Action of matrix metalloproteinases at restricted sites in colon anastomosis repair: an immunohistochemical and biochemical study

Action of matrix metalloproteinases at restricted sites in colon anastomosis repair: an immunohistochemical and biochemical study Magnus S. Ågren, DrMedSci,a,b Thomas L. Andersen, PhD,b,c Ursula Mirastschijski, MD, PhD,d Ingvar Syk, MD, PhD,e Christine Bruun Schiødt, PhD,b Vikas Surve, PhD,b Jan Lindebjerg, MD,f and Jean-Marie Delaissé, PhD,b,c Copenhagen, Herlev, and Vejle, Denmark, Magdeburg, Germany, and Malmö, Sweden

Background. Dehiscence of colon anastomosis is a common, serious and potentially life-threatening complication after colorectal operation. In experimental models, impaired biomechanic strength of colon anastomoses is preventable by general inhibitors of matrix metalloproteinases (MMPs) and associated with collagen loss, which indicates a possible link between MMP-mediated collagen degradation and dehiscence. The precise localization of collagen degradation within the anastomotic area and the specific MMPs responsible are unknown. Methods. We have analyzed distinct zones within anastomoses using a novel microdissection technique for collagen levels, collagenolytic activity exerted directly by endogenous proteinases, and MMP-8 and MMP-9 immunoreactivity and their collagenolytic activity. Results. The most pronounced collagen loss was observed in the suture-holding zone, showing a 29% drop compared with adjacent micro-areas of 3-day-old anastomoses. Only this specific tissue compartment underwent a dramatic and significant increase in collagenolysis, amounting to a loss of 10% of existing collagen molecules in 24 hours, and was abolished by metalloproteinase inhibitors. The tissue surrounding suture channels was heavily infiltrated with CD68-positive histiocytes that expressed MMP-8 and to a lesser extent MMP-9. The collagenolytic effect of the interstitial collagenase MMP-8 was synergistically potentiated by the gelatinase MMP-9 when added to colon biopsies incubated in vitro. Conclusions. The unique finding of this study was that the specific tissue holding the sutures of a colon anastomosis lost the most collagen presumably through induction and activation of multiple MMPs that may explain the beneficial effects of treatment with non-selective MMP antagonists. (Surgery 2006;140:72-82.) From the Department of Surgery K, Bispebjerg Hospital, Copenhagen University Hospital, Denmark,a Nordic Bioscience A/S, Herlev, Denmark,b Clinical Cell Biology, Vejle Hospital, Denmark,c Department of Plastic, Reconstructive and Hand Surgery, University Hospital, Otto-von-Guericke-University, Magdeburg, Germany,d Department of Surgery, Malmö University Hospital, Sweden,e and the Department of Pathology, Vejle Hospital, Denmarkf

Anastomotic dehiscence that is associated with high morbidity and increased mortality is one of the most feared complications after operation for

Supported by a Marie Curie Fellowship of the European Community programme Human Potential under contract number HPMF-CT-2001-01429 and from the Danish Medical Research Council (22-02-0287). Accepted for publication December 2, 2005. Reprint requests: Magnus S. Ågren, DrMedSci, Bispebjerg Hospital, Department of Surgery K, Bispebjerg Bakke 23, Copenhagen DK-2400, Denmark. E-mail: [email protected] 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2005.12.013

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colorectal cancer.1,2 Its etiology is multifactorial.3,4 In elective operation, clinically proven leakage is reported to occur in up to 11% of colonic anastomosis, more often in male than in female patients.1,5,6 The biochemical and molecular processes responsible for anastomotic dehiscence are complex.7 The integrity of a colonic anastomosis is reflected in its biomechanic properties. In experimental models, anastomotic strength declines in the early postoperative course and is minimal on postoperative day 3.7–10 The sutures of a colonic anastomosis are retained primarily by the diagonally arranged collagen network in the submucosal layer of the bowel wall.11 The reduced tensile

