Induction of pulmonary fibrosis by methotrexate treatment in mice lung in vivo and in vitro

Ohbayashi, Masayuki; Suzuki, Masanori; Yashiro, Yoshiki; Fukuwaka, Sayaka; Yasuda, Masako; Kohyama, Noriko; Kobayashi, Yasuna; Yamamoto, Toshinori

doi:10.2131/jts.35.653

Cited by 60 publications

(60 citation statements)

References 36 publications

(36 reference statements)

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“…We have previously reported that hyperplasia of interstitial tissues was observed in the alveolar septa and the increase of collagen content in the alveolar wall and fibrosis area after consecutive administration of MTX for 35 days (Ohbayashi et al, 2010). In addition, we have also revealed that the E-cadherin (epithelial cell marker) is significantly detected, whereas α-SMA (mesenchymal marker) is faintly observed in the alveolar septa and, which is restricted to vascular smooth muscle cells in control mice.…”

Section: Immunohistochemical Analyses For the Expression Of E-cadherimentioning

confidence: 62%

“…Primary cell culture of lung fibroblasts was achieved using the protocol described previously (Ohbayashi et al, 2010). Briefly, the lungs removed from mice were minced into pieces of 2-3 mm 3 , agitated, and digested enzy-matically for 80 min at 37°C.…”

Section: Preparation Of Lung Alveolar Epithelial and Fibroblast Cellsmentioning

confidence: 99%

“…These results indicate that fibroblasts indeed can be derived from epithelial cells through EMT and may contribute to the development of pulmonary fibrosis. We have previously demonstrated that treatment of MTX resulted in a decrease of cytokeratin (alveolar epithelial cell marker) and an increase of α-SMA (myofibroblast marker) in fibrosis foci (Ohbayashi et al, 2010). However, there is still limited data concerning the involvement of EMT in MTX-induced pulmonary fibrosis.…”

Section: Introductionmentioning

confidence: 99%

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Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis

et al. 2014

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-Epithelial-mesenchymal transition (EMT) plays a pivotal event in the development of pulmonary fibrosis. We have previously reported that methotrexate (MTX)-induced alveolar epithelial cell injury followed by pulmonary fibrosis as a result of the recruitment and proliferation of myofibroblasts. However, there is no data concerning whether EMT occurs in MTX-induced pulmonary fibrosis. In the present study, therefore, we investigated the expression of EMT markers such as E-cadherin, α-SMA, and vimentin by immunofluorescence analysis in mouse lung tissues after administration of MTX. We found that vimentin and α-SMA-positive cells of the MTX-induced pulmonary fibrosis were increased; on the other hand, E-cadherin was decreased, indicating that epithelial cells act as the main source of mesenchymal expansion. These results exhibited the down-regulation of E-cadherin expression and the up-regulation of α-smooth muscle actin (α-SMA) in primary mouse alveolar epithelial cells (MAECs) and A549 cell lines. Additionally, MTX-induced A549 cells exhibited an EMT-like phenotype accompanied by the elevation of the expression of interleukin-6 (IL-6) and transforming growth factor (TGF)-β1, as well as an enhancement of migration. All of these findings suggest that MTX-induced pulmonary fibrosis occurs via EMT.

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Section: Immunohistochemical Analyses For the Expression Of E-cadherimentioning

confidence: 62%

Section: Preparation Of Lung Alveolar Epithelial and Fibroblast Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis

et al. 2014

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“…Then 5-mm slices were cut and stained with hematoxylin and eosin following deparaffinization. Different histologic staining was used according to the tissue sources, for example, azan for collagen staining in the lung (28), and PAS for basement membrane staining in the kidney were performed (29). Then the tumor tissues were stained overnight with antibodies directed against Ki-67 (Novus Biologicals) and CREB (Cell Signaling Technology), using suitable secondary antibodies (SignalStain Boost IHC Detection Reagent; Cell Signaling Technology) in combination with diaminobenzidine (Applichem) as a substrate and counterstained with methylene blue.…”

