Mechanical stress induces lung fibrosis by epithelial–mesenchymal transition*
Nuria E. Cabrera-BenítezMatteo ParottoMartin PostBing HanPeter M. SpiethWei-Erh ChengFrancisco ValladaresJesús VillarMingyao LiuMasaaki SatoHaibo ZhangArthur S. Slutsky
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In Brief Objectives: Many mechanically ventilated patients with acute respiratory distress syndrome develop pulmonary fibrosis. Stresses induced by mechanical ventilation may explain the development of fibrosis by a number of mechanisms (e.g., damage the alveolar epithelium, biotrauma). The objective of this study was t test the hypothesis that mechanical ventilation plays an important role in the pathogenesis of lung fibrosis. Methods: C57BL/6 mice were randomized into four groups: healthy controls; hydrochloric acid aspiration alone; vehicle control solution followed 24 hrs later by mechanical ventilation (peak inspiratory pressure 22 cm H2O and positive end-expiratory pressure 2 cm H2O for 2 hrs); and acid aspiration followed 24 hrs later by mechanical ventilation. The animals were monitored for up to 15 days after acid aspiration. To explore the direct effects of mechanical stress on lung fibrotic formation, human lung epithelial cells (BEAS-2B) were exposed to mechanical stretch for up to 48 hrs. Measurement and Main Results: Impaired lung mechanics after mechanical ventilation was associated with increased lung hydroxyproline content, and increased expression of transforming growth factor-β, β-catenin, and mesenchymal markers (α-smooth muscle actin and vimentin) at both the gene and protein levels. Expression of epithelial markers including cytokeratin-8, E-cadherin, and prosurfactant protein B decreased. Lung histology demonstrated fibrosis formation and potential epithelia–mesenchymal transition. In vitro direct mechanical stretch of BEAS-2B cells resulted in similar fibrotic and epithelia–mesenchymal transition formation. Conclusions: Mechanical stress induces lung fibrosis, and epithelia–mesenchymal transition may play an important role in mediating the ventilator-induced lung fibrosis. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.ccmjournal.com).Keywords:
Hydroxyproline
Purpose: Epithelial mesenchymal transition (EMT) is a well characterized embryological process thought to play a vital role in tumor progression. The purpose of this study was to evaluate the expression of EMT-related factors and then use EMT status to identify high metastatic potential in commonly used human colorectal cancer cell lines. Methods: In 11 commonly used cell lines, total mRNA expression levels were checked for the cell markers E- cadherin, vimentin, N-cadherin, and fibronectin, and the transcription factors snail, slug, twist, and SIP1 using real time PCR. Results: E-cadherin expression was positive in COLO205, DLD1, and SW48 cell lines. Vimentin expression was positive in COLO205, DLD1, HCT15, KM20, and SW480. Three different EMT status groups were defined according to mRNA expression of the cell markers. The epithelial phenotype expressed E-cadherin without vimentin (SW48), incomplete EMT phenotypes either concurrently expressed or did not express both E-cadherin and vimentin (CACO2, COLO205, DLD1, KM12C, KM12SM, LoVo, and RKO), and complete EMT phenotypes expressed vimentin without E-cadherin (HCT15, KM20, and SW480). The mRNA expression of transcription factors had no correlation with expression of vimentin, N-cadherin, and fibronectin, but there was an inverse directional correlation between mRNA expression levels of transcription factors and E-cadherin. There were no statistically significant correlations, however. Conclusion: Ten cell lines among the 11 were included in the incomplete and complete EMT groups. These findings could provide evidence of the high tumorigenesis and metastatic properties. The levels of mRNA expression and EMT status of human colorectal cancer cell lines can be applied to further investigations into cancer progression. Keywords: Epithelial mesenchymal transition, Colorectal cancer, Cell line
Twist transcription factor
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Objective: To investigate the role of TGF-β1 on Epithelial Mesenchymal Transition (EMT) and invasion ability in human bladder cancer cell line T24.
Methods: After pre-treatment with TGF-β1 of different concentrations for 24 h, we observed the morphological changes of T24 cells under phase-contrast microscopy. The mRNA and protein expression levels of EMT relative marker E-cadherin and Vimentin were detected by RT-PCR and Western blot. The effect of TGF-β1 on migration and invasion abilities of T2 cell line was detected with transwell migration and invasion assay.
