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. 2017 Dec;21(12):3821-3835.
doi: 10.1111/jcmm.13304. Epub 2017 Aug 7.

MMP-13 deletion decreases profibrogenic molecules and attenuates N-nitrosodimethylamine-induced liver injury and fibrosis in mice

Joseph George  1   2 , Mikihiro Tsutsumi  2 , Mutsumi Tsuchishima  2
Affiliations

MMP-13 deletion decreases profibrogenic molecules and attenuates N-nitrosodimethylamine-induced liver injury and fibrosis in mice

Joseph George et al. J Cell Mol Med. 2017 Dec.

Abstract

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase-13 (MMP-13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP-13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N-nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild-type (WT) and MMP-13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF-β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP-13 knockout mice. Protein and mRNA levels of CTGF, TGF-β1, α-SMA and type I collagen and the levels of MMP-2, MMP-9 and cleaved products of CTGF were markedly increased in NDMA-treated WT mice compared to the MMP-13 knockout mice. Blocking of MMP-13 with CL-82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full-length CTGF and its C-terminal fragments and active TGF-β1. The data demonstrate that MMP-13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP-13 and CTGF has potential therapeutic implications to arrest liver fibrosis.

Keywords: NDMA; MMP-13; N-nitrosodimethylamine; connective tissue growth factor; hepatic fibrosis.

