Intest Res.  2020 Apr;18(2):219-228. 10.5217/ir.2019.00037.

Inhibition of plasminogen activator inhibitor-1 attenuates against intestinal fibrosis in mice

• Imai J
• Yahata T
• Ichikawa H
• Ibrahim AA
• Yazawa M
• Sumiyoshi
• Inagaki Y
• Matsushima M
• Suzuki T
• Mine T
• Ando K
• Miyata T
• Hozumi K

• KMID: 2501387
• DOI: http://doi.org/10.5217/ir.2019.00037

Abstract

Background/Aims
Intestinal fibrosis is a major complication of Crohn’s disease (CD). The profibrotic protein transforming growth factor-β (TGF-β) has been considered to be critical for the induction of the fibrotic program. TGF-β has the ability to induce not only the expression of extracellular matrix (ECM) including collagen, but also the production of plasminogen activator inhibitor-1 (PAI-1) that prevents enzymatic degradation of the ECM during the onset of fibrotic diseases. However, the significance of PAI-1 in the developing intestinal fibrosis has not been fully understood. In the present study, we examined the actual expression of PAI-1 in fibrotic legion of intestinal inflammation and its correlation with the abnormal ECM deposition.
Methods
Chronic intestinal inflammation was induced in BALB/c mice using 8 repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). TM5275, a PAI-1 inhibitor, was orally administered as a carboxymethyl cellulose suspension each day for 2 weeks after the sixth TNBS injection.
Results
Using a publicly available dataset (accession number, GSE75214) and TNBS-treated mice, we observed increases in PAI-1 transcripts at active fibrotic lesions in both patients with CD and mice with chronic intestinal inflammation. Oral administration of TM5275 immediately after the onset of intestinal fibrosis upregulated MMP-9 (matrix metalloproteinase 9) and decreased collagen accumulation, resulting in attenuation of the fibrogenesis in TNBS-treated mice.
Conclusions
PAI-1-mediated fibrinolytic system facilitates collagen degradation suppression. Hence, PAI-1 inhibitor could be applied as an anti-fibrotic drug in CD treatment.

Keyword

Plasminogen activator inhibitor-1; Matrix metalloproteinase 9; Intestinal fibrosis; Crohn disease; 2,4,6-Trinitrobenzene sulfonic acid

Figure

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  Fig. 1. The mRNA expression of PAI-1 is elevated in the terminal ileum of CD patients. The mRNA expression of PAI-1 (PAI-1), Col1A1 (COL1A1), Col1A2 (COL1A2) and TGF-β1 (TGF-β1) genes in the ileal tissue from control subjects (n=11), patients with inactive CD (n=16) and active CD (n=51). Data were derived from a Gene Expression Omnibus (GEO) dataset GSE75214. (A) The expression of PAI-1 (PAI-1) was significantly upregulated in the terminal ileal mucosa of active CD patients compared with that of normal controls or inactive CD patients. (B) COL1A1 and TGF-β1 are positively correlated. Correlation coefficient between PAI-1 and Col1A1 or Col1A2 are 0.73 or 0.75. P-value by Kruskal-Wallis test. Correlation of 2 values using Pearson correlation coefficient. a P<0.001. PAI-1, plasminogen activator inhibitor-1; TFG-β, transforming growth factor β.

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  Fig. 2. TNBS-induced fibrotic model demonstrated the enhancement of plasminogen activator inhibitor-1 (Pai-1) expression. BALB/c mice were intrarectal injected with 2% TNBS. (A) Representative histological examples of each group (H&E and Masson trichrome staining). (B) Collagen deposition was determined by the Sircol collagen assay. (C) The mRNA expression of murine Pai-1 in the colon samples was assessed by real-time PCR. Expression of the Pai-1 gene was normalized against that of the Gapdh gene in the same RNA preparation (5 ETOH, 10 TNBS). Bars represent the median. P-value by Mann-Whitney U-test. a P<0.05; b P<0.01. TNBS, 2,4,6-trinitrobenzene sulfonic acid; ETOH, ethanol.

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  Fig. 3. PAI-1 inhibitor (TM5275) suppressed intestinal fibrosis in TNBS-induced murine colitis. (A) Representative histological examples of each group (H&E and Masson trichrome staining). (B, C) Analysis of macroscopic score and microscopic score. (D) Collagen deposition was determined by the Sircol collagen assay. (E) Body weight changes of each group. (F) Colon length of each group. (G) Local production of MMP-9 in the colonic mucosa of chronic colitis by ELISA. Bars represent the median. P-value by two-way ANOVA with Bonferroni post-hoc test. a P<0.05; b P<0.001. PAI-1, plasminogen activator inhibitor-1; TNBS, 2,4,6-trinitrobenzene sulfonic acid; MMP-9, matrix metalloproteinase 9; ETOH, ethanol; CMC, carboxymethyl cellulose.

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  Fig. 4. The anti-fibrotic mechanism of plasminogen activator inhibitor-1 (PAI-1) inhibitor. (A) Induced transforming growth factor β (TGF-β) and its autocrine signaling (straight and semicircular arrows with +) promote the deposition of collagen during colonic fibrosis in mice as shown in previous report.7 Simultaneously, it induces PAI-1 (arrow with red +), which inactivates tissue plasminogen activator (tPA)/plasmin axis, and reduces the total amount and active matrix metalloproteinase 9 (MMP-9) (downward red arrows), resulting in the accumulation of collagen (upward red arrows). (B) Conversely, PAI-1 inhibitor activates tPA/plasmin axis, up-regulates MMP-9 (upward blue arrows) and causes the degradation of collagen (downward blue arrows).

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