Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Cigarette smoke (CS) drives disease development and progression. The epithelial barrier is damaged by CS with increased monolayer permeability. However, the molecular changes that cause this barrier disruption and the interaction between adhesion proteins and the cytoskeleton are not well defined. We hypothesized that CS alters monolayer integrity by increasing cell contractility and decreasing cell adhesion in epithelia. Normal human airway epithelial cells and primary COPD epithelial cells were exposed to air or CS, and changes measured in protein levels. We measured the cortical tension of individual cells and the stiffness of cells in a monolayer. We confirmed that the changes in acute and subacute in vitro smoke exposure reflect protein changes seen in cell monolayers and tissue sections from COPD patients. Epithelial cells exposed to repetitive CS and those derived from COPD patients have increased monolayer permeability. E-cadherin and β-catenin were reduced in smoke exposed cells as well as in lung tissue sections from patients with COPD. Moreover, repetitive CS caused increased tension in individual cells and cells in a monolayer, which corresponded with increased polymerized actin without changes in myosin IIA and IIB total abundance. Repetitive CS exposure impacts the adhesive intercellular junctions and the tension of epithelial cells by increased actin polymer levels, to further destabilize cell adhesion. Similar changes are seen in epithelial cells from COPD patients indicating that these findings likely contribute to COPD pathology.

Chronic obstructive pulmonary disease (COPD) was the fourth leading cause of death worldwide in 2015. Current treatments for patients ease discomfort and help decrease disease progression; however, none improve lung function or change mortality. COPD is heterogeneous in its molecular and clinical presentation, making it difficult to understand disease aetiology and define robust therapeutic strategies. Given the complexity of the disease we propose a precision medicine approach to understanding and better treating COPD. It is possible that multiOMICs can be used as a tool to integrate data from multiple fields. Moreover, analysis of electronic medical records could aid in the treatment of patients and in the predictions of outcomes. The Precision Medicine Initiative created in 2015 has made precision medicine approaches to treat disease a reality; one of these diseases being COPD.

Background Taking into consideration a recent surge of a lung injury condition associated with electronic cigarette use, we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface, to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS), e-cigarette aerosol (EC), and tobacco waterpipe exposures (TW). Methods Products analyzed include commercially available e-liquid, with 0% or 1.2% concentration of nicotine, tobacco blend (shisha), and reference-grade cigarette (3R4F). In one set of experiments, HBECs were exposed to EC (0 and 1.2%), CS or control air for 10 days using 1 cigarette/day. In the second set of experiments, exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day, every other day, until 3 exposures were performed. After 16–18 h of last exposure, we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate–dextran flux assay (FITC-Dextran), measurements of trans-electrical epithelial resistance (TEER), assessment of the percentage of moving cilia, cilia beat frequency (CBF), cell motion, and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Results When compared to air control, CS increased fluorescence (FITC-Dextran assay) by 5.6 times, whereby CS and EC (1.2%) reduced TEER to 49 and 60% respectively. CS and EC (1.2%) exposure reduced CBF to 62 and 59%, and cilia moving to 47 and 52%, respectively, when compared to control air. CS and EC (1.2%) increased cell velocity compared to air control by 2.5 and 2.6 times, respectively. The expression of E-cadherin reduced to 39% of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether, EC (0%) and TW exposures resulted in more moderate decreases in epithelial integrity, while EC (1.2%) substantially decreased airway epithelial barrier function comparable with CS exposure. Conclusions The results support a toxic effect of sub-chronic exposure to EC (1.2%) as evident by disruption of the bronchial epithelial cell barrier integrity, whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0%) toxicity in chronic exposures.

Epithelial cells line the lung mucosal surface and are the first line of defense against toxic exposures to environmental insults, and their integrity is critical to lung health. An early finding in the lung epithelium of patients with chronic obstructive pulmonary disease (COPD) is the loss of a key component of the adherens junction protein called E-cadherin. The cause of this decrease is not known and could be due to luminal insults or structural changes in the small airways. Irrespective, it is unknown whether the loss of E-cadherin is a marker or a driver of disease. Here we report that loss of E-cadherin is causal to the development of chronic lung disease. Using cell-type-specific promoters, we find that knockout of E-cadherin in alveolar epithelial type II but not type 1 cells in adult mouse models results in airspace enlargement. Furthermore, the knockout of E-cadherin in airway ciliated cells, but not club cells, increase airway hyperreactivity. We demonstrate that strategies to upregulate E-cadherin rescue monolayer integrity and serve as a potential therapeutic target.

Air-liquid interface (ALI) cultures are ex vivo models that are used extensively to study the epithelium of patients with chronic respiratory diseases. However, the in vitro conditions impose a milieu different from that encountered in the patient in vivo, and the degree to which this alters gene expression remains unclear. In this study we employed RNA sequencing to compare the transcriptome of fresh brushings of nasal epithelial cells with that of ALI-cultured epithelial cells from the same patients. We observed a strong correlation between cells cultured at the ALI and cells obtained from the brushed nasal epithelia: 96% of expressed genes showed similar expression profiles, although there was greater similarity between the brushed samples. We observed that while the ALI model provides an excellent representation of the in vivo airway epithelial transcriptome for mechanistic studies, several pathways are affected by the change in milieu.

The airway epithelium is subjected to insults such as cigarette smoke (CS), a primary cause of Chronic Obstructive Pulmonary Disease (COPD) and serves as an excellent model to study cell plasticity. Both CS-exposed and COPD-patient derived epithelia (CHBE) display quantitative evidence of cellular plasticity, with loss of specialized apical features and a transcriptional profile suggestive of partial epithelial to mesenchymal transition, albeit with distinct cell motion indicative of cellular unjamming. These injured/diseased cells have an increased fraction of polymerized actin, due to loss of the actin-severing protein, cofilin-1. Decreasing polymerized actin restores the jammed state in both CHBE and CS exposed epithelia, indicating that the fraction of polymerized actin is critical in unjamming the epithelia. Kinetic energy spectral analysis suggests that loss of cofilin-1 results in unjamming, similar to that seen with both CS exposure and in CHBE cells. Our data suggest that in response to chronic injury, although epithelial cells display evidence of pEMT, their movement is more consistent with cellular unjamming. Inhibitors of actin polymerization rectify the unjamming features of the monolayer.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.

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