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P006. Demonstration of functional matrix metalloproteinase activity in Crohn's disease fistulas: Effect of ramiprilate

E. Efsen1, T. Sœrmark2, A. Hansen3, E. Bruun3, J. Brynskov3

1Rigshospitalet, Copenhagen, Denmark; 2University Hospital Linköping, Linköping, Sweden; 3Herlev Hospital, University of Copenhagen, Copenhagen, Denmark

Aim: Increased expression of matrix metalloproteinase (MMP)-2, -3, and -9 has been demonstrated in Crohn's fistulas, but it is unknown whether they are biologically active and represent a therapeutic target. Therefore, we investigated the proteolytic activity of MMPs in fistula tissue and examined the effect of inhibitors, including clinically available drugs that beside their main action also suppress MMPs.

Materials and Methods: Fistula specimens were obtained by surgical excision from 22 patients with Crohn's disease and from 10 patients with fistulas due to other causes. Colonic endoscopic biopsies from six controls were also included. Total functional MMP-activity was measured by a high pressure liquid chromatography (HPLC)-based, fluorogenic MMP-substrate cleavage assay; the specific activity of MMP-2, -3 and -9 by the MMP Biotrak Activity Assay. The MMP inhibitors comprised ethylene-diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline.

Results: In Crohn's fistulas, about 50% of the total protease activity was attributable to MMP-activity. The average total MMP-activity was significantly higher (about 3.5-fold) in Crohn's fistulas (471 FU/μg protein, range 49–2661) compared to non-Crohn's fistulas (134 FU/μg protein, range 0–495, (p < 0.05)) and normal colon (153 FU/μg protein, range 77–243, (p < 0.01)). MMP-3 activity was increased in Crohn's fistulas (1.4 ng/ml, range 0–9.83) compared to non-Crohn's fistulas, (0.32 ng/ml, range 0–2.66, (p < 0.02)). The same applied to MMP-9 activity (0.64 ng/ml, range 0–5.66 and 0.17 ng/ml, range 0–1.1, respectively (p < 0.04)). Ramiprilate significantly decreased the average total MMP activity level by 42% and suppressed the specific MMP-3 activity by 72% which is comparable to the effect of GM6001 (87%). Moreover, MMP-9-activity was completely blunted by ramiprilate. Doxycycline had no effect on MMP-activity.

Conclusion: Increased functional MMP-activity, notably MMP-3 and -9, is present in Crohn's fistulas and may be inhibited by ramiprilate, a widely available ACE inhibitor.