Prospects for macrolide therapy of asthma and COPD.

Macrolide compounds, many of which are derived from natural sources, all share a lactone ring structure, but of varying sizes. Their biological activities differ with structure and size but tend to overlap. Marketed macrolide drugs include immunosuppressives and antibiotics. Some of the latter have been shown to exert anti-inflammatory activities, due to direct effects on inflammatory cells and processes when used for respiratory infections. Consequently, azithromycin is included in clinical guidelines for COPD and asthma treatment, though it has the disadvantage, as an antibiotic, of increasing bacterial resistance. COPD and asthma, however, like several chronic inflammatory diseases involving other organs, are driven to a large extent by epithelial barrier dysfunction. Recently, azithromycin was shown to directly enhance epithelial barrier function and a new class of derivatives, barriolides, is under development with the lead indication COPD. It is thus likely that by circumventing antibiosis and acting on a crucial etiological disease process, this type of agent will open up a new, safer approach to COPD and asthma therapy with macrolides.

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.

Factors Determining Plasticity of Responses to Drugs.

The plasticity of responses to drugs is an ever-present confounding factor for all aspects of pharmacology, influencing drug discovery and development, clinical use and the expectations of the patient. As an introduction to this Special Issue of the journal IJMS on pharmacological plasticity, we address the various levels at which plasticity appears and how such variability can be controlled, describing the ways in which drug responses can be affected with examples. The various levels include the molecular structures of drugs and their receptors, expression of genes for drug receptors and enzymes involved in metabolism, plasticity of cells targeted by drugs, tissues and clinical variables affected by whole body processes, changes in geography and the environment, and the influence of time and duration of changes. The article provides a rarely considered bird's eye view of the problem and is intended to emphasize the need for increased awareness of pharmacological plasticity and to encourage further debate.

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.

Nonantimicrobial Actions of Macrolides: Overview and Perspectives for Future Development.

Macrolides are among the most widely prescribed broad spectrum antibacterials, particularly for respiratory infections. It is now recognized that these drugs, in particular azithromycin, also exert time-dependent immunomodulatory actions that contribute to their therapeutic benefit in both infectious and other chronic inflammatory diseases. Their increased chronic use in airway inflammation and, more recently, of azithromycin in COVID-19, however, has led to a rise in bacterial resistance. An additional crucial aspect of chronic airway inflammation, such as chronic obstructive pulmonary disease, as well as other inflammatory disorders, is the loss of epithelial barrier protection against pathogens and pollutants. In recent years, azithromycin has been shown with time to enhance the barrier properties of airway epithelial cells, an action that makes an important contribution to its therapeutic efficacy. In this article, we review the background and evidence for various immunomodulatory and time-dependent actions of macrolides on inflammatory processes and on the epithelium and highlight novel nonantibacterial macrolides that are being studied for immunomodulatory and barrier-strengthening properties to circumvent the risk of bacterial resistance that occurs with macrolide antibacterials. We also briefly review the clinical effects of macrolides in respiratory and other inflammatory diseases associated with epithelial injury and propose that the beneficial epithelial effects of nonantibacterial azithromycin derivatives in chronic inflammation, even given prophylactically, are likely to gain increasing attention in the future. SIGNIFICANCE STATEMENT: Based on its immunomodulatory properties and ability to enhance the protective role of the lung epithelium against pathogens, azithromycin has proven superior to other macrolides in treating chronic respiratory inflammation. A nonantibiotic azithromycin derivative is likely to offer prophylactic benefits against inflammation and epithelial damage of differing causes while preserving the use of macrolides as antibiotics.

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.

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.

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

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.

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.

MiR-203a is differentially expressed during branching morphogenesis and EMT in breast progenitor cells and is a repressor of peroxidasin.

