Extracellular Matrix as a Driver of Chronic Lung Diseases.

The extracellular matrix (ECM) is not just a three-dimensional scaffold that provides stable support for all cells in the lungs, but also an important component of chronic fibrotic airway, vascular, and interstitial diseases. It is a bioactive entity that is dynamically modulated during tissue homeostasis and disease, that controls structural and immune cell functions and drug responses, and that can release fragments that have biological activity and that can be used to monitor disease activity. There is a growing recognition of the importance of considering ECM changes in chronic airway, vascular, and interstitial diseases, including 1) compositional changes, 2) structural and organizational changes, and 3) mechanical changes and how these affect disease pathogenesis. As altered ECM biology is an important component of many lung diseases, disease models must incorporate this factor to fully recapitulate disease-driver pathways and to study potential novel therapeutic interventions. Although novel models are evolving that capture some or all of the elements of the altered ECM microenvironment in lung diseases, opportunities exist to more fully understand cell-ECM interactions that will help devise future therapeutic targets to restore function in chronic lung diseases. In this perspective article, we review evolving knowledge about the ECM's role in homeostasis and disease in the lung.

Simple Models of Lung Development.

Models are essential to further our understanding of lung development and regeneration and to facilitate identification and testing of potential treatments for lung diseases. A wide variety of rodent and human models are available that recapitulate one or more stages of lung development. This chapter describes the existing 'simple' in vitro, in silico and ex vivo models of lung development. We define which stage(s) of development each model recapitulates and highlight their pros and cons.

Lung Development Genes and Adult Lung Function.

Rationale: Poor lung health in adult life may occur partly through suboptimal growth and development, as suggested by epidemiological evidence pointing to early life risk factors.Objectives: To systematically investigate the effects of lung development genes on adult lung function.Methods: Using UK Biobank data, we tested the association of 391 genes known to influence lung development with FVC and FEV1/FVC. We split the dataset into two random subsets of 207,616 and 138,411 individuals, using the larger subset to select the most promising signals and the smaller subset for replication.Measurements and Main Results: We identified 55 genes, of which 36 (16 for FVC, 19 for FEV1/FVC, and one for both) had not been identified in the largest, most recent genome-wide study of lung function. Most of these 36 signals were intronic variants; expression data from blood and lung tissue showed that the majority affect the expression of the genes they lie within. Further testing of 34 of these 36 signals in the CHARGE and SpiroMeta consortia showed that 16 replicated after Bonferroni correction and another 12 replicated at nominal significance level. Of the 55 genes, 53 fell into four biological categories whose function is to regulate organ size and cell integrity (growth factors; transcriptional regulators; cell-to-cell adhesion; extracellular matrix), suggesting that these specific processes are important for adult lung health.Conclusions: Our study demonstrates the importance of lung development genes in regulating adult lung function and influencing both restrictive and obstructive patterns. Further investigation of these developmental pathways could lead to druggable targets.

Mechanism of lung development in the aetiology of adult congenital pulmonary airway malformations.

Congenital pulmonary airway malformations (CPAMs) are rare lung abnormalities that result in cyst formation and are associated with respiratory distress in infants and malignant potential in adults. The pathogenesis of CPAMs remains unknown but data suggest disruption of the normal proximo-distal programme of airway branching and differentiation. Here, we demonstrate that adult human CPAM are lined with epithelium that retains SOX-2 and thyroid transcription factor-1 immunohistochemical markers, characteristic of the developing lung. However, RALDH-1, another key marker, is absent. This suggests a more complex aetiology for CPAM than complete focal arrest of lung development and may provide insight to the associated risk of malignancy.

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways.

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

Vangl2, a planar cell polarity molecule, is implicated in irreversible and reversible kidney glomerular injury.

Planar cell polarity (PCP) pathways control the orientation and alignment of epithelial cells within tissues. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the normal differentiation of kidney glomeruli and tubules. Vangl2 has also been implicated in modifying the course of acquired glomerular disease, and here, we further explored how Vangl2 impacts on glomerular pathobiology in this context. Targeted genetic deletion of Vangl2 in mouse glomerular epithelial podocytes enhanced the severity of not only irreversible accelerated nephrotoxic nephritis but also lipopolysaccharide-induced reversible glomerular damage. In each proteinuric model, genetic deletion of Vangl2 in podocytes was associated with an increased ratio of active-MMP9 to inactive MMP9, an enzyme involved in tissue remodelling. In addition, by interrogating microarray data from two cohorts of renal patients, we report increased VANGL2 transcript levels in the glomeruli of individuals with focal segmental glomerulosclerosis, suggesting that the molecule may also be involved in certain human glomerular diseases. These observations support the conclusion that Vangl2 modulates glomerular injury, at least in part by acting as a brake on MMP9, a potentially harmful endogenous enzyme. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Pulmonary ORMDL3 is critical for induction of Alternaria-induced allergic airways disease.

