruction. The objective of this study is to validate computational fluid dynamics (CFD) and computational aero-acoustics (CAA) simulations in a constricted tube at low Mach numbers. Different turbulence models were employed to simulate the flow field. Models included Reynolds Average Navier-Stokes (RANS), Detached eddy simulation (DES) and Large eddy simulation (LES). The models were validated by comparing study results with laser doppler anemometry (LDA) velocity measurements. The comparison showed that experimental data agreed best with the LES model results. Although RANS Reynolds stress transport (RST) model showed good agreement with mean velocity measurements, it was unable to capture velocity fluctuations. RANS shear stress transport (SST) k-{\omega} model and DES models were unable to predict the location of high fluctuating flow region accurately. CAA simulation was performed in parallel with LES using Acoustic Perturbation Equation (APE) based hybrid CAA method. CAA simulation results agreed well with measured wall sound pressure spectra. The APE acoustic sources were found in jet core breakdown region downstream of the constriction, which was also characterized by high flow fluctuations. Proper Orthogonal Decomposition (POD) is used to study the coherent flow structures at the different frequencies corresponding to the peaks of the measured sound pressure spectra. The study results will help enhance our understanding of sound generation mechanisms in constricted tubes including biomedical applications

Modeling of flow generated sound in a constricted duct at low Mach number flow

Modelling flow and acoustics in a constricted duct at low Mach numbers is important for investigating many physiological phenomena such as phonation, generation of arterial murmurs, and pulmonary conditions involving airway obstruction. The objective of this study is to validate computational fluid dynamics (CFD) and computational aero-acoustics (CAA) simulations in a constricted tube at low Mach numbers. Different turbulence models were employed to simulate the flow field. Models included Reynolds Average Navier-Stokes (RANS), Detached eddy simulation (DES) and Large eddy simulation (LES). The models were validated by comparing study results with laser doppler anemometry (LDA) velocity measurements. The comparison showed that experimental data agreed best with the LES model results. Although RANS Reynolds stress transport (RST) model showed good agreement with mean velocity measurements, it was unable to capture velocity fluctuations. RANS shear stress transport (SST) k-ω model and DES models were unable to predict the location of high fluctuating flow region accurately. CAA simulation was performed in parallel with LES using Acoustic Perturbation Equation (APE) based hybrid CAA method. CAA simulation results agreed well with measured wall sound pressure spectra. The APE acoustic sources were found in jet core breakdown region downstream of the constriction, which was also characterized by high flow fluctuations. Proper Orthogonal Decomposition (POD) is used to study the coherent flow structures at the different frequencies corresponding to the peaks of the measured sound pressure spectra. The study results will help enhance our understanding of sound generation mechanisms in constricted tubes including biomedical applications