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strength is also paralleled by loss of existing and mature collagen fibers of the anastomosis.8,9,12,13 This implies involvement of collagen degrading enzymes in the pathogenesis of anastomotic dehiscence. After day 3, the anastomotic strength and collagen synthesis increase rapidly.8,9,12,14 –16 Matrix metalloproteinases (MMPs) comprise a family of at least 25 vertebrate zinc-dependent endopeptidases capable of degrading collagens and other extracellular matrix (ECM) molecules.17 These enzymes are centrally involved in normal tissue remodeling and in pathologic processes such as osteoarthritis and periodontitis.18,19 The expression and activity of MMPs increase after tissue injury although the temporal and spatial pattern varies among the different MMPs during anastomotic wound repair.20 –22 Collagenase-2 (MMP-8) and gelatinase-B (MMP-9) seem to contribute significantly to tissue destruction in inflamed connective tissues.18,19,23,24 Other MMPs are expressed at later stages of anastomosis wound healing.21 The beneficial effects of broad-acting MMP inhibitors on the early anastomosis healing suggest that MMPs are indeed detrimental to the integrity of anastomoses.9,10,25 The mechanism has not been elucidated and intriguingly, the early improvements in biomechanic properties with MMP activity blockage were not accompanied by an increased collagen concentration in the anastomosis.9,10 It has been suggested that this is due to the fact that only the tissue in close proximity to the sutures is involved, which is too subtle to detect by analysis of the whole anastomosis segment that also includes adjacent non-involved tissue.10 The objective of this study was to examine the changes in collagen levels and collagenolytic activity in the direct vicinity of sutures by histology and by biochemical assays in a standardized colon anastomosis model in the rat. In addition, MMP-8 and MMP-9 protein localizations were studied by immunohistochemistry in vivo and their collagenolytic effects on endogenous collagens of normal rat colon biopsies incubated in vitro were investigated. MATERIALS AND METHODS Animals. Six-week old male Sprague–Dawley rats (Tac: SPRD N@Mol, M&B A/S, Silkeborg, Denmark) were housed in standard type III cages with aspen wood-chip bedding. Cages were kept in isolated cabinets with built-in ventilation and filtration kept at 20 to 24°C and relative humidity 50% to 60%. Light cycle was 12-hour light/dark. Rats were fed pellets (Altromin 1324) and tap water ad lib. The study was approved by the Danish National Experimental Animal Inspectorate.

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Operative Procedure. Animals were acclimatized for 7 days before being operated on. Five of 22 rats were allocated to the day 0 group (just after construction of the anastomosis), 12 to the postoperative day 3 group and 5 to the postoperative day 7 group. Anesthesia was induced by a subcutaneous injection of fentanyl citrate (0.18 mg/kg), fluanisone (5.6 mg/kg), and midazolam (2.8 mg/kg) mixture. A 3-cm midline laparotomy was made under aseptic conditions. A rubber catheter was inserted into the left colon 6 cm from the anus and a 10-mm segment of the colon was resected, corresponding to about 3 cm from the peritoneal reflection. End-to-end anastomosis was constructed using 8 single-layer interrupted 6/0 monofilament polyamide (P-1 Ethilon; Ethicon, Johnson & Johnson, Brussels, Belgium) sutures placed about 2 mm from the resection margins. The muscle layer of the laparotomy wound was closed by continuous 3/0 Vicryl (FS-2; Ethicon) and the skin with metal clips. Carprofen (5 mg/kg) was injected subcutaneously for analgesia (Rimadyl; Vericore, Dundee, Scotland). Tissue sampling. On day 0, and on postoperative days 3 and 7, rats were asphyxiated in carbon dioxide gas and killed by cervical dislocation. A 30-mm segment of the colon with the anastomosis in the middle was resected, cleared of mesentery, fat, and feces, washed gently in 3 ⫻ 10 ml sterile saline (0.9% NaCl) and bisected longitudinally into two-thirds and one-third segments. For the biochemical analyses, the two-thirds longitudinal segment was positioned with the serosal side onto the flat surface of a culture dish and stored at ⫺80°C. From the sutured area of a 2-mm wide segment of the anastomosis 4 standardized biopsies were obtained from the suture line using a 2-mm trephine (Miltex, Bethpage, NY) at ⫺20°C. The remaining anastomotic tissue on the culture dish represented the suture-free zone of the anastomosis. The colon tissue immediately proximal and distal to the anastomosis, each 14 mm long, represented non-injured colon (Fig 1). The tissue specimens were kept at ⫺80°C until analyzed for hydroxyproline, a biochemical marker for collagen, and collagenolysis. For the histopathologic and immunohistochemical analyses, the one-third longitudinal segment was pinned to Styrofoam and immersed into 4% paraformaldehyde for 2 hours at room temperature and overnight at 4°C. To examine the tissue response to the sutures, colon anastomoses obtained from 2 of the day 3 rats, were positioned on a wooden stick and fixed

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Biopsies Non-injured colon Proximal (14 mm)

3

1 Anastomosis (2 mm)

X

X

X

Non-injured colon Distal (14 mm)

X

2

3

Fig 1. Sampling sites for the biochemical analyses. Suture line is dotted, suture knots are shown by (X), and anastomosis area is indicated in dark grey. Biopsies comprised: 1) 2-mm punch biopsies around the suture knots (white circles); 2) remaining non-sutured area of the anastomosis; and 3) areas of non-injured colon (light grey), proximal and distal to the anastomosis.

in 4% paraformaldehyde overnight at room temperature. The fixed segments were cut transversely. Microassay for collagenolysis. The method of Delaissé and colleagues,26 which measures the degradation of physiologic substrates by endogenous collagenolytic enzymes was used. Biopsies were weighed to 0.1 mg in 2-ml polypropylene microcentrifuge tubes (Safe-T-Seal; USA/Scientific Plastics, Milton Keynes, England) and 200 ␮l of assay buffer composed of 10 mM Tris, 0.1 M NaCl, 10 mM CaCl2, 50 ␮M ZnCl2, 0.05% Brij®35 (Sigma-Aldrich, Munich, Germany), 3 mM NaN3, pH 7.50, was added. From each sample, 2 biopsies were used to measure the activity of the endogenous proteinases. The remaining 2 biopsies from each sample served as blank by adding to the assay buffer a metal chelator mixture composed of 20 mM ethylenediaminetetraacetic acid (EDTA) and 2 mM 1,10-phenanthroline: these block metal-dependent collagenolytic proteinases. Measurement of synergism between MMP-8 and MMP-9 in collagenolytic activity was carried out essentially as described by Engsig and colleagues.27 Twenty-eight 3-mm discs of full-thickness bowel wall, weighing 8.6 ⫾ 0.6 mg and containing 25.2 ⫾ 2.5 ␮g hydroxyproline, were excised at ⫺20°C from a 30-mm long segment of normal colon 6 cm proximal to the anus. Colon discs were incubated in 200 ␮l of assay buffer without exogenous enzymes, with MMP-8 or MMP-9 alone or with MMP-8 and MMP-9 combined in the absence or presence of the metal chelator mixture. Tubes were incubated at 37°C for 24 hours with gentle agitation and then centrifuged at 20,000g for 30 minutes at 4°C. To the superna-

tants (150 ␮l) the same volume of 12 M HCl was added, and to the pellet, containing insoluble collagens, 1,000 ␮l of 6 M HCl. The samples were hydrolyzed for 18 hours at 110°C in sealed tubes and the hydroxyproline contents determined colorimetrically.28 Collagen degradation was taken as the ratio between the amount of hydroxyproline in supernatant to the total hydroxyproline content of the biopsy multiplied by 100 and expressed as a percentage. Hydroxyproline and protein concentration determinations. Hydroxyproline concentration of biopsies was calculated as the total hydroxyproline content divided by the fresh weight of the biopsies. In 2 day 3 anastomoses, the total protein content was also measured according to a modification of the Lowry protocol.29 Histology and immunohistochemistry. The paraformaldehyde-fixed tissues were embedded in paraffin and 5-␮m thick sections were cut from each paraffin block. The progress of healing of the anastomoses was evaluated in Masson trichrome and hematoxylin-eosin-stained sections. MMP-8, MMP-9, and histiocytes (CD68) were assessed by immunohistochemistry. Sections were first washed in 99% ethanol, endogenous peroxidase quenched with 0.45% hydrogen peroxide in 99% ethanol, and rehydrated. Subsequently, the sections for MMP-8 immunostaining were treated with 10 mM sodium citrate (pH 6.0) overnight, and for MMP-9 and CD68 immunostaining with a proprietary retrieval solution (S2369; Dakocytomation, Glostrup, Denmark) overnight at 4°C followed by blocking with 0.5% casein in Tris-buffered saline (CAS/TBS). The sections were incubated overnight at 4°C with a rabbit anti-rat MMP-8 polyclonal antibody (SA370; Biomol Research Laboratories, Plymouth Meeting, Pa) diluted 1:500, a mouse anti-rat MMP-9 monoclonal antibody (RDI-MMP9abm2A5; Research Diagnostics, Flanders, NJ) diluted 1:250 or with a mouse anti-rat CD68 monoclonal antibody (clone ED1, MCA341R; Serotec, Oxford, UK) diluted 1:100. Sections treated with the mouse MMP-9 and CD68 antibodies were then incubated for 2 hours at room temperature with rabbit polyclonal anti-mouse immunoglobulins with less than 3% cross-reactivity to rat immunoglobulins (Z0456; Dakocytomation) and diluted 1:1,000 in CAS/TBS. These sections and those treated with the primary rabbit MMP-8 antibody were incubated with the peroxidase conjugated anti-rabbit EnVision⫹ system (K4003; Dakocytomation). Finally, the sections were stained with diaminobenzidine dihydrochloride (DAB⫹),