BACKGROUND: Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma. OBJECTIVE: We sought to investigate in vivo the functional role of ORMDL3 in disease inception. METHODS: An Ormdl3-deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno-associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. RESULTS: Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6-mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum-associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen-induced allergic airways disease. CONCLUSIONS: This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses.

Deficient retinoid-driven angiogenesis may contribute to failure of adult human lung regeneration in emphysema.

BACKGROUND: Molecular pathways that regulate alveolar development and adult repair represent potential therapeutic targets for emphysema. Signalling via retinoic acid (RA), derived from vitamin A, is required for mammalian alveologenesis, and exogenous RA can induce alveolar regeneration in rodents. Little is known about RA signalling in the human lung and its potential role in lung disease. OBJECTIVES: To examine regulation of human alveolar epithelial and endothelial repair by RA, and characterise RA signalling in human emphysema. METHODS: The role of RA signalling in alveolar epithelial repair was investigated with a scratch assay using an alveolar cell line (A549) and primary human alveolar type 2 (AT2) cells from resected lung, and the role in angiogenesis using a tube formation assay with human lung microvascular endothelial cells (HLMVEC). Localisation of RA synthetic (RALDH-1) and degrading (cytochrome P450 subfamily 26 A1 (CYP26A1)) enzymes in human lung was determined by immunofluorescence. Regulation of RA pathway components was investigated in emphysematous and control human lung tissue by quantitative real-time PCR and Western analysis. RESULTS: RA stimulated HLMVEC angiogenesis in vitro; this was partially reproduced with a RAR-α agonist. RA induced mRNA expression of vascular endothelial growth factor A (VEGFA) and VEGFR2. RA did not modulate AT2 repair. CYP26A1 protein was identified in human lung microvasculature, whereas RALDH-1 partially co-localised with vimentin-positive fibroblasts. CYP26A1 mRNA and protein were increased in emphysema. CONCLUSIONS: RA regulates lung microvascular angiogenesis; the endothelium produces CYP26A1 which is increased in emphysema, possibly leading to reduced RA availability. These data highlight a role for RA in maintenance of the human pulmonary microvascular endothelium.

Atmin mediates kidney morphogenesis by modulating Wnt signaling.

The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in Atmin(Gpg6/Gpg6) kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including ß-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, Atmin(Gpg6/Gpg6) mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between Atmin(Gpg6/+) and Vangl2(Lp/+) mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.

Scribble is required for normal epithelial cell-cell contacts and lumen morphogenesis in the mammalian lung.

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell-cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical-basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, 'open' lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib(Crc/Crc) lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell-cell association, we show that Scrib associates with ß-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell-cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.

Planar cell polarity and the kidney.

Planar cell polarity (PCP) is the uniform orientation and alignment of a group of cells orthogonal to the apical-basal axis within a tissue. Originally described in insects, it is now known that PCP is required for many processes in vertebrates, including directional cell movement, polarized cell division, ciliary orientation, neural tube closure, heart development and lung branching. In this review, we outline the evidence implicating PCP in kidney development and disease focusing initially on the function of PCP in ureteric bud branching and elongation. We then describe how defects in PCP may lead to polycystic kidney disease and discuss a newly identified role for PCP in the kidney filtration barrier.

A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS).

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

Deciphering the impacts of modulating the Wnt-planar cell polarity (PCP) pathway on alveolar repair.

Many adult lung diseases involve dysregulated lung repair. Deciphering the molecular and cellular mechanisms that govern intrinsic lung repair is essential to develop new treatments to repair/regenerate the lungs. Aberrant Wnt signalling is associated with lung diseases including emphysema, idiopathic pulmonary fibrosis and pulmonary arterial hypertension but how Wnt signalling contributes to these diseases is still unclear. There are several alternative pathways that can be stimulated upon Wnt ligand binding, one of these is the Planar Cell Polarity (PCP) pathway which induces actin cytoskeleton remodelling. Wnt5a is known to stimulate the PCP pathway and this ligand is of particular interest in regenerative lung biology because of its association with lung diseases and its role in the alveolar stem cell niche. To decipher the cellular mechanisms through which Wnt5a and the PCP pathway affect alveolar repair we utilised a 3-D ex-vivo model of lung injury and repair, the AIR model. Our results show that Wnt5a specifically enhances the alveolar epithelial progenitor cell population following injury and surprisingly, this function is attenuated but not abolished in Looptail (Lp) mouse lungs in which the PCP pathway is dysfunctional. However, Lp tracheal epithelial cells show reduced stiffness and Lp alveolar epithelial cells are less migratory than wildtype (WT), indicating that Lp lung epithelial cells have a reduced capacity for repair. These findings provide important mechanistic insight into how Wnt5a and the PCP pathway contribute to lung repair and indicate that these components of Wnt signalling may be viable targets for the development of pro-repair treatments.

