GM6001 Increases Anastomotic Leakage following Colonic Obstruction Possibly by Impeding Epithelialization

Abstract

Background: Emergency operations performed on an obstructed colon are accompanied by an increased risk of anastomotic insufficiency. Tissue-destructive matrix metalloproteinase (MMP) activity is elevated in the obstructed colon and contributes to a loss of suture-holding submucosal collagen, which may be mediated by tumor necrosis factor (TNF)-α. Our aim was to study the effect of the non-selective MMP and TNF-α converting enzyme (TACE) inhibitor GM6001 (30 mg/kg) on anastomosis repair in obstructed left colon. GM6001 has been proved to be highly efficacious in elective anastomosis rodent models.

Methods: A partial obstruction of the distal colon was induced in male Sprague-Dawley rats. After 4 d the obstructed colonic segment was resected, and an end-to-end anastomosis was constructed. Seven days later, the anastomoses were evaluated for clinical leakage. Histopathological and immunohistochemical assessments were also performed. Finally, the direct effect of GM6001 on epithelialization was studied in cultured colonic epithelial cells.

Results: Unlike the robust beneficial effect on anastomosis under uncomplicated conditions, here GM6001 had a negative impact on anastomotic wound healing following colonic obstruction and substantially (p=0.004) more rats in the GM6001 group (75%) than in the control group (11%) had developed anastomotic leakage. In the anastomotic wounds, the myofibroblast abundance and cell proliferation were similar in the two groups. Histologically, GM6001 treatment resulted in wider and minimally epithelialized wounds that were commonly necrotic on the luminal side and infiltrated with numerous granulocytes. In vitro, GM6001 also delayed (p=0.026) epithelialization of denuded intestinal epithelium grown on type I collagen.

Conclusions: Non-selective MMP/TACE inhibition with GM6001 increased the anastomotic complications following colon obstruction. Inhibition of epithelialization is one possible mechanism responsible for the increased leakage following GM6001 treatment.

FIG. 1.

Design of the animal experiment (A) and effect of GM6001 on body weight changes (B) and food intake (C). (A) An obstructing silicone ring was applied around the colon day −4 and removed on day 0. End-to-end colonic anastomoses were made on day 0 and assessed on post-operative day 7. (B) Body weight changes relative to the initial weight on day −4 in the GM6001 and control groups. The box represents the interquartile range; the whiskers represent the range; and the filled square (GM6001)/circle (control) within the box represents the median. *p<0.05, **p<0.01. (C) Median food consumption after the obstruction period in the GM6001 (solid line) and control groups (dotted line).

FIG. 2.

Effect of GM6001 on the healing of 7-day-old anastomoses using the criteria in Table 1 on a scale from 0 to 4. GM6001 (n=12) treatment increased (p=0.004, χ² test) anastomotic leakage compared with the control treatment (n=9).

FIG. 3.

Effect of the MMP/TACE inhibitor GM6001 on morphology, myofibroblast abundance and cell proliferation in 7-day-old colon anastomoses (A, C, E, and G) compared with control group (B, D, F, and H). Representative sections of the typically wide, non-epithelialized anastomotic wounds in the GM6001 group (A and C) in contrast to the narrow wound gap that was completely covered with epithelium in the control group (B and D), as indicated by the double-headed arrows in A, B and D. Immunohistochemical staining for α-smooth muscle actin (E and F) and Ki-67 (G and H). (E) Superficial necrosis (arrow) is indicated by the negative α-smooth muscle actin staining in the GM6001-treated rats. (G and H) A high Ki-67 proliferation index was observed in the crypt epithelium on the granulation tissue (arrows). Hematoxylin and eosin (A and B). Alcian blue and Van Gieson (C and D). (Original magnifications: A and B, ×20; C and D, ×40; E and F, ×100; G and H, ×200). Color image is available online at www.liebertpub.com/sur

FIG. 4.

The effect of GM6001 on epithelial restitution using Caco-2 cells. Defects (0.50 mm² in surface area) in the confluent epithelial layer were caused by applying suction to a micropipette tip. The cultures (n=6) were incubated in the presence or absence of 10 μM GM6001 in 0.1% DMSO (control). The epithelial defects were quantified by image analysis. GM6001 treatment impaired the overall epithelial restitution (p=0.026, Mann-Whitney two-sided test). The box represents the interquartile range; the whiskers represent the range; and the filled square (GM6001)/circle (control) within the box represents the median value.