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. 2017 Apr;14(2):228-237.
doi: 10.1080/15412555.2016.1262340. Epub 2016 Dec 27.

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity

Jun-Ho Jang  1   2 , Hitendra S Chand  3 , Shannon Bruse  4 , Melanie Doyle-Eisele  5 , Christopher Royer  5 , Jacob McDonald  5 , Clifford Qualls  6 , Aloysius J Klingelhutz  7 , Yong Lin  5 , Rama Mallampalli  1   2 , Yohannes Tesfaigzi  5 , Toru Nyunoya  1   2
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V体育平台登录 - Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity

Jun-Ho Jang et al. COPD. 2017 Apr.

Abstract

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs VSports手机版. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema. .

Keywords: Airway epithelial cells; alveolar epithelial cells; cellular senescence; cigarette smoke; connective tissue growth factor; non-human primates. V体育安卓版.

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Conflict of interest statement

DELARATION OF INTEREST

All authors state there is no conflicts of interest.

Figures

Figure 1
Figure 1. CTGF expression is increased in lung epithelial cells of ex-smokers with increasing COPD severity
A. The demographic and clinical data for ex-smokers based on the clinical stage of COPD (GOLD 0: n=6, GOLD 2: n=3, GOLD 3–4: n=8) analyzed in this study (**p <0.01). GOLD: The Global Initiative for Chronic Obstructive Lung Disease, FEV1: Forced expiratory volume in 1 second, FVC: Forced vital capacity % FEV1: % predicted value of FEV1. B. Analysis of CTGF expression in airway epithelium of ex-smokers with COPD. Representative micrographs showing CTGF-immunopositive cells (red) in airway epithelial cells from ex-smokers with COPD at GOLD stage 0, 2 and 3–4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Quantitative analysis of CTGF-positive airway cells in the three clinical groups are also shown (*p < 0.05; ***p < 0.001). C. Analysis of CTGF expression in alveolar cells of ex-smokers with COPD. Representative micrographs showing CTGF-immunopositive cells (red) in alveolar cells from ex-smokers with COPD as mentioned above. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Quantitative analysis of CTGF-positive airway cells in the three clinical groups are also shown (*p < 0.05; ***p < 0.001).
Figure 1
Figure 1. CTGF expression is increased in lung epithelial cells of ex-smokers with increasing COPD severity
A. The demographic and clinical data for ex-smokers based on the clinical stage of COPD (GOLD 0: n=6, GOLD 2: n=3, GOLD 3–4: n=8) analyzed in this study (**p <0.01). GOLD: The Global Initiative for Chronic Obstructive Lung Disease, FEV1: Forced expiratory volume in 1 second, FVC: Forced vital capacity % FEV1: % predicted value of FEV1. B. Analysis of CTGF expression in airway epithelium of ex-smokers with COPD. Representative micrographs showing CTGF-immunopositive cells (red) in airway epithelial cells from ex-smokers with COPD at GOLD stage 0, 2 and 3–4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Quantitative analysis of CTGF-positive airway cells in the three clinical groups are also shown (*p < 0.05; ***p < 0.001). C. Analysis of CTGF expression in alveolar cells of ex-smokers with COPD. Representative micrographs showing CTGF-immunopositive cells (red) in alveolar cells from ex-smokers with COPD as mentioned above. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Quantitative analysis of CTGF-positive airway cells in the three clinical groups are also shown (*p < 0.05; ***p < 0.001).
Figure 2
Figure 2. Influenza virus infection induces CTGF expression in lung epithelial cells of non-human primates exposed to cigarette smoke
A. CTGF mRNA levels in epithelial cells obtained by bronchial brushings of IAV- or mock-infected NHPs following 4 wks of CS exposure were analyzed by quantitative RT-PCR. Data are shown as mean ± SEM (n = 3 per group; *p < 0.05). B. Analysis of CTGF expression in airway epithelium of NHPs exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in airway tissues from NHPs exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of NHPs (**p< 0.01). C. Analysis of CTGF expression in alveolar cells of NHPs exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in alveolar cells from NHPs exposed to CS + IAV or CS + mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of NHPs (***p< 0.001).
Figure 2
Figure 2. Influenza virus infection induces CTGF expression in lung epithelial cells of non-human primates exposed to cigarette smoke
A. CTGF mRNA levels in epithelial cells obtained by bronchial brushings of IAV- or mock-infected NHPs following 4 wks of CS exposure were analyzed by quantitative RT-PCR. Data are shown as mean ± SEM (n = 3 per group; *p < 0.05). B. Analysis of CTGF expression in airway epithelium of NHPs exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in airway tissues from NHPs exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of NHPs (**p< 0.01). C. Analysis of CTGF expression in alveolar cells of NHPs exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in alveolar cells from NHPs exposed to CS + IAV or CS + mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of NHPs (***p< 0.001).
Figure 2
Figure 2. Influenza virus infection induces CTGF expression in lung epithelial cells of non-human primates exposed to cigarette smoke
A. CTGF mRNA levels in epithelial cells obtained by bronchial brushings of IAV- or mock-infected NHPs following 4 wks of CS exposure were analyzed by quantitative RT-PCR. Data are shown as mean ± SEM (n = 3 per group; *p < 0.05). B. Analysis of CTGF expression in airway epithelium of NHPs exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in airway tissues from NHPs exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of NHPs (**p< 0.01). C. Analysis of CTGF expression in alveolar cells of NHPs exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in alveolar cells from NHPs exposed to CS + IAV or CS + mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of NHPs (***p< 0.001).
Figure 3
Figure 3. Influenza virus infection induces CTGF expression in lung epithelial cells of mice exposed to cigarette smoke