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Fig 2. Anastomosis healing from the day of construction (day 0) to postoperative day 7 as viewed in longitudinal sections days 0 (A, F), day 3 (B, D, G) and day 7 (C, E, H). A-C, Morphology of the anastomosis day 0 (A), day 3 (B) and day 7 (C). D, E, High-power view of frames in B and C of the anastomotic wound illustrating the profound collagen deposition day 7 (E) compared with the lack of collagen deposition day 3 (D). F-H, Increased edema and accumulation of inflammatory cells in the submucosa day 3 (G) about 0.5 to 1.5 mm from the margin of the anastomotic wound compared with day 0 (F) and day 7 (H). Scale bars: A-C, F-H, 400 ␮m; D-E, 200 ␮m. Masson’s trichrome stains cell nuclei black, cytoplasm red, muscular fibers red and collagen blue. m, mucosa; mp, muscularis propria; sm, submucosa. Anastomotic wound marked with asterisks in (A) and (B).

counterstained with Ehrlich hematoxylin and mounted in DPX resin mountant. Control sections, processed analogously but without the primary antibodies, showed no staining. The presence of MMP-8 and MMP-9 positive cells in

the longitudinal sections of the anastomotic wound, defined as the repair tissue formed between the resected colon ends, and adjacent histologically normal colon 5 to 10 mm from the edges of the wound was assessed. The numbers of

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was considered statistically significant. Numeric data are presented as mean ⫾ SEM.

Fig 3. Hydroxyproline concentration of full-thickness bowel wall adjacent to the anastomosis (open bars) and of the non-suture (hatched bars) and suture-holding (filled bars) tissue of the anastomosis day 0 (n ⫽ 5) and on postoperative day 3 (n ⫽ 10) and day 7 (n ⫽ 4). Mean ⫾ SEM. *P ⬍ .05; ***P ⬍ .001.

Fig 4. Collagen solubilization in biopsies from the nonsuture (hatched bars) and suture-holding (filled bars) tissue of colon anastomosis day 0 (n ⫽ 5) and on postoperative days 3 (n ⫽ 10) and 7 (n ⫽ 4) and of adjacent full-thickness large bowel wall day 3 (open bar). The values were derived after subtraction of the mean blank value of the EDTA/1,10-phenanthroline-treated paired biopsies. The group means of EDTA/1,10-phenanthroline-treated samples are presented in insert. Mean ⫾ SEM. *P ⬍ .05; **P ⬍ .01.

immunopositive cells in representative areas of the wound, and of the submucosa of the adjacent colon were graded from 0 to 4, without prior knowledge of the postoperative time point, where 0 ⫽ no positive cells, 1 ⫽ 1 to 19 positive cells, 2 ⫽ 20 to 79 positive cells, 3 ⫽ 80 to 249 positive cells, and 4 is greater than or equal to 250 positive cells per field of view (0.33 mm2). Statistical analyses. Student t test for unpaired or paired observations was applied to the data. P ⬍ .05

