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Effects of cigarette smoke extract on human airway smooth muscle cells in COPD

Ling Chen, Qi Ge, Gavin Tjin, Hatem Alkhouri, Linghong Deng, Corry-Anke Brandsma, Ian Adcock, Wim Timens, Dirkje Postma, Janette K. Burgess, Judith L. Black, Brian G.G. Oliver
European Respiratory Journal 2014 44: 634-646; DOI: 10.1183/09031936.00171313
Ling Chen
1Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Shapingba, Chongqing, China
2Woolcock Institute of Medical Research, The University of Sydney, Sydney, NSW, Australia
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Qi Ge
2Woolcock Institute of Medical Research, The University of Sydney, Sydney, NSW, Australia
3Discipline of Pharmacology, The University of Sydney, Sydney, NSW, Australia
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Gavin Tjin
2Woolcock Institute of Medical Research, The University of Sydney, Sydney, NSW, Australia
4Central Clinical School, The University of Sydney, Sydney, NSW, Australia
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Hatem Alkhouri
5Respiratory Research Group, Faculty of Pharmacy, The University of Sydney, Sydney, NSW, Australia
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Linghong Deng
1Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Shapingba, Chongqing, China
6Institute of Biomedical Engineering and Health Science, Changzhou University, Changzhou, Jiangsu, China
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Corry-Anke Brandsma
7Dept of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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Ian Adcock
8Thoracic Medicine, Imperial College London, National Heart and Lung Institute, London, UK
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Wim Timens
7Dept of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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Dirkje Postma
9Dept of Pulmonology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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Janette K. Burgess
2Woolcock Institute of Medical Research, The University of Sydney, Sydney, NSW, Australia
3Discipline of Pharmacology, The University of Sydney, Sydney, NSW, Australia
4Central Clinical School, The University of Sydney, Sydney, NSW, Australia
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Judith L. Black
2Woolcock Institute of Medical Research, The University of Sydney, Sydney, NSW, Australia
3Discipline of Pharmacology, The University of Sydney, Sydney, NSW, Australia
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Brian G.G. Oliver
2Woolcock Institute of Medical Research, The University of Sydney, Sydney, NSW, Australia
10School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, NSW, Australia
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  • For correspondence: brian.oliver@uts.edu.au
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  • Figure 1–
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    Figure 1–

    The effect of cigarette smoke extract (CSE) on cell viability. The cytotoxicity of CSE on a) the mitochondrial activity and b) lactate dehydrogenase (LDH) release of human airway smooth muscle (ASM) cells was measured by Thaizolyl blue tetrazolium bromide (MTT) and LDH assays at an absorbance of 570 nm/690 nm and 490 nm/690 nm, respectively. Human ASM cells were stimulated with serial dilution of CSE for 72 h (n=4). Data are presented as mean±sem. One-way ANOVA plus Bonferroni post-test was used to determine statistical significance. *: p<0.05; **: p<0.01; ***: p<0.001, compared with control.

  • Figure 2–
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    Figure 2–

    Release of CXCL8 and CXCL1 from human airway smooth muscle (ASM) cells. The concentrations of a, b) CXCL8 and c, d) CXCL1 in supernatant from human ASM cells from subjects with (n=9) and without (n=9) chronic obstructive pulmonary disease (COPD) after 72 h stimulation with cigarette smoke extract (CSE) or transforming growth factor (TGF)-β1 were measured by ELISA. Data are presented as mean±sem. Two-way ANOVA plus Bonferroni post-test was used to determine statistical significance. **: p<0.01, ***: p<0.001, compared with control.

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    Figure 3–

    Production of matrix metalloproteinases (MMPs) from human airway smooth muscle (ASM) cells. The concentrations of a ,b) MMP-1, c, d) MMP-2, e, f) MMP-3, and g, h) MMP-10 in supernatant from human ASM cells from subjects with (n=8) and without (n=7) chronic obstructive pulmonary disease (COPD) after 72 h stimulation with cigarette smoke extract (CSE) or transforming growth factor (TGF-β1) were measured by ELISA. Data are presented as mean±sem. Two-way ANOVA plus Bonferroni post-test was used to determine statistical significance. *: p<0.05; **: p<0.01; ***: p<0.001, compared with control. #: p<0.05, comparison between two groups.

  • Figure 4–
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    Figure 4–

    Production of active matrix metalloproteinase (MMP)-1 from human airway smooth muscle (ASM) cells. The concentrations of active MMP-1 in the supernatants from human ASM cells from subjects with (n=5) and without (n=5) chronic obstructive pulmonary disease (COPD) after 72 h stimulation with cigarette smoke extract (CSE) or transforming growth factor (TGF)-β1 were measured by ELISA. Data are presented as mean±sem. Two-way ANOVA plus Bonferroni post-test was used to determine statistical significance. *: p<0.05; **: p<0.01, compared with control.

  • Figure 5–
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    Figure 5–

    The deposition of collagen VIII alpha 1 (COL8A1) from human airway smooth muscle (ASM) cells. The deposited COL8A1 in the extracellular matrix (ECM) from human ASM cells from subjects with (n=7) and without (n=7) chronic obstructive pulmonary disease (COPD) after 72 h stimulation with cigarette smoke extract (CSE) or transforming growth factor (TGF)-β1 was measured by ECM ELISA at an absorbance of 450 nm/570 nm. Data are presented as mean±sem. Two-way ANOVA plus Bonferroni post-test was used to determine statistical significance. *: p<0.05; ***: p<0.001, compared with control. #: p<0.05, comparison between two groups.

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    Figure 6–

    Collagen VIII alpha 1 (COL8A1) airway tissue staining. a) COL8A1 in airway bronchus from subjects with (n=10) and without (n=7) chronic obstructive pulmonary disease (COPD) was measured by immunohistochemistry. Specific staining was detected using a chemical chromophore, DAB (brown), and the cell nucleus was counterstained with haematoxylin (blue). Tissue structure was stained with haematoxylin and eosin. Scale bars=100 μm. b) Immunostaining of COL8A1 in non-COPD and COPD groups were quantified by positive staining area and corrected with isotype control. Data are presented as median. The Mann–Whitney test was used to determine statistical significance. *: p<0.05.

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    Figure 7–

    The effect of cigarette smoke extract (CSE) on 2-h cell attachment at an absorbance of 595 nm. Cell attachment on a CSE treated plate of human airway smooth muscle cells from subjects with (n=5) and without (n=5) chronic obstructive pulmonary disease (COPD) were measured by toluidine blue assay. Data are presented as mean±sem. Two-way ANOVA plus Bonferroni post-test was used to determine statistical significance. *: p<0.05; **: p<0.01; ***: p<0.001, compared with control.

Additional Files

  • Figures
  • Supplementary material

    Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.

    Files in this Data Supplement:

    • Supplementary figures -
      Figures S1
      Figures S2
      Figures S3
      Figures S4
      Figures S5
      Figures S6
      Figures S7
    • Supplementary methods -
      Immunohistochemistry
      Cell attachment assay
      Would healing assay
    • Supplementary tables -
      Table S1
  • Disclosures

    Files in this Data Supplement:

    • C-A. Brandsma
    • D. Postma
    • W. Timens
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Effects of cigarette smoke extract on human airway smooth muscle cells in COPD
Ling Chen, Qi Ge, Gavin Tjin, Hatem Alkhouri, Linghong Deng, Corry-Anke Brandsma, Ian Adcock, Wim Timens, Dirkje Postma, Janette K. Burgess, Judith L. Black, Brian G.G. Oliver
European Respiratory Journal Sep 2014, 44 (3) 634-646; DOI: 10.1183/09031936.00171313

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Effects of cigarette smoke extract on human airway smooth muscle cells in COPD
Ling Chen, Qi Ge, Gavin Tjin, Hatem Alkhouri, Linghong Deng, Corry-Anke Brandsma, Ian Adcock, Wim Timens, Dirkje Postma, Janette K. Burgess, Judith L. Black, Brian G.G. Oliver
European Respiratory Journal Sep 2014, 44 (3) 634-646; DOI: 10.1183/09031936.00171313
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