MicroRNAs regulate developmental events such as branching morphogenesis, epithelial to mesenchymal transition (EMT) and its reverse process mesenchymal to epithelial transition (MET). In this study, we performed small RNA sequencing of a breast epithelial progenitor cell line (D492), and its mesenchymal derivative (D492M) cultured in three-dimensional microenvironment. Among the most downregulated miRNAs in D492M was miR-203a, a miRNA that plays an important role in epithelial differentiation. Increased expression of miR-203a was seen in D492, concomitant with increased complexity of branching. When miR-203a was overexpressed in D492M, a partial reversion towards epithelial phenotype was seen. Gene expression analysis of D492M and D492MmiR-203a revealed peroxidasin, a collagen IV cross-linker, as the most significantly downregulated gene in D492MmiR-203a. Collectively, we demonstrate that miR-203a expression temporally correlates with branching morphogenesis and is suppressed in D492M. Overexpression of miR-203a in D492M induces a partial MET and reduces the expression of peroxidasin. Furthermore, we demonstrate that miR-203a is a novel repressor of peroxidasin. MiR-203-peroxidasin axis may be an important regulator in branching morphogenesis, EMT/MET and basement membrane remodeling.

Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

Context-Dependent Function of Myoepithelial Cells in Breast Morphogenesis and Neoplasia.

Myoepithelial cells (MEPs) are specialized cells derived from epithelial progenitor cells, yet they also express the contractile machinery of smooth muscle cells. MEPs are prominent in glandular tissues where their function is to help expel secretions generated by the glandular epithelial cells. In the breast, MEPs are part of the bi-layered breast epithelium that line ducts and alveoli positioned perpendicular to the luminal epithelial cells (LEPs), separated from the surrounding stroma by the basement membrane. Researchers have recognized MEPs as important regulators of structural and functional behavior of LEPs, namely having role in polarization of LEPs, and regulating milk production. Furthermore, they have also been proposed to act as tumor suppressors as their presence inhibits invasion of cancer cells into the surrounding stroma. There is, however, accumulating evidence that MEPs in normal breast, carcinoma in situ and in invasive breast cancer differ significantly in terms of marker expression and this may truly interfere with their ability to behave as tumor suppressors. The term myoepithelial cell is often used synonymously with basal cell. While all MEPs, due to their position, can be referred to as basal cells, some basal cells do not fulfill the criteria of being MEPs. Synonymous use of these terms may hold true under normal conditions but careful interpretation of these terms should be used in breast cancer. In recent years, partial myoepithelial differentiation and epithelial to mesenchymal transition (EMT) have been shown to be associated with, and in some cases, necessary for cancer invasion and metastasis. In this review, we will discuss the context-dependent role of MEPs in breast morphogenesis, tumor suppression, and also the appearance of basal or partial myoepithelial differentiation in aggressive forms of breast cancer.

Mechanistic investigations into interactions between IGF-I and IGFBPs and their impact on facilitating cell migration on vitronectin.

Numerous studies have reported links between insulin-like growth factors (IGFs) and the extra-cellular matrix protein vitronectin (VN). We ourselves have reported that IGF-I binds to VN via IGF-binding proteins (IGFBPs) to stimulate HaCaT and MCF-7 cell migration. Here, we detail the functional evaluation of IGFBP-1, -2, -3, -4 and -6 in the presence and absence of IGF-I and VN. The data presented here, combined with our prior data on IGFBP-5, suggest that IGFBP-3, -4 and -5 are the most effective at stimulating cell migration in combination with IGF-I and VN. In addition, we demonstrate that different regions within IGFBP-3 and -4 are critical for complex formation. Furthermore, we examine whether multi-protein complexes of IGF-I and IGFBPs associated with fibronectin and collagen IV are also able to enhance functional biological responses.

Substrate-bound insulin-like growth factor (IGF)-I-IGF binding protein-vitronectin-stimulated breast cell migration is enhanced by coactivation of the phosphatidylinositide 3-Kinase/AKT pathway by alphav-integrins and the IGF-I receptor.

IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.

Structure and function of a cellulase gene in redclaw crayfish, Cherax quadricarinatus.

The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes. Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al. [Gene 239 (1999) 317-324.]. Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene. Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase. Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes. The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2). Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region. Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene. These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes.

Structural and functional evidence for the interaction of insulin-like growth factors (IGFs) and IGF binding proteins with vitronectin.

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and (125)iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells.