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. 2023 Apr 3;24(7):6695.
doi: 10.3390/ijms24076695.

Amelioration of Pulmonary Fibrosis by Matrix Metalloproteinase-2 Overexpression

Ryo Inoue  1   2 , Taro Yasuma  1   3 , Valeria Fridman D'Alessandro  1 , Masaaki Toda  1 , Toshiyuki Ito  4 , Atsushi Tomaru  4 , Corina N D'Alessandro-Gabazza  1 , Tatsuki Tsuruga  4 , Tomohito Okano  4 , Atsuro Takeshita  1   3 , Kota Nishihama  3 , Hajime Fujimoto  4 , Tetsu Kobayashi  4 , Esteban C Gabazza  1
Affiliations

Amelioration of Pulmonary Fibrosis by Matrix Metalloproteinase-2 Overexpression

Ryo Inoue et al. Int J Mol Sci. .

Abstract

Idiopathic pulmonary fibrosis is a progressive and fatal disease with a poor prognosis. Matrix metalloproteinase-2 is involved in the pathogenesis of organ fibrosis. The role of matrix metalloproteinase-2 in lung fibrosis is unclear. This study evaluated whether overexpression of matrix metalloproteinase-2 affects the development of pulmonary fibrosis. Lung fibrosis was induced by bleomycin in wild-type mice and transgenic mice overexpressing human matrix metalloproteinase-2. Mice expressing human matrix metalloproteinase-2 showed significantly decreased infiltration of inflammatory cells and inflammatory and fibrotic cytokines in the lungs compared to wild-type mice after induction of lung injury and fibrosis with bleomycin. The computed tomography score, Ashcroft score of fibrosis, and lung collagen deposition were significantly reduced in human matrix metalloproteinase transgenic mice compared to wild-type mice. The expression of anti-apoptotic genes was significantly increased, while caspase-3 activity was significantly reduced in the lungs of matrix metalloproteinase-2 transgenic mice compared to wild-type mice. Active matrix metalloproteinase-2 significantly decreased bleomycin-induced apoptosis in alveolar epithelial cells. Matrix metalloproteinase-2 appears to protect against pulmonary fibrosis by inhibiting apoptosis of lung epithelial cells.

Keywords: apoptosis; collagen deposition; matrix degradation; matrix metalloproteinases; pulmonary fibrosis.

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Conflict of interest statement

E.C.G. and T.K. have a patent (WO2015087916A1) on the human MMP-2 TG mouse reported in this study. The other authors have declared no conflict of interest regarding the work reported in the current study.

