Ventilator-induced lung injury results in oxidative stress response and mitochondrial swelling in a mouse model

© 2022. The Author(s).BACKGROUND: Mechanical ventilation is a life-saving therapy for critically ill patients, providing rest to the respiratory muscles and facilitating gas exchange in the lungs. Ventilator-induced lung injury (VILI) is an unfortunate side effect of mechanical ventilation that may lead to serious consequences for the patient and increase mortality. The four main injury mechanisms associated with VILI are: baro/volutrauma caused by overstretching the lung tissues; atelectrauma, caused by repeated opening and closing of the alveoli resulting in shear stress; oxygen toxicity due to use of high ratio of oxygen in inspired air, causing formation of free radicals; and biotrauma, the resulting biological response to tissue injury, that leads to a cascade of events due to excessive inflammatory reactions and may cause multi-organ failure. An often-overlooked part of the inflammatory reaction is oxidative stress. In this research, a mouse model of VILI was set up with three tidal volume settings (10, 20 and 30 mL/kg) at atmospheric oxygen level. Airway pressures and heart rate were monitored and bronchoalveolar lavage fluid (BALF) and lung tissue samples were taken. RESULTS: We show a correlation between increased inflammation and barrier failure, and higher tidal volumes, evidenced by increased IL-6 expression, high concentration of proteins in BALF along with changes in expression of adhesion molecules. Furthermore, swelling of mitochondria in alveolar type II cells was seen indicating their dysfunction and senescence-like state. RNA sequencing data present clear increases in inflammation, mitochondrial biogenesis and oxidative stress as tidal volume is increased, supported by degradation of Keap1, a redox-regulated substrate adaptor protein. CONCLUSIONS: Oxidative stress seems to be a more prominent mechanism of VILI than previously considered, indicating that possible treatment methods against VILI might be identified by impeding oxidative pathways.Peer reviewe

Azithromycin ameliorates sulfur dioxide-induced airway epithelial damage and inflammatory responses

Publisher's version (útgefin grein)Background: The airway epithelium (AE) forms the first line of defence against harmful particles and pathogens. Barrier failure of the airway epithelium contributes to exacerbations of a range of lung diseases that are commonly treated with Azithromycin (AZM). In addition to its anti-bacterial function, AZM has immunomodulatory effects which are proposed to contribute to its clinical effectiveness. In vitro studies have shown the AE barrier-enhancing effects of AZM. The aim of this study was to analyze whether AE damage caused by inhalation of sulfur dioxide (SO2) in a murine model could be reduced by pre-treatment with AZM. Methods: The leakiness of the AE barrier was evaluated after SO2 exposure by measuring levels of human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). Protein composition in BALF was also assessed and lung tissues were evaluated across treatments using histology and gene expression analysis. Results: AZM pre-treatment (2 mg/kg p.o. 5 times/week for 2 weeks) resulted in reduced glutathione-S-transferases in BALF of SO2 injured mice compared to control (without AZM treatment). AZM treated mice had increased intracellular vacuolization including lamellar bodies and a reduction in epithelial shedding after injury in addition to a dampened SO2-induced inflammatory response. Conclusions: Using a mouse model of AE barrier dysfunction we provide evidence for the protective effects of AZM in vivo, possibly through stabilizing the intracellular microenvironment and reducing inflammatory responses. Our data provide insight into the mechanisms contributing to the efficacy of AZM in the treatment of airway diseases.This work was supported by the Icelandic Research Council Technical Fund (Rannís Grant numbers: 186943–0611) and EpiEndo Pharmaceuticals.Peer Reviewe

Azithromycin induces epidermal differentiation and multivesicular bodies in airway epithelia.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadBACKGROUND: Azithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression. METHODS: VA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present. RESULTS: Bioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives. CONCLUSIONS: Taken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.Icelandic Research Council EpiEndo Pharmaceuticals, Reykjavik, Icelan

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

<p>Abstract</p> <p>Background</p> <p>Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D) co-culture assay.</p> <p>Methods</p> <p>Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy.</p> <p>Results</p> <p>In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors.</p> <p>Conclusion</p> <p>Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.</p

A man and woman crossing the road near Shillidays building, Mildura, Victoria, 1961 [picture] /

Title devised by cataloguer based on information from acquisition documentation.; Also available in an electronic version via the Internet at: http://nla.gov.au/nla.pic-vn3995269; Purchased from the photographer, 2007

