Female (6-week-old) BALB/c mice were from CLEA Japan Inc. (Tokyo, Japan). Mice were maintained in a specific pathogen-free environment. All experiments were approved by the Animal Experimentation Committee of Tokai University, Kanagawa, Japan (approved Nos. #183031 and #194027). Fibrotic inflammation was produced via intrarectal injection by 3.5-F catheter instillation of a 2% solution of TNBS (Research Organics, Cleveland, OH, USA) in 50% ethanol under light anesthesia with isoflurane. TNBS was provided as 8 weekly injections, with the dose sequentially increasing in the 0.4-1.6 mg range (0.4, 0.4, 0.8, 0.8, 1.2, 1.2, 1.6, and 1.6 mg). The mice were picked up food for 24 hours before TNBS injection and were held vertically for 60 seconds after the intrarectal injection. The ethanol group mice injected a similar volume of 50% ethanol. Tissues were harvested 3 days after the completion of the 8th times injections. The colon was opened longitudinally and snap frozen for preparation of frozen sections or RNA extraction. The formalin-fixed paraffin-embedded sections were prepared for H&E or Masson trichrome staining as collagen detection.

TM5275 is a specific inhibitor of PAI-1 molecules, and does not interfere with other serpin/serine protease systems [12].

TM5275 was orally administered as a carboxymethyl cellulose (CMC) suspension each day for 2 weeks after the 6th TNBS injection. The treatment had no effect on body weight or food intake throughout the experimental duration.

The following groups of mice were included in the present chronic study: (1) ethanol-treated mice (n = 10); (2) TNBS and CMC-treated mice (n = 10); (3) TNBS and 10 mg/kg/day TM5275-treated mice (n = 9); or (4) TNBS and 50 mg/kg/day TM5275-treated mice (n = 10).

The evaluator blinded to the groups scored the macroscopic colonic lesions which is shown in Table 1 [7,13].

Specimens obtained from the colons of all groups were immersed in 10% buffered formalin in phosphate-buffered saline overnight at room temperature, and followed by the standard procedure for paraffin embedding. H&E stain assess the degree of inflammation and then with collagen staining to detect tissue fibrosis. The evaluator who was unaware of the group assignments, scored all the microscopic histological scores which is shown in Table 1 [14].

Intestinal collagen content was evaluated using the Sircol Collagen Assay Kit (Biocolor, Carrickfergus, UK). The colonic specimens obtained from of all mice were homogenized in 0.5 M acetic acid containing 1% pepsin. The resulting mixture was stirred over night at 4°C. The total soluble collagen content of the mixture was determined. The acid-soluble type-I collagen supplied with the kit was used to generate a standard curve [15].

mRNA expressions of murine Pai-1 in the snap-frozen samples were assessed using quantitative PCR (qPCR). The amounts of target mRNA were quantified as described previously [16]. Total RNA (50 ng) was reverse transcribed using ImProm-II reverse transcriptase (Promega). The primers used for amplification of Pai-1 was 5′- GACACCCTCAGCATGTTCATC-3′ (forward primer) and 5′- AGGGTTGCACTAAACATGTCAG-3′ (reverse primer). The relative mRNA expressions of Pai-1 was normalized against that of the glyceraldehyde-3-phosphate dehydrogenase (Gapdh), gene in the same RNA preparation. qPCR was performed using SYBR Green qPCR Kits.

MMP-9 expression in the colon was evaluated using an ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). Frozen colon tissues were grounded in phosphate-buffered saline with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. After centrifugation using the Shake Master NEO system (15,000 rpm, 90 seconds, 4°C; Biomedical Science, Kobe, Japan), the supernatants of the homogenate were collected. The protein concentrations were determined using DC protein assays (Bio-Rad, Hercules, CA, USA).

All analyses were performed using Graph Prism5.0 (GraphPad Software Inc. San Diego, CA, USA). Between-group differences were evaluated using the Mann-Whitney U-test (nonparametric). For the comparison of more than 3 groups, statistical analyses were performed using two-way ANOVA (parametric) or the Kruskal-Wallis test (nonparametric), followed by Bonferroni correction for parametric samples as a post-hoc test. P< 0.05 was considered statistically significant.