RESULTS One rat in the day 7 group died due to the anesthesia before being operated on. Body weights did not differ significantly among the day 0 (265 ⫾ 5 g, mean ⫾ SEM, n ⫽ 5), day 3 (252 ⫾ 7 g, n ⫽ 10) and day 7 (268 ⫾ 5 g, n ⫽ 4) groups of the 19 rats used for the biochemical analyses. Histopathologic evaluation of anastomosis repair. The sequential tissue responses and repair processes of the anastomosis were evaluated in longitudinal sections (Fig 2). At day 3, the collagenpoor anastomotic wound showed an extensive inflammatory infiltrate, mainly composed of plump and spindle-shaped cells (Figs 2 B, D). At day 7, collagen-rich granulation tissue, with proliferating fibroblasts and small vessels, had formed in the anastomosis gap (Figs 2 C, E). In the vicinity of the anastomosis, edema of the submucosa was noted on day 3 (Fig 2 G). In the edematous submucosa mononuclear inflammatory cells and few polymorphonuclear inflammatory cells were present. Edema was not evident on day 0 (Fig 2 F), and had essentially disappeared by day 7 (Fig 2 H). Collagen levels. Hydroxyproline, as an indicator of collagen, was measured in adjacent non-injured colon, in the proximity of the suture channels and in the tissue adjacent to the sutures of the anastomosis. In non-injured colon proximal and distal to the anastomosis, the hydroxyproline level on wet tissue weight basis was about 25% lower on postoperative day 3 compared with the normal colon day 0 just after the anastomosis was made (P ⬍ .001). This difference was also significant (P ⬍ .01) on dry tissue weight basis. By postoperative day 7, the hydroxyproline level in normal colon had returned to day 0 baseline concentrations (Fig 3). The hydroxyproline concentration of the whole anastomosis was lowered in the 3-day-old anastomosis by 43% compared with day 0. The most pronounced decline was observed in sutured area of the anastomosis, showing 29% lower values on a wet weight basis than that of adjacent non-sutured tissue and 63% lower than the corresponding site day 0. A similar relation was found when hydroxyproline was expressed per total protein content of the biopsies. For example, in one anastomosis the hydroxyproline content was reduced by 26% in sutured compared with nonsutured area when expressed per wet weight and by 32% when expressed per total protein content. In the second anastomosis, the corresponding values

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Fig 5. Number of MMP-8 and MMP-9 immunopositive cells per field of view (0.33 mm2) in longitudinal sections of the submucosa of the large bowel wall (䡩) 5 to 10 mm from the anastomotic wound (●) on day 0, and 3 and 7 days after operation. The number of MMP-8 (A) and MMP-9 (B) positive cells were increased days 3 and 7 in the wound compared with adjacent non-injured submucosa. Each point corresponds to a different rat. C, MMP-8-immunostained section of submucosa (grade 0) and anastomotic wound (grade 4) day 3. D, MMP-9-immunostained section of submucosa (grade 0) and anastomotic wound (grade 4) day 3. Scale bars: 200 ␮m.

were 39% and 42%, respectively. By postoperative day 7, the anastomotic hydroxyproline level had returned to the day 0 level and there was no significant difference in hydroxyproline concentration between suture and suture-free areas day 7 (Fig 3). Collagen degradation. The extent of collagen degradation was measured by the release of solubilized collagen, detected as hydroxyproline, from colon tissue incubated in vitro. On day 0, collagenolysis was minimal in all three tissue types and there was no significant difference in collagenolysis. On postoperative days 3 and 7, the collagen degradation was elevated significantly and severalfold only in suture-holding tissues of the anastomosis (Fig 4). Collagenolysis was significantly (P ⬍ .01) higher day 7 compared with day 3, and was high enough to degrade 30% of its collagen content in 24 hours. The presence of metal chelators during the incubations reduced greatly but not completely collagen solubilization (Fig 4, insert). Inactivation of aspartic, cysteine and serine proteinases with a gen-

eral proteinase inhibitor cocktail (EDTA-free Complete, 1836170; Roche Diagnostics, Mannheim, Germany) containing 1 ␮M pepstatin did not reduce the hydroxyproline background further. This indicates that the residual hydroxyproline in medium from incubated tissues most likely represents extracted rather than enzymatically solubilized collagen molecules. Evolution of MMP-8 and MMP-9 during the repair process. MMP-8 and MMP-9 were examined by immunohistochemistry. On day 0, scattered MMP-8 immunoreactivity was observed in blood cells and tissue debris in the anastomotic wound, and in adjacent submucosa. At this stage, all sections were negative for MMP-9 immunoreactivity. On postoperative days 3 and 7, more MMP-8 and MMP-9 positive cells were observed in the anastomotic wound compared with the submucosa proximal and distal to the wound, and compared with day 0 anastomotic wound. At all time points, MMP-8 seemed to be present at higher levels than MMP-9 in adjacent submucosa (Fig 5).

