Wu Yao

Role of Insulin-like Growth Factor II Receptor in Transdifferentiation of Free Silica-induced Primary Rat Lung Fibroblasts *

OBJECTIVE To study the role of insulin-like growth factor II receptor in free silica-induced transdifferentiation of primary rat lung fibroblasts. METHODS Rat lung fibroblasts and rat alveolar macrophages were cultured. A transdifferentiation model of primary rat lung fibroblasts was induced by free silica. Levels of α-SMA protein, IGF-IIR protein and mRNA were measured by immunocytochemistry, Western blot and RT-PCR, respectively. Lung fibroblasts were treated with Wortmannin. RESULTS The expression levels of α-SMA and IGF-IIR increased with the increasing free silica concentration and decreased after Wortmannin was used. CONCLUSION The IGF-IIR plays an important role in free silica-induced transdifferentiation of primary rat lung fibroblasts.

Bioinformatics methods for identifying differentially expressed genes and signaling pathways in nano-silica stimulated macrophages.

The incidence of disease relating to nanoparticle exposure has been rising rapidly in recent years, for which there is no effective treatment. Macrophage is suggested to play a crucial role in the development of pulmonary disease. To investigate the changes in macrophage after being stimulated by nanometer silica dust and to explore potential biomarkers and signaling pathways, the gene chip GSE13005 was downloaded from Gene Expression Omnibus database, which contained 21 samples: 3 samples per group and 7 groups in total. Macrophages in the control group were cultured in serum-free medium, while the experimental groups were treated with nanometer silica dust in different sizes and concentrations, respectively. To identify the differentially expressed genes and explore their potential functions, we adopted the gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and also constructed protein–protein interaction network. As a result, 1972 differentially expressed genes were identified from 22,690 microarray data in the gene chip, 1069 genes were upregulated and 903 genes were downregulated. Results of the gene ontology analysis indicated that the differentially expressed genes were widely distributed in intracellular and extracellular regions, regulating macrophage apoptosis, inflammatory response, and cell differentiation. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the majority of differentially expressed genes were enriched in cytokine–cytokine receptor interaction, cancer or phagosome transcriptional misregulation. The top 10 hub genes, S100a9, Nos3, Psmd14, Psmd4, Lck, Atp6v1h, Jun, Foxh1, Pex14, and Fadd were identified from protein–protein interaction network. In addition, Nos3, Psmd14, Atp6v1h, and Jun were clustered into module M2 (rc = 0.74, p < 0.01), which mainly regulates cell carcinogenesis and antivirus process. In conclusion, differentially expressed genes screened from this study may provide new insights into the exploration of mechanisms, biomarkers, and therapeutic targets for diseases relating to nanoparticle exposure.

Protein Expression in Silica Dust-induced Transdifferentiated Rats Lung Fibroblasts

OBJECTIVE To analyze the expression of different proteins in free silica-induced transdifferentiated rat lung fibroblasts. METHODS Rat lung fibroblasts and alveolar macrophages were cultured. A transdifferentiation model of rat lung fibroblasts was established. Free silica was used as a stimulator for rat lung fibroblasts. Changes in α-SMA were detected by immunohistochemistry and Western blot, respectively. Protein of lung fibroblasts was extracted and analyzed by two-dimensional electrophoresis (2-DE). RESULTS Six protein spots were identified by mass spectrometry, including glyceraldehyde 3-phosphate-dehydrogenase, peroxiredoxin 5, heterogeneous nuclear ribonucleoprotein A2, transgelin 2, keratin K6 and vimentin. CONCLUSION Some proteins are changed in free silica-induced transdifferentiated rat lung fibroblasts.

Exosomal miRNA Profiling to Identify Nanoparticle Phagocytic Mechanisms

Inhaling a dangerous amount of nanoparticles leads to pulmonary inflammatory and immune disorders, which integrates several kinds of cells. Exosomes are suggested to play a crucial role in intercellular communication via miRNA transmission. To investigate the role of exosomal miRNA in nanoparticle phagocytosis, a total of 54 pneumoconiosis patients along with 100 healthy controls are recruited, exosomes derived from their venous blood are collected, and then exosomal miRNAs are profiled with high-throughput sequencing technology. miRNAs which are differentially expressed are used to predict target genes and conduct functional annotation. Interactions between miRNA hsa-let-7a-5p, hsa-let-7i-5p, and their cotarget gene WASL are found that can affect nanoparticle phagocytosis. The follow-up analysis of gene structure, tissue specificity, and miRNA-target gene regulatory mode supports the findings. Specially, the assumption is further confirmed via a series of cellular experiments, and the fibroblast transdifferentiate rate that is used as an indicator of nanoparticle phagocytosis decreased when elevating miRNA expression level. Thus, data in this study indicate that downregulation of miRNA hsa-let-7a-5p and hsa-let-7i-5p contributes to WASL elevation, promoting WASL and VASP complex formation, which is necessary for initiating Arp2/3 induced phagocytosis.