Section: Patient Selection and Tissue Microarraysmentioning

confidence: 99%

HER-2/neu Mediates Oncogenic Transformation via Altered CREB Expression and Function

Steven

Leisz

Massa

et al. 2013

Molecular Cancer Research

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The cyclic (c)AMP responsive element binding protein (CREB) plays a key role in many cellular processes, including differentiation, proliferation, and signal transduction. Furthermore, CREB overexpression was found in tumors of distinct origin and evidence suggests an association with tumorigenicity. To establish a mechanistic link between HER-2/neu-mediated transformation and CREB protein expression and function, in vitro models of HER-2/neu-overexpressing and HER-2/neu-negative/silenced counterparts as well as human mammary carcinoma lesions with defined HER-2/neu status were used. HER-2/neu overexpression resulted in the induction and activation of CREB protein in vitro and in vivo, whereas short hairpin RNA (shRNA)-mediated inhibition of HER-2/neu correlated with downregulated CREB activity. CREB activation in HER-2/neu-transformed cells enhanced distinct signal transduction pathways, whereas their inhibition negatively interfered with CREB expression and/or activation. CREB downregulation in HER-2/neu-transformed cells by shRNA and by the inhibitors KG-501 and lapatinib caused morphologic changes, reduced cell proliferation with G 0 -G 1 cell-cycle arrest, which was rescued by CREB expression. This was accompanied by reduced cell migration, wound healing, an increased fibronectin adherence, invasion, and matrix metalloproteinase expression. In vivo shCREB-HER-2/neu þ cells, but not control cells, exerted a significantly decreased tumorgenicity that was associated with decreased proliferative capacity, enhanced apoptosis, and increased frequency of T lymphocytes in peripheral blood mononuclear cells. Thus, CREB plays an important role in the HER-2/neu-mediated transformation by altering in vitro and in vivo growth characteristics.

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“…In many studies, OH-P levels were measured to assess wound healing and fibrosis (1,16,17). In the study by O'Sullivan et al (18), higher OH-P levels were observed in subjects showing improved wound healing.…”

Section: Discussionmentioning

confidence: 99%

Investigation of wound healing in rat lung tissues in the postpartum period

Karadayı¹,

Şahin²,

Nadir³

et al. 2012

Experimental and Therapeutic Medicine

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Abstract. To assess the wound healing capabilities of damaged lung tissue in the postpartum period, we investigated the parameters related to wound healing in a rat model of lung damage. Rats were divided into six groups: IA, IB, II, IIIA, IIIB and IV (n=7 in each group). Group IA included rats not in the postpartum period that were sacrificed on the third day after lung injury, group IB included rats not in the postpartum period that were sacrificed on the tenth day after lung injury, group II included rats not in the postpartum period that did not receive lung injury, group IIIA included rats in the postpartum period that were sacrificed on the third day after lung injury, group IIIB included rats in the postpartum period that were sacrificed on the tenth day after lung injury and group IV included rats in the postpartum period without lung injury. Wound healing was evaluated histopathologically and measurements of hydroxyproline levels, serum alanine and glutamine were taken. A significant difference in serum alanine levels was evident between groups IA and IIIA. Significant differences were also observed between serum alanine and glutamine levels in groups IB and IIIB. In conclusion, we demonstrated that serum alanine levels were reduced in the postpartum period following lung injury, which may be expected to negatively impact wound healing in this period. The administration of exogenous alanine for traumatic events occurring during the postpartum period may thus contribute positively to wound healing capabilities during this period.

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Induction of pulmonary fibrosis by methotrexate treatment in mice lung in vivo and in vitro

Cited by 60 publications

References 36 publications

Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis

Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis

HER-2/neu Mediates Oncogenic Transformation via Altered CREB Expression and Function

Investigation of wound healing in rat lung tissues in the postpartum period

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