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Tissue response to sutures and associated MMP-8 and MMP-9 localization. The local cellular events around the sutures were assessed in transverse sections of 3-day-old anastomoses. In the submucosa of the non-sutured zone bordering the anastomotic wound collagen was observed whereas no collagen was observed in the vicinity of sutures in Masson’s trichrome-stained sections (Figs 6 A, B). Histiocytes dominated, verified with CD68 immunostaining, and only few neutrophils were noted in the non-sutured submucosa (Fig 6 C). The majority of the cells contained MMP-8 whereas MMP-9 expression was limited to a subpopulation (Figs 6 E, G). Around the sutures, the cell infiltrate, predominantly composed of spindle-shaped and plump cells and only sparsely by neutrophils, was markedly increased as opposed to adjacent nonsutured tissue of the anastomosis. Most of the spindle-shaped and plump cells were CD68 positive (Fig 6 D). A pronounced increase in the number of MMP-8 and MMP-9 positive cells was observed around the suture channels (Figs 6 F, H) compared with adjacent suture-free area (Figs 6 E, G). No foreign body giant cells were observed. Collagenolytic synergism between MMP-8 and MMP-9. To test the functional role of MMP-8 and MMP-9 on degradation of collagen molecules present in the colon, recombinant active MMP-8 and MMP-9 were added individually or together to biopsies of normal rat colon incubated in vitro at 37°C for 24 hours. Although both MMP-8 (0.3 ⫾ 0.2%) and MMP-9 (1.4 ⫾ 0.5%) displayed some collagenolytic activity separately compared with basal levels (0.1 ⫾ 0.1%), their combination showed a synergistic effect because collagenolysis was augmented more than 3-fold (5.4 ⫾ 2.5%) compared with the contribution to collagenolysis of MMP-8 and MMP-9 alone (Fig 7A). The positive control, purified bacterial collagenase derived from Clostridium histolyticum (Advance Biofactures, Lynbrook, NY) at 1 ng/␮l, caused 10.9 ⫾ 0.6% (n ⫽ 3) collagenolysis. Notably MMP-9 alone exhibited higher collagen solubilization activity than MMP-8 alone at 37°C. The specificity of the enzymes used was examined using native triple helical collagen as substrate. MMP-8 caused cleavage of intact collagen into the expected three-fourth and one-fourth (not shown) ␣-chain fragments whereas MMP-9 by itself did not cleave intact collagen molecules at 23°C. MMP-9 combined with MMP-8 degraded the collagen fragments into even smaller-sized collagen fragments (Fig 7 B). Thus, the most likely explanations for more collagen solubilization with MMP-9 were the presence of denatured collagens in the colon biopsies and the

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Fig 6. Tissue responses to sutures (asterisks) postoperative day 3 as viewed in Masson trichrome-stained (A, B), and CD68 (C, D), MMP-8 (E, F), and MMP-9 (G, H) immunostained transversal sections. The microphotographs are arranged with non-sutured areas to the left (A, C, E, G) and sutured areas of the submucosa to the right (B, D, F, H) in the panel. A and B, Lack of bluestained submucosal collagen in tissue surrounding a suture (B) compared with adjacent suture-free area (A). C, E, and G, Scanty cell infiltration with about half of the cells being CD68⫹ (C). Virtually all cells contain MMP-8 (E) whereas only a fraction of them contain MMP-9 (G). D, F, and H, Suture channel in the submucosa surrounded by numerous and predominantly CD68-immunopositive cells (D) and only by few neutrophils. The majority of CD68⫹ cells express MMP-8 (F) and only a few of them MMP-9 (H). Scale bars: 50 ␮m.

potential of MMP-9 to act as a telopeptidase thereby solubilizing single triple helical collagen molecules from the fibers. DISCUSSION Our study shows a series of very local changes specifically in the tissue holding the sutures in co-