Figures

Figure 1
Figure 1
Decreased inflammatory cells in the lungs from hMMP-2 TG mice. The total number of cells in bronchoalveolar lavage fluid was measured with a nucleocounter (A). The cells were stained with Giemsa to perform differential cell count. Differential cell count is expressed as the total number of each cell type (B). The number of mice: n = 8 in WT/SAL, n = 9 in hMMP-2 TG/SAL, n = 16 in WT/BLM, and n = 11 in hMMP-2 TG/BLM. Scale bars indicate 20 µm. All mice were female. Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.0001; *** p < 0.0001; ** p < 0.0001. WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin; ns, not significant.
Figure 2
Figure 2
Reduced inflammatory cytokines and total protein in hMMP-2 TG mice. Inflammatory cytokines and chemokines were measured by commercially available enzyme immunoassay kits following the manufacturer’s instructions. The number of mice: n = 8 in WT/SAL, n = 9 in hMMP-2 TG/SAL, n = 16 in WT/BLM, and n = 11 in hMMP-2 TG/BLM. Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin; ns, not significant.
Figure 3
Figure 3
Reduced BALF and relative mRNA levels of TNFα, IL-6, and MMP-9 in hMMP-2 TG mice. Inflammatory cytokines and chemokines were measured by commercially available enzyme immunoassay kits following the manufacturer’s instructions. The number of mice: n = 8 in WT/SAL, n = 9 in hMMP-2 TG/SAL, n = 16 in WT/BLM, and n = 11 in hMMP-2 TG/BLM. Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. BALF, bronchoalveolar lavage fluid; TNFα, tumor necrosis factor α; WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin.
Figure 4
Figure 4
Substrates of hMMP-2 are reduced in the lungs of hMMP-2 TG mice. IL-1β, osteopontin, MCP-3, and CTGF were measured by commercially available enzyme immunoassay kits following the manufacturer’s instructions. The number of mice: n = 8 in WT/SAL, n = 9 in hMMP-2 TG/SAL, n = 16 in WT/BLM, and n = 11 in hMMP-2 TG/BLM. Data are the mean ± S.D. All mice were female. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin, MCP-3, monocyte chemoattractant protein-3; CTGF, connective tissue growth factor; ns, not significant.
Figure 5
Figure 5
Amelioration of CT score of lung fibrosis and lung volumes in hMMP-2 TG mice. MicroCT was performed, and the radiological findings of fibrosis were scored by experts blinded to the treatment groups (A,B). Lung volumes were measured using microCT software (C). The number of mice: n = 4 in WT/SAL and hMMP-2 TG/SAL, n = 6 in WT/BLM, and n = 7 in hMMP-2 TG/BLM. Data are the mean ± S.D. All mice were female. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.0001; * p < 0.05. CT computed tomography; WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin; TG, transgenic.
Figure 6
Figure 6
Amelioration of histopathological and biochemical findings of pulmonary fibrosis in hMMP-2 TG mice. Ashcroft scoring was performed in lung tissue stained with hematoxylin & eosin by blinded experts for the treatment groups (A,B). Collagen deposition was evaluated after trichrome staining, and the total lung collagen volume fraction was calculated (C,D). The lung tissue hydroxyproline content was measured by a colorimetric assay (E). Scale bars indicate 200 µm. The number of mice in (A–C,E): n = 8 in WT/SAL, n = 9 in hMMP-2 TG/SAL, n = 16 in WT/BLM, and n = 11 in hMMP-2 TG/BLM. Data are the mean ± S.D. The number of mice in D: n = 4 in WT/SAL, n = 5 in hMMP-2 TG/SAL, n = 6 in WT/BLM, and n = 7 in hMMP-2 TG/BLM. All mice were female. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.001; *** p < 0.001; * p < 0.05. WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin; ns, not significant.
Figure 7
Figure 7
Inhibition of lung cell apoptosis by hMMP-2 overexpression. The total mRNA from the lungs of each mouse was extracted, and cDNA was synthesized using commercially available reagents. The number of mice: n = 6 in WT/SAL and hMMP-2 TG/SAL, n = 6 in WT/BLM, and n = 7 in hMMP-2 TG/BLM. Data are the mean ± S.D. All mice were female. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.001; *** p < 0.001; ** p < 0.01; * p < 0.05. WT, wild-type; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin; ns, not significant.
Figure 8
Figure 8
Reduced caspase-3 activity in mice with hMMP-2 overexpression. DNA fragmentation was evaluated by TUNEl assay, and the TUNEL-positive area was quantified using the WindRoof image processing software. Arrows indicate apoptotic cells (A,B). Caspase 3 activity was measured by a colorimetric assay (C). Data are the mean ± S.D. All mice were female. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.0001; * p < 0.05; hM MP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin.
Figure 9
Figure 9
Active hMMP-2 inhibits apoptosis of lung epithelial cells. A549 cells (A,B) and normal human bronchial epithelial (NHBE) cells (C,D) were cultured and pretreated with active hMMP-2 (0.5 µg/mL) or doxycycline (25 µg/mL) before stimulating with BLM (50 mU/mL) to evaluate apoptosis by flow cytometry. n = 4 in each treatment group. Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.0001; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin. DOXY, doxycycline.
Figure 10
Figure 10
Anti-integrin-β3 antibody blocks the anti-apoptotic activity of hMMP-2 in lung epithelial cells. A549 cells were pretreated with 5 µg/mL of anti-integrin-β3 or control IgG for 30 min before adding 0.5 μg/mL hMMP-2. The cells were then treated with BLM (50 mU/mL) or saline and cultured for 24 h, and the number of apoptotic cells was evaluated by flow cytometry (A,B). Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. **** p < 0.0001; hMMP-2, human matrix metalloproteinase-2; SAL, saline; BLM, bleomycin.
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