Biology subjects

Rowe, S. M.

Publications and source records attributed to Rowe, S. M..

8 recordsLinked to original sources

bioRxiv2024

ACE-2 Blockade & TMPRSS2 Inhibition Mitigate SARS-CoV-2 Severity Following Cigarette Smoke Exposure in Airway Epithelial Cells In Vitro

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

cell biology↗

bioRxiv2024

Loss of cell junctional components and matrix alterations drive cell desquamation and fibrotic changes in Idiopathic Pulmonary Fibrosis

The distal bronchioles in Idiopathic Pulmonary Fibrosis (IPF) exhibit histopathological abnormalities such as bronchiolization, peribronchiolar fibrosis and honeycomb cysts that contribute to the overall architectural remodeling of lung tissue seen in the disease. Here we describe an additional histopathologic finding of epithelial desquamation in patients with IPF, wherein epithelial cells detach from the basement membrane of the distal bronchioles. To understand the mechanism driving this pathology, we performed spatial transcriptomics of the epithelial cells and spatial proteomics of the basement membrane of the distal bronchioles from IPF patients and patients with no prior history of lung disease. Our findings reveal a downregulation of cell junctional components, upregulation of epithelial-mesenchymal transition signatures and dysregulated basement membrane matrix in IPF distal bronchioles, facilitating epithelial desquamation. Further, functional assays identified regulation between Collagen IV in the matrix, and the junctional genes JUP and PLEC, that is crucial for maintaining distal bronchiolar homeostasis. In IPF, this balanced regulation between matrix and cell-junctions is disrupted, leading to loss of epithelial adhesion, peribronchiolar fibrosis and epithelial desquamation. Overall, our study suggests that in IPF the interplay between the loss of cell junctions and a dysregulated matrix results in desquamation of distal bronchiolar epithelium and lung remodeling, exacerbating the disease. One Sentence SummaryTwo-way regulation of cell junctional proteins and matrix proteins drives cellular desquamation and fibrosis in the distal bronchioles of patients with Idiopathic Pulmonary Fibrosis.

pathology↗

bioRxiv2024

Pulmonary Fibrosis Ferret Model Demonstrates Sustained Fibrosis, Restrictive Physiology, and Aberrant Repair

RationaleThe role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. MethodsBleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with {micro}CT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. ResultsBleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric {micro}CT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant proximalization repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. ConclusionsBleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

physiology↗

bioRxiv2024

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells

BackgroundCystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. MethodsA novel small molecule potentiator (SK-POT1), previously identified in CFTR binding studies, was tested for its activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. ResultsWe showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

physiology↗

bioRxiv2023

Mucociliary Clearance Augmenting Drugs Block SARS-Cov-2 Replication in Human Airway Epithelial Cells

The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve barrier function of the airway epithelium, reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury, thus, it was further studied using biochemical, genetic and biophysical methods to ascertain mechanism of action via improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression and motion was required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that Intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.

microbiology↗

bioRxiv2022

COVID-19 Causes Ciliary Dysfunction as Demonstrated by Human Intranasal Micro-Optical Coherence Tomography Imaging

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), causative agent of coronavirus disease 2019 (COVID-19), binds via ACE2 receptors, highly expressed in ciliated cells of the nasal epithelium. Micro-optical coherence tomography (OCT) is a minimally invasive intranasal imaging technique that can determine cellular and functional dynamics of respiratory epithelia at 1-m resolution, enabling real time visualization and quantification of epithelial anatomy, ciliary motion, and mucus transport. We hypothesized that respiratory epithelial cell dysfunction in COVID-19 will manifest as reduced ciliated cell function and mucociliary abnormalities, features readily visualized by OCT. Symptomatic outpatients with SARS-CoV-2 aged [≥] 18 years were recruited within 14 days of symptom onset. Data was interpreted for subjects with COVID-19 (n=13) in comparison to healthy controls (n=8). Significant reduction in functional cilia, diminished ciliary beat frequency, and abnormal ciliary activity were evident. Other abnormalities included denuded epithelium, presence of mucus rafts, and increased inflammatory cells. Our results indicate that subjects with mild but symptomatic COVID-19 exhibit functional abnormalities of the respiratory mucosa underscoring the importance of mucociliary health in viral illness and disease transmission. Ciliary imaging enables investigation of early pathogenic mechanisms of COVID-19 and may be useful for evaluating disease progression and therapeutic response. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/499336v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1f4bb17org.highwire.dtl.DTLVardef@271438org.highwire.dtl.DTLVardef@1a08762org.highwire.dtl.DTLVardef@184847_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

microbiology↗

bioRxiv2021

Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10kb C-to-T splicing mutation

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently been approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10kb C-to-T splicing mutation in the CFTR gene. We have screened, in FRT cells expressing this mutation, ~30 ASOs chemically modified with 2'-O-Methyl on a phosphrothioate backbone, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The screening identified five ASO candidates able to promote CFTR correct splicing and rescue channel activity. Further analyses in well differentiated primary human nasal and bronchial epithelial cells (HNEs, HBEs), derived from patients carrying at least one 3849+10kb C-to-T allele, led to the identification of a highly potent lead ASO. The ASO was efficiently delivered by free uptake into patients HNEs and HBEs and completely restored CFTR function to wild type levels in cells from a homozygous patient and led to 43{+/-}8% of wild type levels in cells from various heterozygous patients. Optimized efficiency was further obtained with 2-Methoxy Ethyl chemical modification. The results demonstrate the therapeutic potential and clinical benefit of ASO-based splicing modulation for genetic diseases caused by splicing mutations.

genetics↗