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lon anastomoses. These changes include reduction of collagen content, concentration collagenolysis, histiocyte infiltration, and increased MMP-8 and MMP-9 levels postoperative day 3 and contribute to diminished matrix integrity. Previous studies on collagen metabolism during anastomotic repair have been restricted to analysis of whole segments of anastomoses. Hydroxyproline reductions of 30% to 40% compared with initial concentrations have been reported.9 We developed a microdissection technique that enabled isolation of sutured tissue from adjacent non-sutured tissue of the anastomosis. This tissue underwent even a more pronounced degradation of existing collagen fibers and amounted to a more than 60% drop in hydroxyproline concentration. About 25% of this hydroxyproline loss was attributed to a general reduction of collagen in the colon proximal and distal to the anastomotic area due to the operative trauma to the colon as indicated in earlier reports.14,30 Concurrently, collagenolysis was increased in this specific tissue compartment compared with

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adjacent suture-free area of the anastomosis. Because metal chelators inhibited collagenolytic activity completely, presumably one or, more likely, multiple MMPs were responsible for degradation of the existing anastomotic collagen. This further underscores the possible detrimental role of MMPs to the integrity of anastomosis, which is supported by the confined localization of MMPs to the suture line22 and preservation of biomechanic properties of colon anastomoses by non-selective hydroxamate MMP inhibitors.9,10,25 We focused on the possible pathogenic roles of neutrophil collagenase or MMP-8 and gelatinase-B or MMP-9. MMP-8, originally cloned and sequenced from human neutrophils,31 is also expressed by other cells such as epithelial cells, endothelial cells, chondrocytes, odontoblasts, smooth muscle cells, rheumatoid synovial fibroblasts, and monocytes/macrophages.24,32 MMP-8 is

Fig 7. Effect of MMP-8 and MMP-9 alone or combined on degradation of rat colon collagens (A) and native, intact triple helical collagen (B). Full-length recombinant human latent MMP-8 (CC067; Chemicon International, Temecula, Calif) was completely activated with 1 mM aminophenylmercuric acetate (APMA) for 60 minutes at 37°C and 30 minutes at ambient temperature. Full-length recombinant mouse MMP-9, expressed in baby hamster kidney cells, was purified by means of 2 consecutive chromatographic steps on gelatin Sepharose and concanavalin A Sepharose columns. MMP-9 was fully activated with 1 mM APMA for 60 minutes at 37°C. MMP-8 and MMP-9 were used at 50 nM (final concentrations) determined by active site titration with the tightbinding hydroxamate MMP inhibitor BB-9427 using a synthetic peptidic fluorogenic substrate (M-1895; Bachem, Bubendorf, Switzerland). A, Synergism between MMP-8 and MMP-9 on collagen solubilization of normal left colon biopsies incubated for 24 hours at 37°C. Individual values, with the mean blank value of the EDTA/ 1,10-phenanthroline-treated paired biopsies subtracted, are presented. B, Specific digestion of native rat collagen fibrils (50 ␮g), prepared as described by Dean and Woessner48 after incubation without MMPs (lane 1), with MMP-8 alone (lane 2), MMP-9 alone (lane 3), MMP-8 and MMP-9 together without (lane 4), or with 20 mM EDTA/2 mM 1,10 phenanthroline (lane 5) for 4 hours at 23°C in assay buffer totalling 50 ␮l. The reactions were stopped by the addition of 40 mM EDTA (final concentration). Samples were denatured in Laemmli buffer containing 2.5% ␤-mercaptoethanol for 10 minutes at 95°C, electrophoresed in 7.5% sodium dodecyl sulfate-polyacrylamide gel, which was then stained with Coomassie Blue. The positions of molecular size standards are indicated in kilodaltons. Fifteen micrograms of collagen were loaded in each lane.

