YKL-40/CHI3L1 facilitates migration and invasion in HER2 overexpressing breast epithelial progenitor cells and generates a niche for capillary-like network formation

Publisher's version (útgefin grein).Epithelial to mesenchymal transition (EMT) is a developmental event that is hijacked in some diseases such as fibrosis and cancer. In cancer, EMT has been linked to increased invasion and metastasis and is generally associated with a poor prognosis. In this study, we have compared phenotypic and functional differences between two isogenic cell lines with an EMT profile: D492M and D492HER2 that are both derived from D492, a breast epithelial cell line with stem cell properties. D492M is non-tumorigenic while D492HER2 is tumorigenic. Thus, the aim of this study was to analyze the expression profile of these cell lines, identify potential oncogenes, and evaluate their effects on cellular phenotype. We performed transcriptome and secretome analyses of D492M and D492HER2 and verified expression of selected genes at the RNA and protein level. One candidate, YKL-40 (also known as CHI3L1), was selected for further studies due to its differential expression between D492M and D492HER2, being considerably higher in D492HER2. YKL-40 has been linked to chronic inflammation diseases and cancer, yet its function is not fully understood. Knock-down experiments of YKL-40 in D492HER2 resulted in reduced migration and invasion as well as reduced ability to induce angiogenesis in an in vitro assay, plus changes in the EMT-phenotype. In summary, our data suggest that YKL-40 may provide D492HER2 with increased aggressiveness, supporting cancer progression and facilitating angiogenesis.This work was supported by Grants from Landspitali University Hospital Science Fund, University of Iceland Research Fund, and Icelandic Science and Technology Policy—Grant of Excellence: 152144051. ‘Göngum saman,’ a supporting group for breast cancer research in Iceland ( www.gongumsaman.is ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

YKL-40/CHI3L1 facilitates migration and invasion in HER2 overexpressing breast epithelial progenitor cells and generates a niche for capillary-like network formation

Publisher's version (útgefin grein).Epithelial to mesenchymal transition (EMT) is a developmental event that is hijacked in some diseases such as fibrosis and cancer. In cancer, EMT has been linked to increased invasion and metastasis and is generally associated with a poor prognosis. In this study, we have compared phenotypic and functional differences between two isogenic cell lines with an EMT profile: D492M and D492HER2 that are both derived from D492, a breast epithelial cell line with stem cell properties. D492M is non-tumorigenic while D492HER2 is tumorigenic. Thus, the aim of this study was to analyze the expression profile of these cell lines, identify potential oncogenes, and evaluate their effects on cellular phenotype. We performed transcriptome and secretome analyses of D492M and D492HER2 and verified expression of selected genes at the RNA and protein level. One candidate, YKL-40 (also known as CHI3L1), was selected for further studies due to its differential expression between D492M and D492HER2, being considerably higher in D492HER2. YKL-40 has been linked to chronic inflammation diseases and cancer, yet its function is not fully understood. Knock-down experiments of YKL-40 in D492HER2 resulted in reduced migration and invasion as well as reduced ability to induce angiogenesis in an in vitro assay, plus changes in the EMT-phenotype. In summary, our data suggest that YKL-40 may provide D492HER2 with increased aggressiveness, supporting cancer progression and facilitating angiogenesis.This work was supported by Grants from Landspitali University Hospital Science Fund, University of Iceland Research Fund, and Icelandic Science and Technology Policy—Grant of Excellence: 152144051. ‘Göngum saman,’ a supporting group for breast cancer research in Iceland ( www.gongumsaman.is ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

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

Innovative in vitro method to study ventilator induced lung injury.

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 DownloadMechanical ventilation (MV) is a life-saving therapy for critically ill patients, alleviating the work of breathing and supporting adequate gas exchange. However, MV can cause ventilator induced lung injury (VILI) by baro/volu- and atelectrauma, even lead to acute respiratory distress syndrome (ARDS), and substantially augment mortality. There is a need for specific biomarkers and novel research platforms for VILI/ARDS research to study these detrimental disorders and seek ways to avoid or prevent them. Previous in vitro studies on bronchial epithelium, cultured in air-liquid interface (ALI) conditions, have generally utilized static or constant pressure. We have developed a Cyclical Pressure ALI Device (CPAD) that enables cyclical stress on ALI cultured human bronchial cells, with the aim of mimicking the effects of MV. Using CPAD we were able to analyze differentially expressed VILI/ARDS and innate immunity associated genes along with increased expression of associated proteins. CPAD provides an easy and accessible way to analyze functional and phenotypic changes that occur during VILI and may provide a platform for future drug testing.Technology development fund - Icelandic research council University of Iceland Land-spitali, University Hospital, Science fun

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

Evanescent-wave fluorescence microscopy using symmetric planar waveguides

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldWe describe a new evanescent-wave fluorescence excitation method, ideally suited for imaging of biological samples. The excitation light propagates in a planar optical waveguide, consisting of a thin waveguide core sandwiched between a sample in an aqueous solution and a polymer with a matching refractive index, forming a symmetric cladding environment. This configuration offers clear advantages over other waveguide-excitation methods, such as superior image quality, wide tunability of the evanescent field penetration depth and compatibility with optical fibers. The method is well suited for cell membrane imaging on cells in culture, including cell-cell and cell-matrix interaction, monitoring of surface binding events and similar applications involving aqueous solutions

Ventilator-induced lung-injury in mouse models: Is there a trap?

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 DownloadVentilator-induced lung injury (VILI) is a serious acute injury to the lung tissue that can develop during mechanical ventilation of patients. Due to the mechanical strain of ventilation, damage can occur in the bronchiolar and alveolar epithelium resulting in a cascade of events that may be fatal to the patients. Patients requiring mechanical ventilation are often critically ill, which limits the possibility of obtaining patient samples, making VILI research challenging. In vitro models are very important for VILI research, but the complexity of the cellular interactions in multi-organ animals, necessitates in vivo studies where the mouse model is a common choice. However, the settings and duration of ventilation used to create VILI in mice vary greatly, causing uncertainty in interpretation and comparison of results. This review examines approaches to induce VILI in mouse models for the last 10 years, to our best knowledge, summarizing methods and key parameters presented across the studies. The results imply that a more standardized approach is warranted. Keywords: Acute lung injury; Animal models; Mouse studies; Ventilator-induced lung injury.Landspitali research fun