Results: Results demonstrated that TGF-β1 could induce epithelial-mesenchymal transition in T24 cells. The mRNA and protein levels of epithelial marker E-cadherin were downregulated by TGF-β1, whereas those of mesenchymal maker protein Vimentin were upregulated. Furthermore, it is noted that TGF-β1 could significantly enhance the migration and invasion ability of T24 cells, which, however, could be effectively reversed by TGF-β1 inhibitor LY2109761.
Conclusion: TGF-β1 can enhance migration and invasion ability of T24 cells by inducing epithelial mesenchymal transition.
Slug
Gentamicin protection assay
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IH764-3, an effective component isolated from Salvae milliorrhize (a component of traditional Chinese medicine) was used against pulmonary fibrosis in this study. The results indicated that in the treated group, lung coefficient, surfactant, hydroxyproline content and FGF activity were significantly lower than those in the model group. Electron microscopic observation confirmed that the pulmonary ultrastructure was improved remarkably in the treated group: the proliferation of collagen-forming cells and infiltration of inflammatory cells, collagen and elastic fibers were obviously less. All the results demonstrated that IH764-3 has prophylactic and therapeutic effects on the development of pneumonitis and pulmonary fibrosis.
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Infiltration (HVAC)
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Objective To study the therapeutic effects of total salvianolic acid on Bleomycin-induced pulmonary fibrosis in mice.Methods The pulmonary fibrosis model was established by intratracheal injection of Bleomycin.The lung index,pathomorphology,and level of hydroxyproline(Hyp) in lung tissue were measured to observe the effect of total salvianolic acid on pulmonary fibrosis of mice.Results Total salvianolic acid could obviously reduced alveolitis and fibrosis.The lung index was significantly decreased in total salvianolic acid group compared with that in model group,and the level of Hyp was significantly lower than that in model group(P0.05 and 0.01).Conclusion Total salvianolic acid could have therapeutic effects on Bleomycin-induced pulmonary fibrosis in mice.
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Therapeutic index
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Epithelial mesenchymal transition (EMT) is a biological process in which epithelial cells undergo extensive molecular reprogramming allowing these cells to undergo numerous biochemical changes and acquire a mesenchymal phenotype. This is accompanied by progressive loss of epithelial markers, gain in migratory potential and invasiveness, and enhanced capacity to produce extracellular matrix components. The digestion of underlying basement membrane facilitates the process of EMT and the fragmented anatomical appearance of the reticular basement membrane (Rbm) has been reported as a marker of completion of the process. EMT is considered critical during cancer metastasis and fibrosis. The intermediate filament protein vimentin is also an important marker of EMT and increases in abundance in epithelial cells undergoing EMT. It is also seen in tumour invasion, suggesting a particular role in cell migration. Vimentin has been most commonly used as marker to identify cells undergoing EMT in cancers; a positive correlation has been reported between vimentin and increased metastatic potential in breast cancer. This chapter reviews these issues, and highlights recent advances in understanding the role of vimentin in EMT as part of disease processes.
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Reticular connective tissue
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Abstract The process of epithelial mesenchymal transition, whereby cells acquire molecular alterations and fibroblastic features, is a fundamental process of embryogenesis and cancer invasion/metastasis. The mechanisms responsible for epithelial mesenchymal transition remain elusive. Human tumors frequently establish constitutively activated RAS signaling, which contributes to the malignant phenotype. In an effort to dissect distinct RAS isoform specific functions, we previously established human colon cell lines stably overexpressing activated Harvey‐RAS (Ha‐RAS) and Kirsten‐RAS (Ki‐RAS). Using these, we observed that only oncogenic Ha‐RAS overexpression resulted in morphologic and molecular changes suggestive of epithelial to mesenchymal transition. We showed that vimentin, a key molecule of epithelial mesenchymal transition, was differentially regulated between Ha‐RAS and Ki‐RAS leading to a Ha‐RAS specific induction of a migrative phenotype and eventually epithelial to mesenchymal transition. We demonstrated that the AP‐1 sites in vimentin promoter could be involved in this regulation. A potential role of FRA‐1 was suggested in the regulation of vimentin during the Ha‐RAS‐induced epithelial to mesenchymal transition, in association with colon cell migration. Our results therefore propose that in colon cells, the induction of epithelial mesenchymal transition by oncogenic Ha‐RAS could occur through the overexpression of proteins like FRA‐1 and vimentin. © 2007 Wiley‐Liss, Inc.