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the most efficient type I collagenolytic enzyme and is closely involved in rupture of atherosclerotic plaques, abdominal aortic aneurysms, faulty wound healing, and other tissue-destructive processes.18,19,24,31,33,34 MMP-9, expressed mainly by neutrophils and macrophages,35 is unable to digest interstitial collagens, and is upregulated in healing colon anastomoses.22,36 Moreover, stromal MMP-9 overexpression is associated with increased anastomotic leakage,4 decreased collagen deposition,23 and poor tissue repair.37 In the present study, both MMP-8 and MMP-9 were increased in the anastomotic wound and were concentrated in close proximity to the sutures in the submucosa. Previous in vitro studies indicate that collagenases and gelatinases act in a synergistic manner in collagenolysis.27,38,39 In our study, a synergistic collagenolytic effect of MMP-8 and MMP-9 on normal colon biopsies incubated in vitro was observed and collagenolysis was augmented three-fold compared with the contributions of MMP-8 and MMP-9 separately. A similar synergistic mechanism has been demonstrated previously between collagenase-1 or MMP-1 and MMP-9.39 However, the contributions of other members of the MMP family to collagenolysis directly or via MMP activation40,41 cannot be excluded, eg, the stromelysins (MMP-3 and MMP-10) and matrilysin-1 (MMP-7) that are expressed in intestinal wounds.21,22,42 Macrophages (CD68⫹) seemed to be the major source of MMP-8 and partially of MMP-9. Maximal macrophage infiltration has also been observed on the third postoperative day in colonic anastomoses.43 One possible mechanism for the accumulation of MMP-expressing macrophages is local hypoxia, induced by the sutures. These conditions may attract macrophages that subsequently secrete MMPs.22 Anastomosis hydroxyproline concentration is also severely depressed in ischemic rat colon.44 Collagen loss in the anastomosis is gradually compensated for by the synthesis of new collagen molecules.8,14 A burst of type I collagen and less so of type III collagen mRNA expression also occurs from postoperative days 3 to 4 and reaches maximal levels by postoperative day 7.13,16 In situ hybridization studies indicate that fibroblast-like cells in the submucosa and repair tissue between the colon cut ends are the source of the new collagen molecules.16 Accordingly, no collagen was observed in the genuine anastomotic area during the first 3 postoperative days whereas a massive collagen deposition was apparent day 7 and hydroxyproline concentrations were normalized both in adjacent uninjured colon and in the anastomosis. This indi-

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cates that the collagen synthetic pathway exceeded the degradative one resulting in net hydroxyproline accumulation. Surprisingly, collagenolysis was significantly elevated on day 7 compared with day 3, in our assay, suggesting still high collagenolytic potential despite the apparently unaltered MMP-8 and MMP-9 levels by the immunohistochemical analyses. In contrast, similar collagenolytic45 and gelatinolytic36 activities have been reported on days 3 and 7 of anastomotic extracts using non-colonic substrates. The discrepancy may be explained by differences in methods used to determine collagenolytic activities in tissues. Our method preserves the spatial relation between endogenous tissue proteinases and inhibitors, circumvents the disadvantages of incomplete or selective extraction of collagenolytic proteinases and the use of non-physiologic substrates, and takes into account the joint contribution of endogenous proteinases to collagen degradation. Possibly the elevated collagenolysis may reflect increased susceptibility of the newly deposited immature collagen molecules to proteolytic attack by MMPs.46 Admittedly we did not assess the quality of the formed collagen, ie, degree of cross-linking and thickness of the collagen fibrils. For example, attenuation of cross-linking with ␤-aminopropionitrile decreased average collagen fibril diameter and strength on postoperative day 7 in rat colon anastomosis.47 Our in vivo and in vitro data indicate that the critical event for the diminished postoperative integrity of a colon anastomosis is the degradation of existent collagen in the narrow zone around the sutures. This collagenolysis is mediated by at least 2 types of MMPs, the interstitial collagenase MMP-8 that makes the initial sitespecific cut of the intact collagen molecules followed by further degradation of the denatured collagens by the gelatinase MMP-9. These very local biochemical processes may contribute to increased risk of dehiscence. REFERENCES 1. Walker KG, Bell SW, Rickard MJ, Mehanna D, Dent OF, Chapuis PH, et al. Anastomotic leakage is predictive of diminished survival after potentially curative resection for colorectal cancer. Ann Surg 2004;240:255-9. 2. McArdle CS, McMillan DC, Hole DJ. Impact of anastomotic leakage on long-term survival of patients undergoing curative resection for colorectal cancer. Br J Surg 2005;92:1150-4. 3. Witte MB, Barbul A. Repair of full-thickness bowel injury. Crit Care Med 2003;31:S538-46. 4. Stumpf M, Klinge U, Wilms A, Zabrocki R, Rosch R, Junge K, et al. Changes of the extracellular matrix as a risk factor

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