development-an-vitro-assay-to-evaluate-contractile-function

Development of an <em>In Vitro</em> Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

The University of Tokyo Hospital

We systematically investigated serial efficacy of granulocyte colony-stimulating factor (G-CSF) therapy upon experimental autoimmune myocarditis (EAM) in rats treated with and without the inhibition of nitric oxide (NO) with the analyses of tissue regeneration. G-CSF could mobilize multipotent progenitor cells of bone marrow into the peripheral blood and may improve ventricular function. A rat model of porcine myosin-induced EAM was used. After the immunization of myosin, G-CSF (10 microg/kg/day) or saline was injected intraperitoneally on days 0-21 in experiment 1 and on days 21-42 in experiment 2. Additional myosin-immunized rats were orally given 25 mg/kg/day of N(G)-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase (NOS), in each experiment (each group; n=8-21). Serum cytokines and peripheral blood cell counts were measured in each group. In experiment 1, G-CSF treatment aggravated cardiac pathology associated with increased macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) levels and enhanced superoxide production. In experiment 2, G-CSF treatment reduced the severity of myocarditis with increased capillary density and improved left ventricular ejection fraction. In the rats with EAM treated with G-CSF associated with oral L-NAME treatment in experiment 2, the severity of myocarditis was not reduced. Myocardial c-kit(+) cells were demonstrated only in G-CSF-treated group in experiment 2 but not in other groups. G-CSF has differential effects on EAM in rats associated with the modulation of cytokine network. The overwhelming superoxide production by G-CSF administration in the acute stage may worsen the disease. G-CSF therapy improved cardiac function via NO system in a rat model of myocarditis in the chronic stage, but not in the acute stage, possibly through the myocardial regeneration and acceleration of healing process.

Therapy with granulocyte colony-stimulating factor in the chronic stage, but not in the acute stage, improves experimental autoimmune myocarditis in rats via nitric oxide.

We assessed the hypothesis whether behavioral stress may affect the development of atherosclerosis and whether regular exercise training may influence the composition of atherosclerotic plaques in apolipoprotein (apo) E-deficient mice.

Atherosclerotic plaques induced by marble-burying behavior are stabilized by exercise training in experimental atherosclerosis.

The first case in Japan of 2009 pandemic influenza A (H1N1) was reported in May, with pediatric hospitalization exceeding that of adults. We evaluated six adults hospitalized for 2009 H1N1 respiratory complications and compared the pandemic to seasonal influenza. Hospitalization was due to aggravated asthma in four of the six, two of whom had simultaneous pneumonia probably virus-caused, and two cases of bacterial pneumonia. Among the three seasons examined, the number of adults increased slightly but the hospitalization rate was low in 2009-2010. Respiratory complications such as viral pneumonia were not seen outside of 2009-2010. Attention should therefore be paid to respiratory complications in adults with pandemic 2009 influenza A (H1N1) virus.

[Six adult cases of respiratory complications caused by pandemic 2009 influenza A (H1N1) compared to seasonal influenza].

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model.

Fibrosis is an abnormal response to organ injury, characterized by accumulation of activated fibroblasts at the sites of injury. Fibroblasts arise from several sources, including resident fibroblasts and circulating fibrocytes that infiltrate organ tissue. Recently, epithelial-mesenchymal transition (EMT) has been recognized as a source of mesenchymal cells. EMT is induced by various growth factors, such as transforming growth factor (TGF)-β1, and enhanced by inflammatory cytokines. Recently the tumor necrosis factor superfamily member LIGHT has been implicated in the pathogenesis of inflammatory disease and airway remodeling in severe asthma. We hypothesized that LIGHT might contribute to the pathogenesis of airway fibrosis via enhancement of EMT. Therefore, we investigated LIGHT's ability to induce EMT. A549 cells were stimulated with LIGHT, TGF-β1 or both for 48h. To estimate EMT, we evaluated the expression of epithelial and mesenchymal markers using immunocytochemistry, Western blotting and quantitative RT-PCR. Signaling pathways for EMT were characterized by Western analysis to detect phosphorylation of Erk1/2 and smad2. LIGHT enhanced TGF-β1-induced EMT both morphologically, by suppressing E-cadherin and enhancing vimentin, and functionally, by enhancing cell contractility. Additionally, LIGHT induced EMT without TGF-β1. Evaluation of the mechanism showed that LIGHT did not induce TGF-β1 production or affect the smad-snai1 pathway. Inhibition of Erk1/2 phosphorylation reduced LIGHT-induced EMT, indicating the Erk1/2 pathway to be a key pathway in LIGHT-induced EMT. In summary, LIGHT enhanced TGF-β1-induced EMT but also induced EMT via the Erk1/2 pathway by itself, without TGF-β1 signaling. LIGHT may contribute to the pathogenesis of airway fibrosis through enhancement of EMT.