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Figures

Figure 1
Figure 1
Serological and histological findings of NDMA‐induced liver injury in WT and MMP‐13 KO mice. (A and B) Serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) in WT and MMP‐13 KO mice treated with or without NDMA. The data are mean ± S.D. of eight animals. ***P < 0.001 NDMA‐treated mice versus untreated control mice; # P < 0.001 NDMA‐treated WT versus MMP‐13 KO mice. (C–F) Haematoxylin and eosin staining of liver tissues from WT and MMP‐13 KO mice without NDMA treatment (C and D) and with NDMA treatment (E and F). Note that the marked contrast of hepatic necrosis with haemorrhage and inflammatory cell infiltration in NDMA‐treated WT and MMP‐13 KO mouse livers shown in E and F, respectively. Original magnification, ×100 for C, D, and E; ×40 for F.
Figure 2
Figure 2
Masson's trichrome staining of liver tissues from WT and MMP‐13 KO mice without (A and B) and with NDMA treatment (C and D). Note the definite deposition of collagen fibres in NDMA‐treated WT mouse liver (C, arrows) and negligible staining in MMP‐13 KO mouse liver (D). Original magnification, ×100 for A, B, and C; ×40 for D. (E). Total collagen content in WT and MMP‐13 KO mice liver without and with NDMA treatment. The total content in the liver tissue was determined by estimating the hydroxyproline content, a characteristic imino acid present in collagen, and multiplied the value with 7.46. The data are mean ± S.D. of eight animals. ***P < 0.001 NDMA‐treated WT mice versus untreated WT mice and **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated WT versus MMP‐13 KO mice.
Figure 3
Figure 3
Immunohistochemical staining of α‐SMA in the liver tissues from WT and MMP‐13 KO mice without (A and B) and with NDMA treatment (C and D). The images are representative of eight mice per group. Note the marked contrast of α‐SMA‐positive stellate cells in NDMA‐treated WT (C) and MMP‐13 KO mouse livers (D). (E) Quantitative evaluation of activated stellate cells. Staining intensity of α‐SMA‐positive cells was analysed using Image‐pro discovery software. The data are mean ± S.D. of 10 randomly selected microscopic fields from eight mice per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice. Original magnification, ×100.
Figure 4
Figure 4
Immunohistochemical staining of CTGF in the liver tissues from WT and MMP‐13 KO mice without and with NDMA treatment. The images are representative of eight mice per group. (A and B) WT and MMP‐13 KO mice without NDMA treatment, respectively. Arrows, weak staining of CTGF in one row of the peri‐central hepatocytes. (C and D) NDMA‐treated WT and MMP13 KO mice, respectively. Note that marked contrast in number of the CTGF‐stained cells between WT (C) and MMP‐13 KO mice (D). (E) Quantification of CTGF staining using Image‐pro discovery software. The data are mean ± S.D. of 10 randomly selected microscopic fields from eight mice per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice. Original magnification, ×100.
Figure 5
Figure 5
Serum levels of IL‐6, TGF‐β1 and HA in WT and MMP‐13 KO mice treated without and with NDMA. The data are mean ± S.D. of eight mice per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; *P < 0.05 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice.
Figure 6
Figure 6
Expression of MMP‐9 and MMP‐2 in WT and MMP‐13 KO mice treated without and with NDMA. (A) Gelatin zymography for MMP‐9 and MMP‐2 in supernatants of the liver tissues from WT and MMP‐13 KO mice. Pro‐MMP‐9 of 105 kD, active MMP‐9 of 97 kD, pro‐MMP‐2 of 72 kD and active MMP‐2 of 64 kD are indicated. (B) Western blotting for proteins of MMP‐9 and MMP‐2 in the liver tissue. β‐Actin was used as a loading control. (C) Quantitative densitometric analyses of MMP‐9 activity and protein level. The data are mean ± S.D. of eight mice per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice. (D) Real‐time PCR analysis for the quantitative expression of MMP‐9 mRNA in WT and MMP‐13 KO. The data are mean ± S.D. of eight mice per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice.
Figure 7
Figure 7
Expression of CTGF and TGF‐β1 in WT and MMP‐13 KO mice treated without and with NDMA. (A) Real‐time PCR analysis for the quantitative expression of CTGF, TGF‐β1, α‐SMA and collagen type 1 mRNA in WT and MMP‐13 KO mice. The data are mean ± S.D. of six samples per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; *P < 0.05 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice. (B) Semiquantitative RT‐PCR for the expression of CTGF, TGF‐β1, α‐SMA and type 1 collagen mRNA in WT and MMP‐13 KO mice treated without and with NDMA. GAPDH was used as a loading control. The data are representative of six samples in each group.
Figure 8
Figure 8
Protein expression of CTGF, TIMP‐1, TGF‐β1, α‐SMA and collagen type 1 in WT and MMP‐13 KO mice treated without and with NDMA. (A) Western blotting for the proteins of full‐length CTGF and its C‐terminal fragments, TIMP‐1, active TGF‐β1, α‐SMA and collagen type 1 in the liver tissues from WT and MMP‐13 KO mice. The images are representative of six Western blots per each group. (B) Quantitative analysis of Western blot images. The data are mean ± S.D. of six images per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice and NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; *P < 0.05 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; # P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice; @, P < 0.05 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice.
Figure 9
Figure 9
Protein expression of CTGF and TGF‐β1 in cultured rat hepatic stellate cells. (A) Immunohistochemical staining for CTGF in cultured rat hepatic stellate cells. Marked and strong staining for CTGF was present in the activated hepatic stellate cells. Treatment with 10 μM CL‐82198 (final concentration) in the culture media resulted in a marked decrease of CTGF staining intensity. Original magnification, ×200. (B) Quantification of the staining intensity of CTGF in cultured stellate cells using Image‐pro discovery software. The data are mean ± S.D. of 10 randomly selected microscopic fields from five culture slides. ***P < 0.001 CL‐82198 treated cultures versus untreated cultures. (C) Western blotting for the proteins of full‐length CTGF and its C‐terminal fragments and active TGF‐β1 in hepatic stellate cells untreated and treated with CL‐82198. The images are representative of five Westerns per group. (D) Quantitative analysis of CTGF and TGF‐β1 Western blot images. The data are mean ± S.D. of five Western blot images per group. ***P < 0.001 CL‐82198‐treated cultures versus untreated cultures.
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