Crystalline Silica Promotes Rat Fibrocyte Differentiation in Vitro, and Fibrocytes Participate in Silicosis in Vivo

OBJECTIVE The aim of this study was to investigate the effects of SiO2 on fibrocytes and whether fibrocytes participate in silicosis in vivo. METHODS A macrophagocyte (AM)/fibrocyte coculture system was established, and AMs were treated with 100 μg/mL SiO2. Flow cytometry was used to detect the number of fibrocytes. Real-time PCR was performed to measure the expression of collagen I, collagen III, and α-SMA mRNA. The levels of collagen I, collagen III, and TGF-β1 protein were determined by ELISA. Immunohistochemical staining was performed to measure α-SMA protein expression. A rat silicosis model was induced by intratracheal instillation of SiO2. Lung histopathological evaluation was conducted using HE and Massons trichrome staining after 1 and 9 weeks. The number of fibrocytes in peripheral blood or lung tissue of rat was detected by flow cytometry. Double-color immunofluorescence was applied to identify fibrocytes in the lung tissue. RESULTS Peripheral blood monocytes were found to differentiate into fibrocytes in vitro in a time-dependent manner, and exposure to crystalline silica might potentiate fibrocyte differentiation. In addition, fibrocytes were able to migrate from peripheral blood to the lung tissue, and the number of fibrocytes was increased after SiO2 exposure. CONCLUSION Silica exposure potentiates fibrocyte differentiation, and fibrocytes may participate in silicosis in vivo.

Dendritic cells trigger imbalance of Th1/Th2 cells in silica dust exposure rat model via MHC-II, CD80, CD86 and IL-12

Silicosis is one of the most common occupational respiratory diseases caused by inhaling silica dust over a prolonged period of time, and the progression of silicosis is accompanied with chronic inflammation and progressive pulmonary fibrosis, in which dendritic cells (DCs), the most powerful antigen presentation cell (APC) in the immune response, play a crucial role. To investigate the role of DCs in the development of silicosis, we established an experimental silicosis rat model and examined the number of DCs and alveolar macrophages (AMs) in lung tissues using immunofluorescence over 84 days. Additionally, to obtain an overview of the immunological changes in rat lung tissues, a series of indicators including Th1/Th2 cells, IFN-γ, IL-4, MHC-II, CD80/86 and IL-12 were detected using flow cytometry and an enzyme-linked immunosorbent assay (ELISA) as well as a real-time polymerase chain reaction (PCR) assay. We observed that the number of DCs slightly increased at the inflammatory stage, and it increased significantly at the final stage of fibrosis. Polarization of Th1 cells and IFN-γ expressions were dominant during the inflammatory stage, whereas polarization of Th2 cells and IL-4 expressions were dominant during the fibrotic stage. The subsequent mechanistic study found that the expressions of MHC-II, CD80/86 and IL-12, which are the key molecules that connect DCs and Th cells, changed dynamically in the experimental silicosis rat model. The data obtained in this study indicated that the increase in DCs may contribute to polarization of Th1/Th2 cells via MHC-II, CD80/86, and IL-12 in silica dust-exposed rats.

Up-regulation of exosomal miR-125a in pneumoconiosis inhibits lung cancer development by suppressing expressions of EZH2 and hnRNPK

Exposure to nanoparticles may lead to pneumoconiosis and lung cancer; however, whether patients suffering from pneumoconiosis also face a high risk of lung cancer has been under debate for decades. Recently, exosomes have been found to play critical roles in many diseases via intercellular cargo transportation, which has provided a new insight into the mechanistic investigation of nanoparticle-induced respiratory disorders. Herein, we isolated exosomes from the venous blood of patients with pneumoconiosis and healthy controls and then, we profiled the expression signatures of exosomal miRNAs using high-throughput sequencing technology. A total of 14 aberrantly expressed miRNAs were identified and used to process target gene prediction and functional annotation. Specially, miR-125a along with its target genes EZH2 and hnRNPK was found to play a significant role in the development of lung cancer. We then adopted a series of cellular experiments to validate the role of miR-125a in lung cancer. From the results obtained, we found that the suppression of EZH2 and hnRNPK by high levels of miR-125a inhibited the development of nanoparticle-induced lung adenocarcinoma, which contributed to the clarification of the relation between pneumoconiosis and lung cancer.