An increase in TGF-β production, which directly induces PAI-1 transcription, is correlated with the abnormal deposition of collagen during the onset of fibrotic disease [6]. Therefore, we validated the expression of PAI-1 in patients with CD. Intestinal fibrotic strictures are most commonly located in the terminal ileum [17], therefore, using a publicly available dataset (accession number GSE75214) [18], we discovered that PAI-1 transcript was significantly upregulated in the terminal ileal mucosa of active patients with CD than in that of normal controls or inactive patients with CD (Fig. 1A). In addition, we confirmed the correlation between the expressions of PAI-1 and TGF-β1 and demonstrated that PAI-1 transcript was positively regulated by TGF-β1 in patients with CD (Fig. 1B). Of note, the correlation coefficients between PAI-1 and COL1A1 and between PAI-1 and COL1A2 were 0.73 and 0.75, respectively (Fig. 1B). These results indicated that PAI-1 expression increased with fibrosis-related genes in the fibrotic ileal lesion of patients with CD.

To examine whether Pai-1 expression facilitates intestinal fibrosis, we employed a TNBS-induced murine chronic colitis model. In the model, intestinal fibrosis gradually emerges with transition from the acute inflammatory phase to the chronic phase [7,15]. As demonstrated in our previous report, collagen deposition is completed following intrarectal injection of TNBS in the 8th week after the initial administration [7]. TNBStreated mice exhibited obvious collagen deposition, particularly in the submucosal layer (H&E and Masson trichrome staining) (Fig. 2A). A similar trend was observed in the collagen assay for quantifying actual deposition (Fig. 2B). We previously revealed that TGF-β1 production is increased in the colonic mucosa of TNBS-treated mice [7]. In addition, we detected an increase in local Pai-1 expression in fibrotic lesions when compared with the control mice (Fig. 2C). The results suggested that repeated intrarectal injection of TNBS-induced intestinal fibrosis along with local high expression of Pai-1, which was similar to the findings in patients with CD.

To assess the role of PAI-1 in TNBS-elicited intestinal fibrosis and to improve outcomes, we attempted to inhibit the signal by administering a PAI-1 inhibitor TM5275 after the collagen had accumulated (5 weeks later from the first treatment of TNBS) [7,12]. TM5275 is originally developed by ourselves and prevents PAI-1/tPA complex formation, thereby preserving active tPA [12,19]. Histological analysis revealed that TM5275 administration decreased TNBS-induced collagen deposition only in the 50 mg/kg group, compared with that in the CMCadministered control group (Fig. 3A). The effects of TM5275 on the macroscopic and microscopic score of colitis were clearly observed in the thickness, strictures and dilation of colon (Table 2) and the depth of lesion, the degree of fibrosis (Table 3), which is also shown as the score in Fig. 3B and C. This indicated that TM5275 attenuated the TNBS-induced colonic fibrosis. Consistently, tissue collagen content was significantly reduced in the 50 mg/kg TM5275 group but not in the 15 mg/kg group (Fig. 3D). These results suggested that the PAI-1 induction promoted collagen deposition and exacerbated colorectal fibrosis. In contrast, neither the loss of body weight nor the shortening of the gut was attenuated by the administration of TM5275 (Fig. 3E and F). These could be realized that the blockade of PAI-1 does not affect the chronic inflammation in the gut. PAI-1 reportedly promotes ECM deposition in fibrotic tissue by inhibiting MMP-9 production, and PAI-1 inhibition increases MMP-9 production [10,20,21]. Consistent with these reports, we detected the MMP-9 upregulation in the 50 mg/kg TM5275 group (Fig. 3G). The results indicated that targeting PAI-1 inhibition reduced collagen deposition via actual MMP-9 induction and that PAI-1 inhibitor could be useful in the treatment of intestinal fibrosis in patients with CD (Fig. 4).