Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases

Abstract

Background and aims: Crohn's disease [CD] and ulcerative colitis [UC] require lifelong treatment and patient monitoring. Current biomarkers have several limitations; therefore, there is an unmet need to identify novel biomarkers in inflammatory bowel disease [IBD]. Previously, the role of plasminogen activator inhibitor 1 [PAI-1] was established in the pathogenesis of IBD and suggested as a potential biomarker. Therefore, we aimed to comprehensively analyse the selectivity of PAI-1 in IBD, its correlation with disease activity, and its potential to predict therapeutic response.

Methods: Blood, colon biopsy, organoid cultures [OC], and faecal samples were used from active and inactive IBD patients and control subjects. Serpin E1 gene expressions and PAI-1 protein levels and localisation in serum, biopsy, and faecal samples were evaluated by qRT-PCR, ELISA, and immunostaining, respectively.

Results: The study population comprised 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients. We demonstrated that the serum, mucosal, and faecal PAI-1 concentrations are elevated in IBD patients, showing clinical and endoscopic activity. In responders [decrease of eMayo ≥3 in UC; or SES-CD 50% in CD], the initial PAI-1 level decreased significantly upon successful therapy. OCs derived from active IBD patients produced higher concentrations of PAI-1 than the controls, suggesting that epithelial cells could be a source of PAI-1. Moreover, faecal PAI-1 selectively increases in active IBD but not in other organic gastrointestinal diseases.

Conclusions: The serum, mucosal, and faecal PAI-1 concentration correlates with disease activity and therapeutic response in IBD, suggesting that PAI-1 could be used as a novel, non-invasive, disease-specific, faecal biomarker in patient follow-up.

Keywords: Inflammatory bowel disease; PAI-1; faecal marker.

Graphical Abstract

Figure 1.

Determination of the PAI-1 level in serum. A. The serum PAI-1 concentration was significantly higher in the patients with IBD [N = 29] compared with control [N = 14] subjects [p = 0.0409]. B. No significant difference was detected in the UC [N = 41] and CD [N = 22] patients [p = 0.9915]. C–D. Significantly increased concentration of PAI-1 was measured in the endoscopically [C] and clinically [D] active IBD [endoscopically: N = 61, clinically: N = 52] patients compared with the inactive patients [endoscopically: N = 27, clinically: N = 34] and with controls [N = 14] [endoscopically: inactive vs active p = 0.0018, control vs active p = 0.0239; clinically: inactive vs active p = 0.0069, control vs active p = 0.0273]. E. Correlation analysis between disease activity and the serum PAI-1 concentration. F–G. ROC analysis between the inactive [N = 27] and active [N = 61] IBD [F] and control [N = 14] vs active [N = 61] IBD patients [G]. H–I. The serum PAI-1 concentration significantly decreased after the induction of therapy in the responders [N = 10] [p = 0.047 and p = 0.002]. *p <0.05; **p <0.01. IBD-Inflammatory Bowel Disease, CD-Crohn's disease, UC-Ulcerative colitis, AUC-Area under curve.

Figure 2.

Gene expression pattern of Serpin E1 in mucosa. A. Relative gene expression fold change [Fc] in the IBD patients [N = 32] compared with controls [N = 13] of the Serpin E1 gene. B. Comparison of the gene expression patterns of Serpin E1 in CD [N = 12] and UC patients [N = 46]. C. Comparison of the gene expression patterns of Serpin E1 in inactive [N = 20] and endoscopically active IBD patients [N = 35]. D. Comparison of the gene expression patterns of Serpin E1 in controls [N = 13], untreated IBD patients [N = 23], responders [N = 16], and non-responders [N = 16]. *p <0.05; **p <0.01; ***p <0.001. IBD-Inflammatory Bowel Disease, CD-Crohn's disease, UC-Ulcerative colitis, GAPDH-Glyceraldehyde 3-phosphate dehydrogenase.

Figure 3.