Paul Petroff as Ivan Tsarevitch (centre in group on left), Tamara Grigorieva as The Beautiful Tsarevna (kneeling centre), David Lichine as The Immortal Kostchei (hunched over on right), and artists of the company, in L'oiseau de feu, Original Ballet Russe, Australian tour, His Majesty's Theatre, Melbourne, April 1940 [picture] /

Part of the collection: Hugh P. Hall collection of photographs, 1938-1940.; From: L'oiseau de feu (The fire bird) : ballet in two scenes / by Michel Fokine, drawn from Russian folk tales, music by Igor Stravinsky.; Inscription: "4K/19".; Choreography by Michel Fokine ; scenery and costumes by Nathalie Gontcharova.; Also available in an electronic version via the Internet at: http://nla.gov.au/nla.pic-vn3995905. One of a collection of photographs taken by Hugh P. Hall of 28 ballet productions performed by the Covent Garden Russian Ballet (toured Australia 1938-1939) and the Original Ballet Russe (toured Australia 1939-1940). These are the second and third of the three Ballets Russes companies which toured Australasia between 1936 and 1940. The photographs were taken from the auditorium during a live performance in His Majesty's Theatre, Melbourne and mounted on cardboard for display purposes. For conservation and storage, the photographs have been demounted. The original arrangement of the photographs has been recorded, and details are available from the Pictures Branch of the National Library

Epithelial integrity is disturbed in SPRY2 KD cells when co-cultured with endothelial cells.

<p><i>A) Endothelial cells stimulate growth of D492 cells.</i> When plated in 3D rBM culture with breast endothelial cells (BRENCs), D492 cells can form complex branching colonies from as little as 100–1000 cells compared to 7×10³ –10⁴ in 3D monoculture. <i>B) D492-derived branching structures form bi-layered epithelium with BRENCs positioned extralobular.</i> The branching colonies are bi-layered and polarized structures as evidenced by the expression of the myoepithelial marker CK14 on the outer side and the luminal epithelial marker CK19 on the inner side (upper figure). In co-cultures endothelial cells stay as single cells positioned outside the branching structures as seen with CD31 staining (lower figure, arrows). Bar  =  100 µm. Sections counterstained with TOPRO-3 nuclear stain. <i>C) Phenotypes of D492 in co-culture with BRENCs.</i> In co-culture with endothelial cells D492 cells form both branching- and spindle-like colonies. The branching colonies show strong expression of E-cadherin, while spindle like colonies have undergone EMT as evidenced by cadherin switch from E- to N-cadherin. Staining for the proliferation marker ki67 shows that both the branching and spindle like colonies are viable and growing. Bar  =  100 µm. Sections counterstained with TOPRO-3 nuclear stain. <i>D) Spry2-KD cells show an increase in the spindle-like morphology.</i> While D492^NS cells form about 40% spindle-like colonies there is a significant increase in the D492^Spry2-KD3 cells up to 65%. The D492^Spry2-KD3A form almost exclusively spindle-like colonies in co-culture with endothelial cells.</p

SPRY2 Knockdown in D492 breast epithelial stem cell line.

<p><i>A) D492 cells show significant knockdown of SPRY2.</i> D492 were transfected with non-silencing (NS) shRNA and different version of knockdown (KD) shRNA against SPRY2. KD3 showed most efficient knockdown (70%) measured by western blot. KD3A is a single cell cloned subline from KD3. <i>B) D492SPRY2^-KD3 retains an epithelial phenotype in monolayer culture.</i> No phenotypic differences were observed in monolayer of D492^NS and <i>D492^SPRY2-KD3</i> (upper row). Transfection efficacy was evaluated by GFP (lower row). <i>C) D492^SPRY2-KD cells have acquired increased migration potential.</i> When plated on porous transwell filter <i>D492^SPRY2-KD3</i> showed increased migration compared to D492^NS. Single cell derived clone KD3A from KD3 had the highest migration potential. <i>D) SPRY2 knockdown has no effect on cell proliferation</i>. Monolayer proliferation of D492^NS, D492^SPRY2-KD3 and D492^SPR2-KD3A was evaluated at different time points, as indicated. There was no remarkable difference in the proliferation rate of the NS and KD cells, although at day 4 D492^SPRY2-KD3A seemed to proliferate slightly less.</p