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. 2017 Jul 14;7(1):5392.
doi: 10.1038/s41598-017-05624-2.

Inhibition of CTGF ameliorates peritoneal fibrosis through suppression of fibroblast and myofibroblast accumulation and angiogenesis

Norihiko Sakai  1   2 , Miki Nakamura  3 , Kenneth E Lipson  4 , Taito Miyake  5 , Yasutaka Kamikawa  5 , Akihiro Sagara  5 , Yasuyuki Shinozaki  5 , Shinji Kitajima  5 , Tadashi Toyama  5 , Akinori Hara  5 , Yasunori Iwata  5 , Miho Shimizu  5 , Kengo Furuichi  5   6 , Shuichi Kaneko  7 , Andrew M Tager  8   9 , Takashi Wada  5   3
Affiliations

Inhibition of CTGF ameliorates peritoneal fibrosis through suppression of fibroblast and myofibroblast accumulation and angiogenesis

Norihiko Sakai et al. Sci Rep. .

Abstract

Peritoneal fibrosis (PF) is a serious complication in various clinical settings, but the mechanisms driving it remain to be fully determined. Connective tissue growth factor (CTGF) is known to regulate fibroblast activities. We therefore examined if CTGF inhibition has anti-fibrotic effects in PF. PF was induced by repetitive intraperitoneal injections of chlorhexidine gluconate (CG) in mice with type I pro-collagen promoter-driven green fluorescent protein (GFP) expression to identify fibroblasts. FG-3019, an anti-CTGF monoclonal antibody, was used to inhibit CTGF. CG-induced PF was significantly attenuated in FG-3019-treated mice. CG challenges induced marked accumulations of proliferating fibroblasts and of myofibroblasts, which were both reduced by FG-3019. Levels of peritoneal CTGF expression were increased by CG challenges, and suppressed in FG-3019-treated mice. FG-3019 treatment also reduced the number of CD31+ vessels and VEGF-A-positive cells in fibrotic peritoneum. In vitro studies using NIH 3T3 fibroblasts and peritoneal mesothelial cells (PMCs) showed that CTGF blockade suppressed TGF-β1-induced fibroblast proliferation and myofibroblast differentiation, PMC mesothelial-to-mesenchymal transition, and VEGF-A production. These findings suggest that the inhibition of CTGF by FG-3019 might be a novel treatment for PF through the regulation of fibroblast and myofibroblast accumulation and angiogenesis.

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Conflict of interest statement

Dr. Lipson receives income and owns equity in FibroGen Inc.