Tumor necrosis factor superfamily member LIGHT induces epithelial-mesenchymal transition in A549 human alveolar epithelial cells.

Airway remodeling is implicated in irreversible airflow limitation of refractory asthma, which includes increased smooth muscle mass and subepithelial fibrosis. Activated fibroblasts acquire contractile phenotype to participate in tissue contraction and structural alteration of extracellular matrices. Histamine is a potent mediator of allergic inflammation, substantially involved in asthmatic pathophysiology.

Histamine induces human lung fibroblast-mediated collagen gel contraction via histamine H1 receptor.

TAZ, also known as WWTR1, has recently been suggested as an oncogene in non-small cell lung cancer (NSCLC). We investigated the clinical relevance of TAZ expression and its functional role in NSCLC tumorigenesis.

An integrative analysis of the tumorigenic role of TAZ in human non-small cell lung cancer.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available.

Novel swine model of transfusion-related acute lung injury.

Asthma-related mortality has been decreasing due to inhaled corticosteroid use, but severe asthma remains a major clinical problem. One characteristic of severe asthma is resistance to steroid therapy, which is related to neutrophilic inflammation. Recently, the tumor necrosis factor superfamily member (TNFSF) 14/LIGHT has been recognized as a key mediator in severe asthmatic airway inflammation. However, the profiles and intracellular mechanisms of cytokine/chemokine production induced in cells by LIGHT are poorly understood. We aimed to elucidate the molecular mechanism of LIGHT-induced cytokine/chemokine production by bronchial epithelial cells. Human bronchial epithelial cells express lymphotoxin β receptor (LTβR), but not herpesvirus entry mediator, which are receptors for LIGHT. LIGHT induced various cytokines/chemokines, such as interleukin (IL)-6, oncostatin M, monocyte chemotactic protein-1, growth-regulated protein α and IL-8. Specific siRNA for LTβR attenuated IL-6 and IL-8 production by BEAS-2B and normal human bronchial epithelial cells. LIGHT activated intracellular signaling, such as mitogen-activated protein kinase and nuclear factor-κB (NF-κB) signaling. LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element. Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways. LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.

Lymphotoxin β Receptor Signaling Induces IL-8 Production in Human Bronchial Epithelial Cells.

Chronic inflammatory airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease, are common respiratory disorders worldwide. Exacerbations of these diseases are frequent and worsen patients' respiratory condition and overall health. However, the mechanisms of exacerbation have not been fully elucidated. Recently, it was reported that interleukin (IL)-17A might play an important role in neutrophilic inflammation, which is characteristic of such exacerbations, through increased production of neutrophil chemoattractants. Therefore, we hypothesized that IL-17A was involved in the pathogenesis of acute exacerbation, due to viral infection in chronic inflammatory airway diseases. In this study, we assessed chemokine production by bronchial epithelial cells and investigated the underlying mechanisms. Comprehensive chemokine analysis showed that, compared with poly(I:C) alone, co-stimulation of BEAS-2B cells with IL-17A and poly(I:C) strongly induced production of such neutrophil chemoattractants as CXC chemokine ligand (CXCL)8, growth-related oncogene (GRO), and CXCL1. Co-stimulation synergistically induced CXCL8 and CXCL1 mRNA and protein production by BEAS-2B cells and normal human bronchial epithelial cells. Poly(I:C) induced chemokine expression by BEAS-2B cells mainly via Toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-β-mediated signals. The co-stimulation with IL-17A and poly(I:C) markedly activated the p38 and extracellular-signal-regulated kinase 1/2 pathway, compared with poly(I:C), although there was little change in nuclear factor-κB translocation into the nucleus or the transcriptional activities of nuclear factor-κB and activator protein 1. IL-17A promoted stabilization of CXCL8 mRNA in BEAS-2B cells treated with poly(I:C). In conclusion, IL-17A appears to be involved in the pathogenesis of chronic inflammatory airway disease exacerbation, due to viral infection by promoting release of neutrophil chemoattractants from bronchial epithelial cells.

Yu Mikami

Department of Clinical Laboratory,
Department of Respiratory Medicine

The University of Tokyo Hospital

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Yu Mikami

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Clinical Laboratory,Department of Respiratory Medicine

The University of Tokyo Hospital

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Department of Clinical Laboratory,
Department of Respiratory Medicine