Defective Fibrillar Collagen Organization by Fibroblasts Contributes to Airway Remodeling in Asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function.Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma.Methods: Nontransplantable donor lungs from donors with asthma (n = 13) and control (n = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments.Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy.Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo.

A Heterotopic Xenograft Model of Human Airways for Investigating Fibrosis in Asthma.

Limited in vivo models exist to investigate the lung airway epithelial role in repair, regeneration, and pathology of chronic lung diseases. Herein, we introduce a novel animal model in asthma-a xenograft system integrating a differentiating human asthmatic airway epithelium with an actively remodeling rodent mesenchyme in an immunocompromised murine host. Human asthmatic and nonasthmatic airway epithelial cells were seeded into decellularized rat tracheas. Tracheas were ligated to a sterile cassette and implanted subcutaneously in the flanks of nude mice. Grafts were harvested at 2, 4, or 6 weeks for tissue histology, fibrillar collagen, and transforming growth factor-ß activation analysis. We compared immunostaining in these xenografts to human lungs. Grafted epithelial cells generated a differentiated epithelium containing basal, ciliated, and mucus-expressing cells. By 4 weeks postengraftment, asthmatic epithelia showed decreased numbers of ciliated cells and decreased E-cadherin expression compared with nonasthmatic grafts, similar to human lungs. Grafts seeded with asthmatic epithelial cells had three times more fibrillar collagen and induction of transforming growth factor-ß isoforms at 6 weeks postengraftment compared with nonasthmatic grafts. Asthmatic epithelium alone is sufficient to drive aberrant mesenchymal remodeling with fibrillar collagen deposition in asthmatic xenografts. Moreover, this xenograft system represents an advance over current asthma models in that it permits direct assessment of the epithelial-mesenchymal trophic unit.

Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts.

BACKGROUND: Recognition of the airway epithelium as a central mediator in the pathogenesis of asthma has necessitated greater understanding of the aberrant cellular mechanisms of the epithelium in asthma. The architecture of chromatin is integral to the regulation of gene expression and is determined by modifications to the surrounding histones and DNA. The acetylation, methylation, phosphorylation, and ubiquitination of histone tail residues has the potential to greatly alter the accessibility of DNA to the cells transcriptional machinery. DNA methylation can also interrupt binding of transcription factors and recruit chromatin remodelers resulting in general gene silencing. Although previous studies have found numerous irregularities in the expression of genes involved in asthma, the contribution of epigenetic regulation of these genes is less well known. We propose that the gene expression of epigenetic modifying enzymes is cell-specific and influenced by asthma status in tissues derived from the airways. METHODS: Airway epithelial cells (AECs) isolated by pronase digestion or endobronchial brushings and airway fibroblasts obtained by outgrowth technique from healthy and asthmatic donors were maintained in monolayer culture. RNA was analyzed for the expression of 82 epigenetic enzymes across 5 families of epigenetic modifying enzymes. Western blot and immunohistochemistry were also used to examine expression of 3 genes. RESULTS: Between AECs and airway fibroblasts, we identified cell-specific gene expression in each of the families of epigenetic modifying enzymes; specifically 24 of the 82 genes analyzed showed differential expression. We found that 6 histone modifiers in AECs and one in fibroblasts were differentially expressed in cells from asthmatic compared to healthy donors however, not all passed correction. In addition, we identified a corresponding increase in Aurora Kinase A (AURKA) protein expression in epithelial cells from asthmatics compared to those from non-asthmatics. CONCLUSIONS: In summary, we have identified cell-specific variation in gene expression in each of the families of epigenetic modifying enzymes in airway epithelial cells and airway fibroblasts. These data provide insight into the cell-specific variation in epigenetic regulation which may be relevant to cell fate and function, and disease susceptibility.

Elevated H3K18 acetylation in airway epithelial cells of asthmatic subjects.

BACKGROUND: Epigenetic adjustments of the chromatin architecture through histone modifications are reactive to the environment and can establish chromatin states which are permissive or repressive to gene expression. Epigenetic regulation of gene expression is cell specific and therefore, it is important to understand its contribution to individual cellular responses in tissues like the airway epithelium which forms the mucosal barrier to the inhaled environment within the lung. The airway epithelium of asthmatics is abnormal with dysregulation of genes such as epidermal growth factor receptor (EGFR), the ΔN isoform of the transcription factor p63 (ΔNp63), and signal transducer and activator of transcription 6 (STAT6), integral to differentiation, proliferation, and inflammation. It is important to establish in diseases like asthma how histone modifications affect tissue responses such as proliferation and differentiation. OBJECTIVES: To characterize the global histone acetylation and methylation status in the epithelium of asthmatic compared to healthy subjects and to identify the impact of these variations on genes involved in epithelial functions. METHODS: Whole lungs were obtained from healthy and asthmatic subjects (n = 6) from which airway epithelial cells (AECs) were isolated and airway sections were taken for analysis of histone lysine acetylation and methylation by immunohistochemistry. AECs were subjected to chromatin immunoprecipitation (ChIP) using anti-H3K18ac and anti-H3K4me2 antibodies followed by RT-PCR targeting ΔNp63, EGFR, and STAT6. AECs were also treated with TSA and changes in ΔNp63, EGFR, and STAT6 expression were determined. RESULTS: We identified an increase in the acetylation of lysine 18 on histone 3 (H3K18ac) and trimethylation of lysine 9 on histone 3 (H3K9me3) in the airway epithelium of asthmatic compared to healthy subjects. We found increased association of H3K18ac around the transcription start site of ΔNp63, EGFR, and STAT6 in AECs of asthmatics. However, we were unable to modify the expression of these genes with the use of the HDAC inhibitor TSA in healthy subjects. DISCUSSION: The airway epithelium from asthmatic subjects displays increased acetylation of H3K18 and association of this mark around the transcription start site of ΔNp63, EGFR, and STAT6. These findings suggest a complex interaction between histone modifications and gene regulation in asthma.

Fibroblast signal transducer and activator of transcription 4 drives cigarette smoke-induced airway fibrosis.

Cigarette smoke-induced emphysema and small airway remodeling are the anatomic bases of chronic obstructive pulmonary disease (COPD), but the pathogenesis of these changes is unclear, and current treatments for COPD are minimally effective. To evaluate the role of signal transducer and activator of transcription (STAT)-4 in cigarette smoke-induced small airway remodeling, we used C57BL/6J (wild type [WT]) and STAT4-/- mice exposed to air or cigarette smoke for 6 months and isolated airway and parenchymal fibroblasts. We also compared the results with those obtained with human fibroblasts. We found that STAT4-/- mice were protected against smoke-induced small airway remodeling but not emphysema. STAT4 is abundantly expressed in airway compared with parenchymal-derived fibroblasts isolated from normal human and murine lung. WT airway fibroblasts proliferate faster than STAT4-/- airway fibroblasts, whereas there is no difference between strains for parenchymal fibroblasts. IL-12 is up-regulated in the lung after smoke exposure, and IL-12 receptor B2 is expressed on airway and parenchymal fibroblasts in mouse and human lung. Treatment with IL-12 causes phosphorylation of STAT4 in WT airway fibroblasts. Exposure of WT airway, but not parenchymal, fibroblasts to IL-12 causes increased expression of collagen 1α1 and transforming growth factor ß1, factors involved in small airway remodeling, whereas STAT4-/- fibroblasts are unresponsive to IL-12. These results indicate that IL-12 can drive small airway remodeling via STAT4 signaling and suggest that treatment with clinically available anti-IL-12p40 drugs might provide a new approach to preventing small airway remodeling in cigarette smokers.

Transcription factor p63 regulates key genes and wound repair in human airway epithelial basal cells.

The airway epithelium in asthma displays altered repair and incomplete barrier formation. Basal cells are the progenitor cells of the airway epithelium, and can repopulate other cell types after injury. We previously reported increased numbers of basal cells expressing the transcription factor p63 in the airway epithelium of patients with asthma. Here we sought to determine the molecular consequences of p63 expression in basal human airway epithelial cells during wound repair. Because at least six isoforms of p63 exist (N-terminally truncated [ΔN] versus transcriptional activation promoter variants and α, ß, or γ 3' splice variants), the expression of all isoforms was investigated in primary human airway epithelial cells (pHAECs). We modulated p63 expression, using small interfering RNA (siRNA) and adenoviral constructs to determine the effects of p63 on 21 candidate target genes by RT-PCR, and on repair using a scratch wound assay. We found that basal pHAECs from asthmatic and nonasthmatic donors predominantly expressed the N-terminally truncated p63α variant (ΔNp63α) isoform, with no disease-specific differences in expression. The knockdown of ΔNp63, using specific siRNA, decreased the expression of 11 out of 21 genes associated with epithelial repair and differentiation, including ß-catenin, epidermal growth factor receptor, and Jagged1. The loss of ΔNp63 significantly inhibited wound closure (which was associated with the decreased expression of ß-catenin and Jagged1), reduced epithelial proliferation as measured by Ki-67 staining, and increased E-cadherin expression, potentially preventing cytokinesis. In conclusion, ΔNp63α is the major isoform expressed in basal pHAECs, and is essential for epithelial wound repair. The role of ΔNp63α in epithelial barrier integrity requires further study to understand its role in health and disease.

Caveolin-1 controls airway epithelial barrier function. Implications for asthma.

The molecular basis for airway epithelial fragility in asthma has remained unclear. We investigated whether the loss of caveolin-1, the major component of caveolae and a known stabilizer of adherens junctions, contributes to epithelial barrier dysfunction in asthma. We studied the expression of caveolin-1 and adhesion molecules E-cadherin and ß-catenin in airway sections, and we cultured bronchial epithelial cells from patients with asthma and from healthy control subjects. To determine the functional role of caveolin-1, we investigated the effects of caveolin-1 up-regulation and down-regulation on E-cadherin expression, barrier function, and proallergic activity in the human bronchial epithelial cell lines 16HBE and BEAS-2B. The membrane expression of caveolin-1 was significantly lower in airway epithelia from patients with asthma than from subjects without asthma, and this lower expression was maintained in vitro upon air-liquid interface and submerged culturing. Importantly, reduced caveolin-1 expression was accompanied by a loss of junctional E-cadherin and ß-catenin expression, disrupted epithelial barrier function, and increased levels of the proallergic cytokine thymic stromal lymphopoietin (TSLP). Furthermore, E-cadherin redistribution upon exposure to epidermal growth factor or house dust mite was paralleled by the internalization of caveolin-1 in 16HBE cells. These effects appear to be causally related, because the short, interfering RNA down-regulation of caveolin-1 resulted in the delocalization of E-cadherin and barrier dysfunction in 16HBE cells. Moreover, caveolin-1 overexpression improved barrier function and reduced TSLP expression in BEAS-2B cells. Together, our data demonstrate a crucial role for caveolin-1 in epithelial cell-cell adhesion, with important consequences for epithelial barrier function and the promotion of Th2 responses in asthma.

The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter.

BACKGROUND: The airway epithelium is the first line of defense against inhaled insults and therefore must be capable of coordinating appropriate inflammatory and immune responses. OBJECTIVE: We sought to test the hypothesis that the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome, an intracellular danger-sensing complex, plays a critical role in airway epithelium-mediated immune responses to urban particulate matter (PM) exposure. METHODS: In this study we (1) identified NLRP3 and caspase-1 expression in human airway epithelium bronchus and primary cells, (2) characterized NLRP3 inflammasome-mediated IL-1ß production from human airway epithelium in response to PM, and (3) performed in vivo PM exposure experiments with wild-type and Nlrp3(-/-) mice. RESULTS: Our results demonstrate that human airway epithelium contains a functional NLRP3 inflammasome that responds to PM exposure with caspase-1 cleavage and production of IL-1ß. Exposure of Nlrp3(-/-) and wild-type mice to PM in vivo demonstrates NLRP3-dependent production of IL-1ß in the lung, airway neutrophilia, and increases in CD11c(+hi)/MHC class II(+hi) cell numbers in intrathoracic lymph nodes. CONCLUSION: Our study is the first to characterize airway epithelial NLRP3 inflammasome-mediated immune responses to PM exposure, which might have implications in patients with asthma and other lung diseases.

Intrinsic phenotypic differences of asthmatic epithelium and its inflammatory responses to respiratory syncytial virus and air pollution.

A substantial proportion of healthcare cost associated with asthma is attributable to exacerbations of the disease. Within the airway, the epithelium forms the mucosal immune barrier, the first structural cell defense against common environmental insults such as respiratory syncytial virus (RSV) and particulate matter. We sought to characterize the phenotype of differentiated asthmatic-derived airway epithelial cultures and their intrinsic inflammatory responses to environmental challenges. Air-liquid interface (ALI) cultures were generated from asthmatic (n = 6) and nonasthmatic (n = 6) airway epithelial cells. Airway tissue and ALI cultures were analyzed by immunohistochemistry for cytokeratin-5, E-cadherin, Ki67, Muc5AC, NF-κB, the activation of p38, and apoptosis. ALI cultures were exposed to RSV (4 × 10(6) plaque forming unit/ml), particulate matter collected by Environmental Health Canada (EHC-93, 100 µg/ml), or mechanically wounded for 24, 48, and 96 hours and basolateral supernatants analyzed for inflammatory cytokines, using Luminex and ELISA. The airway epithelium in airway sections of patients with asthma as well as in vitro ALI cultures demonstrated a less differentiated epithelium, characterized by elevated numbers of basal cells marked by the expression of cytokeratin-5, increased phosphorylation of p38 mitogen-activated protein kinase, and less adherens junction protein E-cadherin. Transepithelial resistance was not different between asthmatic and nonasthmatic cultures. In response to infection with RSV, exposure to EHC-93, or mechanical wounding, asthmatic ALI cultures released greater concentrations of IL-6, IL-8, and granulocyte macrophage colony-stimulating factor, compared with nonasthmatic cultures (P < 0.05). This parallel ex vivo and in vitro study of the asthmatic epithelium demonstrates an intrinsically altered phenotype and aberrant inflammatory response to common environmental challenges, compared with nonasthmatic epithelium.

A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis.

OBJECTIVE: Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC. METHODS AND RESULTS: EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing. CONCLUSIONS: Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.