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MicroRNA profiling implicates the insulin-like growth factor pathway in bleomycin-induced pulmonary fibrosis in mice.

ABSTRACT: BACKGROUND:Idiopathic pulmonary fibrosis is a disease characterized by alveolar epithelial cell injury, inflammatory cell infiltration and deposition of extracellular matrix in lung tissue. As mouse models of bleomycin-induced pulmonary fibrosis display many of the same phenotypes observed in patients with idiopathic pulmonary fibrosis, they have been used to study various aspects of the disease, including altered expression of microRNAs. RESULTS:In this work, microRNA expression profiling of the lungs from treated C57BL/6J mice, relative to that of untreated controls, was undertaken to determine which alterations in microRNAs could in part regulate the fibrosis phenotype induced by bleomycin delivered through mini-osmotic pumps. We identified 11 microRNAs, including miR-21 and miR-34a, to be significantly differentially expressed (P < 0.01) in lungs of bleomycin treated mice and confirmed these data with real time PCR measurements. In situ hybridization of both miR-21 and miR-34a indicated that they were expressed in alveolar macrophages. Using a previously reported gene expression profile, we identified 195 genes to be both predicted targets of the 11 microRNAs and of altered expression in bleomycin-induced lung disease of C57BL/6J mice. Pathway analysis with these 195 genes indicated that altered microRNA expression may be associated with hepatocyte growth factor signaling, cholecystokinin/gastrin-mediated signaling, and insulin-like growth factor (IGF-1) signaling, among others, in fibrotic lung disease. The relevance of the IGF-1 pathway in this model was then demonstrated by showing lung tissue of bleomycin treated C57BL/6J mice had increased expression of Igf1 and that increased numbers of Igf-1 positive cells, predominantly in macrophages, were detected in the lungs. CONCLUSIONS:We conclude that altered microRNA expression in macrophages is a feature which putatively influences the insulin-like growth factor signaling component of bleomycin-induced pulmonary fibrosis.

SUBMITTER: Honeyman L

PROVIDER: S-EPMC3766165 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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MicroRNA profiling implicates the insulin-like growth factor pathway in bleomycin-induced pulmonary fibrosis in mice.

Honeyman Lisa L Bazett Mark M Tomko Tomasz G TG Haston Christina K CK

Fibrogenesis & tissue repair 20130829 1

<h4>Background</h4>Idiopathic pulmonary fibrosis is a disease characterized by alveolar epithelial cell injury, inflammatory cell infiltration and deposition of extracellular matrix in lung tissue. As mouse models of bleomycin-induced pulmonary fibrosis display many of the same phenotypes observed in patients with idiopathic pulmonary fibrosis, they have been used to study various aspects of the disease, including altered expression of microRNAs.<h4>Results</h4>In this work, microRNA expression ...[more]

PMID: 23987664

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Project description:Pulmonary fibrosis is a disease characterized by inflammatory cell infiltration, scar formation, deposition of extracellular matrix, alveolar epithelial cell injury and hyperplasia. To determine if alterations in microRNA expression contribute to these phenotypes, microRNA expression profiling of the lungs from bleomycin treated C57Bl/6J mice, relative to that of untreated controls, was undertaken. Mice were treated at 8 weeks old with 100 Units/kg of bleomycin delivered subcutaneously with osmotic minipumps. At 42 days post treatment mice were euthanized and lung microRNA isolated. We identified 11 microRNA's to be significantly differentially expressed (FDR threshold of 0.01) in the lungs of bleomycin treated mice and confirmed these data with real time PCR measurements. These included bleomycin upregulated miR-34a, 335-5p, 207, 21, 301a, 146b, 199a-5p, and 449a and bleomycin downregulated miR-151-3p, 26a and 676. We have previously shown that 1558 genes are differentially expressed in the lungs of bleomycin treated mice. Of the 1412 targets of upregulated microRNAs, 142 were confirmed to be downregulated in the gene expression profile (GEP). Of the 583 targets of downregulated microRNAs, 53 were confirmed to be upregulated in the gene expression profile. Pathway analysis of the microRNA targets and GEP overlapping genes indicated that altered microRNA expression is associated with cellular development, cellular growth, cellular proliferation and changed tissue/cell morphology. Specific pathways include HGF signaling, Cholecystokinin/Gastrin-mediated signaling, Endothelin-1 signaling, RAR activation, Phospholipase C signaling and IGF1 signaling. We conclude that altered microRNA expression is a feature of pulmonary fibrosis which putatively influences components of the altered airway disease. Two condition study, C57Bl/6J mice treated with 100 Units/kg bleomycin and untreated controls. Biological replicated n =3 for each group. Left lung tissue.

Project description:: The incidence of idiopathic pulmonary fibrosis is on the rise and existing treatments have failed to halt or reverse disease progression. Mesenchymal stromal cells (MSCs) have potent cytoprotective effects, can promote tissue repair, and have demonstrated efficacy in a range of fibrotic lung diseases; however, the exact mechanisms of action remain to be elucidated. Chemical antagonists and short hairpin RNA knockdown were used to identify the mechanisms of action used by MSCs in promoting wound healing, proliferation, and inhibiting apoptosis. Using the bleomycin induced fibrosis model, the protective effects of early or late MSC administration were examined. The role for hepatocyte growth factor (HGF) in MSC protection against bleomycin lung injury was examined using HGF knockdown MSC. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling assay was performed on ex vivo lung sections to examine the effects of MSC on apoptosis. MSC conditioned media (CM) enhanced wound closure and inhibited apoptosis of pulmonary cells in vitro. HGF was required for MSC CM enhancement of epithelial cell proliferation and inhibition of apoptosis. In contrast, MSC required COX-2 for CM to inhibit fibroblast proliferation. In a murine model, early administration of MSC protected against bleomycin induced lung fibrosis and correlated with reduced levels of the proinflammatory cytokine interleukin-1β, reduced levels of apoptosis, and significantly increased levels of HGF. These protective effects were in part mediated by MSC derived HGF as HGF knockdown MSC were unable to protect against fibrosis in vivo. These findings delineate the mechanisms of MSC protection in a preclinical model of fibrotic lung disease.SignificanceThe mechanisms used by mesenchymal stromal cells (MSCs) in mediating protective effects in chronic models of lung disease are not understood and remain to be elucidated. These findings from in vitro studies highlight an important role for the MSC-derived soluble factors hepatocyte growth factor (HGF) and prostaglandin E2 in promoting wound healing and inhibiting apoptosis. Furthermore, this study translates these findings demonstrating an important role for HGF in the protective effects mediated by MSC in vivo in the bleomycin model. These findings support a targeted approach to enhancing MSC therapy for fibrotic disease and highlight the importance of timing of MSC therapy.

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MicroRNA profiling implicates the insulin-like growth factor pathway in bleomycin-induced pulmonary fibrosis in mice.

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MicroRNA profiling implicates the insulin-like growth factor pathway in bleomycin-induced pulmonary fibrosis in mice.

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