Figures

Figure 1
Figure 1
Pharmacological inhibition of CTGF protects mice from CG-induced peritoneal fibrosis. (a) Hydroxyproline content in the peritoneum from control IgG-, preventive FG-3019- or therapeutic FG-3019-treated mice following CG (Day 21, n = 7 mice/group, P; preventive regimen, T; therapeutic regimen). (b) Representative Mallory Azan-stained peritoneal sections of control IgG-treated or preventive FG-3019-treated mice (magnification x 200). Bars, 100 μm. (c) Peritoneal thickness from control IgG- or preventive FG-3019-treated mice following CG (Day 21, n = 5 mice/group). (d) Peritoneal expression of COLIα1 mRNA following CG (Day 21, n = 5 mice/group).
Figure 2
Figure 2
CG-induced peritoneal fibroblast accumulation and proliferation is dependent on CTGF. (a) Accumulation of proliferating fibroblasts (GFP+PCNA+). Peritoneal sections at day 21 were obtained from mice treated with control IgG or FG-3019. Representative tissue sections stained with anti-GFP antibody (green)/anti-PCNA antibody (red) are shown. Bars, 50 μm. (b) Numbers of GFP+ cells in the peritoneum are expressed as the mean number ± SEM per HPF (n = 5 mice/group). (c) Numbers of peritoneal GFP+PCNA+ cells (proliferating fibroblasts) are expressed as mean number ± SEM per HPF. (d) Percentages of peritoneal fibroblasts that are proliferating (GFP+PCNA+ cells/total GFP+ cells).
Figure 3
Figure 3
The accumulation of peritoneal αSMA+ myofibroblasts is mediated by CTGF in peritoneal fibrosis induced by CG. (a) Peritoneal expression of αSMA mRNA (n = 5 mice/each group). Data are expressed as mean copies of αSMA mRNA relative to copies of GAPDH mRNA ± SEM. (b) Accumulation of peritoneal myofibroblasts (αSMA+GFP+). Peritoneal sections at day 21 were obtained from mice treated with control IgG or FG-3019. Representative tissue sections stained with anti-αSMA antibody (red)/anti-GFP antibody (green) are shown. Bars, 50 μm. (c) Numbers of αSMA+GFP+ cells in the peritoneum are expressed as the mean number ± SEM per HPF (n = 5 mice/group). (d) Percentages of peritoneal myofibroblasts among total fibroblasts (αSMA+GFP+ cells/total GFP+ cells).
Figure 4
Figure 4
Peritoneal CTGF expression induced by CG challenge was down-regulated by FG-3019. (a) Peritoneal expression of CTGF mRNA at day 21 (n = 5 mice/group). Data are expressed as mean copies of CTGF mRNA relative to copies of GAPDH mRNA ± SEM. (b) The localization of CTGF protein in the peritoneum. Representative tissue sections are shown (magnification x 200). Bars, 100 μm. (c) CTGF+ areas in the peritoneum are expressed as mean ± SEM per HPF (n = 5 mice/group). (d) Representative tissue sections of dual-immunostainings of GFP (green) and CTGF (red) 21 days after CG onset. Bars, 50 μm. (e) Numbers of TGF-β1 + cells in the peritoneum are expressed as the mean number ± SEM per HPF (n = 5 mice/group). (f) Representative tissue sections of TGF-β1 immunostainings at day 21. Bars, 50 μm.
Figure 5
Figure 5
Blockade of CTGF suppressed fibroblast proliferation induced by TGF-β1. (a) Stimulation with 5 ng/ml TGF-β1 enhanced fibroblast proliferation in a time-dependent manner. (n = 3 cell preparations/group). (b) Validation of siRNA inhibition of CTGF. NIH3T3 cells were transfected with CTGF siRNA or control siRNA, and CTGF expression in response to TGF-β1 was determined 2 hours after the stimulation. Data are expressed as copies of CTGF mRNA relative to copies of β2 microglobulin mRNA ± SEM. (n = 3 cell preparations/group). (c) The effect of CTGF knockdown on TGF-β1-induced fibroblast proliferation. NIH3T3 cells were transfected with control siRNA or siRNA targeting CTGF, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). Data are expressed as mean ± SEM. (n = 6 cell preparations/group). (d) Effect of CTGF inhibition on TGF-β1-induced fibroblast proliferation. NIH3T3 cells were preincubated with FG-3019 (20 μg/ml) or control IgG (20 μg/ml) for 1 h, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). Data are expressed as mean ± SEM. (n = 7 cell preparations/group).
Figure 6
Figure 6
