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

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.

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

Ventilator-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.

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

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.

Transforming growth factor ß-mediated micromechanics modulates disease progression in primary myelofibrosis.

Primary myelofibrosis (PMF) is a Ph-negative myeloproliferative neoplasm (MPN), characterized by advanced bone marrow fibrosis and extramedullary haematopoiesis. The bone marrow fibrosis results from excessive proliferation of fibroblasts that are influenced by several cytokines in the microenvironment, of which transforming growth factor-ß (TGF-ß) is the most important. Micromechanics related to the niche has not yet been elucidated. In this study, we hypothesized that mechanical stress modulates TGF-ß signalling leading to further activation and subsequent proliferation and invasion of bone marrow fibroblasts, thus showing the important role of micromechanics in the development and progression of PMF, both in the bone marrow and in extramedullary sites. Using three PMF-derived fibroblast cell lines and transforming growth factor-ß receptor (TGFBR) 1 and 2 knock-down PMF-derived fibroblasts, we showed that mechanical stress does stimulate the collagen synthesis by the fibroblasts in patients with myelofibrosis, through the TGFBR1, which however seems to be activated through alternative pathways, other than TGFBR2.

Melflufen, a peptide-conjugated alkylator, is an efficient anti-neo-plastic drug in breast cancer cell lines.

Melphalan flufenamide (hereinafter referred to as "melflufen") is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins Æ´H2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.

Azithromycin induces epidermal differentiation and multivesicular bodies in airway epithelia.

BACKGROUND: 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.