A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices.

Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.

The planar cell polarity gene Vangl2 is required for mammalian kidney-branching morphogenesis and glomerular maturation.

The planar cell polarity (PCP) pathway, incorporating non-canonical Wnt signalling, controls embryonic convergent (CE) extension, polarized cell division and ciliary orientation. It also limits diameters of differentiating renal tubules, with mutation of certain components of the pathway causing cystic kidneys. Mutations in mouse Vangl genes encoding core PCP proteins cause neural tube defects (NTDs) and Vangl2 mutations also impair branching of embryonic mouse lung airways. Embryonic metanephric kidneys also undergo branching morphogenesis and Vangl2 is known to be expressed in ureteric bud/collecting duct and metanephric mesenchymal/nephron lineages. These observations led us to investigate metanephroi in Vangl2 mutant mice, Loop-tail (Lp). Although ureteric bud formation is normal in Vangl2(Lp/Lp) embryos, subsequent in vivo and in vitro branching morphogenesis is impaired. Null mutant kidneys are short, consistent with a CE defect. Differentiating glomerular epithelia express several PCP genes (Vangl1/2, Celsr1, Scrib, Mpk1/2 and Fat4) and glomeruli in Vangl2(Lp/Lp) fetuses are smaller and contain less prominent capillary loops than wild-type littermates. Furthermore, Vangl2(Lp/+) kidneys had modest reduction in glomerular numbers postnatally. Vangl2(Lp/Lp) metanephroi contained occasional dilated tubules but no overt cystic phenotype. These data show for the first time that a PCP gene is required for normal morphogenesis of both the ureteric bud and metanephric mesenchyme-derived structures. It has long been recognized that certain individuals with NTDs are born with malformed kidneys, and recent studies have discovered VANGL mutations in some NTD patients. On the basis of our mutant mouse study, we suggest that PCP pathway mutations should be sought when NTD and renal malformation co-exist.

The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis.

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

The Biological Significance and Implications of Planar Cell Polarity for Nephrology.

The orientation of cells in two-dimensional and three-dimensional space underpins how the kidney develops and responds to disease. The process by which cells orientate themselves within the plane of a tissue is termed planar cell polarity. In this Review, we discuss how planar cell polarity and the proteins that underpin it govern kidney organogenesis and pathology. The importance of planar cell polarity and its constituent proteins in multiple facets of kidney development is emphasised, including ureteric bud branching, tubular morphogenesis and nephron maturation. An overview is given of the relevance of planar cell polarity and its proteins for inherited human renal diseases, including congenital malformations with unknown aetiology and polycystic kidney disease. Finally, recent work is described outlining the influence of planar cell polarity proteins on glomerular diseases and highlight how this fundamental pathway could yield a new treatment paradigm for nephrology.

The acid injury and repair (AIR) model: A novel ex-vivo tool to understand lung repair.

Research into mechanisms underlying lung injury and subsequent repair responses is currently of paramount importance. There is a paucity of models that bridge the gap between in vitro and in vivo research. Such intermediate models are critical for researchers to decipher the mechanisms that drive repair and to test potential new treatments for lung repair and regeneration. Here we report the establishment of a new tool, the Acid Injury and Repair (AIR) model, that will facilitate studies of lung tissue repair. In this model, injury is applied to a restricted area of a precision-cut lung slice using hydrochloric acid, a clinically relevant driver. The surrounding area remains uninjured, thus mimicking the heterogeneous pattern of injury frequently observed in lung diseases. We show that in response to injury, the percentage of progenitor cells (pro surfactant protein C, proSP-C and TM4SF1 positive) significantly increases in the injured region. Whereas in the uninjured area, the percentage of proSP-C/TM4SF1 cells remains unchanged but proliferating cells (Ki67 positive) increase. These effects are modified in the presence of inhibitors of proliferation (Cytochalasin D) and Wnt secretion (C59) demonstrating that the AIR model is an important new tool for research into lung disease pathogenesis and potential regenerative medicine strategies.