TGF-β1-induced myofibroblast differentiation was inhibited by CTGF blockade. (a) The effect of CTGF knockdown on TGF-β1-induced αSMA expression. NIH3T3 cells were transfected with control siRNA or siRNA targeting CTGF, and then stimulated with TGF-β1 (5 ng/ml for 6 hrs). Data are expressed as copies of αSMA mRNA relative to copies of β2 microglobulin mRNA ± SEM. (n = 3 cell preparations/group). (b) Effect of CTGF inhibition on TGF-β1-induced αSMA expression. NIH3T3 cells were preincubated with the indicated concentration of FG-3019 or control IgG for 1 h, and then stimulated with TGF-β1 (5 ng/ml for 6 hrs). Data are expressed as copies of αSMA mRNA relative to copies of β2 microglobulin mRNA ± SEM. (n = 15 cell preparations/group).
Figure 7
Figure 7
Inhibition of CTGF suppressed TGF-β1-induced MMT in PMCs. (a) αSMA expression upon stimulation of PMCs with 5 ng/ml TGF-β1. (n = 3 cell preparations/group). (b) Validation of siRNA inhibition of CTGF. PMCs were transfected with CTGF siRNA or control siRNA, and CTGF expression in response to TGF-β1 was determined 2 hours after the stimulation. (n = 3 cell preparations/group). (c) The effect of CTGF knockdown on TGF-β1-induced αSMA expression. PMCs were transfected with control siRNA or CTGF siRNA, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (n = 3 cell preparations/group). (d) Effect of CTGF inhibition on TGF-β1-induced αSMA expression. PMCs were preincubated with FG-3019 (10 μg/ml) or control IgG (10 μg/ml) for 1 h, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (n = 6 cell preparations/group). (e) E-cadherin expression upon stimulation of PMCs with 5 ng/ml TGF-β1. (n = 3 cell preparations/group). (f) The effect of CTGF knockdown on TGF-β1-induced E-cadherin expression. PMCs were transfected with control siRNA or CTGF siRNA, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (n = 3 cell preparations/group). (g) Effect of CTGF inhibition on TGF-β1-induced E-cadherin expression. PMCs were preincubated with FG-3019 (20 μg/ml) or control IgG (20 μg/ml) for 1 h, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (n = 3 cell preparations/group). All data are expressed as mean ± SEM.
Figure 8
Figure 8
CG-induced peritoneal angiogenesis and VEGF-A expression required CTGF. (a) The localization of CD31+ vessels in fibrotic peritoneum. Peritoneal sections at day 21 were obtained from mice treated with control IgG or FG-3019. Representative tissue sections stained with anti-CD31 antibody are shown. Bars, 50 μm. (b) Numbers of CD31+ vessels in the peritoneum are expressed as the mean number ± SEM per HPF (n = 5 mice/group). (c) Immunostainings of VEGF-A+ cells in fibrotic peritoneum at day 21. Representative tissue sections stained with anti-VEGF-A antibody are shown. Bars, 50 μm. (d) Numbers of VEGF-A+ vessels in the peritoneum are expressed as the mean number ± SEM per HPF (n = 5 mice/group). (e) Representative tissue sections of dual-immunostainings of GFP (green) and VEGF-A (red) at 21 days. Bars, 50 μm. (f) VEGF-A expression in PMCs stimulated with 5 ng/ml TGF-β1. (g) VEGF-A expression in PMCs. PMCs were transfected with control siRNA or CTGF siRNA, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (h) VEGF-A expression in PMCs. PMCs were preincubated with FG-3019 (10 μg/ml) or control IgG (10 μg/ml) for 1 h, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (i) VEGF-A expression in NIH3T3 cells stimulated with 5 ng/ml TGF-β1. (j) VEGF-A expression in NIH3T3 cells. NIH3T3 cells were transfected with control siRNA or CTGF siRNA, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). (k) NIH3T3 cells were preincubated with FG-3019 (10 μg/ml) or control IgG (10 μg/ml) for 1 h, and then stimulated with TGF-β1 (5 ng/ml for 24 hrs). In in vitro studies, all data are expressed as copies of VEGF-A mRNA relative to copies of β2 microglobulin mRNA ± SEM (n = 3 cell preparations/group).
Figure 9
Figure 9
Proposed schema for the development of peritoneal fibrosis regulated by CTGF. CTGF contributes to multiple processes fundamentally involved in the pathogenesis of peritoneal fibrosis, including the induction of fibroblast proliferation, myofibroblast differentiation, MMT and VEGF-A production, in an autocrine and/or paracrine manner. Targeting CTGF using FG-3019 may therefore be an effective therapeutic strategy for peritoneal fibrosis.
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