A. Analysis of CTGF expression in airway epithelium of mice exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in airway tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of mice (***p< 0.001). B. Analysis of CTGF expression in alveolar cells of mice exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in alveolar cells from mice exposed to CS + IAV or CS + mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Left panel shows quantitative analysis of CTGF–positive cells in the two groups of mice (*p< 0.05).
Figure 3
Figure 3. Influenza virus infection induces CTGF expression in lung epithelial cells of mice exposed to cigarette smoke
A. Analysis of CTGF expression in airway epithelium of mice exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in airway tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of CTGF–positive cells in the two groups of mice (***p< 0.001). B. Analysis of CTGF expression in alveolar cells of mice exposed to CS and IAV infection. Representative micrographs showing CTGF–immunopositive cells (red) in alveolar cells from mice exposed to CS + IAV or CS + mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Left panel shows quantitative analysis of CTGF–positive cells in the two groups of mice (*p< 0.05).
Figure 4
Figure 4. Transgenic overexpression of CTGF induces cellular senescence in human airway epithelial cells
A. Effect of CTGF overexpression on cell viability. Primary HBECs were transduced with a lentiviral vector (pReceiver) encoding either CTGF cDNA or an empty vector, and transduced cells were selected using hygromycin (5 µg/ml). The viable cell counts monitored at 0, 3 and 6 d post-transduction showed a significant (**p < 0.01) attenuation of cell growth in cells with CTGF-overexpression (CTGF-OE). B. CTGF overexpression induces cellular senescence as measured by SA-β-galactosidase activity. The percentage of SA β-gal positive cells/total cell number was measured for the transduced cells at 6 d. Data are expressed as mean ± SEM for three independent experiments (**p < 0.01). Representative photomicrographs of cells transduced with either empty or CTGF-OE vector and stained for β-gal activity (blue) are shown (scale bar, 10 µm). C. CTGF-OE induces p16 protein levels. HBECs treated as in A were lysed for immunoblot analysis of p53, p21, and p16 proteins after 3 d of culture. Immunoblotting data are representative of three experiments. D. HBECs were treated as in A. Complete medium obtained from the transduced cells were concentrated using a SpeedVac. Immunoblot analysis of CTGF was performed. E. HBECs were cultured for 3 d in conditioned medium obtained from CTGF-overexpressing cells or control cells. Cell proliferation was determined by MTT assay. Data are expressed as the mean ± SEM for two independent experiments with triplicate samples (**p < 0.01).
Figure 5
Figure 5. CTGF expression is positively associated with p16 accumulation in lung epithelial cells in vivo
A. Analysis of CTGF and p16 co-expression in airway epithelium of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). B. Analysis of CTGF and p16 co-expression in alveolar cells of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). C. Analysis of CTGF and p16 co-expression in airway epithelium of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway epithelium of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001). D. Analysis of CTGF and p16 co-expression in alveolar cells of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar cells of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001).
Figure 5
Figure 5. CTGF expression is positively associated with p16 accumulation in lung epithelial cells in vivo
A. Analysis of CTGF and p16 co-expression in airway epithelium of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). B. Analysis of CTGF and p16 co-expression in alveolar cells of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). C. Analysis of CTGF and p16 co-expression in airway epithelium of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway epithelium of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001). D. Analysis of CTGF and p16 co-expression in alveolar cells of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar cells of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001).
Figure 5
Figure 5. CTGF expression is positively associated with p16 accumulation in lung epithelial cells in vivo
A. Analysis of CTGF and p16 co-expression in airway epithelium of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). B. Analysis of CTGF and p16 co-expression in alveolar cells of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). C. Analysis of CTGF and p16 co-expression in airway epithelium of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway epithelium of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001). D. Analysis of CTGF and p16 co-expression in alveolar cells of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar cells of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001).
Figure 5
Figure 5. CTGF expression is positively associated with p16 accumulation in lung epithelial cells in vivo
A. Analysis of CTGF and p16 co-expression in airway epithelium of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue). (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). B. Analysis of CTGF and p16 co-expression in alveolar cells of mice exposed to CS and IAV infection. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar tissues from mice exposed to CS and IAV infection compared with CS and mock infection. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µM). Lower panel shows quantitative analysis of p16-positive cells (***p< 0.001). C. Analysis of CTGF and p16 co-expression in airway epithelium of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in airway epithelium of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001). D. Analysis of CTGF and p16 co-expression in alveolar cells of ex-smokers with COPD. Representative micrographs showing CTGF- (red) and p16 (green)-positive cells in alveolar cells of ex-smokers with COPD at GOLD stage 0, 2 and 3 or 4. Nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). Right panel shows quantitative analysis of p16-positive cells from the three clinical groups (**p < 0.01; ***p < 0.001).
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