1070 Contribution of bone marrow-derived fibrocytes to silicosis

Introduction Exposure to free silica induces silicosis and its mechanism is less clear. Myofibroblast is regarded as a primary effector cell which is highly synthetic for collagen and lead to extensive fibrosis in lung. However, its origin is still controversial. Fibrocyte is one source of myofibroblast and proved to play a pivotal role in lung fibrogenesis, but whether fibrocyte participates in the process of silicosis is rarely reported. Therefore, the present study was designed to investigate the contribution of fibrocytes in silicosis. Method The rat model of silicosis was established by single intratracheal instillation of SiO2 solution (100 mg/0.5 ml/rat). HE and Masson staining were used to evaluate the histopathology and collagen deposition. Flow cytometry and immunofluorescence were performed to detect number of fibrocytes and contribution to myofibroblasts. Results During experimental silicosis (from week 1, 2, 3, 6, 9, 12), the number of fibrocyte is markedly increased in peripheral blood and lung tissue by using flow cytometry.Further study found that CD45+ collagen I+ fibrocyte is existed in lung tissue by using double-colour immunofluorescence analysis. Meanwhile, fibrocyte and lung type II epithelial cells contribute 15%˜35% and 9%˜21% of myofibroblasts, respectively. The trend analysis of different sources of myofibroblast during silicosis indicates that fibrocyte and lung type II epithelial cell derived myofibroblast play an important role at the early stage of silicosis (week 1 to week 3), while resident lung fibroblast-derived myofibroblast mainly do a predominant role during fibrosis formative period (week 6 to week 12). Discussion Taken together, these data suggest that fibrocyte is involved in the pathogenesis of silicosis and it may be useful as an indicator for disease activeity. Different sources of myofibroblasts play roles in different phases of silicosis.

The Role of Fibrocyte in the Pathogenesis of Silicosis

Exposure to free silica induces silicosis and myofibroblasts are regarded as primary effector cells. Fibrocytes can differentiate into myofibroblast. Therefore, the present study was designed to investigate whether fibrocytes participate in silicosis. The rat model of silicosis was established. Hematoxylin-eosin stainings and Masson stainings were used to evaluate the histopathology and collagen deposition. Flow cytometry and immunofluorescence were performed to detect the number of fibrocytes and their contribution to myofibroblasts. Results showed that fibrocytes participate in silicosis. Trend analysis of different sources of myofibroblasts during silicosis indicated that fibrocytes and lung type II epithelial cell-derived myofibroblasts play an important role in the early stage of silicosis, while resident lung fibroblast-derived myofibroblasts play a predominant role during the fibrosis formative period.

Screening and Preliminary Verification of a Phage Display Single-Chain Antibody Library Against Coal Workers' Pneumoconiosis.

Objectives: To construct a phage display human antibody library (PDHAL) against pneumoconiosis for the diagnosis and treatment of coal worker pneumoconiosis (CWP). Methods: The PDHAL was established via CWP blood and six positive antibodies were discovered. 867 coal workers (558 CWP and 309 without CWP) and 393 controls were recruited to validate the results. Results: A PDHAL against CWP was established, from which six strong positive clones were selected, sequenced and identified as VEGF, interleukin-18, HSP70, HER3, Gz-B and RF. Logistic regression analysis revealed that VEGF (OR (95% CI), 0.02 (0.01to 0.07), P < 0.05), RF-Ab (OR (95% CI): 0.46 (0.28 to 0.73), P < 0.05) and HSP70/HSP-70-Ab (OR (95% CI): 0.71 (0.53 to 0.95), P < 0.05) were protective factors for CWP after adjustment of confounding factors. Conclusion: The serum VEGF, RF-Ab and HSP-70/HSP-70 antibodies were potential biomarkers for diagnosis and treatment of CWP.