The concentration of PAI-1 in the colonic mucosa. A. Localisation of PAI-1 in the colonic mucosa [blue: nucleus, green: PAI-1, scale bar: 25 µm]. B. Increased ratio of PAI-1 positive cells was detected in the mucosa of inflamed samples [N = 8] compared with non-inflamed tissues [N = 8, p = 0.0126] and controls [N = 8, p = 0.014]. C. The mucosal PAI-1 concentration was significantly higher in the patients with IBD [N = 49] compared with control [N = 19] subjects [p <0.0001]. D. No significant difference was detected between the CD [N = 12] and UC [N = 45] patients [p = 0.7903]. E–F. Significantly increased concentration of PAI-1 was measured in the endoscopically [E] and clinically [F] active [endoscopically active: N = 74, clinically active: N = 62] IBD patients compared with the inactive patients [endoscopically inactive: N = 24, clinically inactive: N = 36] and controls [N = 19] [endoscopically inactive vs active p <0.0001, control vs endoscopically active p <0.0001; clinically inactive vs active p = 0.0015, control vs clinically active p <0.0001]. G. Correlation analysis between the mucosal PAI-1 level and endoscopic disease activity. H–I. ROC analysis of the PAI-1 levels [H] between the inactive [N = 24] and active IBD patients [N = 74] and [I] between the control [N = 19] and active IBD [N = 74] subjects. J–K. After the effective therapy, the mucosal PAI-1 concentration decreased in responders [N = 26]. *p <0.05; **p <0.01; ***p <0.001; ****p <0.0001. IBD-Inflammatory Bowel Disease, CD-Crohn's disease, UC-Ulcerative colitis, PAI-1-Plasminogen activator inhibitor 1

Figure 4.

Gene and protein expression of Serpin E1 in human colon organoids. A. Relative gene expression fold change [Fc] of Serpin E1 is not significantly altered in organoid cultures [OCs] generated from active IBD patients [N = 4] and controls [N = 4] [p = 0.3094]. B. Transmission and confocal microscope images of control and IBD OCs. Localisation of the PAI-1 in control and IBD OCs [blue: nucleus, green: PAI-1, scale bar: 25 µm]. C–D. Increased isolated [IBD: N = 4, control: N = 4, p = 0.0286] [C] and secreted [IBD: N = 23, control: N = 14, p = 0.0423]. [D] PAI-1 levels in the IBD OCs compared with the controls. *p <0.05. IBD-Inflammatory Bowel Disease, BACT-Beta-actin.

Figure 5.

Stability of the faecal PAI-1 at room temperature and 4°C. RT-Room temperature.

Figure 6.

PAI-1 concentration in faecal samples. A. Faecal PAI-1 concentration was elevated in IBD patients [N = 42] compared with controls [N = 39] [p <0.0001]. B. No significant difference in the faecal PAI-1 levels was detected among the CD [N = 13] and UC [N = 29] patients [p = 0.7319]. C–D. Faecal PAI-1 levels were significantly higher in endoscopically [C] and clinically [D] active IBD patients [endoscopically: N = 90, clinically: N = 78] compared with the inactive patients [endoscopically: N = 60, clinically: N = 72] and controls [N = 29] [endoscopically: inactive vs active p <0.0001, control vs active p <0.0001; clinically: inactive vs active p <0.0001, control vs active p <0.0001]. E. Faecal PAI-1 concentration displayed a positive correlation with the endoscopic disease activity. F–G. ROC analysis of the faecal PAI-1 levels between the inactive [N = 60] and active [N = 90] IBD patients [F] and between the control [N = 29] and active [N = 90] IBD subjects [G]. H. Faecal PAI-1 concentration was elevated in non-responder patients [N = 23] compared with responders [N = 33] [p <0.0001]. IBD-Inflammatory Bowel Disease, CD-Crohn's disease, UC-Ulcerative colitis, AUC-Area under curve.

Figure 7.

Faecal PAI-1 concentration in organic gastrointestinal diseases. Increased PAI-1 levels were detected in faecal samples of active IBD patients [N = 86] compared with patients with organic gastrointestinal diseases [control: N = 29, p <0.0001; inactive IBD: N = 60, p <0.0001; adenoma: N = 15, p = 0.0346; colorectal cancer: N = 8, p <0.0001; diverticulosis: N = 9, p = 0.0005]. IBD-Inflammatory Bowel Disease, CRC-Colorectal cancer.