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Objective To investigate the impact of migration inhibitory factor (MIF) over-expression on the epithelial to mesenchymal transition in human cervical cancer SiHa cells.Methods Recombinant plasmid pEGFP-N1-MIF was transfected into SiHa cells,and then of MIF mRNA relative quantitative expression was tested by RT-PCR.The mRNA and protein expression level of E-cadherin and vimentin were detected by RT-PCR and immunocytochemistry,respectively.Results RT-PCR results showed that MIF mRNA expression quantity in experimental group was higher than that in control group cells (F =2 950.278,P ＜ 0.01).In MIF-overexpressing SiHa cells,vimentin mRNA was increased and E-cadherin mRNA was decreased determined by RT-PCR (Fvalues were 2 135.048,1 893.563,P＜ 0.01).Immunocytochemistry results showed that vimentin expression quantity in experimental group cells were higher than that in control group cells,however,E-cadherin expression quantity was lower than that in control group cells (F values were 2 348.021,1 789.421,P ＜ 0.01).Conclusions The over-expression of MIF gene can significantly up-regulate the expression of vimentin,and down-regulate the expression of E-cadherin.Consequently,MIF over-expression induces epithelial to mesenchymal transition in human cervical cancer SiHa cells.
Key words:
Macrophage migration inhibitory factor; Epithelial-mesenchymal transition; Cervical cancer; E-cadherin; vimentin
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Anaplastic thyroid cancer (ATC) remains a cancer with one of the worst prognoses, despite novel targeted therapies. The median survival rate has not improved for decades. Epithelial‑to‑mesenchymal transition (EMT) is a crucial step in physiological processes and in cancer progression, but the underlying mechanisms are not yet fully understood. The current study examined the role of microRNA (miR)‑200b in mesenchymal‑to‑epithelial transition in ATC. Total RNA and miR isolation were performed from ATC cell lines transfected with a miR‑200b mimic. After miR‑200b mimic transfection, expression levels of E‑cadherin, vimentin and zinc finger E‑box binding homeobox 1 (ZEB1) were confirmed by reverse transcription‑quantitative PCR and western blotting. Additionally, cell migration was evaluated using miR‑200b mimic and scrambled negative control‑transfected cells. A total of 14 human ATC and 15 non‑cancerous human thyroid tissues were immunohistochemically stained and scored as controls for E‑cadherin, vimentin and ZEB1. In ATC tissues and cell lines, the mesenchymal marker ZEB1 was significantly upregulated and the epithelial marker E‑cadherin was significantly downregulated. Additionally, the mesenchymal marker vimentin was significantly upregulated in ATC tissues and in one ATC cell line. MiR‑200b mimic transfection significantly increased vimentin and ZEB1 expression, but E‑cadherin expression remained below the measurement sensitivity. Furthermore, miR‑200b overexpression decreased cell migration. The current study suggested that miR‑200b may regulate the expression levels of mesenchymal markers such as vimentin and ZEB1 in ATC and may promote mesenchymal‑to‑epithelial transition.
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Aim To investigate the expressions of Fas/FasL and apoptosis of pulmonary tissue as well as oxygen free radical in the pulmonary fibrosis induced by bleomycin in rats at inflammation stage and fibrosis stage.Methods Forty-eight rats were divided into three groups randomly,including control group,model group and breviscapine group.On experimental day 0,the rats were intratracheally instilled with bleomycin,and then treated with breviscapine 10 mg·kg-1·d-1 until they were killed.On days 7,28 after stillation,eight rats of each group were killed and the lungs were harvested for examination.Flow cytomagnetometry was used to detect the changes of cell apoptosis and the expressions of Fas/Fasl in rats with pulmonary fibrosis caused by bleomycin at flammation stage and fibrosis stage.Malonaldehyde and hydroxyproline(HYP)of lung tissue were detected by TAB method.Results The apoptosis index of lung cells in the pulmonary fibrosis group was higher than that of control and breviscapine group.The expression of Fas/FasL and contents of Malonaldehyde as well as hydroxyproline in the pulmonary fibrosis group were higher than those of control group and breviscapine group(P0.01).Conclusion Breviscapine may alleviate the process of rat pulmonary fibrosis induced by bleomycin,and has protective effect on rats with pulmonary fibrosis.
Hydroxyproline
Fas ligand
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